Anaerobe Reference Laboratory, National Public Health Service Wales, Microbiology Cardiff, University Hospital of Wales, Cardiff CF14 4XW, UK
Received 30 January 2004; returned 23 March 2004; revised 19 April 2004; accepted 23 April 2004
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Abstract |
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Methods: Isolates were screened for susceptibility to a metronidazole disc and had their MIC determined by the Etest method. They were investigated for the presence of nim genes by PCR. An experiment to determine the effect of prolonged exposure to metronidazole was applied to nim-positive isolates with MICs below the therapeutic breakpoint.
Results: Fifty of 206 isolates (24%) were found to possess nim genes and these had MICs of metronidazole ranging from 1.5 to >256 mg/L with 24 (11.6%) above the therapeutic breakpoint of 16 mg/L. The remaining 26 nim-gene-positive isolates had MICs that were still below the therapeutic breakpoint, ranging from 1.5 to 6.0 mg/L. nim genes were not found in 156 (76%) isolates, and all but seven of these were susceptible to a 5 µg disc of metronidazole. Ten members of the group for which the MICs were below the therapeutic level were found to have slow-growing sub-populations with metronidazole MICs ranging from 8.0 to >256 mg/L that became evident after prolonged exposure to metronidazole in vitro. This resistance selection process was sometimes reversible after passage in the absence of metronidazole; however, seven of the 10 slow-growing mutants converted to stable high-level resistance (MIC >256 mg/L).
Conclusions: Although the presence of nim genes per se does not always equate to therapeutic resistance, and other metronidazole resistance mechanisms may exist, this study has shown that prolonged exposure of nim-gene-carrying Bacteroides spp. to metronidazole can select for therapeutic resistance.
Keywords: anaerobes , antibiotics , nitroimidazoles
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Introduction |
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However, the presence of a nim gene per se does not necessarily confer therapeutic metronidazole resistance as nim genes have been detected in Bacteroides spp. that have MICs below the therapeutic breakpoint of 16 mg/L and are therefore considered susceptible.13 In these cases the nim gene may not be expressed, or expressed only at very low levels. It may also be possible that a mutation in the nim gene or the insertion of an IS element immediately upstream of the nim gene might lead to the expression or upregulation of these previously silent nim genes, and thereby elevate the MIC to levels conveying resistance to therapy. Metronidazole-resistant B. fragilis isolates have been recovered from patients following long-term treatment with metronidazole.19,20
This study examined the distribution of nim genes within Bacteroides spp. that were referred to the ARL for identification, and examined the relationship between strains possessing nim genes and their susceptibility to metronidazole. It also set out to examine the possibility that metronidazole resistance might be selected for by exposure of some Bacteroides isolates to sub-lethal levels of metronidazole and investigated the role of IS elements in this process.
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Materials and methods |
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A total of 206 human clinical isolates identified as Bacteroides spp. submitted to the ARL for identification were selected. Isolates were identified to species level by a combination of conventional phenotypic tests21 and PCRrestriction fragment length polymorphism (RFLP).13
All isolates of Bacteroides spp. had been stored frozen at 80°C on beads (ProLab Diagnostics, Wirral, UK) and were cultured on Fastidious Anaerobe Agar (FAA; Lab M, Bury, UK) supplemented with 6% horse blood in an anaerobic atmosphere [10% CO2, 10% H2, 80% N2 (v/v)] at 37°C. B. fragilis NCTC 9343 was included as a control organism.
The isolates were identified as: B. fragilis (n=142), Bacteroides thetaiotaomicron (n=27), Bacteroides vulgatus (n=11), Bacteroides ovatus (n=7), Bacteroides distasonis (n=6), Bacteroides caccae (n=5), Bacteroides splanchnicus (n=5) and Bacteroides uniformis (n=3).
