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or macrolide–lincosamides–streptogramin resistance in anaerobes
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Antimicrobial susceptibility
Acquired tetracycline and/or macrolide–lincosamides–streptogramin resistance in anaerobes$ Marilyn C. Roberts* Department of Pathobiology, University of Washington, Seattle, WA 98195,USA Received 9 September 2002; received in revised form 28 March 2003; accepted 7 April 2003
Abstract In general bacterial antibiotic resistance is acquired on mobile elements such as plasmids, transposons and/or conjugative transposons. This is also true for many antibiotic resistant anaerobic species described in the literature. Of the 23 different tetracycline resistant efflux genes identified, tet(B), tet(K), tet(L), and tetA(P) have been found in anaerobic species and six of the ten tetracycline resistant genes coding for ribosomal protection proteins, tet(M), tet(O), tetB(P), tet(Q), tet(W), and tet(32), have been identified in anaerobes. There are now three enzymes which inactivate tetracycline, of which the tet(X) has been identified in Bacteroides though is not functional under anaerobic growth conditions. A similar situation exists with the genes conferring macrolide–lincosamide–streptogramin (MLS) resistance. Of the 26 rRNA methylase MLS resistant genes characterized, five genes; erm(B), erm(C), erm(F), erm(G), and erm(Q), have been identified in anaerobes. In contrast, no genes coding for MLS resistant efflux proteins or inactivating enzymes have been described in anaerobic species. This mini-review will summarize what is known about tetracycline and MLS resistance in genera with anaerobic species and the mobile elements associated with acquired tetracycline and/or MLS resistance genes. r 2003 Elsevier Science Ltd. All rights reserved.
1. Introduction Anaerobic bacteria are found in diverse ecological niches; however, when it comes to characterization of antibiotic resistance in anaerobes the majority of species studied are either opportunistic pathogens or commensal bacteria associated with humans or animals. It has been hypothesized that the commensal flora, including the anaerobes, act as a reservoir for mobile antibiotic resistant genes. If this hypothesis is correct, it is important to understand what antibiotic resistance genes the commensal flora, as well as, the opportunistic and pathogenic species carry. Many reports exist on the decrease in susceptiblity to antibiotics especially clindamycin, cephalosporins and penicillins in both the Bacteroides fragilis group and non-Bacteroides anaerobes [1]. Over 50% of the Bacteroides fragilis group isolates are resistant to tetracyclines, while resistance in $
Paper from Anaerobe Olympiad 2002. The 6th Biennial Congress of the Anaerobe Society of the Americas, Park City, Utah, 29 June– 2 July. *Fax: +1-206-543-3873. E-mail address: [email protected] (M.C. Roberts).
non-Bacteroides and other Gram-negative genera are more variable [2]. Anaerobic in vitro susceptiblity tests have difficulties in accuracy, reproducibility and predictive value for therapy. Differences in the methods used, choice of media, and inoculum size all affect results [2]. Most of the information on carriage and distribution of acquired antibiotic resistance genes has been done using DNA probes in selected research laboratories [3–8]. With some bacteria the increase in the MIC correlates with the presence of specific antibiotic resistance genes [4]. This mini-review will summarize what is known about tetracycline and macrolide–lincosamide–streptogramin (MLS) resistance in genera with anaerobic species and the mobile elements associated with acquired tetracycline and/or MLS resistance genes.
2. Tetracycline and the acquired resistance genes Tetracyclines are broad-spectrum antibiotics which have a wide range of activity. Only a limited number of derivatives are currently in use. Tetracycline reversibly
1075-9964/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1075-9964(03)00058-1
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inhibits bacterial protein synthesis by binding to the ribosomal complex. This prevents the association of the aminoacyl-tRNA to the ribosomal acceptor (A) site [9]. Once inside the bacteria, the tetracycline binds reversibly with the prokaryotic 30S ribosomal subunit, stopping protein synthesis. Diluting out the antimicrobial can reverse this process. Three types of acquired tetracycline resistance mechanisms have been described (Table 1). The first and most common mechanism is the tetracycline (tet) resistant efflux proteins which belong to the major facilitator superfamily (MFS). These tet efflux genes code for membrane-associated proteins that
Table 1 Mechanism of resistance for characterized tet and otr genes Efflux
Ribosomal protection
Enzymatic
Unknowna
tet(A), tet(B), tet(C), tet(D), tet(E)
tet(M), tet(O), tet(S), tet(W), tet(32), tet(36) tet(Q), tet (T)
tet(X)
tet(U)
otr(A), tetB(P)b, tet
tet(37)
tet(G), tet(H), tet(J), tet(Z), tet (30) tet(31), tet(33)
otr(C)
tet(K), tet( L) otr(B) tetA(P) tet(V) tet(Y) tet(34), tet(35) tcr3 a tet(U) has been sequenced but does not appear to be related to either efflux or ribosomal protection proteins, otr(C) has not been sequenced; b tetB(P) is not found alone and tetA(P) and tetB(P) are counted as one operon; tet(36) and tet(37) [25] have recently been assigned.
recognize and export tetracycline from the cell. This reduces the intracellular drug concentration and protects the majority of the ribosomes within the cell [9,10]. Efflux genes are found in both Gram-positive and Gram-negative anaerobic species (Table 2). Most of these efflux proteins confer resistance to both tetracycline and doxycycline, but not minocycline or the newer experimental glycylcyclines [9,11,12]. There are 22 different tetracycline resistant efflux genes identified. The efflux proteins have been divided into groups based on amino acid sequence identity [9,13]. Group 1 contains the proteins, Tet (A), (B), (C), (D), (E), (G), (H), (J), (Z) and Tet(30). Tet(Z) has been in a Gram-positive species, while all the others are found only in gram-negative species [12]. In this group, the tetracycline resistance efflux proteins share 41–78% amino acid identity with each other. From this group only the tet(B) gene has been found in one Gramnegative anaerobe, Treponema denticola (Table 2). The other genes have not been found in anaerobes at this time. Group 2 efflux proteins include Tet(K) and Tet(L) with 58–59% amino acid identity. These two genes are of Gram-positive origin but have occasionally been found in Gram-negative species. Among anaerobes the tet(K) gene has been found in Eubacterium and Peptostreptococcus, while the tet(L) gene has been found in Actinomyces, Peptostreptococcus, Veillonella, and Clostridium (Table 2). Group 3 efflux proteins include the otr(B) and tcr3 gene products both found in Streptomyces spp. Group 4 efflux proteins include the tetA(P) gene product described below, while Group 5 efflux protein include the gene products from the tet(V) gene from Mycobacterium smegmatis. None of the genes from Group 3, 4, or 5 have been found in anaerobic species.
Table 2 Distribution of tetracycline resistance genes in anaerobes Efflux
Ribosomal protection and/or efflux
One gene
One gene
Treponema
a
tet(B)
Capnocytophaga
Found in a Clostridum-like micro [22].
