الجمعة، 25 ديسمبر 2009
الأربعاء، 13 مايو 2009
Protein Synthesis in Bacteria
Protein Synthesis in Bacteria
Development and Homeostasis Block
Thursday, September 23, 2004
PROTEIN SYNTHESIS - REGULATION OF TRANSLATION
Topics to be covered
1. Genetic code
7. Protein synthesis in mitochondria
1. Genetic code
Definition: relation between the nucleotide sequence of an mRNA and the amino acid sequence of the protein.
RNA is composed of 4 different nucleotides, adenosine (A), guanosine (G), cytidine (C), uridine (U) and there are 20 amino acids that have to be coded. Therefore, how many nucleotides are necessary to generate 20 amiino acids?
if use 1 then can only code for 4 amino acids
if use 2 then can only code for 16 amino acids
if use 3 then can code for 64 amino acids and
if use 4 can code for 256 amino acids.
Thus 1 or 2 is not enough and 3 or 4 is too many. Therefore the genetic code uses 3 nucleotides. However, since there are 4 bases but only 3 compose the code there are a number of possible formats that could be used, however a “commaless” triplet code is the one that has been universally adopted (Figure 1)
However since there are only 20 amino acids and three stop codons each amino acid (except for tryptophan and methionine) is coded by more than one codon : in other words the genetic code is degenerate which can be demonstrated in tabular form.(Figure 2A). The degree of degeneracy varies from one amino acid to another with arginine, leucine and serine being coded by 6 different nucleotide sequences. This degeneracy means that 20 amino acids can be matched to 61 codons using only 31 tRNAs (Figure 2B).
2. Overview of protein synthesis
While the genetic code itself resides in DNA, DNA is never used directly in the synthesis of proteins. DNA is “transcribed” into messenger RNA (mRNA) that carries information from DNA and it is mRNA that is then used to “translate” this information into a specific sequence of amino acids that constitute proteins.Similarly, amino acids cannot recognize codons directly, an adapter molecule is necessary, a transfer RNA (tRNA). Amino acids are incorporated into a protein in an order predetermined by the mRNA sequence. A tRNA can recognize more than one codon; very often, the first 2 nucleotides of the codon are sufficient to specify an amino acid and the third nucleotide varies. The first two nucleotides of the codon form a standard Watson-Crick pairing with the last two nucleotides of the tRNA. The third nucleotide of the codon forms a non standard Watson-Crick pairing or “wobble”.The overriding constraint in protein synthesis is that theere is little margin for error. Messenger RNA has to be translated correctly hence there are a variety of safeguards built in to the process to minimize errors. Protein synthesis only begins when all the components, appropriate mRNA, tRNAs with loaded amino acids, ribosomal subunits and other auxiliary factors come together to form a functional ribosome, the site of protein synthesis in the cell. Then, as a single mRNA moves stepwise through each ribosome, the sequence of nucleotides in the mRNA is translated into a corresponding sequence of amino acids to produce a nascent polypeptide chain.
3. Transcription (DNA > RNA)
RNA is synthesized on a DNA template in the process known as DNA transcription. Transcription generates the mRNA containing the information to synthesize a specific protein and also the other RNA molecules, ribosomal RNA, tRNA’s involved in the process. The key enzyme(s) involved in the process is RNA polymerase, an incredibly complex enzyme of molecular mass of 500,000kDa. DNA is transcribed by RNA polymerase binding to a specific start site or “promoter” on the DNA and proceeding until it reaches a termination signal.The DNA double helix is partially unwound by the polymerase and transcription always proceeds in a 3’ to 5’ direction on the DNA template so that the RNA produced is extended in a 5’ to 3’ direction.In theory any region of a DNA molecule could be transcribed into two RNA molecules (one form each strand). However only one strand is copied at any given time, although it is not always the same strand for different genes. The strand to be copied is determined by the promoter sequence.Typical mRNA is 70 – 10,000 nucleotides in length and is codeified by RNA splicing before becoming functional. Only one strand of DNA is copied at any one time. However either strand can be copied. The strand that is copied is determined by the promoter.The process is rapid and proceeds at a rate of about 30 nucleotides per second. There are 3 RNA polymerases, one makes mRNA while the other 2 make tRNA and rRNA. (Figure 3).
4. Translation (RNA > Protein)
Amino acids have first to be loaded onto specific tRNAs. The process is energetically unfavorable so amino acids are first activated by adenylation using ATP. The adenylated amino acid is then linked to the 3' end (always a CCA sequence) of the tRNA to form an aminoacyl tRNA. This reaction is catalyzed by a specific aminoacyl-tRNA synthase (Figure 4). There are distinct synthases for each of the 20 amino acids. Each synthase must be able to recognize the correct amino acid and an appropriate tRNA (Figure 5).
Simplistically, a polypeptide can be formed by the stepwise addition of new amino acids to its carboxy-terminal end. If the aminoacyl tRNAs can be correctly aligned, the only additional requirement would be a peptidyl transferase enzyme to synthesize the peptide bond between the incoming amino acid and the polypeptide.(Figure 6). The sequence of amino acids in the polypeptide would then be determined only by the tRNA that delivers the amino acid. (Figure 7).
Since alignment is determined by the mRNA, in theory no additional components are required for protein synthesis to proceed since the specificity is provided by the mRNA and the tRNA. (Figure 8).
However to ensure the absolute fidelity and to make the process much more efficient the whole process occurs on a protein/RNA complex, the ribosome(Figure 9a). Ribosomes and some detail of their structure can often be visualized by electron microscopy (Figure 9b).
The ribosome is composed of 2 subunits, the smaller of which binds to the mRNA and has two specific sites (A and P) that bind tRNA. The larger subunit contains the peptidyl transferase activity required to synthesize the peptide bond. It associates with both the tRNA and the small ribosomal subunit (Figure 10).
Together they ensure that 2 aminoacyl-tRNAs come together on the mRNA to form a peptide bond and that the complex advances smoothly along the mRNA (Figure 11).
5. Initiation, elongation and termination
In theory any mRNA can be translated in any of three different "reading frames" depending on the nucleotide sequence at which translation starts. Thus 3 different amino acid sequences can be obtained from a single mRNA (Figure 12).
However in both eucaryotic and procaryotic cells the correct reading frame is set by several initiation factors and by the fact that the start codon for protein synthesis is always AUG, which codes for methionine. Therefore in all proteins, at least as they are synthesized on the ribosome, their first amino acid is always methionine(Figure 13).
However there are a few significant differences between procaryotes and eucaryotes at this point in the process. Initiation always begins at the 5' end of the mRNA and in eucaryotes the 5' end is codeified by the addtion of a “cap”. In eucaryotes the 3' end of the mRNA is always polyadenylated. Furthermore, procaryotic mRNA is often polycistronic (one mRNA can produce several different proteins). This never occurs in eucaryiotes. Thus eucaryotic mRNA is monocystronic (Figure 14A). Initiation always begins at the 5' end of the mRNA and in eukaryotes the 5' end is codeified by the addtion of a "cap". The cap is composed of a terminal 7-methyl guanosine linked by a 5'-to-5' triphosphate bridge. The 1st and 2nd ribose of the mRNA are also often methylated at the 2' hydroxyl (Figure 14B). The end result is that the 5' end of the mRNA becomes positively charged.
Initiation factors are required in order for the ribosome to locate an initiation site. The first event is association of the small ribosomal subunit with a Met-tRNA. In procryotes IF2 is required to locate the AUG start codon and in eucaryotes eIF4 helps the small ribosomal subunit bind to the mRNA cap and search for an AUG. Once an AUG is located the large ribosomal subunit joins the complex and initiation is complete (Figure 14C). The initiation factors then dissociate from the ribosome and a second amino can be added to start the elongation process (Figure 14D).
Elongation proceeds when a second aminoacyl-tRNA is brought to the A site on the ribosme. Peptide bond formation occurs, the ribosome advances 3 bases along the mRNA, the Met-tRNA is displaced and there is a translocation of the peptidyl-tRNA from the A site to the P site (Figure 15A).
The previous diagram is an oversimplification. Several elongation factors are required to maintain the fidelity of the process. The reason relates to the degeneracy of the genetic code. Because of the similarities between certain codons (unavoidable in a 3 digit code). For example, AAC and AAU code for asparagine whereas AAA and AAG code for lysine. The anticodon of an aminoacyl tRNA loaded with asparagine would be able to bind by Watson-Crick and “wobble” pairing to AAA or AAG and asparagine could therefore become incorporated into a protein instead of lysine. Nevertheless the error rate in protein synthesis is extremely low. One incorrect amino acid is incorporated for every 10,000 correct amino acids. So that one out of every 25 proteins produced could contain an error.To maintain the low error rate an elongation factor termed EF1 (Tu in procaryotes) that is a GTP binding protein associates with any aminoacyl-tRNA that is transiently bound to the ribosomal A-site This association triggers EF1 's GTPase activity and GTP is hydrolysed to GDP and EF1 dissociates from the aminoacyl-tRNA (Figure 15B). Because this requires a finite amount of time, aminoacyl-tRNAs will dissociate from the A-site unless they have the correct anticodon. These GTP-binding/GTPase proteins are used quite often to function as biological "timers" or "switches" and function in kinetic proofreading (Figure 15C).
GTP is used as an energy source for elongation and termination. Overall 4 GTPs are hydrolyzed for each polypeptide bond. Therefore protein synthesis consumes more energy than any other biosynthetic process. Termination of the polypeptide chain occurs when any of three codons (UAA, UAG or UGA) is encountered. These three do not code for any amino acid and therefore do not recruit a tRNA (Figure 16A). Instead cytoplasmic "release factors" bind to the A site on the ribosome and causes the peptidyl transferase to add an H2O instead of forming a peptide bond, thus freeing the carboxy terminous of the polypeptide chain. Since this is the attachment to the peptidyl tRNA and the ribosome the newly synthesized polypeptide is released. The ribosomal subunits and the remaining tRNA dissociate from the mRNA (Figure 16B).
The entire process of protein assembly from individual amino acids and the participation of the various RNAs can be seen in this short animation. Animation courtesy of the Department of Biological Sciences at the University of Southern Mississippi (http://tidepool.st.usm.edu/)
Quite often several copies of a protein are produced by several ribosomes advancing sequentially along a single mRNA to form a "polyribosome" which is large enough to be visualized by electron microscopy (Figure 17).
6. Mechanism of action of antibiotics
Almost all of the widely used antibiotics are inhibitors of some component of the protein synthetic process. Those that are used therepeutically act to inhibit procaryotic protein synthesis and therefore stop bacterial growth. However there are others that inhibit both procaryotic and eucaryotic protein synthesis and some that are affective only in procaryotes (Figure 18). The latter have been useful in elucidating the cellular mechanisms of protein synthesis.The inhibition often involves an interaction between the antibiotic and the ribosome. The mechanism of inhibition of protein synthesis by many antibiotics has been elucidated. For example, the chemical structure of puromycin is similar to an aminoacyl-tRNA. This similarity allows puromycin to fool the ribosome and enables it to bind to the the A-site. The peptidyl transferase activity of the ribosome then catalyses the formation of an amide bond between the growing polypeptide chain and puromycin thereby causing premature termination of polypeptide chain elongation and release of the partially formed polypeptide from the ribosome. As might be expected puromycin inhibits protein synthesis in both procaryotes and eucaryotes.
7. Protein synthesis in mitochondria
Why do mitochondria (and chloroplasts) have their own genetic systems whereas other cytoplasmic organelles do not? There is no simple answer. The maintenance of a distinct protein synthetic system in mitochondria is an expensive proposition for the cell. Almost 100 proteins have to be made specifically for this purpose (Figure 19). It has been hypothesized that since most of the mitochondrial enzymes are associated with its membrane, these proteins are so hydrophobic that it would be impossible to get them from the cytosol to the membrane.
However, this now seems an unlikely explanation since there are many other hydrophobic proteins in the cell that can be transported from their cytoplasmic site of synthesis to a membrane without difficulty. Nonetheless, the mitochondria does have its own genome and because of its small size the entire human mitochondrial genome has been sequenced (Figure 20).There are some interesting differences between the mitochondrial and nuclear genomes. Every nucleotide in mitochondrial DNA appears to be a part of a coding sequence and some codons are unique to the mitochondrial genome (Figure 21). Interestingly, because it is acquired by non-Mendelian or cytoplasmic inheritance the mammalian mitochondrial genome is always maternal.
The End
Click here for a printable version of the lecture in PDF format. Note that this is a large file (approx 2mBytes) and the resolution of the images will depend on the quality of your printer.
For information or questions concerning this site please contact: mailto:djfranks@uottawa.ca
pour information au sujet de ce site s'adresser à:djfranks@uottawa.ca
1. Genetic code
Definition: relation between the nucleotide sequence of an mRNA and the amino acid sequence of the protein.
RNA is composed of 4 different nucleotides, adenosine (A), guanosine (G), cytidine (C), uridine (U) and there are 20 amino acids that have to be coded. Therefore, how many nucleotides are necessary to generate 20 amiino acids?
if use 1 then can only code for 4 amino acids
if use 2 then can only code for 16 amino acids
if use 3 then can code for 64 amino acids and
if use 4 can code for 256 amino acids.
Thus 1 or 2 is not enough and 3 or 4 is too many. Therefore the genetic code uses 3 nucleotides. However, since there are 4 bases but only 3 compose the code there are a number of possible formats that could be used, however a “commaless” triplet code is the one that has been universally adopted (Figure 1)
However since there are only 20 amino acids and three stop codons each amino acid (except for tryptophan and methionine) is coded by more than one codon : in other words the genetic code is degenerate which can be demonstrated in tabular form.(Figure 2A). The degree of degeneracy varies from one amino acid to another with arginine, leucine and serine being coded by 6 different nucleotide sequences. This degeneracy means that 20 amino acids can be matched to 61 codons using only 31 tRNAs (Figure 2B).
2. Overview of protein synthesis
While the genetic code itself resides in DNA, DNA is never used directly in the synthesis of proteins. DNA is “transcribed” into messenger RNA (mRNA) that carries information from DNA and it is mRNA that is then used to “translate” this information into a specific sequence of amino acids that constitute proteins.Similarly, amino acids cannot recognize codons directly, an adapter molecule is necessary, a transfer RNA (tRNA). Amino acids are incorporated into a protein in an order predetermined by the mRNA sequence. A tRNA can recognize more than one codon; very often, the first 2 nucleotides of the codon are sufficient to specify an amino acid and the third nucleotide varies. The first two nucleotides of the codon form a standard Watson-Crick pairing with the last two nucleotides of the tRNA. The third nucleotide of the codon forms a non standard Watson-Crick pairing or “wobble”.The overriding constraint in protein synthesis is that theere is little margin for error. Messenger RNA has to be translated correctly hence there are a variety of safeguards built in to the process to minimize errors. Protein synthesis only begins when all the components, appropriate mRNA, tRNAs with loaded amino acids, ribosomal subunits and other auxiliary factors come together to form a functional ribosome, the site of protein synthesis in the cell. Then, as a single mRNA moves stepwise through each ribosome, the sequence of nucleotides in the mRNA is translated into a corresponding sequence of amino acids to produce a nascent polypeptide chain.
3. Transcription (DNA > RNA)
RNA is synthesized on a DNA template in the process known as DNA transcription. Transcription generates the mRNA containing the information to synthesize a specific protein and also the other RNA molecules, ribosomal RNA, tRNA’s involved in the process. The key enzyme(s) involved in the process is RNA polymerase, an incredibly complex enzyme of molecular mass of 500,000kDa. DNA is transcribed by RNA polymerase binding to a specific start site or “promoter” on the DNA and proceeding until it reaches a termination signal.The DNA double helix is partially unwound by the polymerase and transcription always proceeds in a 3’ to 5’ direction on the DNA template so that the RNA produced is extended in a 5’ to 3’ direction.In theory any region of a DNA molecule could be transcribed into two RNA molecules (one form each strand). However only one strand is copied at any given time, although it is not always the same strand for different genes. The strand to be copied is determined by the promoter sequence.Typical mRNA is 70 – 10,000 nucleotides in length and is codeified by RNA splicing before becoming functional. Only one strand of DNA is copied at any one time. However either strand can be copied. The strand that is copied is determined by the promoter.The process is rapid and proceeds at a rate of about 30 nucleotides per second. There are 3 RNA polymerases, one makes mRNA while the other 2 make tRNA and rRNA. (Figure 3).
4. Translation (RNA > Protein)
Amino acids have first to be loaded onto specific tRNAs. The process is energetically unfavorable so amino acids are first activated by adenylation using ATP. The adenylated amino acid is then linked to the 3' end (always a CCA sequence) of the tRNA to form an aminoacyl tRNA. This reaction is catalyzed by a specific aminoacyl-tRNA synthase (Figure 4). There are distinct synthases for each of the 20 amino acids. Each synthase must be able to recognize the correct amino acid and an appropriate tRNA (Figure 5).
Simplistically, a polypeptide can be formed by the stepwise addition of new amino acids to its carboxy-terminal end. If the aminoacyl tRNAs can be correctly aligned, the only additional requirement would be a peptidyl transferase enzyme to synthesize the peptide bond between the incoming amino acid and the polypeptide.(Figure 6). The sequence of amino acids in the polypeptide would then be determined only by the tRNA that delivers the amino acid. (Figure 7).
Since alignment is determined by the mRNA, in theory no additional components are required for protein synthesis to proceed since the specificity is provided by the mRNA and the tRNA. (Figure 8).
However to ensure the absolute fidelity and to make the process much more efficient the whole process occurs on a protein/RNA complex, the ribosome(Figure 9a). Ribosomes and some detail of their structure can often be visualized by electron microscopy (Figure 9b).
The ribosome is composed of 2 subunits, the smaller of which binds to the mRNA and has two specific sites (A and P) that bind tRNA. The larger subunit contains the peptidyl transferase activity required to synthesize the peptide bond. It associates with both the tRNA and the small ribosomal subunit (Figure 10).
Together they ensure that 2 aminoacyl-tRNAs come together on the mRNA to form a peptide bond and that the complex advances smoothly along the mRNA (Figure 11).
5. Initiation, elongation and termination
In theory any mRNA can be translated in any of three different "reading frames" depending on the nucleotide sequence at which translation starts. Thus 3 different amino acid sequences can be obtained from a single mRNA (Figure 12).
However in both eucaryotic and procaryotic cells the correct reading frame is set by several initiation factors and by the fact that the start codon for protein synthesis is always AUG, which codes for methionine. Therefore in all proteins, at least as they are synthesized on the ribosome, their first amino acid is always methionine(Figure 13).
However there are a few significant differences between procaryotes and eucaryotes at this point in the process. Initiation always begins at the 5' end of the mRNA and in eucaryotes the 5' end is codeified by the addtion of a “cap”. In eucaryotes the 3' end of the mRNA is always polyadenylated. Furthermore, procaryotic mRNA is often polycistronic (one mRNA can produce several different proteins). This never occurs in eucaryiotes. Thus eucaryotic mRNA is monocystronic (Figure 14A). Initiation always begins at the 5' end of the mRNA and in eukaryotes the 5' end is codeified by the addtion of a "cap". The cap is composed of a terminal 7-methyl guanosine linked by a 5'-to-5' triphosphate bridge. The 1st and 2nd ribose of the mRNA are also often methylated at the 2' hydroxyl (Figure 14B). The end result is that the 5' end of the mRNA becomes positively charged.
Initiation factors are required in order for the ribosome to locate an initiation site. The first event is association of the small ribosomal subunit with a Met-tRNA. In procryotes IF2 is required to locate the AUG start codon and in eucaryotes eIF4 helps the small ribosomal subunit bind to the mRNA cap and search for an AUG. Once an AUG is located the large ribosomal subunit joins the complex and initiation is complete (Figure 14C). The initiation factors then dissociate from the ribosome and a second amino can be added to start the elongation process (Figure 14D).
Elongation proceeds when a second aminoacyl-tRNA is brought to the A site on the ribosme. Peptide bond formation occurs, the ribosome advances 3 bases along the mRNA, the Met-tRNA is displaced and there is a translocation of the peptidyl-tRNA from the A site to the P site (Figure 15A).
The previous diagram is an oversimplification. Several elongation factors are required to maintain the fidelity of the process. The reason relates to the degeneracy of the genetic code. Because of the similarities between certain codons (unavoidable in a 3 digit code). For example, AAC and AAU code for asparagine whereas AAA and AAG code for lysine. The anticodon of an aminoacyl tRNA loaded with asparagine would be able to bind by Watson-Crick and “wobble” pairing to AAA or AAG and asparagine could therefore become incorporated into a protein instead of lysine. Nevertheless the error rate in protein synthesis is extremely low. One incorrect amino acid is incorporated for every 10,000 correct amino acids. So that one out of every 25 proteins produced could contain an error.To maintain the low error rate an elongation factor termed EF1 (Tu in procaryotes) that is a GTP binding protein associates with any aminoacyl-tRNA that is transiently bound to the ribosomal A-site This association triggers EF1 's GTPase activity and GTP is hydrolysed to GDP and EF1 dissociates from the aminoacyl-tRNA (Figure 15B). Because this requires a finite amount of time, aminoacyl-tRNAs will dissociate from the A-site unless they have the correct anticodon. These GTP-binding/GTPase proteins are used quite often to function as biological "timers" or "switches" and function in kinetic proofreading (Figure 15C).
