JAC Advance Access originally published online on December 7, 2005
Journal of Antimicrobial Chemotherapy 2006 57(2):212-220; doi:10.1093/jac/dki443
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Molecular investigation of genetic elements contributing to metronidazole resistance in Bacteroides strains
1 Instutute of Clinical Microbiology, University of Szeged, H-6725 Szeged, Somogyi Béla tér 1, Hungary; 2 Anaerobe Reference Laboratory, Department of Medical Microbiology, University Hospital of Wales, Cardiff, UK; 3 Department of Microbiology, Faculty of Medicine, University of Kuwait, Kuwait
* Corresponding author. Tel/Fax: +36-62-545712; E-mail: soki{at}mlab.szote.u-szeged.hu
Received 20 July 2005; returned 9 September 2005; revised 2 November 2005; accepted 8 November 2005
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Abstract |
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Objectives: The aim of this study was to investigate the constitution of nim gene types, their activating insertion sequence (IS) element, their localization (plasmid or chromosome) and cfiA gene status in metronidazole-resistant Bacteroides strains (n = 26) in order to examine their interchangeability.
Methods: Southern hybridization and conjugative plasmid transfer were used to localize the nimA–E genes and plasmid functions. PCR was used to detect the IS elements and the cfiA genes. PCR-mapping was applied to detect the nim gene-associated IS elements. PCR-mapping products and a nimE gene-containing plasmid fragment were sequenced.
Results: Nine of the nimA genes (12) were activated by IS1168 and nine were carried on plasmids, four of which were pIP417-like. The five nimB genes were chromosomal, and two of them were associated with IS1168 and one with IS612. Of the three nimC genes, two were activated by IS1170, and one was carried on a pIP419-like plasmid. The only nimD gene was chromosomal. The five nimE strains harboured the resistance genes on plasmids: one plasmid, pBF388c, 8.3 kb, was characterized, and a novel IS-like element was demonstrated upstream of all the nimE genes. The insertion events of some of these IS elements were restricted to certain nim gene-specific positions. The 11 chromosomal nim genes displayed a positive association with the cfiA gene-specific background.
Conclusions: Fourteen strains harboured the well-known genetic elements: pIP417- and pIP419-like plasmids, chromosomal nimB genes and a common nimE plasmid. However, a rate of interchangeability was also demonstrated, mostly due to combinations of nim genes and their associated IS elements harboured on different replicons.
Keywords: nim genes , insertion sequence (IS) elements , cfiA , plasmid functions
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Introduction |
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The species of the anaerobic Bacteroides sensu stricto genus are the clinically most significant endogenous anaerobic pathogens, often causing life-threatening infections such as intra-abdominal, intrapelvic, lung and brain abscesses and sepsis. They are also amongst the most abundant members of the normal intestinal microbiota. Furthermore, they have the most numerous antibiotic resistance mechanisms and the highest percentage of resistance of all pathogenic anaerobic bacteria. The resistance rates for common ß-lactam agents and tetracyclines are close to 100%, and for cephamycins and macrolide–lincosamide–streptogramin drugs they are high enough for them not to be suitable for empirical therapy. Bacteroides strains resistant to the most useful carbapenems, nitroimidazoles, ß-lactam/ß-lactamase inhibitor combinations and fourth-generation fluoroquinolones have appeared worldwide.1
In the cases of the carbapenems and nitroimidazoles, the best-known resistance mechanisms involve enzymatic modification of the drugs, the expression of the otherwise ‘silent’ resistance genes (cfiA/ccrA for carbapenems and nim for nitroimidazoles) being activated by insertion sequence (IS) elements.2–4 The nitroimidazole resistance genes include several types: nimA–G; these display around 70% sequence identity, they can usually be mobilized and they can occur in all Bacteroides species.5–7 The first four nim gene types (nimA–D) were described on distinct mobile genetic elements specific for each nim gene type and with activating IS elements also highly specific for each nim gene type (nimA pIP417 IS1186, nimB chromosomal IS1186, nimC pIP419 IS1170 and nimD pIP421 IS1169).3,4,8–10