Two of more genes tet(Q)
Actinomyces Butyrivibrio Bifidobacterium Mitsuokella Mobiluncus Selenomonas Porphyromonas Bacteroides Prevotella Fusobacterium Eubacterium Lactobacillus Peptostreptococcus Veillonella Clostridium
tet(L), (M) tet(O), (W) tet(M), (W) tet(Q), (W) tet(O), (Q) tet(Q), (W) tet(Q), (W) tet(M), (Q), (X), (36) tet(M), (Q), (W) tet(L), (M), (Q) tet(K), (M), (Q) tet(M), (O), (S), (Q) tet(K), (L), (M), (O), (Q) tet(A), (L), (M), (S), (Q), (W) tet(K), (L), (M), (P), (Q), (32)a
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The tet(P) operon from Clostridium has a unique structure and appears to be unique to this genus [14]. It consists of two overlapping genes, tetA(P) and tetB(P). The tetA(P) gene encodes for an efflux protein similar to the other tetracycline resistant efflux proteins described above. The tetB(P) gene codes for a protein which is similar to the ribosomal protection proteins described below. When both genes are present, they are transcribed from a single promoter located upstream of the tetA(P) start codon [14–17]. A factor-independent terminator, T1, located downstream from the promoter and upstream of the tetA(P) has recently been described [15] and it has been postulated that the T1 terminator is an intrinsic control element of the tet(P) operon which acts by preventing over-expression of the efflux protein [tetA(P)]. The tetA(P) gene has been found alone in Clostridium spp., and confers tetracycline resistance. However, the tetB(P) gene has not been found alone in nature, but when the tetB(P) gene was separated from the rest of the operon, in the laboratory, it conferred low-level resistance to tetracycline and minocycline in both Clostridium perfringens and Escherichia coli [18]. This resistance level is lower than normally found when other ribosomal protection genes are cloned into these organisms. Thus it is unclear if the tetB(P) gene is independently found and its role in nature. There are 11 tetracycline resistant genes coding for ribosomal protection proteins described (Table 1). These are cytoplasmic proteins that protect the ribosomes from the action of tetracycline in vitro and in vivo. They confer resistance to tetracycline, doxycycline and minocycline and have homology with elongation factors EF-Tu and EF-G [19,20]. The current data suggest that the ribosomal protection proteins bind to the ribosome. This binding is not affected by the presence of tetracycline but is inhibited by thiostrepton, which also inhibits the binding of the EF-G protein. When the ribosomal proteins bind they cause an alteration in ribosomal conformation, which then prevents tetracycline from binding to the ribosome. EF-G and the Tet(M) protein compete for binding to the ribosomes, suggesting that they may have overlapping binding sites. Both the Tet(M) and Tet(O) proteins have been shown to have ribosome-dependent GTPase activity and it has been suggested that the GTP binding is important to the function of the Tet(O) protein. It has been hypothesized that the hydrolysis of GTP provides the energy for the ribosomal conformational change [21]. The tet(M), tet(O), tet(Q), tet(W), tet(32) genes, and the tet(P) operon have all been found in anaerobic species [9, 22] (Table 2). This group of genes have low G+C content and are thought to be originally of Grampositive ancestry. The tet(M) gene has the widest host range among all genera of bacteria [9]. The tet(M) gene is currently found in nine anaerobic genera, the tet(Q)
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gene is found in 12 and the tet(W) gene is found in seven anaerobic genera (Table 2). There are now two genes which code for enzymes which inactivate tetracycline. The tet(37) gene has been found from the oral bacteria in adults and determination of whether this gene is associated with oral anaerobic species has not been done [23]. The tet(X) gene codes an enzyme, which alters the tetracycline molecule. Two closely related anaerobic Bacteroides transposons (Tn4351, Tn4400) containing the tet(X) gene has been described [24]. The tet(X) gene was originally found because it was linked to an erm(F) gene, which codes for a rRNA methylase. The erm(F) gene was cloned into E. coli and the clones were found to confer tetracycline resistance in E. coli when grown aerobically. The tet(X) gene product is a 44 kDa cytoplasmic protein that chemically modifies tetracycline in the presence of both oxygen and NADPH. Sequence analysis indicates that this protein shared amino acid homology with other NADPH-requiring oxidoreductases and should not be able to function in the anaerobic Bacteroides hosts. It has not been found outside Bacteroides, however, no survey has been conducted to assess the distribution of the tet(X) gene in any bacterial population. Neither the tet(U) or the otr(C) gene has been found in anaerobes, though no survey has been conducted to assess the distribution of either gene [9]. These will not be discussed further (Tables 1 and 2).
3. Macrolide–lincosamide–streptogramin and the acquired resistance genes Macrolides, lincosamides, streptogramin B and the new ketolides are a structurally diverse group of antibiotics with overlapping binding sites in the peptidyl transferase region of the 23S rRNA. This group of drugs interacts with the 50S subunit of the bacterial ribosome. They appear to inhibit protein synthesis by inhibiting movement of the peptide chain and/or alter the binding of the peptidyl-tRNA molecule from the ribosomes during elongation. This results in chain termination and a reversible stoppage of protein synthesis. Three types of acquired resistance genes have been described and include rRNA methylases (erm genes) which are a family of related proteins that add one or two methyl groups to a single adenine (A2058 in E. coli) in the 23S rRNA moiety. Modification of the ribosomes by rRNA methylases reduces the binding of macrolides, lincosamides and streptogramin B antibiotics (MLSB) [25–28]. The erm genes are the most commonly found acquired resistance mechanism in bacteria and the only genes currently found in anaerobes [27] (Table 3). All 27 of the erm genes are of Gram-positive origin, they have low G+C contents and can be expressed in
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Table 3 Location of rRNA methylase resistance genes in anaerobes One gene Actinomyces Fusobacterium Propionibacterium Treponema Veillonella
Two or more genes erm(F) erm(F) erm(W) erm(F) erm(F)
Porphyromonas Prevotella Eubacterium Lactobacillus Bacteroides Clostrdium Wolinella
erm(F), erm(G) erm(F), erm(G) erm(C), erm(F) erm(C), erm(G), erm(T) erm(F), erm(G), erm(35) erm(B), erm(F) , erm(Q) erm(B), erm(C) , erm(F), erm(Q)
Table 4 Mechanism of MLS resistance rRNA methylase
Efflux
27 rRNA methylases (erm genes) (A), (B), (C), (D), (E), (F), (G), (H) (I), (N), (O), (Q), (R), (S), (T), (U) (V), (Y), (Z), (30), (31), (32), (33) (34), (35), (36), (37)
11 efflux mef(A), msr(A), (C) car(A), lmr(A), ole(B), (C) srm(B), tlr(C), vga(A), (B)
both Gram-positive and Gram-negative species [27,28]. This is true even for the erm(F) gene which was first described in Gram-negative anaerobes [29]. Seven of the erm genes, erm(B), erm(C), erm(F), erm(Q), erm(T), and erm(35) have been found in anaerobes. The erm(F) gene has the widest host range and is found in 10 anaerobic genera examined. In four of the genera only the erm(F) gene has been identified. However, the current distribution may similarly reflect on what erm genes investigators examined rather than the true distribution in nature (Table 3). There are 11 genes which code for efflux proteins, which pump drug(s) out of the cell, and 17 genes which code for inactivating enzymes including esterases, lyases, transferases, and phosphorylases, which inactivate the drug(s) by adding moieties to them (Table 4). All 28 genes confer resistance to macrolides and/or lincosamides and/or streptogramins. However, none of these genes have been identified in anaerobes, though I could not find any published reports where anaerobes have been screened for these genes.