GTP is used as an energy source for elongation and termination. Overall 4 GTPs are hydrolyzed for each polypeptide bond. Therefore protein synthesis consumes more energy than any other biosynthetic process. Termination of the polypeptide chain occurs when any of three codons (UAA, UAG or UGA) is encountered. These three do not code for any amino acid and therefore do not recruit a tRNA (Figure 16A). Instead cytoplasmic "release factors" bind to the A site on the ribosome and causes the peptidyl transferase to add an H2O instead of forming a peptide bond, thus freeing the carboxy terminous of the polypeptide chain. Since this is the attachment to the peptidyl tRNA and the ribosome the newly synthesized polypeptide is released. The ribosomal subunits and the remaining tRNA dissociate from the mRNA (Figure 16B).
The entire process of protein assembly from individual amino acids and the participation of the various RNAs can be seen in this short animation. Animation courtesy of the Department of Biological Sciences at the University of Southern Mississippi (http://tidepool.st.usm.edu/)
Quite often several copies of a protein are produced by several ribosomes advancing sequentially along a single mRNA to form a "polyribosome" which is large enough to be visualized by electron microscopy (Figure 17).
6. Mechanism of action of antibiotics
Almost all of the widely used antibiotics are inhibitors of some component of the protein synthetic process. Those that are used therepeutically act to inhibit procaryotic protein synthesis and therefore stop bacterial growth. However there are others that inhibit both procaryotic and eucaryotic protein synthesis and some that are affective only in procaryotes (Figure 18). The latter have been useful in elucidating the cellular mechanisms of protein synthesis.The inhibition often involves an interaction between the antibiotic and the ribosome. The mechanism of inhibition of protein synthesis by many antibiotics has been elucidated. For example, the chemical structure of puromycin is similar to an aminoacyl-tRNA. This similarity allows puromycin to fool the ribosome and enables it to bind to the the A-site. The peptidyl transferase activity of the ribosome then catalyses the formation of an amide bond between the growing polypeptide chain and puromycin thereby causing premature termination of polypeptide chain elongation and release of the partially formed polypeptide from the ribosome. As might be expected puromycin inhibits protein synthesis in both procaryotes and eucaryotes.
7. Protein synthesis in mitochondria
Why do mitochondria (and chloroplasts) have their own genetic systems whereas other cytoplasmic organelles do not? There is no simple answer. The maintenance of a distinct protein synthetic system in mitochondria is an expensive proposition for the cell. Almost 100 proteins have to be made specifically for this purpose (Figure 19). It has been hypothesized that since most of the mitochondrial enzymes are associated with its membrane, these proteins are so hydrophobic that it would be impossible to get them from the cytosol to the membrane.
However, this now seems an unlikely explanation since there are many other hydrophobic proteins in the cell that can be transported from their cytoplasmic site of synthesis to a membrane without difficulty. Nonetheless, the mitochondria does have its own genome and because of its small size the entire human mitochondrial genome has been sequenced (Figure 20).There are some interesting differences between the mitochondrial and nuclear genomes. Every nucleotide in mitochondrial DNA appears to be a part of a coding sequence and some codons are unique to the mitochondrial genome (Figure 21). Interestingly, because it is acquired by non-Mendelian or cytoplasmic inheritance the mammalian mitochondrial genome is always maternal.
The End
Click here for a printable version of the lecture in PDF format. Note that this is a large file (approx 2mBytes) and the resolution of the images will depend on the quality of your printer.
For information or questions concerning this site please contact: mailto:djfranks@uottawa.ca
pour information au sujet de ce site s'adresser à:djfranks@uottawa.ca
الثلاثاء، 5 مايو 2009
Actinomycetes
Actinomycetes
Actinomycetes are best known for their ability to produce antibiotics
and are gram positive bacteria which comprise a group of
branching unicellular microorganisms. They produce branching
mycelium which may be of two kinds viz., substrate mycelium and
aerial mycelium. Among actinomycetes, the streptomycetes are the
dominant. The non‐streptomycetes are called rare actinomycetes,
comprising approximately 100 genera.
Members of the actinomycetes, which live in marine
environment, are poorly understood and only few reports are available
pertaining to actinomycetes from mangroves (Siva Kumar, 2001;
Vikineswari et al. 1997; Rathana Kala & Chandrika, 1993;
Lakshmanaperumalsamy, 1978).
Isolation of Actinomycetes from Sediments For isolation actinomycetes, the samples can be collected by inserting a polyvinyl corer (10cm dia.) (previously sterilized with alcohol) into the sediments.
The corer is sterilized with alcohol before sampling at each station. The
central portion of the top 2 cm sediment sample can be taken out with
the help of a sterile spatula. This sample can be transferred to a sterile
polythene bag and transported immediately to the laboratory. The sediment samples thus collected are air‐dried aseptically. After a week, the sediment samples are to be incubated at 550 C for 5 min (Balagurunathan, 1992). Then, 10‐fold serial dilutions of the sediment samples should be prepared, using filtered and sterilized 50% seawater.
One ml of the serially diluted samples should be plated in the Kuster’s
Agar (Siva Kumar, 2001) in triplicate petriplates. To minimize fungal
contamination, all agar plates should be supplemented with 50 ug/ml of nystatin. The actinomycete colonies that appear on the petriplates can be
counted from 5th day onwards, upto 28th day. All the colonies that are growing on the petriplates can be separately streaked in petriplates, subcultured, ensured for their axenicity and maintained in slants.
Identification of Actinomycets.
Various approaches for the identification of actinomycets are given briefly below:
a) Molecular Approach
The most powerful approaches to taxonomy are through the study of
nucleic acids. Because these are either direct gene products or the genes
themselves and comparisons of nucleic aids yield considerable
information about true relatedness.
Molecular systematics, which includes both classification and
identification, has its origin in the early nucleic acid hybridization
studies, but has achieved a new status following the introduction of
nucleic acid sequencing techniques (O’Donnell et al., 1993). Significance
of phylogenetic studies based on 16S rDNA sequences is increasing in
the systematics of bacteria and actinomycetes (Yokota, 1997). Sequences
of 16S ribosomal DNA have provided actinomycetologists with a
phylogenetic tree that allows the investigation of evolution of
actinomycetes and also provides the basis for identification.
Analysis of the 16S rDNA begins by isolating DNA (Hapwood,
1985) and amplifying the gene coding for 16S rRNA using the
polymerase chain reaction (e.g. Siva Kumar, 2001). The purified DNA
fragments are directly sequenced. The sequencing reactions are performed
using DNA sequencer in order to determine the order in which the bases
are arranged within the length of sample (Xu Li‐Hua, et al., 1999) and a
computer is then used for studying the sequence for identification using
phylogenetic analysis procedures. However, analysis of 16S rDNA
generally allows us to identify the organisms upto the genus level only.
b) Chemotaxonomical Approach
Chemotaxonomy is the study of chemical variation in organisms and the
use of chemical characters in the classification and identification. It is
one of the valuable methods to identify the genera of actinomycetes.
Studies of Cummins and Harris (1956) established that
actinomycetes have a cell wall composition akin to that of gram‐positive
bacteria, and also indicated that the chemical composition of the cell
wall might furnish practical methods of differentiating various types of
actinomycetes. This is because of the fact that chemical components of
the organisms that satisfy the following conditions, have significant
meaning in systematics.
K. Sivakumar 199
i. They should be distributed universally among the
microorganisms studied; and,
ii. The components should be homologous among the strains
within a taxon, while significant differences exist between the
taxa to be differentiated.
Presence of Diaminopimelic Acid (DAP) isomers is one of the
most important cell‐wall properties of gram‐positive bacteria and
actinomycetes. Most bacteria have a characteristic wall envelope,
composed of peptidoglycan. The 2, 6‐ Diaminopimelic Acid (DAP) is
widely distributed as a key aminoacid and it has optical isomers. The
systematic significance lies mostly in the key aminoacid with two amino
bases, and determination of the key aminoacid is usually sufficient for
characterisation. If DAP is present, bacteria generally contain one of the
isomers, the LL‐form or the meso‐form, mostly located in the
peptidoglycan. Major constituents of cell wall of actinomycetes (Lechevalier and
Lechevalier, 1970) are as follows:
I + +
II + +**
III +
** hydroxy DAP (may also be present)
The sugar composition often provides valuable information on
the classification and identification of actinomycetes. Actinomycete cells
contain some kinds of sugars, in addition to the glucosamine and
muramic acid of peptidoglycan. The sugar pattern plays a key role in the
identification of sporulating actinomycetes which have meso‐DAP in
their cell walls. However, the actinomycetes which have LL‐DAP along
with glycine (wall chemo type‐I) have no characteristic pattern of sugars
(Lechevalier and Lechevalier, 1970) and hence the whole cell sugar test
has not received much attention here.
c) Classical Approach
Classical approaches for classification make use of morphological,
physiological, and biochemical characters. The classical method
200 Actinomycetes
described in the identification key by Nonomura (1974) and Bergey’s
Manual of Determinative Bacteriology (Buchanan & Gibbons, 1974) is
very much useful in the identification of streptomycetes. These
characteristics have been commonly employed in taxonomy of
streptomycetes for many years. They are quite useful in routine
identification. They are as follows.
- Aerial Mass Colour
The colour of the mature sporulating aerial mycelium is recorded in a
simple way (White, grey, red, green, blue and violet). When the aerial
mass colour falls between two colour series, both the colours are
recorded. If the aerial mass colour of a strain to be studied shows
intermediate tints, then also, both the colour series are noted.
- Melanoid Pigments
The grouping is made on the production of melanoid pigments (i.e.
greenish brown, brownish black or distinct brown, pigment modified by
other colours) on the medium. The strains are grouped as melanoid
pigment produced (+) and not produced (‐).
- Reverse Side Pigments
The strains were divided into two groups, according to their ability to
produce characteristic pigments on the reverse side of the colony,
namely, distinctive (+) and not distinctive or none (‐). In case, a colour
with low chroma such as pale yellow, olive or yellowish brown occurs, it
is included in the latter group (‐).
. Soluble Pigments
The strains are divided into two groups by their ability to produce
soluble pigments other than melanin: namely, produced (+) and not
produced (‐). The colour is recorded (red, orange, green, yellow, blue
and violet).
Spore Chain Morphology
With regard to spore chains, the strains can be grouped into ‘sections’.
The species belonging to the genus Streptomyces are divided into three
sections (Shirling & Gottlieb, 1966), namely rectiflexibiles (RF),
retinaculiaperti (RA) and Spirales (S). When a strain forms two types of
spore chains, both are noted (e.g. SRA).
K. Sivakumar 201
Characteristics of the spore‐bearing hyphae and spore chains
should be determined by using direct microscopic examination of the
culture surface. Adequate magnification (400x) could be used to
establish the presence or absence of spore chains and to observe the
nature of sporophores.
Spore morphological characters of the strains can be studied by
inoculating a loopful of one week old cultures into 1.5% agar medium
contained in test tubes at 370C. The actinomycete should be suspended
and thoroughly mixed in the semisolid agar medium and 1 or 2 drops of
the medium could be aseptically pipetted on to a sterile glass slide. A
drop of agar should be spread well on the slide and allowed to solidify
into a thin film so as to facilitate direct observation under microscope.
The cultures should be incubated at 28 + 20C and examined periodically
for the formation of aerial mycelium, sporophore structure and spore
morphology.
Spore Surface
Spore morphology and its surface features should be observed under the
scanning electron microscope. The cross hatched cultures prepared for
observation under the light microscope can be used for this purpose.
RF Spore chains (400X)
RA Spore chains (400X)
Spiral Spore chains (400X)
202 Actinomycetes
The electron grid should be cleaned and adhesive tape should be placed
on the surface of the grid. The mature spores of the strain should be
carefully placed on the surface of the adhesive tape and gold coating
should be applied for half an hour and the specimen can be examined
under the electron microscope at different magnifications. The spore
silhouettes can be characterized as smooth, spiny, hairy and warty.
Assimilation of Carbon Source
The ability of different actinomycete strains in utilizing various carbon
compounds as source of energy should be studied following the method
recommended by International Streptomyces Project (Shirling and
Gottlieb, 1966). Chemically, pure carbon sources, certified to be free of
admixture with other carbohydrates or contaminating materials, should
be used for this purpose. Carbon sources for this test could be arabinose,
xylose, inositol, mannitol, fructose, rhamnose, sucrose and raffinose.
These carbon sources should be sterilized by ether sterilization without
heating.
Comparing the properties of the isolated strain with the
representative species found in the key of Nonomura (1974) and
Bergey’s Manual of Determinative Bacteriology (1974, 1989, 1994 & 2005) can help in the species level identification. If the isolated strain
could not be assigned to any of the valid representatives listed in the key
of Nonomura (1974) and Bergey’s Manual of Determinative Bacteriology
(Buchanan & Gibbons, 1974), then it can be identified based on the
numerical taxonomic studies.
d) Numerical Taxonomic Approach
Numerical taxonomy involves examining many strains for a large
number of characters prior to assigning the test organism to a cluster
based on shared features. The numerically defined taxa are polythetic;
so, no single property is either indispensable or sufficient to entitle an
organism for membership of a group. Once classification has been
achieved, cluster‐specific or predictive characters can be selected for
identification (Williams et al, 1983).
Numerical taxonomy was first applied to Streptomyces by
Silvestri et al. (1962). The numerical taxonomic study of the genus
Streptomyces by Williams et al. (1983) involves determination of 139 unit
characters for 394 type cultures of Streptomyces; clusters were defined at
77.5% or 81% Ssm and 63% Sj similarity levels, and the former co‐effieient
K. Sivakumar 203
is being used to define the clusters. His study includes 23 major, 20
minor and 25 single member clusters.
The numerical classification of the genus Streptomyces by
Kampfer et al. (1991) involves determination of 329 physiological tests.
His study includes 15 major clusters, 34 minor clusters and 40 single
member clusters which are defined at 81.5% similarity level Ssm using
the simple matching coefficient (Sokal and Michener, 1958) and 59.6 to
64.6% similarity level Sj using Jaccard coefficient (Sneath, 1957). Thus,
numerical taxonomy provides us with an invaluable framework for
Streptomyces taxonomy, including identification of species.
Preservation
The preservation methods are similar to that of bacteria such as subculturing, freezing especially in liquid nitrogen, freeze‐drying and
maintenance of strains in mineral oil.
Conclusion Studies on actinomycetes are very limited and the actinomycetes have been mentioned incidentally, on the microbial community of marine habitats. Further, only little information is available on the actinomycetes of the mangrove environment (which is one among the most productive coastal ecosystems) with regard to their occurrence and distribution (Vikineswari et al., 1997; Rathna Kala and Chandrika, 1993; Lakshmanaperumalsamy, 1978).
Actinomycetes are best known for their ability to produce antibiotics
and are gram positive bacteria which comprise a group of
branching unicellular microorganisms. They produce branching
mycelium which may be of two kinds viz., substrate mycelium and
aerial mycelium. Among actinomycetes, the streptomycetes are the
dominant. The non‐streptomycetes are called rare actinomycetes,
comprising approximately 100 genera.
Members of the actinomycetes, which live in marine
environment, are poorly understood and only few reports are available
pertaining to actinomycetes from mangroves (Siva Kumar, 2001;
Vikineswari et al. 1997; Rathana Kala & Chandrika, 1993;
Lakshmanaperumalsamy, 1978).
Isolation of Actinomycetes from Sediments For isolation actinomycetes, the samples can be collected by inserting a polyvinyl corer (10cm dia.) (previously sterilized with alcohol) into the sediments.
The corer is sterilized with alcohol before sampling at each station. The
central portion of the top 2 cm sediment sample can be taken out with
the help of a sterile spatula. This sample can be transferred to a sterile
polythene bag and transported immediately to the laboratory. The sediment samples thus collected are air‐dried aseptically. After a week, the sediment samples are to be incubated at 550 C for 5 min (Balagurunathan, 1992). Then, 10‐fold serial dilutions of the sediment samples should be prepared, using filtered and sterilized 50% seawater.
One ml of the serially diluted samples should be plated in the Kuster’s
Agar (Siva Kumar, 2001) in triplicate petriplates. To minimize fungal
contamination, all agar plates should be supplemented with 50 ug/ml of nystatin. The actinomycete colonies that appear on the petriplates can be
counted from 5th day onwards, upto 28th day. All the colonies that are growing on the petriplates can be separately streaked in petriplates, subcultured, ensured for their axenicity and maintained in slants.
Identification of Actinomycets.
Various approaches for the identification of actinomycets are given briefly below:
a) Molecular Approach
The most powerful approaches to taxonomy are through the study of
nucleic acids. Because these are either direct gene products or the genes
themselves and comparisons of nucleic aids yield considerable
information about true relatedness.
Molecular systematics, which includes both classification and
identification, has its origin in the early nucleic acid hybridization
studies, but has achieved a new status following the introduction of
nucleic acid sequencing techniques (O’Donnell et al., 1993). Significance
of phylogenetic studies based on 16S rDNA sequences is increasing in
the systematics of bacteria and actinomycetes (Yokota, 1997). Sequences
of 16S ribosomal DNA have provided actinomycetologists with a
phylogenetic tree that allows the investigation of evolution of
actinomycetes and also provides the basis for identification.
Analysis of the 16S rDNA begins by isolating DNA (Hapwood,
1985) and amplifying the gene coding for 16S rRNA using the
polymerase chain reaction (e.g. Siva Kumar, 2001). The purified DNA
fragments are directly sequenced. The sequencing reactions are performed
using DNA sequencer in order to determine the order in which the bases
are arranged within the length of sample (Xu Li‐Hua, et al., 1999) and a
computer is then used for studying the sequence for identification using
phylogenetic analysis procedures. However, analysis of 16S rDNA
generally allows us to identify the organisms upto the genus level only.
b) Chemotaxonomical Approach
Chemotaxonomy is the study of chemical variation in organisms and the
use of chemical characters in the classification and identification. It is
one of the valuable methods to identify the genera of actinomycetes.
Studies of Cummins and Harris (1956) established that
actinomycetes have a cell wall composition akin to that of gram‐positive
bacteria, and also indicated that the chemical composition of the cell
wall might furnish practical methods of differentiating various types of
actinomycetes. This is because of the fact that chemical components of
the organisms that satisfy the following conditions, have significant
meaning in systematics.
K. Sivakumar 199
i. They should be distributed universally among the
microorganisms studied; and,
ii. The components should be homologous among the strains
within a taxon, while significant differences exist between the
taxa to be differentiated.
Presence of Diaminopimelic Acid (DAP) isomers is one of the
most important cell‐wall properties of gram‐positive bacteria and
actinomycetes. Most bacteria have a characteristic wall envelope,
composed of peptidoglycan. The 2, 6‐ Diaminopimelic Acid (DAP) is
widely distributed as a key aminoacid and it has optical isomers. The
systematic significance lies mostly in the key aminoacid with two amino
bases, and determination of the key aminoacid is usually sufficient for
characterisation. If DAP is present, bacteria generally contain one of the
isomers, the LL‐form or the meso‐form, mostly located in the
peptidoglycan. Major constituents of cell wall of actinomycetes (Lechevalier and
Lechevalier, 1970) are as follows:
I + +
II + +**
III +
** hydroxy DAP (may also be present)
The sugar composition often provides valuable information on
the classification and identification of actinomycetes. Actinomycete cells
contain some kinds of sugars, in addition to the glucosamine and
muramic acid of peptidoglycan. The sugar pattern plays a key role in the
identification of sporulating actinomycetes which have meso‐DAP in
their cell walls. However, the actinomycetes which have LL‐DAP along
with glycine (wall chemo type‐I) have no characteristic pattern of sugars
(Lechevalier and Lechevalier, 1970) and hence the whole cell sugar test
has not received much attention here.
c) Classical Approach
Classical approaches for classification make use of morphological,
physiological, and biochemical characters. The classical method
200 Actinomycetes
described in the identification key by Nonomura (1974) and Bergey’s
Manual of Determinative Bacteriology (Buchanan & Gibbons, 1974) is
very much useful in the identification of streptomycetes. These
characteristics have been commonly employed in taxonomy of
streptomycetes for many years. They are quite useful in routine
identification. They are as follows.
- Aerial Mass Colour
The colour of the mature sporulating aerial mycelium is recorded in a
simple way (White, grey, red, green, blue and violet). When the aerial
mass colour falls between two colour series, both the colours are
recorded. If the aerial mass colour of a strain to be studied shows
intermediate tints, then also, both the colour series are noted.
- Melanoid Pigments
The grouping is made on the production of melanoid pigments (i.e.
greenish brown, brownish black or distinct brown, pigment modified by
other colours) on the medium. The strains are grouped as melanoid
pigment produced (+) and not produced (‐).