Three novel nim gene types (E, F and G)5–7 have subsequently been described, but knowledge concerning their mobility and the harbouring of genetic elements is lacking. Additionally, IS elements have proved to be capable of activating other resistance genes of Bacteroides, e.g. ermB and cepA,1 and have been proved to be interchangeable between these genes,2,11 but the majority of cases are of interchanges observed between nim and cfiA genes.12 However, although many nim-positive Bacteroides strains do not develop a high level of resistance, they can be induced to higher levels of resistance by the antibiotic both reversibly and irreversibly.6,7 Interestingly, the cfiA gene has proved to be non-mobile, chromosomal and restricted to Bacteroides fragilis.13,14 The strains with cfiA genes form a subgroup of the B. fragilis population that have been examined by several typing methods.14–17 The nimA and nimC genes have also been found chromosomally.18
The molecular action of a nim nitroimidazole reductase protein of Deinococcus radiodurans has recently been deduced from crystallographic data suggesting a reaction mechanism involving the 2-electron reduction of metronidazole, preventing accumulation of the toxic nitro radical, and an important role for His-71 residue.19
The aim of the present study was to examine the genetic background of metronidazole-resistant Bacteroides strains with respect to the nim gene type, the location of the genes on known or novel plasmids or on chromosomes and demonstration of the activating IS element type. Examinations were made of the extent of heterogeneity in the relation of the nim gene type and the activating IS element. The molecular characteristics of a novel nimE gene-carrying genetic element were determined, and the relation between the localization of the nim genes and the cfiA status was investigated.
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Bacterial isolates and cultivation
Strains belonging to the Bacteroides genus (n = 26) were collected at the Anaerobic Reference Laboratory in Wales. They originated from England and Wales or Kuwait. Identification and preliminary characterization of these strains were described previously.5,20 They are listed in Tables 1 and 2. The strains were stored at –70°C in CryoBank vials (Mast Diagnostica, Rheinfeld, Germany) and were cultivated on Fastidious Anaerobe Agar or Columbia agar supplemented with 5% (v/v) blood, 5 mg/L haemin and 1 mg/L vitamin K1 or in BHIS broth [brain–heart infusion broth supplemented with 0.5% (w/v) yeast extract, 5 mg/L haemin and 1 mg/L vitamin K1] at 37°C in an anaerobic cabinet (85% N2, 10% H2, 5% CO2) for 24 (for BHIS) or 48 h.
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B. fragilis 638R was used as the host strain for conjugation experiments. Positive controls and templates for probe preparation included B. fragilis 638R (pIP417) (nimA), B. fragilis BF-8 (nimB), B. fragilis 638R (pIP419) (nimC), B. fragilis 638R (pIP421) (nimD), B. fragilis ARU6881 (nimE), B. fragilis 1776 (IS1187), B. fragilis BFr902 (IS1188) and B. fragilis 2944 (IS612, IS614, IS614B and IS614C). Escherichia coli V/517 was used to prepare a molecular weight marker plasmid set.
Tetracycline MICs were determined by broth microdilution as recommended by the NCCLS (NLSI),21 and metronidazole MICs were determined via Etests (AB Biodisk, Solna, Sweden).
Bacterial matings were carried out mainly as described by Mays et al.22 For the cultivation of transconjugants containing the pBF388c plasmid, BHIS broth with a metronidazole content of 4 mg/L was used.
Gel electrophoresis, restriction endonuclease treatment and mapping
Restriction enzyme digestions were performed under the conditions recommended by the suppliers, in 20–50 µL final volumes, and restriction maps were determined by means of single and double digestions. PCR, plasmid and total DNA samples were electrophoresed in 0.7–1.5% agarose gels in TAE (40 mM Tris-acetate and 1 mM EDTA) or TBE (45 mM Tris-borate and 1 mM EDTA) buffer containing 0.5 mg/L ethidium bromide; DNA was visualized with UV light, and permanent records were made electronically.
Preparation of plasmid samples, chromosomal DNA and Southern hybridization
The cultivated bacterial cell mass was processed with the Qiagen Plasmid Mini Preparation Kit (Qiagen, Hilden, Germany) or a mild alkali/heat-treatment plasmid extraction procedure to obtain plasmid preparations,23 and the Proteinase K digestion cetyltrimethylammonium bromide extraction procedure was applied to prepare chromosomal DNA.24 Concentrations of DNA preparations were determined by UV spectrophotometry at 280 and 260 nm.