Inactivating enzymes 2 esterase ere(A), (B)
2 lyases vgb (A), (B)
9 transferases lnu(A), (B) vat(A), (B), (C), (D) vat(D), (E), (F)
4 phosphorylases mph(A), (B), (C), (D)
tions can change the adenine (A2058) or one of the adjacent residues in the peptidyl transferase region (A2057 or A2059) to another nucleotide. A number of different pathogenic genera with 23S rRNA mutations are listed in Table 1 of [28]. The authors include a number of different Propionibacterium spp., and microaerophilic Helicobacter pylori. Tetracycline resistances due to mutations in the 16S rRNA have also been described in H. pylori [31]. Mutations in ribosomal proteins L4 and/or L22 that confer erythromycin resistance have been documented in laboratory strains of E. coli and Gram-positive cocci [28], as well as, in clinical Gram-positive cocci. Mutations in intrinsic pumps may confer increased resistance to tetracyclines and/or erythromycin in both Gram-positive and Gramnegative bacteria [28]. Clearly the potential of mutations leading to increased resistance in anaerobes does exist. Therefore it is likely that in time other anaerobes will be identified with mutations conferring increased resistance to tetracycline and/or MLS antibiotics [27].
5. Mobile elements 4. Mutations A less common way for bacteria to become tetracycline and/or MLS resistant is by mutations of chromosomal genes. Tetracycline resistant cutaneous propionibacteria (MICs 2–64 mg/ml of tetracycline, 1– 32 mg/ml doxycycline) have been described [30]. In these isolates, a guanine was switched to a cytosine at position 1058 in the 16S rRNA. This change was associated with the increase in tetracycline resistance. Similarly, muta-
Horizontal (lateral) DNA transfer is thought to be a major player in bacterial evolution [32–41]. It allows for blocks of DNA with multiple genes to be moved as a unit. Mobile elements are able to transfer between unrelated species and ecosystems and allows for the rapid spread of associated genes within and between bacterial populations. Today it is clear that once a gene becomes associated with a mobile element it can move to surprisingly diverse ecosystems and bacterial species,
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including its integration into the chromosome and/or plasmid(s) of its new host. Conjugative elements may also mobilize co-resident plasmids. Mobile elements add significant flexibility to the bacterial host’s ability to cope with changes in its environment with relatively little cost to the bacterial cell. It is the most likely reason that acquired antibiotic resistant genes have spread so rapidly in the last 50 years. Horizontal DNA transfer has also played a role in the development of multi-drug resistant isolates. Some mobile elements contain antibiotic resistance genes and virulence factors. Most acquired antibiotic resistant gene(s) are associated with some type of mobile element (plasmid, transposon, conjugative transposon, and/or integron). A large number of these elements have been described over the past 30 years [15,37–41]. Plasmids were the first to be identified in the 1960s [9]. Plasmids are usually circular DNA molecules, which normally carry non-essential genes. Many can move between related genera, but their host ranges vary significantly from very restricted (only related strains) to a broad-host range (wide range of genera). Plasmids may contain transposons, conjugative transposons, IS elements and integrons as well as a wide range of genes including toxin protein, virulence factors in addition to antibiotic resistance genes [42,43]. Published reports suggest that 20–50% of the Bacteroides spp. examined carry plasmids. In general these plasmids have a limited host range and can replicate in Bacteroides spp. and the closely related Porphyromonas spp. and Prevotella spp., but not in E. coli. Most of the Bacteroides spp. plasmids are small (o 8 kb) and cryptic (lack a detectable phenotype) [38]. These plasmids are mobilizable and belong to one of three homology groups and are of modular construction sharing replication and mobilization gene cassettes. C. perfringens plasmids coding for tetracycline, and/or chloramphenicol and/or erythromycin and clindamycin resistance has been characterized [44]. These large plasmids are ubiquitous in the species and appear to be genetically related to each other. Some of the plasmids carry transposons and similar antibiotic resistance genes in C. perfringens and C. difficile suggesting conjugative transfer between the two species. Some conjugative plasmids (pAMb1, pJH4 and pIP502) carrying the erm(B) resistance gene have been transferred in the laboratory. More information about Bacteroides spp. and Clostridium spp. plasmids and/or conjugative transposons is available from a variety of articles and beyond the scope of this review. Transposons are defined elements with either direct or inverted repeats at the ends, often associated with IS elements flanking the element. Some of these elements integrate into a few sites within some bacteria but randomly in others, variations on this theme do occur. Transposons carry their own enzymes, which allow transposition from one piece of DNA to another piece
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of DNA within the same bacterial cell. These enzymes allow for the direct cleaving and rejoining of the DNA after transposition has occurred. These elements are ubiquitous in both prokaryotes and eukaryotes. Conjugative transposons are defined genetic entities, which can be associated with either plasmids and/or chromosomes. These carry genes for both transposition and conjugation and thus can transfer between DNA within a host cell and transfer between strains by conjugation [45]. These elements are very common in Gram-positive aerobes and anaerobes such as Clostridium spp., and anaerobic Gram-negative species (Bacteroides), but are not commonly found in Gram-negative enteric-like bacteria, such as E. coli [4,9,18,27,37–39]. Some of the best studied conjugative transposons are from Bacteroides spp. [24,38,41,45,46] and Clostridium spp. [16–18,39]. Integrons are relatively newly described genetic elements. It is thought that these elements play an important role in the evolution of antibiotic resistance in Gram-negative bacteria and Gram-positive staphylococci. A few of the integrons have virulence genes associated with them, but most carry antibiotic resistance genes. Integrons acquire and exchange exogenous DNA (gene cassettes) by site-specific recombination. They function as a general gene-capture system. Gene cassettes have a target recombination sequence (attC site ) usually with one open reading frame (orf), can be located in the chromosome or on plasmids, integronintegrases identifies these units. They have been found in Gram-negative enteric-like bacteria and staphylococci, but not in anaerobes, though this may simply be due to the fact that no one has looked. However, as more anaerobic genomes are sequenced these elements may be found. Integrons can carry upwards of 100 different genes. These have been called ‘‘super-integrons’’ [39]. These elements have distinctive features and can be identified directly from the genome sequence. IS elements are often associated with other mobile elements [43]. IS elements are very small, generally do not encode for functions other than those involved in their own mobility (transposase). The majority of IS elements carry short terminal inverted-repeat sequences (IR) of 10–40 bp. IS elements can up-regulate expression of antibiotic resistance genes, supply a new promoter region and can be involved with transposition of transposons. These elements can transpose to a number of locations in the host chromosome and may result in inactivation of genes due to integration. A number of IS elements have been described in Bacteroides spp. Most IS elements were identified because of their ability to upregulate expression of antibioitc resitance genes. Bacteroides IS elements are associated with specific antibiotic resistance genes including the erm(F) gene, and the metallo-b-lactamase gene ccrA, and closely related cfiA genes, another IS element is found with the endogenous
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cephalosporinase cepA gene, while three are associated with resistance to 5-nitroimidazole (nim genes) [44]. It is clear that as more genomes are sequenced IS elements will be found in other anaerobes.