- Reverse Side Pigments
The strains were divided into two groups, according to their ability to
produce characteristic pigments on the reverse side of the colony,
namely, distinctive (+) and not distinctive or none (‐). In case, a colour
with low chroma such as pale yellow, olive or yellowish brown occurs, it
is included in the latter group (‐).
. Soluble Pigments
The strains are divided into two groups by their ability to produce
soluble pigments other than melanin: namely, produced (+) and not
produced (‐). The colour is recorded (red, orange, green, yellow, blue
and violet).
Spore Chain Morphology
With regard to spore chains, the strains can be grouped into ‘sections’.
The species belonging to the genus Streptomyces are divided into three
sections (Shirling & Gottlieb, 1966), namely rectiflexibiles (RF),
retinaculiaperti (RA) and Spirales (S). When a strain forms two types of
spore chains, both are noted (e.g. SRA).
K. Sivakumar 201
Characteristics of the spore‐bearing hyphae and spore chains
should be determined by using direct microscopic examination of the
culture surface. Adequate magnification (400x) could be used to
establish the presence or absence of spore chains and to observe the
nature of sporophores.
Spore morphological characters of the strains can be studied by
inoculating a loopful of one week old cultures into 1.5% agar medium
contained in test tubes at 370C. The actinomycete should be suspended
and thoroughly mixed in the semisolid agar medium and 1 or 2 drops of
the medium could be aseptically pipetted on to a sterile glass slide. A
drop of agar should be spread well on the slide and allowed to solidify
into a thin film so as to facilitate direct observation under microscope.
The cultures should be incubated at 28 + 20C and examined periodically
for the formation of aerial mycelium, sporophore structure and spore
morphology.
Spore Surface
Spore morphology and its surface features should be observed under the
scanning electron microscope. The cross hatched cultures prepared for
observation under the light microscope can be used for this purpose.
RF Spore chains (400X)
RA Spore chains (400X)
Spiral Spore chains (400X)
202 Actinomycetes
The electron grid should be cleaned and adhesive tape should be placed
on the surface of the grid. The mature spores of the strain should be
carefully placed on the surface of the adhesive tape and gold coating
should be applied for half an hour and the specimen can be examined
under the electron microscope at different magnifications. The spore
silhouettes can be characterized as smooth, spiny, hairy and warty.
Assimilation of Carbon Source
The ability of different actinomycete strains in utilizing various carbon
compounds as source of energy should be studied following the method
recommended by International Streptomyces Project (Shirling and
Gottlieb, 1966). Chemically, pure carbon sources, certified to be free of
admixture with other carbohydrates or contaminating materials, should
be used for this purpose. Carbon sources for this test could be arabinose,
xylose, inositol, mannitol, fructose, rhamnose, sucrose and raffinose.
These carbon sources should be sterilized by ether sterilization without
heating.
Comparing the properties of the isolated strain with the
representative species found in the key of Nonomura (1974) and
Bergey’s Manual of Determinative Bacteriology (1974, 1989, 1994 & 2005) can help in the species level identification. If the isolated strain
could not be assigned to any of the valid representatives listed in the key
of Nonomura (1974) and Bergey’s Manual of Determinative Bacteriology
(Buchanan & Gibbons, 1974), then it can be identified based on the
numerical taxonomic studies.
d) Numerical Taxonomic Approach
Numerical taxonomy involves examining many strains for a large
number of characters prior to assigning the test organism to a cluster
based on shared features. The numerically defined taxa are polythetic;
so, no single property is either indispensable or sufficient to entitle an
organism for membership of a group. Once classification has been
achieved, cluster‐specific or predictive characters can be selected for
identification (Williams et al, 1983).
Numerical taxonomy was first applied to Streptomyces by
Silvestri et al. (1962). The numerical taxonomic study of the genus
Streptomyces by Williams et al. (1983) involves determination of 139 unit
characters for 394 type cultures of Streptomyces; clusters were defined at
77.5% or 81% Ssm and 63% Sj similarity levels, and the former co‐effieient
K. Sivakumar 203
is being used to define the clusters. His study includes 23 major, 20
minor and 25 single member clusters.
The numerical classification of the genus Streptomyces by
Kampfer et al. (1991) involves determination of 329 physiological tests.
His study includes 15 major clusters, 34 minor clusters and 40 single
member clusters which are defined at 81.5% similarity level Ssm using
the simple matching coefficient (Sokal and Michener, 1958) and 59.6 to
64.6% similarity level Sj using Jaccard coefficient (Sneath, 1957). Thus,
numerical taxonomy provides us with an invaluable framework for
Streptomyces taxonomy, including identification of species.
Preservation
The preservation methods are similar to that of bacteria such as subculturing, freezing especially in liquid nitrogen, freeze‐drying and
maintenance of strains in mineral oil.
Conclusion Studies on actinomycetes are very limited and the actinomycetes have been mentioned incidentally, on the microbial community of marine habitats. Further, only little information is available on the actinomycetes of the mangrove environment (which is one among the most productive coastal ecosystems) with regard to their occurrence and distribution (Vikineswari et al., 1997; Rathna Kala and Chandrika, 1993; Lakshmanaperumalsamy, 1978).
Actinomycetes from Sediments in the Trondheim Fjord, Norway. Diversity and Biological Activity
Abstract: The marine environment represents a largely untapped source for isolation of new microorganisms with potential to produce biologically active secondary metabolites. Among such microorganisms, Gram-positive actinomycete bacteria are of special interest, since they are known to produce chemically diverse compounds with a wide range of biological activities.
We have set out to isolate and characterize actinomycete bacteria from the sediments in one of the largest Norwegian fjords, the Trondheim fjord, with respect to diversity and antibiotic-producing potential. Approximately 3,200 actinomycete bacteria were isolated using four different agar media from the sediment samples collected at different locations and depths (4.5 to 450 m). Grouping of the isolates first according to the morphology followed by characterization of isolates chosen as group representatives by molecular taxonomy revealed that Micromonospora was the dominating actinomycete genus isolated from the sediments.
The deep water sediments contained a higher relative amount of Micromonospora compared to the shallow water samples. Nine percent of the isolates clearly required sea water for normal growth, suggesting that these strains represent obligate marine organisms. Extensive screening of the extracts from all collected isolates for antibacterial and antifungal activities revealed strong antibiotic-producing potential among them. The latter implies that actinomycetes from marine sediments in Norwegian fjords can be potential sources for the discovery of novel anti-infective agents.
Keywords: Actinomycete bacteria, fjord sediments, molecular taxonomy, antimicrobial activities
The demand for new antibiotics continues to grow due to the rapid spread of antibiotic-resistant pathogens causing life-threatening infections. Although considerable progress is being made within the fields of chemical synthesis and engineered biosynthesis of antimicrobial compounds, nature still remains the richest and the most versatile source for new antibiotics [1,2,3].
Bacteria belonging to the family Actinomycetaceae are well known for their ability to produce secondary metabolites, many of which are active against pathogenic microorganisms. Traditionally, these bacteria have been isolated from terrestrial sources although the first report of mycelium-forming actinomycetes being recovered from marine sediments appeared several decades ago [4]. It is only more recently that marine-derived actinomycetes have become recognized as a source of novel antibiotics and anti-cancer agents with unusual structures and properties [5].
Many microbiologists believe that free-living bacteria are cosmopolitan due to their easy dispersal [6]. However, chemical and physical factors contribute to selection of species and strains that are best adapted to that particular environment. Due to the broad bacterial species definition one may find members of one species in two very different environments [7,8]. However, comprehensive analysis of the recent studies strongly suggests that free-living microbial taxa exhibit biogeographic patterns [7,9]. Some of the unusual structures and properties of compounds isolated from marine sources and the fact that 58 % of the isolated actinomycetes from sediments collected around Guam in the Pacific ocean required sea water for growth [5] implies that one may find microorganisms adapted to the marine environment and producing compounds not found among microorganisms adapted to the terrestrial sources.
Fjords are narrow inlets of the sea, which have been formed as a result of marine inundation of glaciated valleys. Typical characteristics of a fjord include a relatively narrow inlet, significantly eroded bottom and communication with the open sea. The ecology of the microorganisms, especially bacteria, inhabiting the fjords is poorly studied. The Trondheim fjord (135 km long) differs from many other fjords by a large fresh water supplement from six major river systems. In one year these rivers bring fresh water to the fjord corresponding to 6.5 % of it total water volume [10]. From May until September the fjord contains a 5-25m thick layer of brackish surface water, depending on time, weather and location. In the brackish water layer the salt content varies from approximately 18 to 32 practical salinity units (PSU). The dissolved and particulate organic matter in the sediments from the fjord has both marine origin from phytoplankton and macroalgae as well as terrestrial from soil due to fresh water run-off, highly influenced by snow melting in April-May.
The Norwegian marine environments are largely unexplored, and may provide a rich source of the microorganisms producing novel and efficient anti-infective compounds. The purpose of this study was to investigate if fjord sediments from temperate areas could be suitable sources for isolation of mycelium-forming actinomycetes producing antimicrobial compounds.
Mar. Drugs 2008, 6(1) 14
Collection and analysis of the sediment samples
Sediment samples from two different locations and five depths were collected and processed (see Table 1 for description and ID). The first site (B-site) was close to the shore and here samples were collected at depths of 4.5, 6, 27 and 28 m. The 4.5 m and 6 m sampling sites were situated in the kelp forest belt and the 6 m sampling site had a brown layer of sedimented micro algae on the surface.
At 27 and 28 m depth, the sediment surface was dominated by sand/silt. The second site (T-site) was located in the middle of the fjord and here samples were taken at 450 m depth, where the sediment was dominated by clay particles. Visual inspection indicated a higher content of fine organic matter than in the other sediments. Similar to the B2 sample the sediments from this location also had a layer of dead micro algae on the surface.
Table 1. Description of sediments and sampling sites.
The contents of organic material in the different sediment samples were measured as total carbon (C) and nitrogen (N), in order to test possible correlation with actinomycete microbiota and the ability to produce antimicrobial compounds. The highest content of organic carbon (1.8 %) and nitrogen (0.15 %) was found in the T1 sediment (Table 1). The contents of organic carbon and nitrogen in the B-site sediments varied from 0.6 to 1.1 % and 0.04 to 0.06 %, respectively, where the B2 sample was clearly the one with highest content of the organic matter. Higher total N over C ratio may indicate a higher content of more rapidly degradable organic material [11]. The highest N/C ratio was found for the B3 and T1 sediments, suggesting the presence of more easily degradable organic matter in these samples.
Isolation of actinomycete bacteria
Settled suspensions of the sediments were plated onto four different selective media adding up to a total of 320 primary isolation plates (diameter 14 cm) of which 248 (78%) yielded myceliumforming actinomycete colonies. The total number of actinomycetes observed on the primary isolation plates was 7874. An average of 24.6 mycelium-forming actinomycete colonies per plate were observed with numbers increasing to 31.8 when only considering those plates that yielded actinomycetes.
3200 colonies were picked based on actinomycete-like morphology, of which around 900 formed powdery colonies with well-developed aerial hyphae fragmented into spore chains. Thes isolates were tentatively termed as Streptomyces-like actinomycetes. The main part of the remaining isolates formed orange to red pigmented colonies with solid colony texture, non fragmenting substrat Mar. Drugs 2008, 6(1) 15 mycelium that lacked aerial hyphae and often turned purple, brown or black upon sporulation, an was tentatively termed as MNSA (mycelium-forming non-streptomycete actinomycetes). The tota CFU (colony forming units) for Streptomyces-like and MNSA isolates were registered (Table 2). Th highest total CFU was found on soil agar from the B1 sample and the highest number of MNS colonies was found in the same sediment when plated onto chitin agar (IM7b). Highest number o Streptomyces-like colonies was found when Biologen 6 m sediments wer plated on to IM6 (modifie Kusters) agar Table 2. Viable counts of bacteria and actinomycetes in sediments from different depths in the Trondheim fjord after plating onto selective agar media. The number are mean values of CFU (colony forming units) per mL wet sediment
The T1 sample contained the highest relative number of MNSA colonies (93 %) and the lowest number of Streptomyces-like colonies (0.7 %) (Table 2). However the total CFU on selective media from this sediment was approximately one tenth of that from the B-site sediments. For the latter sediments the highest relative numbers of MNSA colonies varied from 17 to 30 % when plated on to IM7b agar. In general, the IM7b (colloid chitin) gave the highest numbers of MNSA colonies with the exception of the T1 sample where IM6 was clearly the best in this respect. IM5 (humic acid sea water Mar. Drugs 2008, 6(1) 16 agar) was the isolation medium that gave the lowest number of mycelium-forming actinomycetes from fjord sediments.
Different types of selective pre-treatments were applied in order to increase the number of mycelium-forming actinomycetes relative to the non-actinomycetal heterotrophic microbial flora.
These treatments included dry heat, phenol treatment, dry heat followed by phenol treatment, dry heat followed by benzethonium chloride treatment, and pollen baiting. For the B-site sediment samples (Figure 1) it was possible to obtain increased relative numbers of actinomycetes on the agar plates with all the different types of pretreatments, except for the pollen baiting, which did not yield any isolates. With some of the pretreatments (dry heat followed by phenol treatment), it was possible to obtain isolation plates only containing actinomycetes with morphologies typical for the genera Micromonospora.
Figure 1. The effect of different types of pre-treatments, applied to the sediments samples, on the relative numbers of actinomycetes appearing on the isolation plates
However, for the T1 (450 m) samples these pretreatments were detrimental, and apparently eliminated most of the actinomycete microbiota. We were not able to isolate any mycelium-forming actinomycetes from the sediments with the pollen baiting technique, suggesting that actinomycetes producing zoospores are rare in the fjord sediments we have investigated.
Sea water requirement
All original media for isolation of actinomycetes contained sea water, and we decided to test whether the presence of this media component can be crucial for growth of isolated actinomycetes.
Therefore, the isolates were transferred to the respective media with or without sea water, and their Mar. Drugs 2008, 6(1) 17 growth was monitored over a period of 8 weeks. Growth of approximately 8 % percent of the isolates from T1 sample was found to be completely dependent on the presence of sea water, while the respective average figure for the B-site samples was 9 %. The individual percentages for the B-site samples were: 4.5 m; 8 %; 6 m; 10 %, 27 m; 5 % and 28 m; 8 %. In addition, around 20 % of the isolates grew considerably faster on agar media containing sea water. The major part of the isolates (50 %) did not show any clear preference for media with or without sea water, while around 20 % of the isolates grew better in the absence of sea water.
Figure 2. The percentage of the mycelium-forming actinomycete isolates
displaying antibacterial activities against Micrococcus luteus and antifungal activities against Candida albicans in agar diffusion assays. A. Non-streptomycete isolates. B. Streptomyces-like isolates.
Biological activities
The 3,200 isolated actinomycete bacteria were transferred to three different solid (agar) production media. After an incubation period of one to six weeks depending on growth rate of the isolates, the media and the cells were dried and extracted with DMSO. The extracts were tested for antibacterial and antifungal activity against the Gram-positive bacterium Micrococcus luteus and the yeast Candida albicans using traditional agar diffusion assays. The results of this analysis are presented in Figure 2.
Somewhat surprisingly, the MNSA isolates from the T1 sediments showed the highest frequency of activity against Gram-positive bacterium M. luteus (58 %) and yeast C. albicans (39 %). For the B-site MNSA isolates the frequency of the corresponding activities varied between 25-32%and 13-21%, respectively.
The frequency of activities against M. luteus and C. albicans among the Streptomyces-like strains varied between 34-47 % and 23-42 %, respectively. Within this group, the highest frequency of antibacterial activities was found among the isolates from the B2 sample and the highest antifungal activity among the B1 sample.
Molecular taxonomy
Examination of the colony morphology of the actinomycete isolates suggested that they may tentatively belong to the genera Streptomyces and Micromonospora. In order to confirm preliminary classification, we selected 30 isolates from the T1 sample having different “representative” morphologies.
These isolates were morphologically more homogeneous than the B-site isolates. Fragments of the 16S rRNA genes from these isolates were PCR-amplified, sequenced, and a phylogenetic tree was constructed, allowing the sorting of the sequences into seven different clusters (Figure 3). Cluster 1 consisted of 12 isolates showing 99 % to 100 % homology to Micromonospora matsumotoense. All displayed moderate antibacterial activity, except MP38-F9 and MP38-F5 which displayed strong and no activity respectively. They all also produced a weak antifungal activity, except MP38-F5. Cluster 2 contained four isolates showing 99 to 100 % homology to Micromonospora sp. e24. None of these isolates showed antibacterial or antifungal activity. The six isolates in Cluster 3 showed 99 to 100 % identity to M. chokoriensis. Of these isolates only MP38-A6 showed antimicrobial activity which was moderate antibacterial and weak antifungal. Cluster 4 consisted of one isolate with its partial 16S rDNA sequence showing 99 % identity to M. aurantiaca. This isolate showed no antibacterial or antifungal activity. Cluster 5 contained three isolates that showed 99 % homology to M. marina. Two of the isolates MP38-E11 and MP38-D12 displayed moderate antibacterial activity. Cluster 6 consisted of one isolate showing 100 % homology to Verrucosispora gifhornensis. No antimicrobial activity was detected for this strain. Cluster 7 contained three isolates which showed 99 % homology to M.
chersina, all of them showing antibacterial activity. Despite clear division of the isolates to different clusters according to the partial 16S rDNA sequences, there was just as much morphological variation among the isolates within the clusters as between the different clusters. For example, among 12 Micromonospora isolates from Cluster 1, only three have shown differences in the partial 16S rDNA sequences. However, all isolates in this cluster exhibited differences in morphology, and some of them were clearly different in terms of biological activity profiles.
Mar. Drugs 2008, 6(1) 19
Figure 3. Phylogenetic relationship of partial 16S rDNA sequences generated in
this study, rooted using the 16S rDNA sequence of Streptomyces coelicolor. See
Material and methods for tree description. Numbers at tree nodes represent the
number of times the topology to the right of the node was recovered in 1000
bootstrap re-samplings; values lower than 50 are not shown. Accession numbers
for the sequences are in parentheses. Scale bar represents the number of changes per base position.
Discussion
The Trondheim fjord is approximately 135 km long and is characterized by the large water supply from six rivers entering the fjord. The dissolved and particulate organic matter in the sediments from the fjord originates from both marine phytoplankton and macro algae, and terrestrial material from the Mar. Drugs 2008, 6(1) 20 run-off caused by snow melting in the spring brought to the fjord by the rivers. An average of 9 % of the actinomycete isolates in this investigation required sea water for growth, most of them originating from the deep-water sediment sample. The latter can probably be explained by a better adaptation of the bacteria in this sediment to the sea water environment, since fresh water supply at such depth shall be minimal. Jensen et al. [5] reported that as much as 58 % of the actinomycetes, isolated from sediment samples collected around the island of Guam required sea water for growth. While the Trondheim fjord is a sea inlet into the terrestrial environment, Guam is positioned far out into the Pacific Ocean. These facts suggests that although there is a clear evidence of metabolically active marine actinomycetes in the fjord sediments, there is also a considerable number of actinomycetes ofterrestrial origin present as well.
Micromonospora and Streptomyces-like actinomycetes were the dominating actinomycetes isolated from the near shore shallow water sediments, where the numbers of streptomycetes decreased with depth and distance from the shore, as have also been reported by others [12]. However, a considerable number of isolates displayed morphologies that did not conform with these two genera. Their relative phylogenetic positions are under investigation and will be reported elsewhere.
The use of different types of selective treatments originally designed for selective isolation of actinomycetes from soil increased the relative numbers of actinomycetes on the agar plates inoculated from the shallow water (4.5 to 28 m) near shore samples. Although these techniques have previously been applied with success for isolation of groups of actinomycetes from soil [13-15], no reports exist to our knowledge on effectiveness of such techniques applied to the marine sediments from temperate areas. For the deep-water sediment samples (450 m) these treatments were clearly detrimental, mainly resulting in agar plates free for actinomycetes. This suggests that the selective pre treatments designed to isolate actinomycetes from soil are not optimal for marine samples and more effort is required in order to establish methods allowing specific enrichment of marine actinomycetes. The dominating actinomycete genus isolated from the deep sediments under the conditions tested was Micromonospora.
Some of the selective treatments as for example dry heat at 120 ◦C for 60 min and exposure to 1.5 % phenol is indeed designed for the selective isolation of Micromonospora. However, very few Micromonospora isolates from the deep water samples survived these treatments. This could suggest that although their relative phylogenetic position determined by 16S rDNA is identical or close to known terrestrial organisms, they still have signs of adaptation to their marine environment. Micromonospora species growing at 450 m experience an environment with relatively low and constant ambient temperature (8 ◦C), stable and high salinity, and are not exposed to desiccation and may therefore differ significantly from their terrestrial counterparts. Without the use of selective pre-treatments the relative numbers of actinomycetes (mainly Micromonospora) could account for as much as 90 % of the colonies on some of the selective agar media. At the moment, however, we can not exclude the possibility that the dominance of Micromonospora on our isolation plates was caused by the chosen isolation procedures, and that other actinomycetes are also present in considerable numbers in these sediments.