Plasmid (200–300 ng) and chromosomal DNA (750 ng) samples were electrophoresed in agarose gels containing 0.5 mg/L ethidium bromide in TAE or TBE buffer with a constant voltage gradient of 5 V/cm.
DNA was transferred by capillary action to nylon membranes (Amersham) according to Sambrook et al.25 and as recommended by the supplier of the Gene Images hybridization kit (Amersham). PCR fragments to be labelled non-isotopically were gel-purified (Qiagen) and labelled with the Amersham Gene Images random primer labelling kit. Prehybridizations, hybridizations and high-stringency washes (first and second washes with 1 x SSC/0.1% SDS and 0.5% SSC/0.1% SDS, respectively) were carried out at 60°C as described for the Gene Images hybridization modules. Detection was performed as described for the Gene Images detection module, using alkaline phosphatase-conjugated anti-fluorescein antibody and CDP-star chemiluminescent substrate and Hyperfilm exposure for 4 min for plasmid preparations and for 4–16 h for total DNA blots.
PCR detection of insertion sequences
PCR templates and reactions were as described previously, and the PCR strategy to detect resistance genes and associated IS elements was also the same.12,26 Primers were selected to attempt to cover all IS element types already found in Europe for IS detection in the strains (IS1168, IS1169, IS1170, IS942, IS4351, IS1187, IS1188, IS612, IS614, IS614B IS614C and the newly described ISBf3) by PCR of the genome. (For the detection of IS612, IS614, IS614B and IS614C, a single primer pair could be used and sequencing served for identification.) The primers newly used in this study were designed using the Primer 3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and are listed in Table 3. The cycling conditions were the same as described previously, or were those indicated in Table 3. For the PCR-mapping of nim genes and upstream IS elements with IS forward and NIM-5 primers, the conditions as for cfiA genes and IS elements were used, as described previously.12
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Nucleotide sequencing
PCR fragments and plasmids were sequenced as described previously.12 The 1.5 kb EcoRV fragment of pBF388c was cloned to the EcoRV site of the Bluescript(+)KS vector and was mutagenized with the Template Generation System II (Finnzymes, Espoo, Finland) to obtain terminal (pUC19 fwd/rev primers) and internal (SeqE and SeqW primers) readings on this DNA fragment. The different sequence reads of pBF388c were assembled and analysed by the Lasergene suite (DNAStar, Inc.). The DNA sequences obtained were compared with nucleotide sequences in the GenBank at the Internet site of the National Library of Biotechnical Information (http://www.ncbi.nlm.nih.gov/) by means of the available BLAST client program. The EMBL Database accession number for the newly determined ISBf6 element and nimE gene is AM042593 [GenBank] and ISBf6 was also registered at the IS element database IS Finder (http://www-is.biotoul.fr/is/is_infos.html).
Statistical testing
Fisher's exact test calculation was carried out with the SPSS program package (SPSS, Inc., Chicago, IL, USA).
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Results and discussion |
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Localization of nim genes and determination of the activating IS elements
The study involved 26 Bacteroides strains whose nim gene types were determined previously,5 or according to Stubbs et al.5 (B. fragilis 388/1).20 To examine the locations of these nimA–E gene types, plasmids were isolated from the test strains and were hybridized with nimA–E gene probes (representative plasmid profiles from nimA and nimC strains can be seen in Figure 1a, and representative hybridization with nimA probes is depicted in Figure 1c). To confirm the chromosomal localization of nim genes in strains where no plasmid reacted with the appropriate probe, hybridizations were performed on chromosomal blots of all strains using undigested and EcoRI-digested chromosomal DNA. In this way the chromosomal localization of the nim genes was established if the probes reacted with the undigested chromosomal DNA bands distinguishable from the similarly reacting plasmids. Via the EcoRI-digested chromosomal DNA hybridizations the plasmid or chromosomal localizations were confirmed and the sizes of the chromosomal DNA fragments could be determined.