6. Conclusions Resistance has increased significantly over the last 30 years [9] and is becoming a major concern in treatment of antibiotic resistant bacterial disease. It is becoming clear that acquistion of foreign genes (horizontal gene transfer) has played a significant role in shaping bacterial genomes through out their long history. Mobile elements currently carry a variety of genes which are then able to disseminate between bacterial populations and between different ecosystems. Recombination and linkage of different genes most likely has had significant influence on bacterial development in the past, and clearly is a major factor in current bacterial development, especially as it relates to acquisition of antibiotic resistance genes. Many of the mobile elements discussed in this review are ancient. These elements have provided bacteria with flexibility to adapt to unpredictable changes in their environment. It is hypothesized that horizontal gene transfer allows bacteria to acquire foreign genes that may endow them with increased fitness to survive. These elements also provide a means for non-expressed genes to be maintained in a population without direct selective pressure. How bacteria have responded to the increasing use of antibiotics over the last 50 years clearly illustrates what a central role these elements can play in bacterial survival in the face of a new selective pressure. Better understanding of the types of mobile elements, mechanisms of transfer of these elements, how these elements have helped shape bacterial evolution and ultimately what biological significance horizontal gene transfer has on the future coexistence between man and microbe are all important areas for future research. Conjugation is thought to be the most common way for antibiotic resistance genes to spread in bacterial populations. Conjugation requires cell-to-cell contact, live donor and recipient cells, and cell growth. A variety of different types of mobile elements have been described. Most of which have been found in Bacteroides spp. [24,38,41,45,46] and Clostridium spp. [16– 18,39] and most likely many other anaerobes. Perhaps the most important property of these elements is their ability to transfer between unrelated species and ecosystems much more rapidly than what can occur by bacterial replication. This allows for the rapid spread of particular genes within and between bacterial populations. Today it is clear that once a gene becomes associated with a mobile element it can move to surprisingly diverse ecosystems and bacterial species,
including its integration into the chromosomes of plasmids of its new host. Conjugative elements may also mobilize co-resident plasmids. These mobile elements add significant flexibility to the bacterial host’s ability to cope with changes in its environment with relatively little cost to the bacterial cell. They allow for mixing and matching of genes including antibiotic and heavy metal resistance, virulence, and alternative carbon and sugar source pathways. Once collected on an element the genes can then move as a single unit. This allows genes to survive in the population without direct selective pressure. In this mini-review, I have summarized what is currently known about the various genes which confer tetracycline and MLS resistance in bacteria and listed the subset of the tet and erm genes that have been identified in anaerobic strains (Tables 1–4). Because of the limited number of studies and genes screened in the literature, it is unlikely that the distributions shown in Tables 2 and 3 represent the true distribution of these types of genes in anaerobes. This field is moving rapidly, new genes are discovered every year and increases in host ranges for the previously characterized genes occur rapidly. Therefore a web site has been created which updates the general information about host range and new tetracycline and MLS resistance genes. The web site can be accessed through the following URL http:// faculty.washington.edu/marilynr/.
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ARTICLE IN PRESS M.C. Roberts / Anaerobe 9 (2003) 63–69 [9] Chopra I, Roberts MC. Tetracycline antibiotics: mode of action, applications, molecular biology and epidemiology of bacterial resistance. Microbiol Mol Biol Rev 2001;65:232–60. [10] Paulsen IT, Brown MH, Skurray RA. Proton-dependent multidrug efflux systems. Microbiol Rev 1996;60:575–608. [11] Testa RT, Petersen PJ, Jacobus NL, Sum P-E, Lee VJ, Tally FP. In vitro and in vivo antibacterial activities of the glycylcyclines, a new class of semisynthetic tetracyclines. Antimicrob Agents Chemother 1993;37:2270–7. [12] Chopra I, Hawkey PM, Hinton M. Tetracyclines, molecular and clinical aspects. J Antimicrob Chemother 1992;29:245–77. [13] McMurry LM, Levy SB. Tetracycline resistance in Gram-positive bacteria. In: Fischetti VA, Novick RP, Ferretti JJ, Portnoy DA, Rood JI, (editors). Gram-positive pathogens. Washington DC: American Society of Microbiology, 2000. p. 660–7. [14] Bannam T, Rood JI. Identification of structural and functional domains of the tetracycline efflux protein TetA(P) from Clostridium perfringens. Microbiology 1999;145:2947–55. [15] Johanesen PA, Lyras D, Bannam TL, Rood JI. Transcriptional analysis of the tet(P) operon from Clostridium perfringens. J Bacteriol 2001;183:7110–9. [16] Lyras D, Rood JI. Genetic organization and distribution of tetracycline resistance determinants in Clostridium perfringens. Antimicrob Agents Chemother 1996;40:2500–4. [17] Sasaki Y, Yamamoto K, Tamura Y, Takahashi T. Tetracyclineresistance genes of Clostridium perfringens, Clostridium septicum and Clostridium sordellii isolated from cattle affected with malignant edema. Vet Microbiol 2001;83:61–9. [18] Sloan J, McMurry LM, Lyras D, Levy SB, Rood JI. The Clostridium perfringens Tet P determinant comprises two overlapping genes: tetA(P), which mediates active tetracycline efflux, and tetB(P), which is related to the ribosomal protection family of tetracycline-resitance determinants. Mol Microbiol 1994;11: 403–15. [19] Sanchez-Pescador R, Brown JT, Roberts M, Urdea MS. Homology of the TetM with translational elongation factors: implications for potential modes of tetM conferred tetracycline resistance. Nucl Acid Res 1988;16:1218. [20] Taylor DE, Chau A. Tetracycline resistance mediated by ribosomal protection. Antimicrob Agents Chemother 1996;40: 1–5. [21] Trieber CA, Burkhardt N, Nierhaus KH, Taylor DE. Ribosomal protection from tetracycline mediated by Tet(O): Tet(O) interaction with ribosomes is GTP-dependent. Biol Chem 1998;379: 847–55. [22] Melville CM, Scott KP, Mercer DK, Flint HJ. Novel tetracycline resistance gene, tet(32), in the Clostridium-related human colonic anaerobe K10 and its transmission in vitro to the rumen anaerobe Butyrivibrio fibrisolvens. Antimicrob Agents Chemother 2001;45: 3246–9. [23] Diaz-Torres ML, McNab R, Spratt DA, Villedieu A, Hunt N, Wilson M, Mullany P. Novel tetracycline resistance determinant from the oral metagenome. Antimicrob Agents Chemother 2003;47:1430–2. [24] Speer BS, Bedzyk L, Salyers AA. Evidence that a novel tetracycline resistance gene found on two Bacteroides transposons encodes an NADP-requiring oxidoreductase. J Bacteriol 1991; 173:176–83. [25] Weisblum B. Erythromycin resistance by ribosomal modification. Antimicrob Agents Chemother 1995;39:5775–85.