Although we have analyzed too few samples to draw any solid conclusions, the trend in our results was that the percentage of isolates displaying activity against M. luteus among the MNSA isolates was roughly proportional to the carbon content in the different sediments. That is, the percentage of mycelium-forming actinomycetes with this activity increased with the content of organic carbon Mar. Drugs 2008, 6(1) 21 in the sediments (Table 1 and Figure 2). No correlation could be seen between sampling depth and antimicrobial activity among MNSA isolates. With the exception of the T1 sample (collected from 450 m depth), there was a decrease in the percentage of Streptomyces-like isolates displaying activity against C. albicans with increasing sampling depth. At the same time, we are aware of the fact that our test organism, Candida albicans, is of terrestrial origin, and thus we might have missed antibiotic activities directed against marine fungi.
Despite the fact that the T1 sample had the highest content of organic carbon and nitrogen, it gave the lowest CFU number on our selective media. It also contained less morphological diversity among the actinomycete isolates than what was found for the shallow water B-site samples. The reason for this is most probably our failure to cultivate many of the actinomycetes present in the sample due to their need for special cultivation conditions. This is supported by previous reports where high actinobacterial diversity was found in marine sediments by constructing actinobacteriumspecific 16S rDNA clone libraries. Furthermore, the information from the cultivation-independent techniques could be used to improve the recovery of novel actinobacteria [16-18]. Even though, the diversity and biological activities of actinomycetes from the Trondheim fjord sediments unraveled so far suggests that they might be a rich source for discovery of new anti-infective agents.
Experimental Section
Sample collection and isolation of bacteria
Sediments from 4.5, 6.0, 27 and 28 m depths were collected by scuba divers, while sediments from 450 m depth were sampled with a box-corer. The upper 5 cm of the sediments were collected in zip-lock bags (scuba) or with a sterile spade (box –corer) and transferred to 1 liter sterile plastic containers. Approximately 10 % of the container volume was filled with 60 % sediment and 40 % sea water from the sampling site. This was done in order to ensure aerobic conditions under storage upon processing. Samples were processed the same day or the day after sampling. All storage was done in the dark at 4 oC. Sediments were diluted 1:10 v/v with sterile sea water and vigorously shaken with glass beads for 30 sec. Settled (5 min) sediment suspensions were plated on to different selective agar media and incubated at 20 ◦C for two to six weeks. Selective treatments were performed on dried sediments (Speedvac 30 ◦C, 16 h) and included dry heat (120 ◦C, 60 min), phenol (1.5 %, 30 min at 30 ◦C), dry heat and phenol, dry heat and benzethonium chloride (0.02 %, 30 min at 30 ◦C), as well as pollen baiting [19,20].
Carbon and nitrogen content analysis
Sediment samples were dried and ground with a pester and mortar, before the total carbon (C) and nitrogen (N) content in the sediment samples were measured using a NA 1500 Nitrogen/Carbon/Sulphur analyzer from Carlo Erba Instruments. To ensure good average values each sediment sample was analyzed in five parallels.
Mar. Drugs 2008, 6(1) 22
Isolation and production media
Isolation media consisted of the following: IM5 (humic acid agar [21], with sea water), humic acid (1 g), K2HPO4 (0.5 g), FeSO4•7H2O (1 mg), agar (20 g), vitamin B solution (1 mL), natural sea water (0.5 L) and distilled water (0.5 L); IM6 glycerol (0.5 g), starch (0.5 g), sodium propionate (0.5 g), KNO3 (0.1 g), asparagine (0.1 g), casein (0.3 g), K2HPO4 (0.5 g), FeSO4•7H2O (1 mg), agar (20 g), vitamin B solution (1 mL), natural sea water (0.5 L) and distilled water(0.5 L); IM7 (chitin agar [9], with sea water) chitin (Sigma), K2HPO4 (0.5 g), FeSO4•7H2O (1 mg), agar (20 g), vitamin B solution (1 mL), natural sea water (0.7 L) and distilled water (0.3 L); IM8, malt extract (1 g), glycerol (1 g), glucose (1 g), peptone (1 g), yeast extract (1 g), agar
(20 g), natural sea water (0.5 L) and distilled water (0.5 L). The pH of the isolation
media was adjusted to pH 8.2. Vitamin B solution consisted of the following:
thiamine-HCl (50 mg), riboflavin (50 mg), niacin (50 mg), pyridoxine-HCl (50 mg),
inositol (50 mg), Ca-pantothenate (50 mg), p-aminobenzoic acid (50 mg), biotin
(25 mg) and distilled water (100 mL). All isolation media were amended with filtered (0.2-µm pore size) cycloheximide (50 µg/mL), nystatin (75 µg/mL) and nalidixic acid (30 µg/mL).
Production media: PM2, mannitol (5.0 g), soya bean flour (5.0 g), Clerol (antifoam, 0.1 g), dry yeast (0.9 g), agarose (10.0 g), tap water (1 L); PM3, oatmeal (20 g), glycerol (2.5 g), FeSO4•7H2O (0.1 mg), MnCl2•4H2O (0.1 mg), ZnSO4•7H2O (0.1 mg), agarose (10 g), tap water (1 L); PM4, glucose (0.5 g), glycerol (2.5 g), oatmeal (5.0 g), soybean meal (5.0 g), yeast extract (0.5 g), casaminoacids (2.0 g), CaCO3 (2.0 g), Clerol (0.1 g), agarose (10 g) and tap water (1 L).
Media for nucleic acid extraction: organic agar Gause 2 (modified), glucose (10 g), trypton (3 g), peptone (5 g), agar (20 g), tap water (0.5 L) and sea water (0.5 L).
Plates with isolation and production media were incubated at 20 oC for periods of 2 to 6 weeks.
Cultivation, extraction and bioactivity testing
The 3200 isolated mycelium-forming actinomycetes were transferred to three different solid (agar) production media PM2, PM3 and PM4 in 96-well plates. After an incubation at 20 ◦C for one to six weeks, depending on growth rate of the isolates, the media and the cells were dried directly in the plates, and extracted with dimethylsulfoxide (DMSO, Sigma, 200 µL). The extracts were tested for antibacterial and antifungal activity against the Gram-positive bacterium Micrococcus luteus ATCC 9341 and the yeast Candida albicans ATCC 10231 using traditional agar diffusion assays. Each DMSO extract (1 µL) was applied onto the surface of the agar inoculated with the test organism and activity registered as inhibition zones after 16 hours of incubation at 34 ◦C.
Nucleic acid extraction,
16S rDNA amplification, sequencing and analysis Fresh colonies grown on Gause 2 organic agar [22] were macerated and transferred to sterile distilled water (100 µL) and heated to 98 ◦C for 10 min. The suspensions were centrifuged (5000 x g, 1 min) and DNA from the clear supernatant precipitated with three volumes of ethanol, centrifuged (12000 rpm, 15 min) and pellets dissolved in distilled water to the original volume.
16S ribosomal DNA (rDNA) sequencing templates were amplified from genomic
Mar. Drugs 2008, 6(1) 23 DNA by PCR using previously described [18] actinomycete specific primers
S-C-Act-235-S-20 (5’-CGCGGCCTATCAGCTTGTTG-3’) and S-C-Act-878-A-19
(5’-CCGTACTCCCCAGGCGGGG-3’). Reaction mixture (50 µL) contained genomic DNA extract (1 µL), Thermopol Buffer (New England Biolabs), DMSO (2 µL), Bovine serum albumin (2 µL), deoxynucleoside triphosphate mixture (2.5 pmol), each primer (20 pmol), and Taq DNA polymerase (2.5 U). All sequencing reactions were carried out with an ABI PRISM 3100 genetic analyzer at the Department of Biology, The Norwegian University of Science and Technology. DNA sequences were deposited to GenBank under accession numbers DQ645597 to DQ645626. The 16S rDNA sequences (500-625 bp) were used to search the GeneBank database with the BlastN algorithm to reveal closest matches to the 16S rDNA sequences for known species. Sequences were aligned with representative actinomycete 16S rDNA sequences and a phylogenetic tree was constructed using the Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0 [23].
Actinomycetes
CHARACTERISTICS OF SOIL ACTINOMYCETES
FROM ANTARCTICA
Forty-seven actinomyces strains were isolated from Antarctic soils – nineteen of them showed antagonistic activity against Gram-positive and Gram-negative bacteria.
Six of the strains possessed a broad spectrum of antibacterial activity. Results obtained from the physiological and biochemical analyses including determination of 39 characteristics proved that two of the strains (23 and 29) were similar whereas all the rest differed among each other. Morphological studies indicated that the strains belonged to the genera Streptomyces, Actinomadura and Kitasatosporia.
Antibacterial activity of three actinomycetes strains (designed as 29, 30 and 47) was confirmed in batch culture. They were active against clinical isolates from the species Staphylococcus aureus and Streptococcus pneumoniae. The three strains also showed antibacterial activity against the phytopathogenic bacteria Xanthomonas axonopodis pv. glycines, X. vesicatoria, X. axonopodis pv. phaseoli, Pseudomonas syringae pv. tomato and Clavibacter michiganensis, for which no biological means for control, had been developed yet. The broadest spectrum of antibacterial action had the strain 29. The antibacterial compounds produced by these strains probably possessed non-polar structure and consisted of several active components.
ACTINOMYCETES ISOLATED FROM SOIL SAMPLES FROM THE CROCKER RANGE SABAH
A diversity of actinomycetes was isolated from various sites of top soils throughout the Crocker Range in Sabah. The soils were mainly collected during the expedition (15—25 October 1999) together with 2 soil samples collected on 28 November 1999 under Rafflesia keithii in the Rafflesia Reserve Forest, Gunung Mas. A total of 78 strains of actinomycetes, probably mostly Streptomyces, were obtained from different sites. Amongst these strains 20 have been aerobically grown in shaking liquid cultures. Acetone extracts of these cultures were screened for MAPK Kinase and MAP Kinase Phosphatase in a yeast system in the preliminary screening of novel cancer drugs. This screening system is based on the fact that the MAP kinase pathway is homologues from yeast to human. However, no such inhibitors were found. A few strains with pigmentation were collected from specific locations.
INTRODUCTION
Actinomycetes are gram positive bacteria frequently filamentous and sporulating with DNA rich in G+C from 57—75%. Some of their secondary metabolites have employed as useful microbial compounds (Prescott, Harley & Klein, 1993). Examples include streptomycin from Streptomyces griseus for treatment of tuberculosis caused by Mycobacterium tuberculosis and the immunosuppress drug, tacrolimus (FK506) produced by S. tsukubaensis. Actinomycetes of about 100 genera exist in soils (Yokota, 1997). In their natural habitat, such as forests, the actinomycetes interact in various ways with the higher plants.
The fallen tress, barks and flowers first provide nutrients both to the microbes and plants through microbial degradation of carbohydrates, lipids and proteins to sugars, fatty acids, glycerol and amino acids and ultimately to mineralisation. Besides providing these nutrients, plant secondary metabolites (such as dipterocarp resins) that are generally toxic to microorganisms, will need to be degraded or detoxified by certain microbes. These degraders (microbes) are selectively pressured and ultimately evolve to produce novel secondary metabolites of their possibly to counteract the toxic plant secondary metabolites (Park et al, 1999 and Ho et al, 2000).
In this study, soil samples were collected in different habitats in the Crocker Range National Park to investigate the diversity of actinomycetes. Actinomycetes were then isolated on selective medium humic-acid + modified B vitamins and extracts were screened for biological activities of the secondary metabolites.
Soil samples: Soil samples were collected by sterile method from various locations visited throughout this scientific expedition to Crocker Range Park (Figure 1), from an area of mist forest (1400—1500m from sea level), submontane rain forest (Mahua), hill forest (uphill of Mensalog River, Ulu Senagang) to cultivated areas (of introduced Theobroma cacao and Tectonia grandis). Soil samples were air-dried under room temperature for about 30 days before isolation (Table 1). A second set (Table 2) used air-dried soils stored at room temperature over a long period (9-11 months).
Isolation of actinomycetes: 0.5g of soil samples was suspended in 9.5m1 of sterile distilled water and was 1000-fold diluted. 0. lml of the dilutions was spread on humic acid + modified B vitamins agar (HV) medium, pH 7.2, supplemented with cycloheximide. The plates were incubated at 280C for 2 weeks.
Classification of actinomycetes: Isolated strains were transferred from HV medium onto oatmeal agar medium, pH 7.2 and incubated at 280C for 14 days. Colouration of aerial mycelium (on the surface of agar), substrate mycelium (underside of plate) and diffusible pigment were observed.
Extraction of secondary metabolites: Submerged fermentation of purified cultures were carried out in liquid medium of 2% mannitol, 2% peptone and 1% glucose, pH 7.2, for 5 days at 280C, 220rpm. Resultant broths were added with equal volume of acetone to extract secondary metabolites (final concentration of extract in 50% acetone).
Screening:The acetone extracts were tested for inhibitory activity against MAPK kinase and MAP kinase phosphatase inhibitors into yeast strains Saccharomyces cerevisiae MKKlP386 and S. cerevisiae MKK1P386— MSG5 respectively.
RESULTS AND DISCUSSION:
A total of 78 isolates of actinomycetes were isolated from 22 soil samples (Table 1 & 2) while 16 other soil samples without any isolate (Table 3). Some strains were isolated from soil under Theobroma cacao, Rhododendron sp. and particularly Rafflesia kethii (Figure 2) and R. pricei (Figure 3). Most of the isolates were presumed to be of the genera Streptomyces as they showed good sporulation with compact, chalk-like dry colonies of different colours. A few pigmented strains, unique to individual sites were observed.
• All of the isolates were recovered from humic acid + B vitamins* agar plates (pH 7.2) which had been incubated for 14-30 days at 280C.
*B vitamins: thiamine-HC1, pyridoxin-HC1 and inositol.
• The isolates from E1-El1 were recovered from humic acid + B vitamins** agar plates (pH7.2) which had been incubated for 14-30 days at 280C.
*B vitamins: thiamine-HC1, pyridoxin-HC1, ribiflavin, niacin, inositol, Ca-pantothenate & p-aminobenzoic acid.
• Isolated by ‘L’-Lo, C.W; ‘C’- Cheah, H-Y; ‘W’-Wong, N.K. ‘E’- Lai, N.S. Eric
• All of the isolates were recovered from humic acid + B vitamins** agar plates (pH 7.2)
which had been incubated for 14-30 days at 280C. Isolated by Lo.C.W.
** B vitamins: thiamine-HC1, pyridoxin-HC1, ribiflavin, niacin, inositol, Capantothenate, paminobenzoic acid and biotin
All the isolates were grouped into 3 colour groups (white series, grey series and brown series) based on the colour of aerial mycelium on oatmeal agar, after 14 days incubation at 280C (Figure 4). Majority of the strains were of the grey series, followed by white series and brown the least
(Table 4). The grey series include pale grey, light grey, medium grey and dark grey; white colour group includes yellowish white, milky white and orange white while brown colour group includes greyish orange, brownish orange and greyish brown. As description of colour is quite subjective, a colour chart from Nippon 9000(1997) was used for standardization. A few strains varied according to sites are as follow: L-28 exhibited red pigmentation all over the agar medium, while L-27 exhibited orange colour extracellular pigment. Both strains were isolated from soil samples obtained under Theobroma cacao and Tectonia grandis respectively (Table 5). In the MAP kinase screening, 20 extracts were screened but none was found to be inhibitory.
The search for novel metabolites especially from actinomycetes requires a large number of isolates (over thousands) in order to discover a novel compound of pharmaceutical interest. The search will be more promising if diverse actinomycetes are sampled and screened. For this reason, soils were specifically collected under identified trees. This is based on the hypothesis that actinomycetes diversity may be influenced by the diversity of plant species as these bacteria grow profusely in the humus and leaf litter layer. Furthermore, different plants produce different type of secondary metabolites and some of these chemical compounds are toxic to soil microorganisms including actinomycetes. However, adaptation has in turn lead the actinomycetes to produce their own secondary metabolites.
Although the collection sites have mainly been limited to fairly disturbed forests in the fringes of Crocker Range, yet they possess many actinomycetes in the leaf-litter humus layer. The conservation of this park will ensure the survival of these commercially important industrial microbes of biotechnological and pharmaceutical importance together with striking Rafflesia, orchids and Rhododendron.
Vitamins
VITAMINS
Defination
A vitamin is an organic compound required as a nutrient in tiny amounts by an organism.[1] A compound is called a vitamin when it cannot be synthesized in sufficient quantities by an organism, and must be obtained from the diet..
For example,
ascorbic acid functions as vitamin C for some animals but not others, and vitamins D and K are required in the human diet only in certain circumstances.[2] The term vitamin does not include other essential nutrients such as dietary minerals, essential fatty acids, or essential amino acids
classification
Vitamins are classified by their biological and chemical activity, not their structure. ", such as "vitamin A," which includes the compounds retinal, retinol, and many carotenoids.[4]
Vitamers are often inter-converted in the body.
Vitamins have diverse biochemical functions, including function as hormones (e.g. vitamin D), antioxidants (e.g. vitamin E), and mediators of cell signaling and regulators of cell and tissue growth and differentiation (e.g. vitamin A).[5] The largest number of vitamins (e.g. B complex vitamins) function as precursors for enzyme cofactor bio-molecules (coenzymes), that help act as catalysts and substrates in metabolism. When acting as part of a catalyst, vitamins are bound to enzymes and are called prosthetic groups. For example, biotin is part of enzymes involved in making fatty acids.
Vitamins also act as coenzymes to carry chemical groups between enzymes. For example, folic acid carries various forms of carbon group – methyl, formyl and methylene - in the cell. Although these roles in assisting enzyme reactions are vitamins' best-known function, the other vitamin functions are equally important.[6]
In humans
Vitamins are classified as either water-soluble or fat soluble.
In humans there are 13 vitamins: 4 fat-soluble (A, D, E and K) and 9 water-soluble (8 B vitamins and vitamin C).
Water-soluble
Water-soluble vitamins dissolve easily in water, and in general, are readily excreted from the body, to the degree that urinary output is a strong predictor of vitamin consumption.[13] Because they are not readily stored, consistent daily intake is important.[14] Many types of water-soluble vitamins are synthesized by bacteria.[15]
Fat-soluble
Fat-soluble vitamins are absorbed through the intestinal tract with the help of lipids (fats). Because they are more likely to accumulate in the body, they are more likely to lead to hypervitaminosis than are water-soluble vitamins. Fat-soluble vitamin regulation is of particular significance in cystic fibrosis.[16]
In nutrition and diseases
Vitamins are essential for the normal growth and development of a multicellular organism. Using the genetic blueprint inherited from its parents, a fetus begins to develop, at the moment of conception, from the nutrients it absorbs. It requires certain vitamins and minerals to be present at certain times. These nutrients facilitate the chemical reactions that produce among other things, skin, bone, and muscle. If there is serious deficiency in one or more of these nutrients, a child may develop a deficiency disease. Even minor deficiencies may cause permanent damage.[30]
For the most part, vitamins are obtained with food, but a few are obtained by other means. For example, microorganisms in the intestine—commonly known as "gut flora"—produce vitamin K and biotin, while one form of vitamin D is synthesized in the skin with the help of the natural ultraviolet wavelength of sunlight. Humans can produce some vitamins from precursors they consume. Examples include vitamin A, produced from beta carotene, and niacin, from the amino acid tryptophan. Once growth and development are completed, vitamins remain essential nutrients for the healthy maintenance of the cells, tissues, and organs that make up a multicellular organism; they also enable a multicellular life form to efficiently use chemical energy provided by food it eats, and to help process the proteins, carbohydrates, and fats required for respiration.
Deficiencies
Deficiencies of vitamins are classified as either primary or secondary.
A primary deficiency occurs when an organism does not get enough of the vitamin in its food.
A secondary deficiency may be due to an underlying disorder that prevents or limits the absorption or use of the vitamin, due to a “lifestyle factor”, such as smoking, excessive alcohol consumption, or the use of medications that interfere with the absorption or use of the vitamin. People who eat a varied diet are unlikely to develop a severe primary vitamin deficiency. In contrast, restrictive diets have the potential to cause prolonged vitamin deficits, which may result in often painful and potentially deadly diseases.