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Molecular and epidemiological data on strains with nim genes on plasmids are given in Table 1 and data of strains with chromosomal nim genes in Table 2. Of the 12 strains harbouring the nimA gene, nine carried the gene on plasmids (7.7, 8.2 and 10 kb) while three did so on the chromosome (Tables 1 and 2). In the five nimB strains, all the resistance genes were chromosomal. As concerns the three nimC strains included in the study, one carried the nimC gene on a 10 kb plasmid (pIP421-like), whereas in the other two strains the nimC proved to be chromosomal. The only nimD gene was chromosomal (Tables 2 and 3). Five of the five nimE genes were situated on an 8.3 kb plasmid: the other one was on an 11 kb plasmid, but this could have been due to the reinsertion of a 1.5 kb nimE fragment to the same 8.3 kb plasmid (Figure 2c, lanes 3 and 8). One strain was probably a Dnd (which stands for DNA degradation during electrophoresis) phenotype strain, because we could not prepare a DNA preparation without degradation under the electrophoresis conditions (Figure 2c, lanes 3 and 8). A Dnd phenotype involving a sulphur-containing DNA modification leading to DNA degradation under electrophoretic conditions has been described for Streptomyces lividans,27 and among the anaerobes, also for Clostridium difficile.28 On these chromosomal blots, B. fragilis 388/1 gave bands other than the EcoRI-digested plasmid (Figure 2c, lane 10); these most probably relate to the insertion of nimE fragments of the nimE plasmid into the chromosome in this strain.
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The results of PCR detection and mapping of IS elements involved in the nim gene regulation are displayed in Tables 1 and 2. The main rule observed was that the IS elements upstream of the nim genes were more-or-less nim gene-specific: IS1168 for nimA and nimB, IS1170 for nimC and novel IS element (ISBf6) described in the following section for nimE (Tables 2 and 3). Most of the strains (14) formed a group that corresponded to described genetic patterns (pIP417, pIP419 and chromosomal nimB genes)3,4 and a newly described common nimE plasmid. However, besides other non-nim-associated IS elements IS612 was revealed by PCR-mapping and sequencing to be upstream of a nimB gene in B. fragilis 9750 (Table 2). Exceptions to this main rule were observed (Tables 1 and 2), verifying our initial approach.
The above results are in agreement with the findings of Haggoud et al.,18 who demonstrated that nimA and nimC genes could be chromosomal. Another important issue regarding the nim gene IS element connection is the degree of gene activation. In this study, variability in metronidazole MICs was observed among strains with a specific nim gene type and activating IS element. Other factors too may influence the metronidazole MICs of these strains. The variable activity of the cellular redox systems may be one of these factors: other authors have demonstrated that changes in the cellular redox systems give rise to pronounced degrees of metronidazole resistance.29 Löfmark et al.7 recently reported a metronidazole-inducible phenotype of nim-positive strains, where strains with low metronidazole resistance (MICs 0.25–8 mg/L) could be reversibly induced to high metronidazole resistance (MICs 64–256 mg/L); the exact activation mechanism has not been clarified. Accordingly, it seems at present that the term nim gene-associated IS would describe the situation more appropriately than nim-activating IS elements, the more so since we do not have experimental evidence about the action of IS promoters upstream of nim genes yet.
Additionally, in the present study the activating IS elements did not always correspond to those described (nimA, nimB IS1168, nimD IS1169, nimC IS1170). In some cases, no IS could be observed in the upstream region or an IS different from the regular one was identified (IS612 in the B. fragilis 9750 nimB strain). We presume that, similarly to this situation, additional IS elements may be found in strains where no known upstream IS element is detected.
Despite the fact that the results indicated the interchangeability of genetic elements participating in metronidazole resistance of Bacteroides, they also revealed a high degree of gene specificity. IS1168 activated 75% of the nimA genes irrespective of the genetic carrier; IS1170 activated 66% of the nimC genes, and no other nim gene type was activated by it. Similar to our study, a significant degree of nim gene type and IS element association was revealed in the study of Löfmark et al.,7 though with some exceptions. Moreover, a newly identified IS element (ISBf6, see the next section) proved to be associated exclusively with the nimE genes tested.