Determination of metronidazole susceptibility and MICs
Initial determination of the metronidazole susceptibility of each isolate was by a standard disc diffusion test on FAA with a 5 µg metronidazole disc (Mast Laboratories, UK). A zone size of 2830 mm diameter or larger was deemed susceptible, as this is the size of the zone seen using the control strain B. fragilis NCTC 9343. Metronidazole MICs were determined for all nim-positive isolates and five known fully susceptible isolates by the Etest method (AB Biodisk, Solna, Sweden) according to the manufacturer's instruction on FAA supplemented with 6% horse blood at 37°C under anaerobic conditions. In both methods inhibition zones were compared with those of B. fragilis NCTC 9343.
Detection and analysis of nim genes and IS elements
Bacterial colonies were harvested from FAA plates after overnight anaerobic incubation. Template nucleic acid was prepared by suspending a 1 µL loopful of bacterial cells in Chelex-100 resin (Bio-Rad Laboratories, USA) 0.05% in sterile distilled water, boiling for 12 min and centrifuging at 17 000 g for 10 min. The supernatant was decanted and used in PCRs.
PCR amplification was used to screen for the presence of nim genes, and the identity of amplicons confirmed by PCRRFLP with the restriction enzymes HpaII and TaqI as described previously.13 Whenever required, the identification of the nim gene was confirmed by sequence analysis.
The regions upstream of nim were amplified with internal primers homologous to IS1168, IS1169, IS1170 and nim.5,10,2224 For IS element-specific PCR amplifications previously published primers specific for IS1168, IS1169 and IS1170 were used.5,18,23,25 PCR products were visualized and their sizes were estimated, and they were purified with the QIAquick PCR Purification Kit (Qiagen, Crawley, UK). The PCR products were sequenced and the nucleotide sequences were compared with sequences in the GenBank non-redundant nucleotide sequence database at the US National Center for Biotechnology Information (NCBI) by the gapped-basic local alignment search tool (BLAST-N).26 Southern hybridization was performed to determine the presence of nim genes in either plasmid or chromosomal DNA in those metronidazole-resistant strains from which no nim genes could be amplified by PCR. Briefly, genomic and plasmid DNA were extracted separately from these and control strains. Uncut plasmid DNA and EcoRI-digested chromosomal DNA was run on electrophoresis gels. The gels were denatured in 1.5 M NaCl2, 0.5 M NaOH and neutralized in 0.5 M TrisHCl pH 7.4, 0.5 M NaOH. The DNA fragments were transferred to a nylon membrane (Hybond-N+; Amersham International) and covalently linked by heating. The pre-hybridization, hybridization and detection steps were performed with a Gene Images Random Prime Labelling and Detection system according to the protocol recommended by the manufacturer (Amersham Pharmacia UK Ltd). The pre-hybridization and hybridization steps were performed at 60°C. Probes were the nimA and nimB products amplified from control strains using primers described previously.
Positive control strains containing nim genes included B. fragilis BF8 (nimB), B. fragilis 638R(pIP417) (nimA), B. fragilis 638R(pIP419) (nimC), B. fragilis 638R(pIP421) (nimD) and B. fragilis ARU6881 (nimE).5 B. fragilis NCTC 9343 was included as a nim-negative and metronidazole-susceptible control.
To determine whether insertion of IS elements or mutations in IS elements or nim genes were involved in conversion of strains from metronidazole susceptibility to metronidazole resistance, IS elements, nim upstream regions and nim genes were amplified and sequenced in strains pre-induction and post-induction as described above.14,18,25
Selection of metronidazole-resistant sub-populations by disc diffusion
A previously described disc diffusion method for selection or induction of clindamycin resistance was adapted as follows to detect whether metronidazole-resistant sub-populations existed.27 Briefly, a sterile 15 x 50 mm strip of 0.45 µm pore size membrane filter (Millipore, USA) was placed diametrically on the surface of an FAA plate supplemented with 6% horse blood. The total surface of the plate and strip was inoculated with an overnight culture of the isolate of Bacteroides under test by means of a swab. A 5 µg metronidazole disc was then placed in the middle of the strip and the plate was incubated anaerobically for a total of 14 days. Every 24 h (weekends not included) the filter strip was transferred to a fresh FAA plate, and the metronidazole disc was removed and replaced with a new one. Under these circumstances the continuous presence of active antibiotic was ensured. At weekly intervals, any slow-growing colonies that appeared within the initial zone of susceptibility were subcultured and checked for identity and re-tested by Etest to determine whether the MIC had changed from its original value.