Because human bodies do not store most vitamins, humans must consume them regularly to avoid deficiency. Human bodily stores for different vitamins vary widely; vitamins A, D, and B12 are stored in significant amounts in the human body, mainly in the liver,[27] and an adult human's diet may be deficient in vitamins A and B12 for many months before developing a deficiency condition. Vitamin B3 is not stored in the human body in significant amounts, so stores may only last a couple of weeks.[19][27]
Well-known human vitamin deficiencies involve thiamine (beriberi), niacin (pellagra), vitamin C (scurvy) and vitamin D (rickets). In much of the developed world, such deficiencies are rare; this is due to (1) an adequate supply of food; and (2) the addition of vitamins and minerals to common foods, often called fortification.[18][27]
Some evidence also suggests that there is a link between vitamin deficiency and mental disorders.[31]
Side effects and overdose
In large doses, some vitamins have documented side effects that tend to be more severe with a larger dosage. The likelihood of consuming too much of any vitamin from food is remote, but overdosing from vitamin supplementation does occur. At high enough dosages some vitamins cause side effects such as nausea, diarrhea, and vomiting.[19][32]
When side effects emerge, recovery is often accomplished by reducing the dosage. The concentrations of vitamins an individual can tolerate vary widely, and appear to be related to age and state of health
Supplements
Dietary supplements, often containing vitamins, are used to ensure that adequate amounts of nutrients are obtained on a daily basis, if optimal amounts of the nutrients cannot be obtained through a varied diet.
Vitamin A and E supplements not only provide no tangible health benefits for generally healthy individuals, but may actually increase mortality, although two large studies included in the analysis involved smokers, for which it was already known that beta-carotene supplements can be harmful.[37]
In the United States, advertising for dietary supplements is required to include a disclaimer that the product is not intended to treat, diagnose, mitigate, prevent, or cure disease, and that any health claims have not been evaluated by the Food and Drug Administration.[36
In some cases, dietary supplements may have unwanted effects, especially if taken before surgery, with other dietary supplements or medicines, or if the person taking them has certain health conditions.[36] Vitamin supplements may also contain levels of vitamins many times higher, and in different forms, than one may ingest through food.[38]
Intake of excessive quantities can cause vitamin poisoning, often due to overdose of Vitamin A and Vitamin D (The most common poisoning with multinutrient supplement pills does not involve a vitamin, but is rather due to the mineral iron). Due to toxicity, most common vitamins have recommended upper daily intake amounts.
Avitaminosis
Avitaminosis is any disease caused by chronic or long-term vitamin deficiency or caused by a defect in metabolic conversion, such as tryptophan to niacin. They are designated by the same letter as the vitamin.
Conversely hypervitaminosis is the syndrome of symptoms caused by over-retention of fat-soluble vitamins in the body.
Avitaminoses include
• vitamin A deficiency causes xerophthalmia or night blindness
• thiamine deficiency causes beriberi
• niacin deficiency causes pellagra
• vitamin B12 deficiency leads to megaloblastic anemia
• vitamin C deficiency leads to scurvy
• vitamin D deficiency causes rickets
• vitamin K deficiency causes impaired coagulation
•
Vitamin poisoning
Vitamin poisoning, hypervitaminosis or vitamin overdose refers to a condition of high storage levels of vitamins, which can lead to toxic symptoms. The medical names of the different conditions are derived from the vitamin involved: an excess of vitamin A, for example, is called hypervitaminosis A.
With few exceptions, like some vitamins from B complex, hypervitaminosis usually occurs more with fat-soluble vitamins, which remain more time in the body and are harder to be excreted than water soluble vitamins.
High dosage vitamin A; high dosage, slow release vitamin B3; and very high dosage vitamin B6 alone (i.e. without vitamin B complex) are sometimes associated with vitamin side effects that usually rapidly cease with supplement reduction or cessation.
Vitamin C has a brief, pronounced laxative effect when taken in large amounts, typically in the range of 5-20 grams per day in divided doses for a person in normal "good health," although seriously ill people,[1] may take 100-200 grams without inducing vitamin poisoning.
High doses of mineral supplements can also lead to side effects and toxicity. Mineral-supplement poisoning does occur occasionally due to excessive and unusual intake of iron-containing supplements, including some multivitamins, but is not common.
The Dietary Reference Intake recommendations from the United States Department of Agriculture define a "tolerable upper intake level" for most vitamins.
List of vitamins
Each vitamin is typically used in multiple reactions and, therefore, most have multiple functions.[17]
Vitamin generic
descriptor name
Vitamer chemical name(s) (list not complete)
Solubility
Recommended dietary allowances
(male, age 19–70)[18]
Deficiency disease Upper Intake Level
(UL/day)[18]
Overdose disease
Vitamin A
Retinoids
(retinol, retinoids
and carotenoids)
Fat 900 µg Night-blindness and
Keratomalacia[19]
3,000 µg Hypervitaminosis A
Vitamin B1
Thiamine
Water 1.2 mg Beriberi, Wernicke-Korsakoff syndrome
N/D[20]
Rare hypersensitive reactions resembling anaphylactic shock-- injection only;
Drowsiness
Vitamin B2
Riboflavin
Water 1.3 mg Ariboflavinosis
N/D ?
Vitamin B3
Niacin, niacinamide
Water 16.0 mg Pellagra
35.0 mg Liver damage (doses > 2g/day)[21] and other problems
Vitamin B5
Pantothenic acid
Water 5.0 mg[22]
Paresthesia
N/D ?
Vitamin B6
Pyridoxine, pyridoxamine, pyridoxal
Water 1.3-1.7 mg Anemia[23]
100 mg Impairment of proprioception, nerve damage (doses > 100 mg/day)
Vitamin B7
Biotin
Water 30.0 µg Dermatitis, enteritis
N/D ?
Vitamin B9
Folic acid, folinic acid
Water 400 µg Deficiency during pregnancy is associated with birth defects, such as neural tube defects
1,000 µg Possible decrease in seizure threshold
Vitamin B12
Cyanocobalamin, hydroxycobalamin, methylcobalamin
Water 2.4 µg Megaloblastic anemia[24]
N/D No known toxicity[25]
Vitamin C
Ascorbic acid
Water 90.0 mg Scurvy
2,000 mg Vitamin C megadosage
Vitamin D
Ergocalciferol, cholecalciferol
Fat 5.0 µg-10 µg[26]
Rickets and Osteomalacia
50 µg Hypervitaminosis D
Vitamin E
Tocopherols, tocotrienols
Fat 15.0 mg Deficiency is very rare; mild hemolytic anemia in newborn infants.[27]
1,000 mg Increased congestive heart failure seen in one large randomized study.[28]
Vitamin K
phylloquinone, menaquinones
Fat 120 µg
Defination
A vitamin is an organic compound required as a nutrient in tiny amounts by an organism.[1] A compound is called a vitamin when it cannot be synthesized in sufficient quantities by an organism, and must be obtained from the diet..
For example,
ascorbic acid functions as vitamin C for some animals but not others, and vitamins D and K are required in the human diet only in certain circumstances.[2] The term vitamin does not include other essential nutrients such as dietary minerals, essential fatty acids, or essential amino acids
classification
Vitamins are classified by their biological and chemical activity, not their structure. ", such as "vitamin A," which includes the compounds retinal, retinol, and many carotenoids.[4]
Vitamers are often inter-converted in the body.
Vitamins have diverse biochemical functions, including function as hormones (e.g. vitamin D), antioxidants (e.g. vitamin E), and mediators of cell signaling and regulators of cell and tissue growth and differentiation (e.g. vitamin A).[5] The largest number of vitamins (e.g. B complex vitamins) function as precursors for enzyme cofactor bio-molecules (coenzymes), that help act as catalysts and substrates in metabolism. When acting as part of a catalyst, vitamins are bound to enzymes and are called prosthetic groups. For example, biotin is part of enzymes involved in making fatty acids.
Vitamins also act as coenzymes to carry chemical groups between enzymes. For example, folic acid carries various forms of carbon group – methyl, formyl and methylene - in the cell. Although these roles in assisting enzyme reactions are vitamins' best-known function, the other vitamin functions are equally important.[6]
In humans
Vitamins are classified as either water-soluble or fat soluble.
In humans there are 13 vitamins: 4 fat-soluble (A, D, E and K) and 9 water-soluble (8 B vitamins and vitamin C).
Water-soluble
Water-soluble vitamins dissolve easily in water, and in general, are readily excreted from the body, to the degree that urinary output is a strong predictor of vitamin consumption.[13] Because they are not readily stored, consistent daily intake is important.[14] Many types of water-soluble vitamins are synthesized by bacteria.[15]
Fat-soluble
Fat-soluble vitamins are absorbed through the intestinal tract with the help of lipids (fats). Because they are more likely to accumulate in the body, they are more likely to lead to hypervitaminosis than are water-soluble vitamins. Fat-soluble vitamin regulation is of particular significance in cystic fibrosis.[16]
In nutrition and diseases
Vitamins are essential for the normal growth and development of a multicellular organism. Using the genetic blueprint inherited from its parents, a fetus begins to develop, at the moment of conception, from the nutrients it absorbs. It requires certain vitamins and minerals to be present at certain times. These nutrients facilitate the chemical reactions that produce among other things, skin, bone, and muscle. If there is serious deficiency in one or more of these nutrients, a child may develop a deficiency disease. Even minor deficiencies may cause permanent damage.[30]
For the most part, vitamins are obtained with food, but a few are obtained by other means. For example, microorganisms in the intestine—commonly known as "gut flora"—produce vitamin K and biotin, while one form of vitamin D is synthesized in the skin with the help of the natural ultraviolet wavelength of sunlight. Humans can produce some vitamins from precursors they consume. Examples include vitamin A, produced from beta carotene, and niacin, from the amino acid tryptophan. Once growth and development are completed, vitamins remain essential nutrients for the healthy maintenance of the cells, tissues, and organs that make up a multicellular organism; they also enable a multicellular life form to efficiently use chemical energy provided by food it eats, and to help process the proteins, carbohydrates, and fats required for respiration.
Deficiencies
Deficiencies of vitamins are classified as either primary or secondary.
A primary deficiency occurs when an organism does not get enough of the vitamin in its food.
A secondary deficiency may be due to an underlying disorder that prevents or limits the absorption or use of the vitamin, due to a “lifestyle factor”, such as smoking, excessive alcohol consumption, or the use of medications that interfere with the absorption or use of the vitamin. People who eat a varied diet are unlikely to develop a severe primary vitamin deficiency. In contrast, restrictive diets have the potential to cause prolonged vitamin deficits, which may result in often painful and potentially deadly diseases.
Because human bodies do not store most vitamins, humans must consume them regularly to avoid deficiency. Human bodily stores for different vitamins vary widely; vitamins A, D, and B12 are stored in significant amounts in the human body, mainly in the liver,[27] and an adult human's diet may be deficient in vitamins A and B12 for many months before developing a deficiency condition. Vitamin B3 is not stored in the human body in significant amounts, so stores may only last a couple of weeks.[19][27]
Well-known human vitamin deficiencies involve thiamine (beriberi), niacin (pellagra), vitamin C (scurvy) and vitamin D (rickets). In much of the developed world, such deficiencies are rare; this is due to (1) an adequate supply of food; and (2) the addition of vitamins and minerals to common foods, often called fortification.[18][27]
Some evidence also suggests that there is a link between vitamin deficiency and mental disorders.[31]
Side effects and overdose
In large doses, some vitamins have documented side effects that tend to be more severe with a larger dosage. The likelihood of consuming too much of any vitamin from food is remote, but overdosing from vitamin supplementation does occur. At high enough dosages some vitamins cause side effects such as nausea, diarrhea, and vomiting.[19][32]
When side effects emerge, recovery is often accomplished by reducing the dosage. The concentrations of vitamins an individual can tolerate vary widely, and appear to be related to age and state of health
Supplements
Dietary supplements, often containing vitamins, are used to ensure that adequate amounts of nutrients are obtained on a daily basis, if optimal amounts of the nutrients cannot be obtained through a varied diet.
Vitamin A and E supplements not only provide no tangible health benefits for generally healthy individuals, but may actually increase mortality, although two large studies included in the analysis involved smokers, for which it was already known that beta-carotene supplements can be harmful.[37]
In the United States, advertising for dietary supplements is required to include a disclaimer that the product is not intended to treat, diagnose, mitigate, prevent, or cure disease, and that any health claims have not been evaluated by the Food and Drug Administration.[36
In some cases, dietary supplements may have unwanted effects, especially if taken before surgery, with other dietary supplements or medicines, or if the person taking them has certain health conditions.[36] Vitamin supplements may also contain levels of vitamins many times higher, and in different forms, than one may ingest through food.[38]
Intake of excessive quantities can cause vitamin poisoning, often due to overdose of Vitamin A and Vitamin D (The most common poisoning with multinutrient supplement pills does not involve a vitamin, but is rather due to the mineral iron). Due to toxicity, most common vitamins have recommended upper daily intake amounts.
Avitaminosis
Avitaminosis is any disease caused by chronic or long-term vitamin deficiency or caused by a defect in metabolic conversion, such as tryptophan to niacin. They are designated by the same letter as the vitamin.
Conversely hypervitaminosis is the syndrome of symptoms caused by over-retention of fat-soluble vitamins in the body.
Avitaminoses include
• vitamin A deficiency causes xerophthalmia or night blindness
• thiamine deficiency causes beriberi
• niacin deficiency causes pellagra
• vitamin B12 deficiency leads to megaloblastic anemia
• vitamin C deficiency leads to scurvy
• vitamin D deficiency causes rickets
• vitamin K deficiency causes impaired coagulation
•
Vitamin poisoning
Vitamin poisoning, hypervitaminosis or vitamin overdose refers to a condition of high storage levels of vitamins, which can lead to toxic symptoms. The medical names of the different conditions are derived from the vitamin involved: an excess of vitamin A, for example, is called hypervitaminosis A.
With few exceptions, like some vitamins from B complex, hypervitaminosis usually occurs more with fat-soluble vitamins, which remain more time in the body and are harder to be excreted than water soluble vitamins.
High dosage vitamin A; high dosage, slow release vitamin B3; and very high dosage vitamin B6 alone (i.e. without vitamin B complex) are sometimes associated with vitamin side effects that usually rapidly cease with supplement reduction or cessation.
Vitamin C has a brief, pronounced laxative effect when taken in large amounts, typically in the range of 5-20 grams per day in divided doses for a person in normal "good health," although seriously ill people,[1] may take 100-200 grams without inducing vitamin poisoning.
High doses of mineral supplements can also lead to side effects and toxicity. Mineral-supplement poisoning does occur occasionally due to excessive and unusual intake of iron-containing supplements, including some multivitamins, but is not common.
The Dietary Reference Intake recommendations from the United States Department of Agriculture define a "tolerable upper intake level" for most vitamins.
List of vitamins
Each vitamin is typically used in multiple reactions and, therefore, most have multiple functions.[17]
Vitamin generic
descriptor name
Vitamer chemical name(s) (list not complete)
Solubility
Recommended dietary allowances
(male, age 19–70)[18]
Deficiency disease Upper Intake Level
(UL/day)[18]
Overdose disease
Vitamin A
Retinoids
(retinol, retinoids
and carotenoids)
Fat 900 µg Night-blindness and
Keratomalacia[19]
3,000 µg Hypervitaminosis A
Vitamin B1
Thiamine
Water 1.2 mg Beriberi, Wernicke-Korsakoff syndrome
N/D[20]
Rare hypersensitive reactions resembling anaphylactic shock-- injection only;
Drowsiness
Vitamin B2
Riboflavin
Water 1.3 mg Ariboflavinosis
N/D ?
Vitamin B3
Niacin, niacinamide
Water 16.0 mg Pellagra
35.0 mg Liver damage (doses > 2g/day)[21] and other problems
Vitamin B5
Pantothenic acid
Water 5.0 mg[22]
Paresthesia
N/D ?
Vitamin B6
Pyridoxine, pyridoxamine, pyridoxal
Water 1.3-1.7 mg Anemia[23]
100 mg Impairment of proprioception, nerve damage (doses > 100 mg/day)
Vitamin B7
Biotin
Water 30.0 µg Dermatitis, enteritis
N/D ?
Vitamin B9
Folic acid, folinic acid
Water 400 µg Deficiency during pregnancy is associated with birth defects, such as neural tube defects
1,000 µg Possible decrease in seizure threshold
Vitamin B12
Cyanocobalamin, hydroxycobalamin, methylcobalamin
Water 2.4 µg Megaloblastic anemia[24]
N/D No known toxicity[25]
Vitamin C
Ascorbic acid
Water 90.0 mg Scurvy
2,000 mg Vitamin C megadosage
Vitamin D
Ergocalciferol, cholecalciferol
Fat 5.0 µg-10 µg[26]
Rickets and Osteomalacia
50 µg Hypervitaminosis D
Vitamin E
Tocopherols, tocotrienols
Fat 15.0 mg Deficiency is very rare; mild hemolytic anemia in newborn infants.[27]
1,000 mg Increased congestive heart failure seen in one large randomized study.[28]
Vitamin K
phylloquinone, menaquinones
Fat 120 µg
الثلاثاء، 28 أبريل 2009
الثلاثاء، 14 أبريل 2009
المضادات الحيوية
المضادات الحيوية
مقدمة حول المضادات الحيوية
حين بدأ البحث العلمي عن دواء فعال ضد الزهري (السفلس) تم منذالك التاريخ اكتشاف العديد من الادوية الفعالة ضد معظم الامراض الناجمة عن البكتيريا وكان اهم هذه الادويه السلفونامايدات ثم المضادات الحيويهAntibiotics وهي مواد عضوية تنتجها الكائنات الدقيقة كالبكتيريا والفطريات وبعض الطحالب والنباتات الراقية اثناء نموها، وهي عبارة عن نواتج أييضية ثانوية قادرة بتركيز منخفض على أن تبيد أو تثبط نمو الكائنات الدقيقة الأخرى ( غير الكائنات التي انتجتها)
تاريخ اكتشاف المضادات الحيوية:
في عام 1929م ، اكتشف المضادات الحيوية عالم إنجليزي اسمه ( فليمنج ) ( Fleming ) حيث شاهد هذا العالم عفناً أخضراً نامياً على سطح أحد أطباق ( بتري ) التي يجري عليها تجاربه ، و قد سقط هذا العفن من الهواء على شكل أبواغ نمت على المواد الغذائية في الطبق و كونت مستعمرة ( البنسيليوم ) ، و عندما دقق فليمنج النظر في الطبق ، وجد أن المنطقة التي نما فيها العفن لا تحتوي على بكتيريا ، و بعد تجارب عديدة وجد فليمنج أن العفن الذي هو عبارة عن فطر البنسيليوم ينتج مادة تستطيع القضاء على العديد من أنواع البكتيريا ، و سمى فليمنج هذه المادة ( بنسلين ) (Penicillin ) و في البداية أنتج العلماء البنسلين بكميات محدودة ، وما أن جاءت سنة 1938 حتى استطاع العلماء إنتاجه بكميات كبيرة ، و قد كان إنتاجه مفيداً جداً ، و ذلك لأن الحرب العالمية الثانية كانت في بدايتها ، و قد أنقذ البنسلين بقدرة الله حياة الجرحى من موت محتم نتيجة لالتهابات الجروح الناجمة عن الحرب العالمية الثانية، و يستعمل البنسلين على شكل حقن ، أو حبوب ، أو مسحوق ، أو مراهم ...و يستطيع البنسلين القضاء على العديد من الأمراض البكتيرية مثل : الالتهاب الرئوي ، الدفتيريا ، الزهري ( السفلس ) ، السيلان ، و التهابات الجروح . غير أن البنسلين لا يستطيع القضاء على عدد آخر من الأمراض البكتيرية مثل : مرض السل ، كما أنه لا يفيد في علاج أي من الأمراض الفيروسية ، لهذا السبب بحث العلماء عن مضادات حيوية أخرى من أنواع أخرى من الكائنات وفي عام 1940اكتشف العالم الأميريكي واكسمان المضاد الحيوي الستربتوماسين من البكتيريا الخيطية Streptomyces griseus ثم توالت الدراسات العديدة للبحث عن انتاج المضادات الحيوية منالأنواع المختلفة من الأكتينوميسيتات،أ والبكتيريا أو الفطريات أو الطحالب أو الأشنات أو النباتات الراقية، و من المضادات الحيوية المستعملة في الوقت الحاضر بالإضافة إلى البنسلين – ستربتوميسين ( Streptomycin ) ، و اوروميسين ( Aureomycin ) ، و تيراميسين ( Terramycin ) ، و أكرومايسين ( Achromycin ) ، و ... الخ.تستعمل عقاقير السلفا و المضادات الحيوية في الوقت الحاضر على نطاق واسع ، و بهذا العدد يجب أن نتذكر أن المادة التي تقضي على البكتيريا يمكن أن تسبب لخلايا جسمنا الضرر ، و في بعض الحالات فإن تعاطي المضادات الحيوية عن طريق الفم ، يمكن أن يقضي على البكتيريا المفيدة الموجودة في الأمعاء مثل : البكتيريا التي تنتج العديد من فيتامينات ب ( B ) و كذلك البنسلين قد يسبب الموت المفاجئ عند الأشخاص ذوي الحساسية ، و لهذا السبب يجب تعاطي عقاقير السلفا و المضادات الحيوية بحذر شديد كما يجب أن تؤخذ تحت إشراف طبيب ماهر ، و من ناحية أخرى فإن الاستعمال الواسع للمضادات الحيوية ، يمكن أن يؤدي إلى ظهور سلالات من البكتيريا تقاوم بشكل كبير المضادات الحيوية ، و في هذه الحالة يصبح المضاد الحيوي عديم الفائدة بالنسبة للبكتيريا المقاومة ، و لا يستطيع القضاء عليها . فمثلاً نمت في المستشفيات بكتيريا من نوع المكور العنقودي ( البكتيريا الكروية العنقودية ) ( Staphylococci ) ، و هذه البكتريا تقاوم البنسلين ، و قد أصبحت مشكلة كبيرة في العديد من المستشفيات ، حيث تفرز هذه البكتيريا أنزيماً يسمى ( البنسيليز ) ( Penicillinase ) يحطم البنسلين و يبطل مفعوله ، و بذلك يصبح البنسلين عديم الفائدة في هذه الحالة ...