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[26] Weisblum B. Macrolide resistance. Drug Resist Update 1998;1:29–41. [27] Roberts MC, Sutcliffe J, Courvalin P, Jensen LB, Rood J, Seppala H. Nomenclature for macrolide and macrolide–lincosamide–streptogramin B antibiotic resistance determinants. Antimicrob Agents Chemother 1999;43:2823–30. [28] Sutcliffe JA, Leclercq R. Mechanisms of resistance to macrolides, lincosamides , and ketolides. In: Schonfeld W, Kirst HA. (editors) Macrolide antibiotics. Basel, Switzerland: Birhauser, 2002. p. 281–317. [29] Halula M, Manning S, Macrina FL. Nucleotide sequence of ERMFU, macrolide–lincosamide–streptogramin (MLS) resistance gene encoding an RNA methylase from the conjugal element of Bacteroides fragilis V503. Nucc Acid Res 1991;19:3453. [30] Ross JI, Eady EA, Cove JH, Cunliffe WJ. 16S rRNA mutation associated with tetracycline resistance in a Gram-positive bacterium. Antimicrob Agents Chemother. 1998;42:1702–5. [31] Trieber CA, Taylor DE. Mutations in the 16S rRNA genes of Helicobacter pylori mediated resistance to tetracycline. J Bacteriol 2002;184:2131–40. [32] Vester B, Douthwaite S. Macrolide resistance conferred by base substitutions in 23S rRNA. Antimicrob Agents Chemother 2001;45:1–12. [33] Bushman F. Lateral DNA transfer-mechanisms, consequences. ColdSpring Harbour, NY: Cold Spring Harbor Laboratory Press, 2002. p. 1–168, 388–91. [34] Koonin EV, Makaprova KS, Aravind L. Horizontal gene transfer in prokaryotes: quantification and classification. Ann Rev Microbiol 2000;55:709–42. [35] Hughes VM, Datta N. Conjugative plasmids in bacteria of the ‘pre-antibiotic’ era. Nature 1983;302:725–6. [36] Gordon GS, Wright A. DNA segregation in bacteria. Annual Review Microbiology 2000;55:681–708. [37] Clewell DB, Flannagan SE, Jaworski DD. Unconstrained bacterial promiscuity: the tn916-tn1545 family of conjugative transposons. Trends Microbiol. 1995;3:229–36. [38] Smith CJ, Tribble GD, Bayley DP. Genetic elements of Bacteroides species: a moving story. Plasmid 1998;40:12–29. [39] Rood JI, Cole ST. Molecular genetics and pathogenesis of Clostridium perfringens. Microbiol Rev 1991;55:621–48. [40] Rowe-Magnus DA, Guerout A-M, Ploncard P, Dychinco B, Davies J, Mazel D. The evolutionary history of chromosomal super-integrons provides an ancestry for multiresistant integrons. Proc Natl Acad Sci USA 2001;98:653–7. [41] Whittle G, Shoemaker NB, Salyers AA. Characterization of genes involved in modulation of conjugal transfer of the Bacteroides conjugative transposons CTnDOT. J Bacteriol 2002;182:3839–47. [42] Falkow, S. (1975) Infectious multiple drug resistance. London, England: Pion Limited. [43] Mahillon J, Chandler M. Insertion sequences. Microbiol Mol Biol Rev 1998;62:725–74. [44] Sebald M. Genetic basis for antibiotic resitance in anaerobes. Clin Infect Dis 1994;18(Suppl 4):S297–304. [45] Cheng Q, Paszhiet BJ, Shoemaker NB, Gardner JF, Salyers AA. Integration and excision of a Bacteroides conjugative transposon, CTnDOT. J Bacteriol 2002;182:4035–43. [46] Shoemaker NB, Vlamikis H, Hayes K, Salyers AA. Evidence for extensive resistance gene transfer among Bacteroides spp. and between Bacteroides and other genera in the human colon. App Environ Microbiol 2001;67:561–8.
Anaerobe 9 (2003) 63–69
Antimicrobial susceptibility
Acquired tetracycline and/or macrolide–lincosamides–streptogramin resistance in anaerobes$ Marilyn C. Roberts* Department of Pathobiology, University of Washington, Seattle, WA 98195,USA Received 9 September 2002; received in revised form 28 March 2003; accepted 7 April 2003
Abstract In general bacterial antibiotic resistance is acquired on mobile elements such as plasmids, transposons and/or conjugative transposons. This is also true for many antibiotic resistant anaerobic species described in the literature. Of the 23 different tetracycline resistant efflux genes identified, tet(B), tet(K), tet(L), and tetA(P) have been found in anaerobic species and six of the ten tetracycline resistant genes coding for ribosomal protection proteins, tet(M), tet(O), tetB(P), tet(Q), tet(W), and tet(32), have been identified in anaerobes. There are now three enzymes which inactivate tetracycline, of which the tet(X) has been identified in Bacteroides though is not functional under anaerobic growth conditions. A similar situation exists with the genes conferring macrolide–lincosamide–streptogramin (MLS) resistance. Of the 26 rRNA methylase MLS resistant genes characterized, five genes; erm(B), erm(C), erm(F), erm(G), and erm(Q), have been identified in anaerobes. In contrast, no genes coding for MLS resistant efflux proteins or inactivating enzymes have been described in anaerobic species. This mini-review will summarize what is known about tetracycline and MLS resistance in genera with anaerobic species and the mobile elements associated with acquired tetracycline and/or MLS resistance genes. r 2003 Elsevier Science Ltd. All rights reserved.
1. Introduction Anaerobic bacteria are found in diverse ecological niches; however, when it comes to characterization of antibiotic resistance in anaerobes the majority of species studied are either opportunistic pathogens or commensal bacteria associated with humans or animals. It has been hypothesized that the commensal flora, including the anaerobes, act as a reservoir for mobile antibiotic resistant genes. If this hypothesis is correct, it is important to understand what antibiotic resistance genes the commensal flora, as well as, the opportunistic and pathogenic species carry. Many reports exist on the decrease in susceptiblity to antibiotics especially clindamycin, cephalosporins and penicillins in both the Bacteroides fragilis group and non-Bacteroides anaerobes [1]. Over 50% of the Bacteroides fragilis group isolates are resistant to tetracyclines, while resistance in $
Paper from Anaerobe Olympiad 2002. The 6th Biennial Congress of the Anaerobe Society of the Americas, Park City, Utah, 29 June– 2 July. *Fax: +1-206-543-3873. E-mail address: [email protected] (M.C. Roberts).
non-Bacteroides and other Gram-negative genera are more variable [2]. Anaerobic in vitro susceptiblity tests have difficulties in accuracy, reproducibility and predictive value for therapy. Differences in the methods used, choice of media, and inoculum size all affect results [2]. Most of the information on carriage and distribution of acquired antibiotic resistance genes has been done using DNA probes in selected research laboratories [3–8]. With some bacteria the increase in the MIC correlates with the presence of specific antibiotic resistance genes [4]. This mini-review will summarize what is known about tetracycline and macrolide–lincosamide–streptogramin (MLS) resistance in genera with anaerobic species and the mobile elements associated with acquired tetracycline and/or MLS resistance genes.