Molecular analysis of a nimE plasmid and plasmid functions of other nim plasmids
After identification of the nimE gene-carrying genetic element on an 8.3 kb plasmid by Southern hybridization, this result was confirmed by a conjugative transfer of the 8.3 kb plasmid (pBF388c) of B. fragilis 388/1 to B. fragilis 638R. The transfer efficiency was 6.7 x 10–8 per input donor cell. The spontaneously arising rifampicin-resistant mutants with a mutation rate of 1.6 x 10–8 of the donor strain were differentiated from real transconjugants by detection of their non-mobile cfiA gene by PCR and via the original plasmid profile. Transfer of pBF388c could not be enhanced by tetracycline (1 mg/L), despite the fact that the strain was resistant to tetracycline (MIC = 32 mg/L). The conjugative transfer of Bacteroides mobile elements is often enhanced by low-dose tetracycline (1 mg/L),30 but in primary isolates exceptions have been observed.9 The transconjugants containing pBF388c alone also allowed study of this plasmid separately; their metronidazole MICs were the same as that of the parent strain (16 mg/L). The restriction map of pBF388c is shown in Figure 3. By Southern hybridization, the nimE gene was localized to a 1.5 kb EcoRV fragment (data not shown; Figure 3). Subsequent cloning to the pBluescriptKS(+) vector and nucleotide sequencing gave a 1486 nt fragment containing the major portion of the nimE gene and a 962 nt sequence that could be identified as an IS element-like element, termed ISBf6. The nimE gene was 97% identical to those of B. fragilis ARU 6881 and a Veillonella sp. strain.5,31 ISBf6 had a G+C content of 43.4 mol% in the range for the Bacteroides chromosomes, terminal 19 nt inverted repeats, an ORF encoding a putative protein product (TpBf6) of 285 amino acids that had domains for a DDE motif transposase domain and a COG3293 conserved domain termed transposase, and inactivated derivatives, all demonstrating that it is an IS element. However, ISBf6 did not cause target site duplications. BLAST searches identified numerous (
100) IS element transposases exhibiting homology to TpBf6, with scores between 80.1 and 36 and with E-values between 6 x 10–14 and 7 x 10–4. The closest match was found to a Methanosarcina acetivorans strain C2A IS element, ISMac11.32 ISBf6 contains some sequence motifs that resemble Bacteroides promoters; the activity of the outward oriented structure(s) is being tested further.
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The hybridizations of plasmid function probes from pIP417 and pIP421 plasmids are indicated in Tables 2 and 3. The replication probe pIP417 reacted with the control pIP417 plasmid and four other 7.7 kb nimA plasmids from the test strains. No hybridization was obtained for three other-sized plasmids and two 7.7 kb plasmids, but the probe reacted with all five nimE plasmids. The pIP417 mobilization probe had an interestingly high reaction capacity; it reacted with the control pIP417, all eight nimA, one nimC and six nimE plasmids, and hybridization was observed with other non-nim plasmids too (Tables 1 and 2). The pIP421 mobilization probe reacted with the control pIP421 plasmid, one nimC plasmid and additional non-nim plasmids (Tables 1 and 2). These experiments confirmed that the majority of the plasmids corresponded to those described earlier (pIP417, pIP419 and pIP421) on the basis of their sizes and the genetic material contained in them. However, a significant interchangeability rate was detected, which could be explained by the well-known modularity of these bacterial plasmids.2,33 Finding these structures on other than nim plasmids could account for the modularity and additionally for their origin. The plasmid function fragments detected on nim plasmids, and especially the mobilization functions, together with potent and predominant tetracycline resistance conjugative transposons, make their wide distribution possible in the Bacteroides population.9,10,30 In recent years, however, in parallel with the appearance of metronidazole-resistant Bacteroides strains worldwide, the rates of this resistance among Bacteroides seem to be slowly increasing or constant at a very low (<1%) prevalence.34,35
Sites of insertion of nim gene-activating IS elements and aspects of the coincidence of nim and cfiA genes