وتقسم المضادات الحيوية بشكل عام من حي طبيعة تأثيرها في قسمين : مضادات واسعةالمجال الفاعل أو المؤثر(broad spectruim) ,ومنها التراميسين والأريومايسين( أي مجموعة اليتراسيكلين ) والجنتامايسين و الامبيسلين والكلورامفينكول وهي مفيدة في علاج العدوى المختلطه ( mixed infection) . واخرى تسمى مضادات ضيقة المفعول (narrow spectrum) مثل البنسلين والاريثرومايسين
كيف تعمل المضادات الحيوية؟ منها مايثبط نمو البكتريا ( Bacterio static) وذلك بتاثيرها على النمو بمنع التخليق الحيوي لبروتينات الخليه واحماضها النووية. ومنها مبيدة أو قاتلة تماما لخلايا الميكزوبات ( Bacterioidal) وذتك بمنع تكوين جدار الخليه او غشائها السيتوبلازس( cytoplasm) وقد يكون المضاد الحيوي موقف للنمو بجرعته الأدنى ومبيد بجرعته الأعلى ويعتمد استمرار المفعول على الجرعه المناسبه وكذلك نفاذية المضاد الحيوي الى الانسجه. لذا فإنه يفضل قبل البدء في المعالجه فحص حساسية الميكروب للمضادات الحيويه لاستعمال المضاد المؤثر في العلاج.
لمجموعة
آلية العمل
الأمثلة
المجموعة الأولى:
Bactericidal متخصصة في قتل الميكروبات والميكروبات غير المتكاثرة (الساكنةResting )
Streptomycin
Sisomicin
Neomycins
المجموعة الثانية :
متخصصة لقتل الميكروبات المتكاثرة Proliferating
Penicillins
Bacitracin
Fosfomycin
المجموعة الثالثة :
مثبطة لنمو الميكروبات ولكنها بالتراكيز العالية تكون قاتلة Bactericidal
Tetracyclines
Carbomycin
Novobiocin
المجموعة الرابعة :
تعمل فقط على التثبيط دون القتل .
D-Cycloserine
Capreomycn
Viomycin
طريق عمل المضادات الحيوية:
1.منع تكوين جدار الخلية. تحاط خلايا البكتيريا بغشاء يحيط به جدار صلب، يمنع انشقاق الخلية وفتحها. وتدمر مركبات البنسلين وبعض المضادات الحيوية الأخرى الأحياء المجهرية، بإعاقة تكوين هذا الجدار. أما خلايا الإنسان، فإنها ليست بحاجة إليه. ولذلك، فإن هذه المضادات الحيوية لا تتلفها.
2.تصدع غشاء الخلية. تصدع بعض المضادات الحيوية، مثل الأمفوترسين ب، والنستاتين، الغشاء الخلوي لبعض الأحياء المجهرية، الذي يتحكم في حركة المواد الداخلة والخارجة من الخلية. وقد يؤدي تصدع الغشاء الخلوي إلى خروج المغذيات الحيوية من الخلية أو دخول المواد السامة التي تفتك بالخلية. ولا يتأثر الغشاء الخلوي للإنسان بالمضادات الحيوية، ذلك لأن هذه المضادات لا تصدع إلا الأغشية الخلوية المحتوية على عناصر موجودة فقط في خلايا الأحياء المجهرية.
3.اضطراب العمليات الكيميائية. تنتج جميع الخلايا البروتينات والحموض النووية، وهي ضرورية لحياة أي كائن حي. وتكافح بعض المضادات الحيوية المرض، بتداخلها مع العمليات الكيميائية التي تنتج بوساطتها هذه المواد. على سبيل المثال، يمنع الستربتومايسين والتتراسيكلين بعض أنواع الأحياء المجهرية من إنتاج البروتينات، ويعترض الرفامبين تكوين الحموض النووية. وتنتج خلايا الإنسان البروتينات والحموض النووية في الغالب بنفس الطريقة التي تنتجها بها خلايا الميكروبات. ولكن عمليات الإنتاج تختلف اختلافًا كبيرًا، لدرجة أن بعض المضادات الحيوية تعترض الأنشطة الكيميائية في خلايا الميكروبات، بينما لا يحدث هذا في خلايا الإنسان
2.تصدع غشاء الخلية. تصدع بعض المضادات الحيوية، مثل الأمفوترسين ب، والنستاتين، الغشاء الخلوي لبعض الأحياء المجهرية، الذي يتحكم في حركة المواد الداخلة والخارجة من الخلية. وقد يؤدي تصدع الغشاء الخلوي إلى خروج المغذيات الحيوية من الخلية أو دخول المواد السامة التي تفتك بالخلية. ولا يتأثر الغشاء الخلوي للإنسان بالمضادات الحيوية، ذلك لأن هذه المضادات لا تصدع إلا الأغشية الخلوية المحتوية على عناصر موجودة فقط في خلايا الأحياء المجهرية.
3.اضطراب العمليات الكيميائية. تنتج جميع الخلايا البروتينات والحموض النووية، وهي ضرورية لحياة أي كائن حي. وتكافح بعض المضادات الحيوية المرض، بتداخلها مع العمليات الكيميائية التي تنتج بوساطتها هذه المواد. على سبيل المثال، يمنع الستربتومايسين والتتراسيكلين بعض أنواع الأحياء المجهرية من إنتاج البروتينات، ويعترض الرفامبين تكوين الحموض النووية. وتنتج خلايا الإنسان البروتينات والحموض النووية في الغالب بنفس الطريقة التي تنتجها بها خلايا الميكروبات. ولكن عمليات الإنتاج تختلف اختلافًا كبيرًا، لدرجة أن بعض المضادات الحيوية تعترض الأنشطة الكيميائية في خلايا الميكروبات، بينما لا يحدث هذا في خلايا الإنسان
كيفية وشروط استخدام المضادات الحيوية: فيجب عند استخدام هذه المواد أن نستخدمها الاستخدام الصحيح لانها قد تضر: 1. يجب قبل البد في المعالجه عمل مزرعة أجار agar plate (على أطباق بتري ) للكائن المسبب للمرض لمعرفة المضاد المؤثر لها 2. يجب استخدام المضاد الحيوي حتى الشفاء كاملاً وغالبا في الاصابات البسيطه من5ايام_7أيام حتى لايحدث عند المكروب مناعه من الدواء المستخدم 3. مراعات مدة تاثير الجرعه فالبعض يكون كل 6ساعات والبعض كل 8ساعات هذ بالنسبه للدواء الذي يوخذ عن طريق الفم وهي قصيرة المفعول. أنواع المضادات الحيوية: يمكن تقسيم عمل المضادات الحيويه . مع اسماء الادويه: 1. مضادات حيويه تعمل على جدار خلية البكتريا Bacterial cell wall. مثل البنسلين. والسفالوسبورين . وسيكلوسبرين والباستراسين والفانكوميسين والرستوستين. 2. مضادات حيويه تعمل على جدار السيتوبلازمي Cytoplasmic. membrane. مثل البوليكسين. والجراسدين .الامفوتر سين .النيساتين وهذ المضادات الحيويه تؤثر على خلية البكتريا وخلايا العائل ولذا فان لها تاثير ضار على الخليه 3. مضادات حيويه تعمل على تكون البروتينات داخل الخلية. مثل الاستربتوميسين والتتراسكلين.الكلورامفنيكول. الارثروميسين . والنيوميسين . والكانا ميسين .الباروميسين. والاولياندوميسين واللنكوميسين . 4. مضادات حيويه تعمل على حمض النيوكليك (Nucleic acid) مثل الرفيامبسين. والاكيتنوميسين. وهذه المضادات تهبط تكون الدنأ(D.N,A) ولذا فانه يمكن اعتبارهم مضادات للآورام(Cytotoxic Drugs)
تتكون هذه المجموعه من ألادويه الاتيه: 1. الاستربتومايسين 2. الكا ناميسين 3. النيوميسين 4. الباروموميسين(الهيماثين) 5. الأميكاسين 6. الجنتاميسين 7. التوبراميسين(البنسن) 8. الاسيكتينوميسين الاستربتومايسين مضاد حيوى واسع المدى ولانه لايمتص من القناه الهضميه عند تناوله بالفم لذالك لايعطى إلا بالحقن لضمان فاعليته وهو فعال ضدة العديد من البكتريا السالبه والموجبة الغرام ويعمل كقاتل للبكتريا عن طريق تداخله في عملية تخليق بروتيناتها الطبيعيه. ويستخدم الاستربتومايسين معوالديهيدروستروبتوميسينفي علاج النزلات المعويه والزحار المعويه ويتم امتصاص هذه المجموعه اذا اعطت في الحقن بالعضل او الوريد ويتم توزيعها في الانسجه والتجويف البلوري وسائل الجسم وكذالك سائل المفاصل خصوصاً عند حدوث الالتهابات ويمكن ان تنتشر بضعف في غدة البروستات . والعين والصفراء وكذالك في النخاع الشوكي 0ويتحد الاستربتو مايسين ببروتينيات بمقدار 35% اما بقية المجموعه الامنيوجليكزيدات فتتحد بمقدار 10% ويتم اخراج الاستربتومايسين عن طريق الترشيح في محافظ البومان ويقل اخراجه في حاله هبوط وظيفة الكلى ويمكن حدوث تراكم للاستربتومايسين في الدم الاستخدام الطبي (1) يستخدم في علاج الدورن الرئوى ويستخدم في علاج الاصابات الروئية وخارج الرئه ويعطي مع ادوية أخرى منها الابزنيازيد والايثامبيتول أو الريفامبين وذلك لمنع نمو الجراثيم العنيده بسرعة ويعطي 1جم/يوميا بالحقن في العضل أو بعد يوم ويعطى لمدة شهرين على الاقل0 (2) يستخدم في علاج الطاعون والبروسيالات ويعطى مع التتراسيكلين أو الكلورامغنيكول ويعطى 2-4جم/بالعضل يوميا0 (3) يعطى الاستربتومايسين في جرعه مقدارها1-3جم/بالعضل يوميا مع الجنتاميسين 80مجم/كل 8ساعات بالحقن في العضل في علاج التهاب الشفاف القلبي أو مع البنسلين في جرعة 12-20جم/في الوريد يوميا أوالامبيسلين4جم/بالحقن في العضل0 (4) يستخدم في علاج التهابات المجارى البوليه الناجمه عن العصيات القولونية والفطور الكاذبه0 (5) يستخدم بالفم في علاج الزحار العصوى وفي تتهيئه الامعاء قبل الجراعه الهضمية ويعطى الاستلابتومايسين بمقدار500ملج كل 8ساعات لمدة لاتتجاوز3-5 أيام ويعطى الاستربتومايسين بالحقن في العضل أو بالفم لتطهير الامعاء أوبالحقن في الوريد ولكنه يمكن أن يحدث التهاب تجلطى للاورده-واذا اعطى بالحقن في النخاع الشوكي يؤدي الى ازعاج للجهاز العصبي المركزي ويؤدى الى حدوث تشنجات الأعراض الجانبية: (1) حساسيه (2) الم في مكان الحقن (3) تأثير سئ على السمع فيؤدى الاستربتومايسين الى دوار ويؤدي الهديهيدروستربتومايسين على السمع يمكن أن يزيد باستخدام الازكرين واللازكس0 (4) تأثير سئ الكليه (5) يؤدي الى ارتخاء العضلات الادايه مثل الكوراى (6) عند حقنه في النخاع الشوكي يؤدي الى حدوث تشنجات (7) حدوث المناعه (نمو الجراثيم العنيده بسرعه) يلاحظ أن هنالك بعض الادويه يجب عدم اعطائها للمريض الذي يستخدم الاستربتومايسين وهى اللازكس - الادكرين ومرخيات العضلات الاراديه الكاناميسين يشبة بقيه مجموعه الاميتوجليكوزيد –ولكنه غير فعال ضد ميكروبات القطور الكاذبه ويؤثر على بعض العصيات الفطريه السليمه العنيدة للاستربتومايسين والا يزونيازيد و الباس ويستخدم كعلاج للالتهابات الناجمه عن الجراثيم سلبيه الجرام وخاصه المتقلبات والمكورات العنقوديه التى تستجيب للمضادات الاخرى و الا قل سميه0 ويعطى في علاج الدورن بالحقن في العضل½جم كل 6-12ساعات اواجمالى فانه يعطى بمقدار15مجم/كجم/يوميا ويستخدم موضعيات في تطهير الامعاء ويعطى بالفم بجرعه 1جم كل ساعه لمدة4 ثم كل 6 ساعات لمدة36-72ساعة بالعضل0 ومن اعراضة الجانبية الطفح الجلدي والحس والصداع والغثيان والقيء والتلف الكلوي واخطر اعراضه هو الصمم الدائم ومن أسمائة التجارية (Kantrex) النيوميسين Neomycin يستخدم كمضاد حيوى موضعي بمقدار ½ جم با لاضافه الى مظادات حيويه اخر لمنع حدوث المناعه ويعطى النيوميسين مع الباستراسين او النيومسين مع البوليمكسين. ويمكن اعطاء النيوميسين كمطهر للامعاء ويفضل عن الاستربتوميسين لانه فعال ضد ( ميكروب الشيريشيات القولوننيه E.coli والproteus) ويستخدم ضد المكيروبات سلبية الجرام وكذالك ضد المكورات العنقوديه.
تتكون هذه المجموعه من ألادويه الاتيه: 1. الاستربتومايسين 2. الكا ناميسين 3. النيوميسين 4. الباروموميسين(الهيماثين) 5. الأميكاسين 6. الجنتاميسين 7. التوبراميسين(البنسن) 8. الاسيكتينوميسين الاستربتومايسين مضاد حيوى واسع المدى ولانه لايمتص من القناه الهضميه عند تناوله بالفم لذالك لايعطى إلا بالحقن لضمان فاعليته وهو فعال ضدة العديد من البكتريا السالبه والموجبة الغرام ويعمل كقاتل للبكتريا عن طريق تداخله في عملية تخليق بروتيناتها الطبيعيه. ويستخدم الاستربتومايسين معوالديهيدروستروبتوميسينفي علاج النزلات المعويه والزحار المعويه ويتم امتصاص هذه المجموعه اذا اعطت في الحقن بالعضل او الوريد ويتم توزيعها في الانسجه والتجويف البلوري وسائل الجسم وكذالك سائل المفاصل خصوصاً عند حدوث الالتهابات ويمكن ان تنتشر بضعف في غدة البروستات . والعين والصفراء وكذالك في النخاع الشوكي 0ويتحد الاستربتو مايسين ببروتينيات بمقدار 35% اما بقية المجموعه الامنيوجليكزيدات فتتحد بمقدار 10% ويتم اخراج الاستربتومايسين عن طريق الترشيح في محافظ البومان ويقل اخراجه في حاله هبوط وظيفة الكلى ويمكن حدوث تراكم للاستربتومايسين في الدم الاستخدام الطبي (1) يستخدم في علاج الدورن الرئوى ويستخدم في علاج الاصابات الروئية وخارج الرئه ويعطي مع ادوية أخرى منها الابزنيازيد والايثامبيتول أو الريفامبين وذلك لمنع نمو الجراثيم العنيده بسرعة ويعطي 1جم/يوميا بالحقن في العضل أو بعد يوم ويعطى لمدة شهرين على الاقل0 (2) يستخدم في علاج الطاعون والبروسيالات ويعطى مع التتراسيكلين أو الكلورامغنيكول ويعطى 2-4جم/بالعضل يوميا0 (3) يعطى الاستربتومايسين في جرعه مقدارها1-3جم/بالعضل يوميا مع الجنتاميسين 80مجم/كل 8ساعات بالحقن في العضل في علاج التهاب الشفاف القلبي أو مع البنسلين في جرعة 12-20جم/في الوريد يوميا أوالامبيسلين4جم/بالحقن في العضل0 (4) يستخدم في علاج التهابات المجارى البوليه الناجمه عن العصيات القولونية والفطور الكاذبه0 (5) يستخدم بالفم في علاج الزحار العصوى وفي تتهيئه الامعاء قبل الجراعه الهضمية ويعطى الاستلابتومايسين بمقدار500ملج كل 8ساعات لمدة لاتتجاوز3-5 أيام ويعطى الاستربتومايسين بالحقن في العضل أو بالفم لتطهير الامعاء أوبالحقن في الوريد ولكنه يمكن أن يحدث التهاب تجلطى للاورده-واذا اعطى بالحقن في النخاع الشوكي يؤدي الى ازعاج للجهاز العصبي المركزي ويؤدى الى حدوث تشنجات الأعراض الجانبية: (1) حساسيه (2) الم في مكان الحقن (3) تأثير سئ على السمع فيؤدى الاستربتومايسين الى دوار ويؤدي الهديهيدروستربتومايسين على السمع يمكن أن يزيد باستخدام الازكرين واللازكس0 (4) تأثير سئ الكليه (5) يؤدي الى ارتخاء العضلات الادايه مثل الكوراى (6) عند حقنه في النخاع الشوكي يؤدي الى حدوث تشنجات (7) حدوث المناعه (نمو الجراثيم العنيده بسرعه) يلاحظ أن هنالك بعض الادويه يجب عدم اعطائها للمريض الذي يستخدم الاستربتومايسين وهى اللازكس - الادكرين ومرخيات العضلات الاراديه الكاناميسين يشبة بقيه مجموعه الاميتوجليكوزيد –ولكنه غير فعال ضد ميكروبات القطور الكاذبه ويؤثر على بعض العصيات الفطريه السليمه العنيدة للاستربتومايسين والا يزونيازيد و الباس ويستخدم كعلاج للالتهابات الناجمه عن الجراثيم سلبيه الجرام وخاصه المتقلبات والمكورات العنقوديه التى تستجيب للمضادات الاخرى و الا قل سميه0 ويعطى في علاج الدورن بالحقن في العضل½جم كل 6-12ساعات اواجمالى فانه يعطى بمقدار15مجم/كجم/يوميا ويستخدم موضعيات في تطهير الامعاء ويعطى بالفم بجرعه 1جم كل ساعه لمدة4 ثم كل 6 ساعات لمدة36-72ساعة بالعضل0 ومن اعراضة الجانبية الطفح الجلدي والحس والصداع والغثيان والقيء والتلف الكلوي واخطر اعراضه هو الصمم الدائم ومن أسمائة التجارية (Kantrex) النيوميسين Neomycin يستخدم كمضاد حيوى موضعي بمقدار ½ جم با لاضافه الى مظادات حيويه اخر لمنع حدوث المناعه ويعطى النيوميسين مع الباستراسين او النيومسين مع البوليمكسين. ويمكن اعطاء النيوميسين كمطهر للامعاء ويفضل عن الاستربتوميسين لانه فعال ضد ( ميكروب الشيريشيات القولوننيه E.coli والproteus) ويستخدم ضد المكيروبات سلبية الجرام وكذالك ضد المكورات العنقوديه.