2. Tetracycline and the acquired resistance genes Tetracyclines are broad-spectrum antibiotics which have a wide range of activity. Only a limited number of derivatives are currently in use. Tetracycline reversibly
1075-9964/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1075-9964(03)00058-1
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inhibits bacterial protein synthesis by binding to the ribosomal complex. This prevents the association of the aminoacyl-tRNA to the ribosomal acceptor (A) site [9]. Once inside the bacteria, the tetracycline binds reversibly with the prokaryotic 30S ribosomal subunit, stopping protein synthesis. Diluting out the antimicrobial can reverse this process. Three types of acquired tetracycline resistance mechanisms have been described (Table 1). The first and most common mechanism is the tetracycline (tet) resistant efflux proteins which belong to the major facilitator superfamily (MFS). These tet efflux genes code for membrane-associated proteins that
Table 1 Mechanism of resistance for characterized tet and otr genes Efflux
Ribosomal protection
Enzymatic
Unknowna
tet(A), tet(B), tet(C), tet(D), tet(E)
tet(M), tet(O), tet(S), tet(W), tet(32), tet(36) tet(Q), tet (T)
tet(X)
tet(U)
otr(A), tetB(P)b, tet
tet(37)
tet(G), tet(H), tet(J), tet(Z), tet (30) tet(31), tet(33)
otr(C)
tet(K), tet( L) otr(B) tetA(P) tet(V) tet(Y) tet(34), tet(35) tcr3 a tet(U) has been sequenced but does not appear to be related to either efflux or ribosomal protection proteins, otr(C) has not been sequenced; b tetB(P) is not found alone and tetA(P) and tetB(P) are counted as one operon; tet(36) and tet(37) [25] have recently been assigned.
recognize and export tetracycline from the cell. This reduces the intracellular drug concentration and protects the majority of the ribosomes within the cell [9,10]. Efflux genes are found in both Gram-positive and Gram-negative anaerobic species (Table 2). Most of these efflux proteins confer resistance to both tetracycline and doxycycline, but not minocycline or the newer experimental glycylcyclines [9,11,12]. There are 22 different tetracycline resistant efflux genes identified. The efflux proteins have been divided into groups based on amino acid sequence identity [9,13]. Group 1 contains the proteins, Tet (A), (B), (C), (D), (E), (G), (H), (J), (Z) and Tet(30). Tet(Z) has been in a Gram-positive species, while all the others are found only in gram-negative species [12]. In this group, the tetracycline resistance efflux proteins share 41–78% amino acid identity with each other. From this group only the tet(B) gene has been found in one Gramnegative anaerobe, Treponema denticola (Table 2). The other genes have not been found in anaerobes at this time. Group 2 efflux proteins include Tet(K) and Tet(L) with 58–59% amino acid identity. These two genes are of Gram-positive origin but have occasionally been found in Gram-negative species. Among anaerobes the tet(K) gene has been found in Eubacterium and Peptostreptococcus, while the tet(L) gene has been found in Actinomyces, Peptostreptococcus, Veillonella, and Clostridium (Table 2). Group 3 efflux proteins include the otr(B) and tcr3 gene products both found in Streptomyces spp. Group 4 efflux proteins include the tetA(P) gene product described below, while Group 5 efflux protein include the gene products from the tet(V) gene from Mycobacterium smegmatis. None of the genes from Group 3, 4, or 5 have been found in anaerobic species.
Table 2 Distribution of tetracycline resistance genes in anaerobes Efflux
Ribosomal protection and/or efflux
One gene
One gene
Treponema
a
tet(B)
Capnocytophaga
Found in a Clostridum-like micro [22].
Two of more genes tet(Q)
Actinomyces Butyrivibrio Bifidobacterium Mitsuokella Mobiluncus Selenomonas Porphyromonas Bacteroides Prevotella Fusobacterium Eubacterium Lactobacillus Peptostreptococcus Veillonella Clostridium
tet(L), (M) tet(O), (W) tet(M), (W) tet(Q), (W) tet(O), (Q) tet(Q), (W) tet(Q), (W) tet(M), (Q), (X), (36) tet(M), (Q), (W) tet(L), (M), (Q) tet(K), (M), (Q) tet(M), (O), (S), (Q) tet(K), (L), (M), (O), (Q) tet(A), (L), (M), (S), (Q), (W) tet(K), (L), (M), (P), (Q), (32)a
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The tet(P) operon from Clostridium has a unique structure and appears to be unique to this genus [14]. It consists of two overlapping genes, tetA(P) and tetB(P). The tetA(P) gene encodes for an efflux protein similar to the other tetracycline resistant efflux proteins described above. The tetB(P) gene codes for a protein which is similar to the ribosomal protection proteins described below. When both genes are present, they are transcribed from a single promoter located upstream of the tetA(P) start codon [14–17]. A factor-independent terminator, T1, located downstream from the promoter and upstream of the tetA(P) has recently been described [15] and it has been postulated that the T1 terminator is an intrinsic control element of the tet(P) operon which acts by preventing over-expression of the efflux protein [tetA(P)]. The tetA(P) gene has been found alone in Clostridium spp., and confers tetracycline resistance. However, the tetB(P) gene has not been found alone in nature, but when the tetB(P) gene was separated from the rest of the operon, in the laboratory, it conferred low-level resistance to tetracycline and minocycline in both Clostridium perfringens and Escherichia coli [18]. This resistance level is lower than normally found when other ribosomal protection genes are cloned into these organisms. Thus it is unclear if the tetB(P) gene is independently found and its role in nature. There are 11 tetracycline resistant genes coding for ribosomal protection proteins described (Table 1). These are cytoplasmic proteins that protect the ribosomes from the action of tetracycline in vitro and in vivo. They confer resistance to tetracycline, doxycycline and minocycline and have homology with elongation factors EF-Tu and EF-G [19,20]. The current data suggest that the ribosomal protection proteins bind to the ribosome. This binding is not affected by the presence of tetracycline but is inhibited by thiostrepton, which also inhibits the binding of the EF-G protein. When the ribosomal proteins bind they cause an alteration in ribosomal conformation, which then prevents tetracycline from binding to the ribosome. EF-G and the Tet(M) protein compete for binding to the ribosomes, suggesting that they may have overlapping binding sites. Both the Tet(M) and Tet(O) proteins have been shown to have ribosome-dependent GTPase activity and it has been suggested that the GTP binding is important to the function of the Tet(O) protein. It has been hypothesized that the hydrolysis of GTP provides the energy for the ribosomal conformational change [21]. The tet(M), tet(O), tet(Q), tet(W), tet(32) genes, and the tet(P) operon have all been found in anaerobic species [9, 22] (Table 2). This group of genes have low G+C content and are thought to be originally of Grampositive ancestry. The tet(M) gene has the widest host range among all genera of bacteria [9]. The tet(M) gene is currently found in nine anaerobic genera, the tet(Q)
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gene is found in 12 and the tet(W) gene is found in seven anaerobic genera (Table 2). There are now two genes which code for enzymes which inactivate tetracycline. The tet(37) gene has been found from the oral bacteria in adults and determination of whether this gene is associated with oral anaerobic species has not been done [23]. The tet(X) gene codes an enzyme, which alters the tetracycline molecule. Two closely related anaerobic Bacteroides transposons (Tn4351, Tn4400) containing the tet(X) gene has been described [24]. The tet(X) gene was originally found because it was linked to an erm(F) gene, which codes for a rRNA methylase. The erm(F) gene was cloned into E. coli and the clones were found to confer tetracycline resistance in E. coli when grown aerobically. The tet(X) gene product is a 44 kDa cytoplasmic protein that chemically modifies tetracycline in the presence of both oxygen and NADPH. Sequence analysis indicates that this protein shared amino acid homology with other NADPH-requiring oxidoreductases and should not be able to function in the anaerobic Bacteroides hosts. It has not been found outside Bacteroides, however, no survey has been conducted to assess the distribution of the tet(X) gene in any bacterial population. Neither the tet(U) or the otr(C) gene has been found in anaerobes, though no survey has been conducted to assess the distribution of either gene [9]. These will not be discussed further (Tables 1 and 2).