To verify that any increased resistance to metronidazole was not due to subculturing procedures, a series of subcultures of the initial isolates was performed on FAA without antibiotic for a period of 10 days, with the MIC being determined after this time.
Stability of resistance
The stability of the metronidazole resistance of slow-growing isolates was evaluated by performing seven serial passages over 14 days on FAA without antibiotic and the MIC was re-determined.
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Results |
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All 57 isolates that were either nim-positive or resistant to metronidazole were identified by both phenotypic tests and 16S rDNA PCRRFLP (Table 1). The identity of pre- and post-induction strains was confirmed by the 16S rDNA PCRRFLP method.
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Of the 50 nim-positive Bacteroides isolates, 35 (70%) showed either a reduced zone of susceptibility (<2830 mm diameter) or no zone at all to a 5 µg metronidazole disc compared with the B. fragilis control strains and had metronidazole MICs ranging from 6 to >256 mg/L (Table 1). The remaining 15 isolates had a zone size similar to that of the metronidazole-susceptible control strain B. fragilis 9343 and had metronidazole MICs of 1.54.0 mg/L. Seven isolates from which nim genes were not amplified had no zone to a 5 µg metronidazole disc and had raised MICs (Table 1).
Detection and analysis of nim genes
PCR amplification for nim genes gave products of the published 500 bp size from 50 of the 206 isolates. By Southern blotting no hybridization products were detected for the seven nim-negative metronidazole-resistant isolates suggesting a mechanism other than a modified nim gene. PCR products from nim genes were identified by comparison of HpaII and TaqI restriction patterns with those from five previously described nim genes from control stains; 39 nim gene PCR products gave restriction profiles consistent with the control strains. However, restriction profiles of 11 products were inconsistent with those reported and were a mixture of restriction profiles for HpaII-nimC, and TaqI-nimA, representing novel patterns (Figure 1). Direct sequencing of the 10 PCR products indicated one sequence (R17004) with 88% DNA sequence similarity with nimB (Figure 2), seven sequences with 9899% similarity with nimA, two sharing 83% and 90% homology with nimD and one with 98% sequence identity with nimB (Table 1).
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Selection of metronidazole-resistant sub-populations by disc diffusion
Thirteen nim-positive Bacteroides isolates for which the MICs were only moderately raised (1.56.0 mg/L) plus eight nim-negative control strains were used in the disc diffusion study. These isolates showed a zone of inhibition ranging from 14 to 30 mm with a 5 µg metronidazole disc after incubation for 24 h. However, in the nim-positive isolates the diameter of the inhibition zone around the metronidazole disc became successively smaller over time. At 4872 h, small colonies began to appear inside the inhibition zone and became more numerous over the following 24 h. In some isolates the entire zone was covered by small colonies after 57 days of subculture. The slow-growing colonies from around the metronidazole disc were subcultured after incubation for 2 weeks and the MICs were established. These populations showed raised MICs ranging from 8 to >256 mg/L (Table 2). In seven isolates a sub-population of colonies appeared within the ellipse area of the Etest strip consistently after incubation for 4872 h. Isolated colonies within these sub-populations showed MICs ranging from 96 to >256 mg/L (Table 3). No decreased inhibition zone, raised MICs or secondary resistant colonies were found in a similar experiment using the control strain of B. fragilis NCTC 9343 or in seven further metronidazole-susceptible isolates not containing nim genes.