الاستخدام الطبي للنيومسين: (1) يستخدم في غيبوبه الكبد ويعطى 4-6جم / يوميا أو 1جم / كل 4-6 ساعات لتهبيط بكتريا فلورا المعوية ويتقص من أعداد الجراثيم المعويه التي تنتج الامونيا0 (2) يمكن أن يستخدم كمضاد حيوى موضعى على هيئة مراهم أوغسول أو قطرات للعين والاذن بمقدار 25,-5,% ويحتوى على (النيومايسين و الباستراسن أو البوليكسين) ويستخدم في علاج التهاب الجلد والجروح والتهاب الأنف والأذن الخارجيه0ويسبب عند اعطائه بالفم الغثيان والقيء والاسهال وضعف امتصاص الغذاء في الامعاء وأعراض التحسس كالحكه والاحمرار وتقشر الجلد0 ملحوظة: تحدث مقاومه متقاطعه بين النيومايسين والكانامايسين والبارومايسين 0 البارومايسين (الهيمائين) يعطي 1 جم /كل 6ساعات بالفم لمده 6اسابيع في علاج الدوسنتاريا الاميبيه المعويه0 الأميكاسين هو مشتق شبه مصنع من الكانامايسين ولايتأثر بالانزميات التى تؤثر على نشاط الجنتامايسن والتوبرامايسين وهذا المضاد الحيوى فعال ضد البكتريا سالبة الجرام وخصوصا ميكروب القيح الازرق وميكروب الفطور الكاذبه مولده الهواء ويؤثر علىأنواع كثيره من ميكروبات المتقلبات والشيريشيات القلونيه والسيرشيا ويستخدم فى معالجه تجرثم الدم وانتان الدم وفي معالجه الالتهابات الشديدة في الجهاز التنفسى والجهاز العصبي المركزى (بما فى ذلك التهاب السحايا) والعضام المفاصل وفي الجلد والانسجه اللينة والحروق والانتان داخل البطن (بمافي ذلك التهاب البريثون) والانتانات بعد العمليات الجراحيه ويستعم0ايضا في علاج الالتهابات الشديده المختلطه والمتكره في الجهاز البولي التى لاتستجيب لادويه أقل سميه بعد عمل مزرعه وحساسيه للميكروبات والمتسبب في هذا الالتهاب0 الجرعه: يعطى بالحقن في العضل في جرعه 500ملج /كل12 ساعه (15 ملج /كجم/ يوميا)0 الاعراض الجانبيه: (1) تأثير سئ على الكليه (2) تأتير سئ على السمع ويلاحظ عدم اعطاء الاميكاسين مع اللازكس أو الا ركرين ومن اسمائه التجاريه الجنتامايسينهم مضاد حيوى من مجموعه الامنيوجليكوزيد فعال ضد الميكروبات الموجبه والسالبه (gm+ ve& gm-ve) ويذداد غاعليته في الوسط القلوى عنه في الوسط الحامضى0 ويعتبر الجنتامايسين كمضاد قاتل للميكروب عن طريق تهبيط تكوين البروتينات داخل البكتريا في الميكروبات الموجبة والسالبة وخصوصا فهو فعال ضد ميكروب الفطور الكاذبة وميكروب المكورات العنقودية والميكروبات المعوية مثل الشريشبات القولونيه والكلبسيلا والبكتيريا المعويه وفعال ايضا ضد المتقلبات والسيرشيا ويعتبر الجنتامايسين قاتل للميكروب يتيجة لتهبيط تكون البروتينات في خليه البكتريا ويمكن اعطاء الجنامايسين مع الكارنيسلين لعلاج العدوى بلفطور الكاذبه مولده الهواء ولذا يعطى جرعات صغيره من الجنتاميسين لتقليل حدوث المناعه ولتقليل حدوث الاعراض الجانبيه=ويلاحظ ان الميكروب السبحي لايتاثر بالجنتاميسين ولكنه يمكن اعطاء البنسلين مع الجنتاميسين وذلك لمساعدة الجنتاميسين للنفاذ داخل خلية البكتريا لايؤثر الجنتاميسين على الميكروبات اللاهوائيه== يعطى الجنتاميسين بالحقن ويلاحظ انه يتوزع على الانسجه بسرعه ويلاحظ انه يلتحم ببروتينات الدم 25% ويتم اخراجه عن طريق الكلى ولاينفذ عن طريق حاجز المخ الاستخدام الطبي يستخدم بالحقن في علاج العدوى بالمكروبات السالبه الجرام التي لاتتأثر بالمضادات الحيويه الاخرى ويعطى 5-7 مجم/كجم/ يوميا بالحقن في العضل او بالوريد ويعطى على ثلاث جرعات متساويه يونيا لمدة 7-10 أيام (40-80 ملجم كل 8ساعات ويفيد في علاج الانتانات الخطيره التي لاتستجيب للمضادات الحيويه الاخرى مثل. انتانات حديثي الولاده. انتانات الدم . وذات الرئه ويعطى مع البنسلين في علاج التهاب شفاف القلب السبحي ،ويراعى عدم خلط الجنتا ميسين مع البنسلين في حقنه واحده لتجنب حدوث التداخل بينهم ولابد من متابعة وظائف الكليه والسمع اثناء اعطاء الجنتاميسين للمريض ويلاحض في حالة هبوط وظائف الكليه يجب تقليل جرعة الجنتاميسين اوتطويل فترة الحقن للمريض. او إعطى مضاد حيوي اخر غير الجنتاميسين يستخدم لعلاج العدوى بميكروب المكورات العنقوديه. و يستخدم لعلاج التهاب المجاري البوليه، ويعطى في جرعة 8ر—1,2مجم/كجم/ يوميا بالحقن على 2_3مرات يوميا لمده 10 ايام. و يستخدم في علاج التهاب الجهاز التنفسي وكذالك الجهاز الهضمي ( التهاب البريتون) والتهاب الجلد والعضام والانسجه. يستخدم كمضاد حيوي موضعي في الحروق والجروح والتهاب الجلد او التهاب الملتحمه . او يستخدم كنقط للعين او مرهم في تركيز 1, إلى 3,5 الاعراض الجانبيه تأثير سىء على الكليه ولذا لابد من اختبار الكرياتينين وأيضا تأثير سىء على السمع يودي إلى اضطربات سمعيه ودهليزيه تتجلى اعراضها بالدوخه والدوار وطنين الاذن والصم التوبراميسين( البنسن) يشبه التوبراميسين الجنتامايسين في مدى عمله ضد البكتريا وقد ادخل عام 1975 ولكنه اقوى من الجنتاميسين ضد ميكروب الفطور الكاذبه ويعطى في جرعه 3_5ملجم /كجم /يوميا بالحقن في العضل على جرعات مقسمه ثلاث جرعات يونيا كل 8 ساعات، ويتم اخراجه 80%عن طريق الكلى= وفي حالة الفشل الكلوي يجب تقليل الجرعه الاعراض الجانبيه مثل بقية مجموعة الامينو جليكوزيد ( يحدث تأثير سيئا على الكليه والسمع) ويلاحض تأثيره على الكليه اقل من الجنتاميسين ويراعى عدم إعطاءه مع اللازكس او اوكـرين الاسيكتينوميسين يتبع لمجموعة الامينو جليكيوزيد وهو فعال ضد الميكروبات الموجبه والسالبه ويستخدم كدواء بديل في علاج مرض السيلان وفي المرضى الذين تظهر عليهم اعراض التحسس من استخدام البنسلين اوعندما يكون ميكروب المكورات البنيه غير حساس للبنسلين، ويعطى بجرعه 2جم بالحقن في العضل ويعطى نسبة شفاء في مرض السيلان تصل إلى 85__90% الاعراض الجانبيه == اعراض سيئه على الكليه==== انيميا==== الم في مكان وخز الحقنه===== === حمى مع حدوث غثيان==
ميكانيكية عمل البنسلين: يعتبر البنسلين قاتل للميكروب Bactericidal حيث يهبط تكون جدار الخلية للبكتيريا عن طريق تهبيط تكامل تركيب جدار الخلية وتحدث فيها فجوات مما يساعد على دخول السوائل من خارج الخلية الى داخلها نتيجة لزيادة ضغطها الاسموزى فيزيد من حجم البكتيريا وتؤدى في النهاية الى انفجار البكتريا وموتها. ويلاحظ ان البنسلين اكثر نشاطا ضد الميكروبات الموجبة اكثر من الميكروبات السالبة نتيجة للفرق بين مكونات جدار الخلية الموجبة و السالبة ولكن الامبيسلين و الكاربنيسلين لهم نفس الفعل ضد نوعى البكتيريا الموجبة و السالبة. يتم امتصاص البنسلين من الجهاز الهضمي إذا أعطى قبل او بعد الطعام لمدة ساعة ويتم توزيعه في انسجه الجسم وسوائل الجسم المختلفة وتتخذ الأشكال المختلفة للبنسلين مع بروتينات الدم بنسة تترواح 40-90 %0. ويلاحظ ان عند إعطاء البنزاسين بنسلين (Benzathine Penicillin G) بالحقن في العضل في جرعة 2-4 مليون وحدة مدة تتراوح الى 3أسابيع وعند اعطاء بروكاين البنسلين فانة يغطى فترة 24ساعة، ويتم انتشارة في الدم والمفاصل والعين والجهاز العصبي المركزي. ويلاحظ انه في حالة الالتهاب السحائي Meningitis نجد ان درجة نفاذيتة تزداد ولذا يمكن علاج هذه الحالة بإعطاء البنسلين بالحقن دون حقنة في النخاع الشوكي. يتم اخراج البنسلين بسرعة عن طريق الكلية، 10% عن طريق الترشيح في محفظة اليومان و90% عن طريق افراز الأنابيب ويلاحظ ان النافيسلين Nafcillin يتم اخراجة عن طريق القنوات المرارية وايضا30% عن طريق الأنابيب الكلوية ولذا لايحدث له تراكم في حالة الفشل الكلوي. أهم مميزات الانواع المختلفة من البنسلين:1. بنزيل بنسلين (بنسلين ج) - بنسلين سهل الذوبان في المحاليل المائية غير ثابت – يتلف عند إعطائه بالفملذا فإنه يعطى بالحقن. - يعبر المشيمة ويصل إلى دم الجنين. – القليل منة يصل إلى السائل النخاعى الشوكي ويزداد عند التهاب السحايا. 2. بروكايين البنسلين(بروكايين بنزيل بنسلين أو بروكايين بنسلين ج) - قليل الذوبان في الماء – يستعمل على شكل معلق – يبلغ مفعولة حدة الاقصى خلال أربع ساعات ويدوم 12-24 ساعة – يعطى بالحقن العضلي بمقدار(300,000-600,000) وحدة مرة واحدة يوميا ولا يعطى بالفم. - يعطى البنزيل بنسلين مع البروكاين بنسلين ويسمى هذا المزيج بالبنسلين القوي(Fortified procaine penicillin) 3. بنزيل بنسلين: - أقل ذوبان في الماء عن البروكليين – بطىْ الامتصاص بعد الحقن يفيد في الوقاية من الالتهابات الثانوية اثناء عملية استئصال اللوزتين او خلع الأسنان ويعطى قبل أجراء العملية بمقدار 1,300,00 وحدة ويفيد في الوقاية من تكرر حمى الروماتزم(Rheumatic Fever) بمقدار 1,300,000 وحدة كل أربعة أسابيع ويستخدم بجرعة واحدة مقدارها 300,000 وحدة في علاج السيلان الحاد. ويستخدم بجرعة مقدارها 1,300,000 – 2,400,000 وحدة في علاج الزهري ومن اسمائة ال(Penadur). 4. فينوكس مثيل بنسلين ( بنسلين ف) - يعطى بالفم يتأثر بأنزيم البنسلينيز – يفيد في علاج الحمى القرمزية (Scarlet fever)والحمرة البسيطة(erysipelas) ويعطى بالفم بجرعه 250-500ملج كل6-8 ساعات - بعض الاسماء التجاريه للبنسلين (ف) ، اوسبن (ospen) أو (V.Cil.K) (على شكل ملح البوتاسيوم) اوتحت اسم (calcipen) مع ملح الكالسيوم ويلاحظ ان أملاح الكالسيوم والبوتاسيوم أسهل امتصاصا في القناة الهضمية من الحامض الحر ويعطيان تركيزا أعلى في الدم. - يفيد في الوقاية من تكرر حمى الروماتزم(rheumatic fever) والتهاب شغاف القلب (Endocarditis) بجرعة مقدارها 125-250 ملج كل 12 ساعة بشكل مستمر. 5. امبيسلين (Ampicllin) - واسع المفعول - لايتأثر بالعصارة المعدية لذا فهو جيد الإمتصاص - ينتشر عبر المشيمه - يعطى تركيزا عاليا في سائل النخاع الشوكي في التهاب السحايا - يتأثر بانزيم البنسلينيز - يفيد في علاج التهاب الجهاز التنفسي والتهاب الجهاز البولي (الميكروبات السالبه) وفي علاج السيلان والتهاب المجاري الصفراويه والحمى التيفوديه والشيجلات (Shigellae) كالزحار العصوى والشريشيات القولونيه - هواحد بدائل الكلورامفنكول في علاج التهاب السحايا البكترى ويعطى بالفم 350-1500ملج كل 6-8 ساعات حسب شدة الالتهاب ويعطى بالحقن في الوريد أو البريتون أو المفاصل أوالنخاع الشوكي - ومن اسمائة التجارية (Penbritin, Pentrexyl, Omnipen, Ampicillin) 6. الاموكسيلين (Amoxicillin, Amoxycillin) يشبة الامبيسلين في عمله الا انة اقوي قليلا ضد بعض المكورات العقدية والسلمونيات وغيرها – وهو افضل امتصاصا من الامعاء. – ويتلف بانزيم البنسلينيز. - لة نفس استعملات الامبيسلين ويعطى بالفم بجرعة مقدارها 250-15000 ملج ثلاث مرات يوميا ويعطى في علاج السيلان الحاد للذكور والاناث بمقدار 3جم جرعة واحدة. - ومن اسمائة التجارية (Amoxil, Amoxipen) الاستعمال الطبي للبنسلين:1) يستخدم البنسلين في علاج العدوى بميكروبات السبحية مثل التهاب الحلق الحاد Acute Throat Infection والتهاب الجروح حمى النفاس (Purpural Fever) والتهاب الشفاف القلبي تحت الحاد(Subacte Bacterial Endocarditis) في هذه الحالة يعطى البنسلين مع مجموعة الامنيوجليكوزيد. 2) يستخدم في علاج العدوى بميكروبات العنقودية (Staphylococca Infection) 3) يستخدم في علاج العدوى بميكروبات الرئوية (Pneumococcal Infection) 4) يستخدم في علاج الزهري (Syphilis) و السيلان (Gonorrhea) حيث يعطى في علاج الزهري بجرعه مقدارها1,200,000-2,400,000 وحدة وفي علاج السيلان يعطى جرعه واحده مقدارها 300,000 وحده. 5) يستخدم في علاج الالتهاب السحائي (Lieningocoocal Meningitis) ويعطى 20مليون وحدة من البنسلين (ج) يوميا بالحقن لمدة 15 يوم. 6) يستخدم في علاج الجمره الحميدة Anthrax 7) يستخدم في علاج العدوى ببكتيريا الهيمفلس انفلونزا (Haemophilus Influnza) 8) يستخدم في علاج حس التيفود والبارتيفود (Typhoid Fever) 9) يستخدم في علاج الدفيتريا(Diphtheria) والكزار(Tetanus) والغارغرينيا الغازية (Gas Gangrene) ويعطى البنسلين + مصل مضادات السموم. 10) يستخدم في الإصابات النتجة عن الأكتينوميسيتات (Actinomycosis) 11) يستخدم البنسلين كوقاية في بعض الحالات مثل تكرار الحمى الرئويه والتهاب العين السيلاني في الاطفال الحديثي الولادة وذلك بوضع البنزيل بنسلين في الملتحمة. ويفيد في الوقاية من الالتهابات الثانوية اثناء عملية استئصال اللوزتين اوخلع الاسنان ويعطى قبل العملية الجراحية جرعه مقدارها 000؛002،1 وحدة. الجرعة الدوائية وطرق اعطاء البنسلين:بنزيل بنسلين (بنسلين ج) يعطى البنسلين (ج) بالحقن في العضل ويمكن اعطائه في الحالات الشديدة ويعطى كل 4-6ساعات ويعطى من 1-20 مليون وحدة/يوميا بالحقن بالوريد، مقسمه على جرعات تعطى كل 4-6ساعات لمدة على الاقل (2) أسبوع بروكايين البنسلين(بروكايين بنزيل بنسلين أو بروكايين بنسلين ج) يعطى بروكاين البنسلين بالحقن في العضل جرعه من 300,000-600,000 وحده/يوميا كل 12-24 ساعة البنزاسين بنسلين يعطى بالحقن في العضل كل 2-4 اسابيع كجرعه وقائية في حالة الاصابة بحمى الروماتزم (Rheumatic Fever) ويعطى ايضا في علاج المرحله الأولى والثانية والمرحلة الأخيرة من الزهرى ويعطى كجرعه واحده بالعضل 2-4 مليون وحده. فينوكس مثيل بنسلين ( بنسلين ف) ويستخدم بنسلين (ف) في علاج الحمى القزمريه (Scarlet Fever) والحمره البسيطة. يستعمل البنسلين (ف) على شكل الحامض الحر free acid ويعطى كل 250-500مجم كل 6-8 ساعات الكلوكماسلين يعطى بالفم 250-750مجم كل 6 ساعات قبل الطعام بساعة ومن أسمائه التجارية(Orbenin) الامبيكلوكس يعطى بالفم 500مجم (250امبيسلين +250كلوكساسلين) كبسوله كل 6 ساعات او الحقن الامبيسلين (يعطى بالفم) والحقن 350-1500مجم كل 6-8 ساعات الاموكسيساين (الاموكسيل) (Amoxil) يعطى 250-500مجم كل 8ساعات ويلاحظ انه سريع الامتصاص الكارهيسلين والتكارسيلين يعطى بالحقن بالوريد في جرعه كبيرة (ويستخدم ضد الميكروبات الموجيه والسالبه الجرام وكذلك ضدالسيدوموناسPseudomonas وال Proteus ويفيد في علاج التهابات الجهاز البولى الناجمة عن الميكروبات التى لا تستجيب للمضادات الحيوية ويمكن اعطاؤه مع الجنتاميسين (كاربنسلين + جنتاميسين ) في علاج السيدوموناس(Pseudomonas) ويعطى بالحقن في العضل بجرعه 4-8جم يوميا على عده دفعات او بالحقن الوريدى بمقدار 12-30جم على عده دفعات ومن أسمائه التجارية(Pyopen,Geopen) الاعراض الجانبيه للبنسلين:(1) الحساسية (طفح جلدي وضيق في الشعب الهوائية وارتيكاريا) (2) يحدث البنسلين و النافيسلين نقص في عدد كرات الدم البيضاء المحببه (3) تكسير في كرات الدم الحمراء نتيجة لاتحاد الأجسام المضادة للبنسلين مع البنسلين ويتم اتحادهم على سطح كرات الدم الحمراء فيؤدى الى تكسيرها (4) تغير في الملح الدم (5) يؤثر البنسلين على العصاب (6) اضطراب الجهاز الهضمي ويحدث قيء و اسهال خصوصاً مع الامبيسلين والاموكسلين (7) تفاعل الهركس Herixheimer Reaction ويحدث هذا عند مرض الزهرى عند علاجهم بالبنسلين وذلك نتيجه لخروج سموم كثيره من الميكروبات الميته