In further experiments, the association between the nim gene type and each studied IS element was addressed by testing for insertion events. The sequences of upstream fragments of six nimA and two nimC genes were determined after a forward (nimAm and nimCm) and a reverse (NIM-5) primer (Table 3) amplification, and the sequencing data from the IS612 mapping of B. fragilis 9750 were also included. If there had been independent insertion events, multiple insertion sites of the studied IS elements could have been expected. However, all IS elements studied displayed nim gene- and IS-specific insertion sites; IS1168 and nimA, IS1170 and nimC, and IS612 and nimB had insertion sites –19, –25 and –17 nt upstream of the start codons of the nim genes, irrespective of the plasmid type or a chromosomal location (Tables 1 and 2). These numbers also corresponded to those described for the nimA plasmid, pIP417, with IS1168,3 and nimC, the pIP419-like plasmid with IS1170.4 These findings may indicate a common emergence of nim gene IS element structures and argue against independent insertion events. This may indicate that IS insertions occur first and the nim gene IS configuration is dispersed between different replicons afterwards, or that specific nucleotide sequence of the upstream regions of nim genes allows only a few insertion sites. The length of the known conserved upstream region of the cfiA genes is 350 bp, but IS insertions occur only in the closest 100 nt of the cfiA gene.12 These Bacteroides IS elements, however, do not exhibit strict insertion specificity.2 The first possibility considered above is supported by the low rate of transposition of the Bacteroides IS elements, which was estimated to be around 10–8–10–7,13 as compared with the higher conjugation frequency of 10–4 per donor cell for pIP417.9
It has been observed that cfiA genes are quite frequent among Bacteroides strains that carry nim genes. To examine the relationship between the cfiA subgroup and the spread of nim gene-carrying elements, the localization data on the studied nim genes was compared with the cfiA status of these Bacteroides strains. Chi-squared analysis was used to test the hypothesis that the frequency of chromosomal nim genes is higher in strains with cfiA genes. B. fragilis 388/1 had chromosomal insertions of the nimE fragment (Figure 2c), and it was therefore included in the chromosomal group. Fisher's exact test was used to calculate the significance; in this way the prevalence of strains with nim genes on plasmids and with cfiA genes was found to be 1/14 (7.1%), while that of nim genes on chromosomes and with cfiA genes was 6/12 (50.0%); the difference is significant at a 95% confidence level (P = 0.026). Above this significant difference this result implies that this two groups of B. fragilis strains may differently regulate the localization of nim genes.
Bacteroides strains that are cfiA-positive are known to form a subgroup of the B. fragilis population which display different DNA–DNA homology rates,36 and can also be distinguished by molecular typing methods.14–17 Furthermore, the enterotoxin (bft) and the normal cephalosporinase (cep) genes have been reported to occur differently in the cfiA-positive and -negative groups.16,17 The differences at genome and gene levels may indicate that the two groups differentially regulate certain genetic processes. Haggoud et al. suspected that nim gene fragments from plasmids could be inserted into the chromosome in strains with chromosomal nim genes. Similarly, we presume that during horizontal spreading, the nim gene element segments may became integrative under non-permissive conditions, i.e. in the cfiA-positive strains. Moreover, the highly mobilizable phenotype of nim genes is also evidenced by the fact that nim genes have been found in other anaerobic bacteria,29,37,38 and the integrative characteristics of nim genes in species other than Bacteroides have been revealed by plasmid isolation studies of such strains.37 The specificity of activating IS elements for each nim gene type indicates that they may be transferred from strain to strain with higher mobility than that with which the insertion of IS elements occurs into the upstream positions of nim genes as mentioned previously.
Nevertheless, the conclusions of this study are that common nim gene elements such as pIP417, pIP419, pIP421 and pBF388c-like plasmids and chromosomal nimB gene elements are frequent causative agents of the metronidazole resistance of Bacteroides spp. but with interchangeability of the constituting genetic segments, with a highly constant association of each nim gene type and specific IS element.
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None to declare.
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Acknowledgements |
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This study was supported by a Royal Society—Hungarian Academy of Sciences Exchange Fellowship, a Hungarian National Research Foundation (OTKA T037475) grant and a European Society for Clinical Microbiology and Infectious Diseases Research Award to J. S.
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References |
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