3. Macrolide–lincosamide–streptogramin and the acquired resistance genes Macrolides, lincosamides, streptogramin B and the new ketolides are a structurally diverse group of antibiotics with overlapping binding sites in the peptidyl transferase region of the 23S rRNA. This group of drugs interacts with the 50S subunit of the bacterial ribosome. They appear to inhibit protein synthesis by inhibiting movement of the peptide chain and/or alter the binding of the peptidyl-tRNA molecule from the ribosomes during elongation. This results in chain termination and a reversible stoppage of protein synthesis. Three types of acquired resistance genes have been described and include rRNA methylases (erm genes) which are a family of related proteins that add one or two methyl groups to a single adenine (A2058 in E. coli) in the 23S rRNA moiety. Modification of the ribosomes by rRNA methylases reduces the binding of macrolides, lincosamides and streptogramin B antibiotics (MLSB) [25–28]. The erm genes are the most commonly found acquired resistance mechanism in bacteria and the only genes currently found in anaerobes [27] (Table 3). All 27 of the erm genes are of Gram-positive origin, they have low G+C contents and can be expressed in
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Table 3 Location of rRNA methylase resistance genes in anaerobes One gene Actinomyces Fusobacterium Propionibacterium Treponema Veillonella
Two or more genes erm(F) erm(F) erm(W) erm(F) erm(F)
Porphyromonas Prevotella Eubacterium Lactobacillus Bacteroides Clostrdium Wolinella
erm(F), erm(G) erm(F), erm(G) erm(C), erm(F) erm(C), erm(G), erm(T) erm(F), erm(G), erm(35) erm(B), erm(F) , erm(Q) erm(B), erm(C) , erm(F), erm(Q)
Table 4 Mechanism of MLS resistance rRNA methylase
Efflux
27 rRNA methylases (erm genes) (A), (B), (C), (D), (E), (F), (G), (H) (I), (N), (O), (Q), (R), (S), (T), (U) (V), (Y), (Z), (30), (31), (32), (33) (34), (35), (36), (37)
11 efflux mef(A), msr(A), (C) car(A), lmr(A), ole(B), (C) srm(B), tlr(C), vga(A), (B)
both Gram-positive and Gram-negative species [27,28]. This is true even for the erm(F) gene which was first described in Gram-negative anaerobes [29]. Seven of the erm genes, erm(B), erm(C), erm(F), erm(Q), erm(T), and erm(35) have been found in anaerobes. The erm(F) gene has the widest host range and is found in 10 anaerobic genera examined. In four of the genera only the erm(F) gene has been identified. However, the current distribution may similarly reflect on what erm genes investigators examined rather than the true distribution in nature (Table 3). There are 11 genes which code for efflux proteins, which pump drug(s) out of the cell, and 17 genes which code for inactivating enzymes including esterases, lyases, transferases, and phosphorylases, which inactivate the drug(s) by adding moieties to them (Table 4). All 28 genes confer resistance to macrolides and/or lincosamides and/or streptogramins. However, none of these genes have been identified in anaerobes, though I could not find any published reports where anaerobes have been screened for these genes.
Inactivating enzymes 2 esterase ere(A), (B)
2 lyases vgb (A), (B)
9 transferases lnu(A), (B) vat(A), (B), (C), (D) vat(D), (E), (F)
4 phosphorylases mph(A), (B), (C), (D)
tions can change the adenine (A2058) or one of the adjacent residues in the peptidyl transferase region (A2057 or A2059) to another nucleotide. A number of different pathogenic genera with 23S rRNA mutations are listed in Table 1 of [28]. The authors include a number of different Propionibacterium spp., and microaerophilic Helicobacter pylori. Tetracycline resistances due to mutations in the 16S rRNA have also been described in H. pylori [31]. Mutations in ribosomal proteins L4 and/or L22 that confer erythromycin resistance have been documented in laboratory strains of E. coli and Gram-positive cocci [28], as well as, in clinical Gram-positive cocci. Mutations in intrinsic pumps may confer increased resistance to tetracyclines and/or erythromycin in both Gram-positive and Gramnegative bacteria [28]. Clearly the potential of mutations leading to increased resistance in anaerobes does exist. Therefore it is likely that in time other anaerobes will be identified with mutations conferring increased resistance to tetracycline and/or MLS antibiotics [27].