يراعى في علاج التهابات المجارى البولية لابد من عمل مزرعه للبول وحساسه ولكن ليس الضرورى انتظار نتيجة المزرعه لبدايه العلاج... ويلاحظ أن كثيرا من الميكروبات تؤدى الى حدوث التهاب المجارى البولية منها الايشيرشيا القولونية E-coli وال proteus والكليبسيلا Klebsiella والمكورات السبحيه البرازيه strept.faecalis والpseudomanas. ففى حالة الالتهاب الحاد وكذلك وجود البول الحمضى (acidic urine) يبدأ فى العلاج بواسطه مركبات السلفوناميد sulfonamide أو الكويموكسازول co-trimoxazole ويستمر لمده 3أيام واذا لم تحدث استجابه يعطى الامبيسيلين ampicillin او النيتروفيورانتوين nitrofurantion اوينتظر نتيجة المزرعه والحساسية للبول. أما اذا كان البول قلويا (alkaline unine) فتكون الميكروبات المسببه في حدوث التهاب المجارى البوليه هي ميكروب المتقلبات (proteus) ويستخدم في علاج هذه الحاله الكوتريموكسازول co-trimoxazole اوالامبيسلين ampicillin ويمكن استخدام السيفالوسبورين cephalosporin والتتراسيكلين tetracyclin ويلاحظ ان ميكروب المتقلبات لا يستجيب للنيتروفيورانتوين او التتراسيكلين . أى انه يمكن القول بان هناك عامل هام يزيد من فاعليه المضاد الحيوى وهو أ . البول الحامض acidic urine يزيد من فاعليه التتراسيكلين و النيتروفيورانتوين و الكلوكساسلين . ب . البول القلوي alkaline urine يزيد من فعلية الاستربتوميسين (streptomcyin) والجنتاميسين lincomycin والكلنداميسين clindamyin واللنكومايسين lincomycin والسيفالوسبورين cephalosporins ت . ادوية لاتتأثر بدرجة حموضه البول منها البنزيل بنسيلين bnizylpenicillin والكلوستين colistin والكلورامفينول chloramphenicol والفانكوميسين vancomycin يلاحظ انه بتغير درجه الحموضه من الحمضي الى القلوي يمكن ان يساعد في علاج حرقان البول dysurin ويمكن استخدام بيكربونات الصوديوم 3جم كل ساعتين او استخدام سترات اولكتات الصوديوم 3-6 جرام كل ساعات (ويلاحظ ان السترات او الكتات تتحول الى كربونات في الجسم وتحدث قلوية البول. ويمكن احداث حموضه البول بواسطة حمض الاسكوربك (ascorbic acid) 4جم /يوميا بالفم على جرعات مقسمه. اما في حالة الالتهاب المزمن للمجارى البولية (chronic urinary infection) فيعتمد في العلاج على نتائج المزرعة و الحساسية للبول. ومن الادويه المستخدمة في العلاج :1) النيتروانتوين: nitrofurantoin يعمل هذا الدواء مهبطا للمكروبات وقاتلا لكثير من الميكروبات الموجبة الجرام وكذلك السالبة الجرام. ويمتص هذا الدواء امتصاصا كاملا وسريعا من القناة الهضمية ويتم اخراجه بسرعة عبر الكلية ولذا فانه ليس له عمل مضاد للبكتيريا في الدم ويتم اخراجة عن طريق محافظ البومان و الأنابيب الكلوية .ويحدث له تراكم في الدم في حاله الفشل الكلوي وتؤدي الى ظهور اعراض سامه على الجسم . ويعطى هذا في جرعه مقدارها 400 ملج يوميا بالفم في جرعات مقسمه ويعطى للاطفال (5-8ملج/كجم) ويعطي هذا الدواء مع الطعام أو بعده مباشرة لتقليل فعله الموضعى المزعج على الغشاء المخاطى للامعاء ويمكن اعطاء لمدة أسابيع أو شهور أو سنين لعلاج الالتهاب المزمن للمجارى البولية chronic urinary tract infection ويجب أعطاء المزمن ادويه تزيد من حموضه البول لتزيد من فاعلية ضد الجراثيم. ويمكن اعطاء بالحقن في الوريد جرعه مقدارها 360-540 ملج يوميا لمدة بضعه ايام الاعراض الجانبية: 1. اعراض حسيه مباشرة مثل غثيان وقئ. 2. التهاب الاعصاب neuropathies 3. تكسير في كرات الدم ا لحمراء وخصوصا في مرض ناقص انزيم سداس فوسفات الجلوكوز (6 G6.P.D.D.) 4. حساسيه على هيئة طفح جلدى - بقع في الرئة pulmonary infilltration 2.حمض الناليدكسك: nalidixic acid يستخدم في علاج التهاب المجاري البولية الناتج من البكتيريا سالبة الجرام. ويعطى بالفم. واذا اعطى في تركيز كبير فانه يؤدي الى تهبيط البكتيريا الموجبة والايشيرشيا القلولونية (E .coli) وبعض البكتيريا المعوية والكلبسيلا والبروتياس ويلاحظ أن السيدومناس لا تستجيب لحمض الناليدكسك. ويعمل هذا الدواء عن طريق احداث حموضة للبول التي تؤدي بالتالي الى تهبيط البكتيريا وتعتبر مركبات السلفا القصيرة من المضادات الميكروبية العنيدة حيث انها تتركز في المجاري البولية بنسبة عالية وتعتبر فعاله ضد الايشرشيا القولونية (E.coli) والبروتياس (proteus) والكلبسيلا klebsiella والميكروبات الهوائية . aerobacter وميكروبات السيدوموناسpseudomonas مضادات حيوية أخري: الامبيسلين ampicllin السلفالوسبورين cephalo sporin الجنتاميسين Gentamycin الاميكاسين amilkacin التوبراميسين (النبسى) tobramycin(nebcin) التتراسيكلين tetracycline الكلنداسيكلين clindamycin اللنكوميسين lincomycin الكلورلفينكول chloramphenicol
ميكانيكية عمل البنسلين: يعتبر البنسلين قاتل للميكروب Bactericidal حيث يهبط تكون جدار الخلية للبكتيريا عن طريق تهبيط تكامل تركيب جدار الخلية وتحدث فيها فجوات مما يساعد على دخول السوائل من خارج الخلية الى داخلها نتيجة لزيادة ضغطها الاسموزى فيزيد من حجم البكتيريا وتؤدى في النهاية الى انفجار البكتريا وموتها. ويلاحظ ان البنسلين اكثر نشاطا ضد الميكروبات الموجبة اكثر من الميكروبات السالبة نتيجة للفرق بين مكونات جدار الخلية الموجبة و السالبة ولكن الامبيسلين و الكاربنيسلين لهم نفس الفعل ضد نوعى البكتيريا الموجبة و السالبة. يتم امتصاص البنسلين من الجهاز الهضمي إذا أعطى قبل او بعد الطعام لمدة ساعة ويتم توزيعه في انسجه الجسم وسوائل الجسم المختلفة وتتخذ الأشكال المختلفة للبنسلين مع بروتينات الدم بنسة تترواح 40-90 %0. ويلاحظ ان عند إعطاء البنزاسين بنسلين (Benzathine Penicillin G) بالحقن في العضل في جرعة 2-4 مليون وحدة مدة تتراوح الى 3أسابيع وعند اعطاء بروكاين البنسلين فانة يغطى فترة 24ساعة، ويتم انتشارة في الدم والمفاصل والعين والجهاز العصبي المركزي. ويلاحظ انه في حالة الالتهاب السحائي Meningitis نجد ان درجة نفاذيتة تزداد ولذا يمكن علاج هذه الحالة بإعطاء البنسلين بالحقن دون حقنة في النخاع الشوكي. يتم اخراج البنسلين بسرعة عن طريق الكلية، 10% عن طريق الترشيح في محفظة اليومان و90% عن طريق افراز الأنابيب ويلاحظ ان النافيسلين Nafcillin يتم اخراجة عن طريق القنوات المرارية وايضا30% عن طريق الأنابيب الكلوية ولذا لايحدث له تراكم في حالة الفشل الكلوي. أهم مميزات الانواع المختلفة من البنسلين:1. بنزيل بنسلين (بنسلين ج) - بنسلين سهل الذوبان في المحاليل المائية غير ثابت – يتلف عند إعطائه بالفملذا فإنه يعطى بالحقن. - يعبر المشيمة ويصل إلى دم الجنين. – القليل منة يصل إلى السائل النخاعى الشوكي ويزداد عند التهاب السحايا. 2. بروكايين البنسلين(بروكايين بنزيل بنسلين أو بروكايين بنسلين ج) - قليل الذوبان في الماء – يستعمل على شكل معلق – يبلغ مفعولة حدة الاقصى خلال أربع ساعات ويدوم 12-24 ساعة – يعطى بالحقن العضلي بمقدار(300,000-600,000) وحدة مرة واحدة يوميا ولا يعطى بالفم. - يعطى البنزيل بنسلين مع البروكاين بنسلين ويسمى هذا المزيج بالبنسلين القوي(Fortified procaine penicillin) 3. بنزيل بنسلين: - أقل ذوبان في الماء عن البروكليين – بطىْ الامتصاص بعد الحقن يفيد في الوقاية من الالتهابات الثانوية اثناء عملية استئصال اللوزتين او خلع الأسنان ويعطى قبل أجراء العملية بمقدار 1,300,00 وحدة ويفيد في الوقاية من تكرر حمى الروماتزم(Rheumatic Fever) بمقدار 1,300,000 وحدة كل أربعة أسابيع ويستخدم بجرعة واحدة مقدارها 300,000 وحدة في علاج السيلان الحاد. ويستخدم بجرعة مقدارها 1,300,000 – 2,400,000 وحدة في علاج الزهري ومن اسمائة ال(Penadur). 4. فينوكس مثيل بنسلين ( بنسلين ف) - يعطى بالفم يتأثر بأنزيم البنسلينيز – يفيد في علاج الحمى القرمزية (Scarlet fever)والحمرة البسيطة(erysipelas) ويعطى بالفم بجرعه 250-500ملج كل6-8 ساعات - بعض الاسماء التجاريه للبنسلين (ف) ، اوسبن (ospen) أو (V.Cil.K) (على شكل ملح البوتاسيوم) اوتحت اسم (calcipen) مع ملح الكالسيوم ويلاحظ ان أملاح الكالسيوم والبوتاسيوم أسهل امتصاصا في القناة الهضمية من الحامض الحر ويعطيان تركيزا أعلى في الدم. - يفيد في الوقاية من تكرر حمى الروماتزم(rheumatic fever) والتهاب شغاف القلب (Endocarditis) بجرعة مقدارها 125-250 ملج كل 12 ساعة بشكل مستمر. 5. امبيسلين (Ampicllin) - واسع المفعول - لايتأثر بالعصارة المعدية لذا فهو جيد الإمتصاص - ينتشر عبر المشيمه - يعطى تركيزا عاليا في سائل النخاع الشوكي في التهاب السحايا - يتأثر بانزيم البنسلينيز - يفيد في علاج التهاب الجهاز التنفسي والتهاب الجهاز البولي (الميكروبات السالبه) وفي علاج السيلان والتهاب المجاري الصفراويه والحمى التيفوديه والشيجلات (Shigellae) كالزحار العصوى والشريشيات القولونيه - هواحد بدائل الكلورامفنكول في علاج التهاب السحايا البكترى ويعطى بالفم 350-1500ملج كل 6-8 ساعات حسب شدة الالتهاب ويعطى بالحقن في الوريد أو البريتون أو المفاصل أوالنخاع الشوكي - ومن اسمائة التجارية (Penbritin, Pentrexyl, Omnipen, Ampicillin) 6. الاموكسيلين (Amoxicillin, Amoxycillin) يشبة الامبيسلين في عمله الا انة اقوي قليلا ضد بعض المكورات العقدية والسلمونيات وغيرها – وهو افضل امتصاصا من الامعاء. – ويتلف بانزيم البنسلينيز. - لة نفس استعملات الامبيسلين ويعطى بالفم بجرعة مقدارها 250-15000 ملج ثلاث مرات يوميا ويعطى في علاج السيلان الحاد للذكور والاناث بمقدار 3جم جرعة واحدة. - ومن اسمائة التجارية (Amoxil, Amoxipen) الاستعمال الطبي للبنسلين:1) يستخدم البنسلين في علاج العدوى بميكروبات السبحية مثل التهاب الحلق الحاد Acute Throat Infection والتهاب الجروح حمى النفاس (Purpural Fever) والتهاب الشفاف القلبي تحت الحاد(Subacte Bacterial Endocarditis) في هذه الحالة يعطى البنسلين مع مجموعة الامنيوجليكوزيد. 2) يستخدم في علاج العدوى بميكروبات العنقودية (Staphylococca Infection) 3) يستخدم في علاج العدوى بميكروبات الرئوية (Pneumococcal Infection) 4) يستخدم في علاج الزهري (Syphilis) و السيلان (Gonorrhea) حيث يعطى في علاج الزهري بجرعه مقدارها1,200,000-2,400,000 وحدة وفي علاج السيلان يعطى جرعه واحده مقدارها 300,000 وحده. 5) يستخدم في علاج الالتهاب السحائي (Lieningocoocal Meningitis) ويعطى 20مليون وحدة من البنسلين (ج) يوميا بالحقن لمدة 15 يوم. 6) يستخدم في علاج الجمره الحميدة Anthrax 7) يستخدم في علاج العدوى ببكتيريا الهيمفلس انفلونزا (Haemophilus Influnza) 8) يستخدم في علاج حس التيفود والبارتيفود (Typhoid Fever) 9) يستخدم في علاج الدفيتريا(Diphtheria) والكزار(Tetanus) والغارغرينيا الغازية (Gas Gangrene) ويعطى البنسلين + مصل مضادات السموم. 10) يستخدم في الإصابات النتجة عن الأكتينوميسيتات (Actinomycosis) 11) يستخدم البنسلين كوقاية في بعض الحالات مثل تكرار الحمى الرئويه والتهاب العين السيلاني في الاطفال الحديثي الولادة وذلك بوضع البنزيل بنسلين في الملتحمة. ويفيد في الوقاية من الالتهابات الثانوية اثناء عملية استئصال اللوزتين اوخلع الاسنان ويعطى قبل العملية الجراحية جرعه مقدارها 000؛002،1 وحدة. الجرعة الدوائية وطرق اعطاء البنسلين:بنزيل بنسلين (بنسلين ج) يعطى البنسلين (ج) بالحقن في العضل ويمكن اعطائه في الحالات الشديدة ويعطى كل 4-6ساعات ويعطى من 1-20 مليون وحدة/يوميا بالحقن بالوريد، مقسمه على جرعات تعطى كل 4-6ساعات لمدة على الاقل (2) أسبوع بروكايين البنسلين(بروكايين بنزيل بنسلين أو بروكايين بنسلين ج) يعطى بروكاين البنسلين بالحقن في العضل جرعه من 300,000-600,000 وحده/يوميا كل 12-24 ساعة البنزاسين بنسلين يعطى بالحقن في العضل كل 2-4 اسابيع كجرعه وقائية في حالة الاصابة بحمى الروماتزم (Rheumatic Fever) ويعطى ايضا في علاج المرحله الأولى والثانية والمرحلة الأخيرة من الزهرى ويعطى كجرعه واحده بالعضل 2-4 مليون وحده. فينوكس مثيل بنسلين ( بنسلين ف) ويستخدم بنسلين (ف) في علاج الحمى القزمريه (Scarlet Fever) والحمره البسيطة. يستعمل البنسلين (ف) على شكل الحامض الحر free acid ويعطى كل 250-500مجم كل 6-8 ساعات الكلوكماسلين يعطى بالفم 250-750مجم كل 6 ساعات قبل الطعام بساعة ومن أسمائه التجارية(Orbenin) الامبيكلوكس يعطى بالفم 500مجم (250امبيسلين +250كلوكساسلين) كبسوله كل 6 ساعات او الحقن الامبيسلين (يعطى بالفم) والحقن 350-1500مجم كل 6-8 ساعات الاموكسيساين (الاموكسيل) (Amoxil) يعطى 250-500مجم كل 8ساعات ويلاحظ انه سريع الامتصاص الكارهيسلين والتكارسيلين يعطى بالحقن بالوريد في جرعه كبيرة (ويستخدم ضد الميكروبات الموجيه والسالبه الجرام وكذلك ضدالسيدوموناسPseudomonas وال Proteus ويفيد في علاج التهابات الجهاز البولى الناجمة عن الميكروبات التى لا تستجيب للمضادات الحيوية ويمكن اعطاؤه مع الجنتاميسين (كاربنسلين + جنتاميسين ) في علاج السيدوموناس(Pseudomonas) ويعطى بالحقن في العضل بجرعه 4-8جم يوميا على عده دفعات او بالحقن الوريدى بمقدار 12-30جم على عده دفعات ومن أسمائه التجارية(Pyopen,Geopen) الاعراض الجانبيه للبنسلين:(1) الحساسية (طفح جلدي وضيق في الشعب الهوائية وارتيكاريا) (2) يحدث البنسلين و النافيسلين نقص في عدد كرات الدم البيضاء المحببه (3) تكسير في كرات الدم الحمراء نتيجة لاتحاد الأجسام المضادة للبنسلين مع البنسلين ويتم اتحادهم على سطح كرات الدم الحمراء فيؤدى الى تكسيرها (4) تغير في الملح الدم (5) يؤثر البنسلين على العصاب (6) اضطراب الجهاز الهضمي ويحدث قيء و اسهال خصوصاً مع الامبيسلين والاموكسلين (7) تفاعل الهركس Herixheimer Reaction ويحدث هذا عند مرض الزهرى عند علاجهم بالبنسلين وذلك نتيجه لخروج سموم كثيره من الميكروبات الميته
يراعى في علاج التهابات المجارى البولية لابد من عمل مزرعه للبول وحساسه ولكن ليس الضرورى انتظار نتيجة المزرعه لبدايه العلاج... ويلاحظ أن كثيرا من الميكروبات تؤدى الى حدوث التهاب المجارى البولية منها الايشيرشيا القولونية E-coli وال proteus والكليبسيلا Klebsiella والمكورات السبحيه البرازيه strept.faecalis والpseudomanas. ففى حالة الالتهاب الحاد وكذلك وجود البول الحمضى (acidic urine) يبدأ فى العلاج بواسطه مركبات السلفوناميد sulfonamide أو الكويموكسازول co-trimoxazole ويستمر لمده 3أيام واذا لم تحدث استجابه يعطى الامبيسيلين ampicillin او النيتروفيورانتوين nitrofurantion اوينتظر نتيجة المزرعه والحساسية للبول. أما اذا كان البول قلويا (alkaline unine) فتكون الميكروبات المسببه في حدوث التهاب المجارى البوليه هي ميكروب المتقلبات (proteus) ويستخدم في علاج هذه الحاله الكوتريموكسازول co-trimoxazole اوالامبيسلين ampicillin ويمكن استخدام السيفالوسبورين cephalosporin والتتراسيكلين tetracyclin ويلاحظ ان ميكروب المتقلبات لا يستجيب للنيتروفيورانتوين او التتراسيكلين . أى انه يمكن القول بان هناك عامل هام يزيد من فاعليه المضاد الحيوى وهو أ . البول الحامض acidic urine يزيد من فاعليه التتراسيكلين و النيتروفيورانتوين و الكلوكساسلين . ب . البول القلوي alkaline urine يزيد من فعلية الاستربتوميسين (streptomcyin) والجنتاميسين lincomycin والكلنداميسين clindamyin واللنكومايسين lincomycin والسيفالوسبورين cephalosporins ت . ادوية لاتتأثر بدرجة حموضه البول منها البنزيل بنسيلين bnizylpenicillin والكلوستين colistin والكلورامفينول chloramphenicol والفانكوميسين vancomycin يلاحظ انه بتغير درجه الحموضه من الحمضي الى القلوي يمكن ان يساعد في علاج حرقان البول dysurin ويمكن استخدام بيكربونات الصوديوم 3جم كل ساعتين او استخدام سترات اولكتات الصوديوم 3-6 جرام كل ساعات (ويلاحظ ان السترات او الكتات تتحول الى كربونات في الجسم وتحدث قلوية البول. ويمكن احداث حموضه البول بواسطة حمض الاسكوربك (ascorbic acid) 4جم /يوميا بالفم على جرعات مقسمه. اما في حالة الالتهاب المزمن للمجارى البولية (chronic urinary infection) فيعتمد في العلاج على نتائج المزرعة و الحساسية للبول. ومن الادويه المستخدمة في العلاج :1) النيتروانتوين: nitrofurantoin يعمل هذا الدواء مهبطا للمكروبات وقاتلا لكثير من الميكروبات الموجبة الجرام وكذلك السالبة الجرام. ويمتص هذا الدواء امتصاصا كاملا وسريعا من القناة الهضمية ويتم اخراجه بسرعة عبر الكلية ولذا فانه ليس له عمل مضاد للبكتيريا في الدم ويتم اخراجة عن طريق محافظ البومان و الأنابيب الكلوية .ويحدث له تراكم في الدم في حاله الفشل الكلوي وتؤدي الى ظهور اعراض سامه على الجسم . ويعطى هذا في جرعه مقدارها 400 ملج يوميا بالفم في جرعات مقسمه ويعطى للاطفال (5-8ملج/كجم) ويعطي هذا الدواء مع الطعام أو بعده مباشرة لتقليل فعله الموضعى المزعج على الغشاء المخاطى للامعاء ويمكن اعطاء لمدة أسابيع أو شهور أو سنين لعلاج الالتهاب المزمن للمجارى البولية chronic urinary tract infection ويجب أعطاء المزمن ادويه تزيد من حموضه البول لتزيد من فاعلية ضد الجراثيم. ويمكن اعطاء بالحقن في الوريد جرعه مقدارها 360-540 ملج يوميا لمدة بضعه ايام الاعراض الجانبية: 1. اعراض حسيه مباشرة مثل غثيان وقئ. 2. التهاب الاعصاب neuropathies 3. تكسير في كرات الدم ا لحمراء وخصوصا في مرض ناقص انزيم سداس فوسفات الجلوكوز (6 G6.P.D.D.) 4. حساسيه على هيئة طفح جلدى - بقع في الرئة pulmonary infilltration 2.حمض الناليدكسك: nalidixic acid يستخدم في علاج التهاب المجاري البولية الناتج من البكتيريا سالبة الجرام. ويعطى بالفم. واذا اعطى في تركيز كبير فانه يؤدي الى تهبيط البكتيريا الموجبة والايشيرشيا القلولونية (E .coli) وبعض البكتيريا المعوية والكلبسيلا والبروتياس ويلاحظ أن السيدومناس لا تستجيب لحمض الناليدكسك. ويعمل هذا الدواء عن طريق احداث حموضة للبول التي تؤدي بالتالي الى تهبيط البكتيريا وتعتبر مركبات السلفا القصيرة من المضادات الميكروبية العنيدة حيث انها تتركز في المجاري البولية بنسبة عالية وتعتبر فعاله ضد الايشرشيا القولونية (E.coli) والبروتياس (proteus) والكلبسيلا klebsiella والميكروبات الهوائية . aerobacter وميكروبات السيدوموناسpseudomonas مضادات حيوية أخري: الامبيسلين ampicllin السلفالوسبورين cephalo sporin الجنتاميسين Gentamycin الاميكاسين amilkacin التوبراميسين (النبسى) tobramycin(nebcin) التتراسيكلين tetracycline الكلنداسيكلين clindamycin اللنكوميسين lincomycin الكلورلفينكول chloramphenicol
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