5. Mobile elements 4. Mutations A less common way for bacteria to become tetracycline and/or MLS resistant is by mutations of chromosomal genes. Tetracycline resistant cutaneous propionibacteria (MICs 2–64 mg/ml of tetracycline, 1– 32 mg/ml doxycycline) have been described [30]. In these isolates, a guanine was switched to a cytosine at position 1058 in the 16S rRNA. This change was associated with the increase in tetracycline resistance. Similarly, muta-
Horizontal (lateral) DNA transfer is thought to be a major player in bacterial evolution [32–41]. It allows for blocks of DNA with multiple genes to be moved as a unit. Mobile elements are able to transfer between unrelated species and ecosystems and allows for the rapid spread of associated genes within and between bacterial populations. Today it is clear that once a gene becomes associated with a mobile element it can move to surprisingly diverse ecosystems and bacterial species,
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including its integration into the chromosome and/or plasmid(s) of its new host. Conjugative elements may also mobilize co-resident plasmids. Mobile elements add significant flexibility to the bacterial host’s ability to cope with changes in its environment with relatively little cost to the bacterial cell. It is the most likely reason that acquired antibiotic resistant genes have spread so rapidly in the last 50 years. Horizontal DNA transfer has also played a role in the development of multi-drug resistant isolates. Some mobile elements contain antibiotic resistance genes and virulence factors. Most acquired antibiotic resistant gene(s) are associated with some type of mobile element (plasmid, transposon, conjugative transposon, and/or integron). A large number of these elements have been described over the past 30 years [15,37–41]. Plasmids were the first to be identified in the 1960s [9]. Plasmids are usually circular DNA molecules, which normally carry non-essential genes. Many can move between related genera, but their host ranges vary significantly from very restricted (only related strains) to a broad-host range (wide range of genera). Plasmids may contain transposons, conjugative transposons, IS elements and integrons as well as a wide range of genes including toxin protein, virulence factors in addition to antibiotic resistance genes [42,43]. Published reports suggest that 20–50% of the Bacteroides spp. examined carry plasmids. In general these plasmids have a limited host range and can replicate in Bacteroides spp. and the closely related Porphyromonas spp. and Prevotella spp., but not in E. coli. Most of the Bacteroides spp. plasmids are small (o 8 kb) and cryptic (lack a detectable phenotype) [38]. These plasmids are mobilizable and belong to one of three homology groups and are of modular construction sharing replication and mobilization gene cassettes. C. perfringens plasmids coding for tetracycline, and/or chloramphenicol and/or erythromycin and clindamycin resistance has been characterized [44]. These large plasmids are ubiquitous in the species and appear to be genetically related to each other. Some of the plasmids carry transposons and similar antibiotic resistance genes in C. perfringens and C. difficile suggesting conjugative transfer between the two species. Some conjugative plasmids (pAMb1, pJH4 and pIP502) carrying the erm(B) resistance gene have been transferred in the laboratory. More information about Bacteroides spp. and Clostridium spp. plasmids and/or conjugative transposons is available from a variety of articles and beyond the scope of this review. Transposons are defined elements with either direct or inverted repeats at the ends, often associated with IS elements flanking the element. Some of these elements integrate into a few sites within some bacteria but randomly in others, variations on this theme do occur. Transposons carry their own enzymes, which allow transposition from one piece of DNA to another piece
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of DNA within the same bacterial cell. These enzymes allow for the direct cleaving and rejoining of the DNA after transposition has occurred. These elements are ubiquitous in both prokaryotes and eukaryotes. Conjugative transposons are defined genetic entities, which can be associated with either plasmids and/or chromosomes. These carry genes for both transposition and conjugation and thus can transfer between DNA within a host cell and transfer between strains by conjugation [45]. These elements are very common in Gram-positive aerobes and anaerobes such as Clostridium spp., and anaerobic Gram-negative species (Bacteroides), but are not commonly found in Gram-negative enteric-like bacteria, such as E. coli [4,9,18,27,37–39]. Some of the best studied conjugative transposons are from Bacteroides spp. [24,38,41,45,46] and Clostridium spp. [16–18,39]. Integrons are relatively newly described genetic elements. It is thought that these elements play an important role in the evolution of antibiotic resistance in Gram-negative bacteria and Gram-positive staphylococci. A few of the integrons have virulence genes associated with them, but most carry antibiotic resistance genes. Integrons acquire and exchange exogenous DNA (gene cassettes) by site-specific recombination. They function as a general gene-capture system. Gene cassettes have a target recombination sequence (attC site ) usually with one open reading frame (orf), can be located in the chromosome or on plasmids, integronintegrases identifies these units. They have been found in Gram-negative enteric-like bacteria and staphylococci, but not in anaerobes, though this may simply be due to the fact that no one has looked. However, as more anaerobic genomes are sequenced these elements may be found. Integrons can carry upwards of 100 different genes. These have been called ‘‘super-integrons’’ [39]. These elements have distinctive features and can be identified directly from the genome sequence. IS elements are often associated with other mobile elements [43]. IS elements are very small, generally do not encode for functions other than those involved in their own mobility (transposase). The majority of IS elements carry short terminal inverted-repeat sequences (IR) of 10–40 bp. IS elements can up-regulate expression of antibiotic resistance genes, supply a new promoter region and can be involved with transposition of transposons. These elements can transpose to a number of locations in the host chromosome and may result in inactivation of genes due to integration. A number of IS elements have been described in Bacteroides spp. Most IS elements were identified because of their ability to upregulate expression of antibioitc resitance genes. Bacteroides IS elements are associated with specific antibiotic resistance genes including the erm(F) gene, and the metallo-b-lactamase gene ccrA, and closely related cfiA genes, another IS element is found with the endogenous
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cephalosporinase cepA gene, while three are associated with resistance to 5-nitroimidazole (nim genes) [44]. It is clear that as more genomes are sequenced IS elements will be found in other anaerobes.
6. Conclusions Resistance has increased significantly over the last 30 years [9] and is becoming a major concern in treatment of antibiotic resistant bacterial disease. It is becoming clear that acquistion of foreign genes (horizontal gene transfer) has played a significant role in shaping bacterial genomes through out their long history. Mobile elements currently carry a variety of genes which are then able to disseminate between bacterial populations and between different ecosystems. Recombination and linkage of different genes most likely has had significant influence on bacterial development in the past, and clearly is a major factor in current bacterial development, especially as it relates to acquisition of antibiotic resistance genes. Many of the mobile elements discussed in this review are ancient. These elements have provided bacteria with flexibility to adapt to unpredictable changes in their environment. It is hypothesized that horizontal gene transfer allows bacteria to acquire foreign genes that may endow them with increased fitness to survive. These elements also provide a means for non-expressed genes to be maintained in a population without direct selective pressure. How bacteria have responded to the increasing use of antibiotics over the last 50 years clearly illustrates what a central role these elements can play in bacterial survival in the face of a new selective pressure. Better understanding of the types of mobile elements, mechanisms of transfer of these elements, how these elements have helped shape bacterial evolution and ultimately what biological significance horizontal gene transfer has on the future coexistence between man and microbe are all important areas for future research. Conjugation is thought to be the most common way for antibiotic resistance genes to spread in bacterial populations. Conjugation requires cell-to-cell contact, live donor and recipient cells, and cell growth. A variety of different types of mobile elements have been described. Most of which have been found in Bacteroides spp. [24,38,41,45,46] and Clostridium spp. [16– 18,39] and most likely many other anaerobes. Perhaps the most important property of these elements is their ability to transfer between unrelated species and ecosystems much more rapidly than what can occur by bacterial replication. This allows for the rapid spread of particular genes within and between bacterial populations. Today it is clear that once a gene becomes associated with a mobile element it can move to surprisingly diverse ecosystems and bacterial species,
including its integration into the chromosomes of plasmids of its new host. Conjugative elements may also mobilize co-resident plasmids. These mobile elements add significant flexibility to the bacterial host’s ability to cope with changes in its environment with relatively little cost to the bacterial cell. They allow for mixing and matching of genes including antibiotic and heavy metal resistance, virulence, and alternative carbon and sugar source pathways. Once collected on an element the genes can then move as a single unit. This allows genes to survive in the population without direct selective pressure. In this mini-review, I have summarized what is currently known about the various genes which confer tetracycline and MLS resistance in bacteria and listed the subset of the tet and erm genes that have been identified in anaerobic strains (Tables 1–4). Because of the limited number of studies and genes screened in the literature, it is unlikely that the distributions shown in Tables 2 and 3 represent the true distribution of these types of genes in anaerobes. This field is moving rapidly, new genes are discovered every year and increases in host ranges for the previously characterized genes occur rapidly. Therefore a web site has been created which updates the general information about host range and new tetracycline and MLS resistance genes. The web site can be accessed through the following URL http:// faculty.washington.edu/marilynr/.
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