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Ten of the Bacteroides isolates included in the induction study were screened for both the presence of IS elements within the genome and whether they were positioned upstream of the nim genes. Using IS-specific PCR, the insertion sequences IS1168 and IS1170 were detected in the genome of five of the 10 isolates screened both pre- and post-induction (Table 3). However, no IS elements were amplified upstream of the nim genes in eight of the 10 isolates. IS1170 was amplified from the other two isolates R14031 and R15385 upstream of nimC in both pre- and post-induction strains. IS1168 and IS1170 sequences were amplified consistently from the control strains. Sequencing of 500 bp of the IS elements immediately upstream of the nim gene and therefore possible promoter regions in these two isolates, both pre- and post-induction, revealed no sequence differences.
Stability of resistance
After serial passage in the absence of metronidazole, a decrease in MIC was noted for all but one of those isolates with intermediate resistance (MIC 864 mg/L) (Table 2). In the phenotypes for which the metronidazole MIC was high (256 mg/L), this was found to be stable after subculture in the absence of metronidazole and also after storage and freezing.
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Discussion |
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Screening for the presence of nim genes in addition to routine disc testing revealed that 12/206 Bacteroides isolates submitted to the ARL contained nim genes that were silent or were expressed only at levels conveying metronidazole MIC values of 1.56 mg/L. The MIC for these isolates increased when cultured in the presence of sub-inhibitory doses of metronidazole for 2 weeks. Two populations for which metronidazole MICs were raised and which had different phenotypes became evident. First, a non-stable phenotype that reverted to metronidazole susceptibility during growth in the absence of metronidazole and, secondly, a mutant phenotype that remained resistant after removal of the drug. It appears that in some instances exposure to metronidazole can select for sub-populations capable of resistance, with occasional mutation from inducible to a constitutive mutant phenotype. Therefore it may be possible that mutations leading to expression of previously silent nim genes could occur in the clinical setting and lead to clinically relevant levels of metronidazole resistance, i.e. treatment failure.
The serial passage technique demonstrated that the selected resistance to metronidazole was stable in those strains that had converted to high-level metronidazole resistance (MIC 96>256 mg/L) and the role of IS elements in this process needed evaluating. In Bacteroides spp., IS elements have been shown to be involved in modulating the expression of antibiotic resistance genes. For example, the transcription of the metronidazole resistance genes, nim, and the imipenem resistance gene, cfiA, is directed by outward-oriented promoters, carried on the ends of IS elements upstream of nim and cfiA.11,2931
The increase in metronidazole resistance following the prolonged exposure experiment could be a consequence of the activation of the nim gene as a result of point mutations, insertion of an IS element promoter into the upstream region of nim or the formation of a new promoter following insertion. It has been assumed that the transcription of the nim genes is directed by outward-oriented promoters, carried on the right ends of the insertion sequence elements. In our study however, no IS elements preceding the nim gene was amplified either pre- or post-induction with primers designed to amplify IS1168, IS1169 and IS1170, in five of the seven isolates examined. This could be due to the absence of IS elements or because the IS elements were not recognized by the primers used. Three of the isolates contained IS elements elsewhere in the chromosome, and two of the isolates contained the IS1170 element preceding the nimC gene. Sequencing of the 5' end of these IS elements in isolates post- and pre-induction confirmed that these were indistinguishable. The possibility that mutation and reversion within the nim gene itself was responsible for conversion from silent to constitutive expression was ruled out by sequencing of the nim genes pre- and post-induction.
Metronidazole resistance in Bacteroides isolates is routinely detected by a reduced or absent inhibition zone around a 5 µg metronidazole disc. However, 15 of the 50 isolates containing nim genes were initially found to have metronidazole inhibition zones quite similar in size to the metronidazole-susceptible control strain (B. fragilis NCTC 9343). Therefore, routine disc testing may allow strains with the potential to convert to metronidazole resistance to go undetected.
Resistance to metronidazole in anaerobic bacteria in the UK is not currently perceived as a clinical problem; however, metronidazole-resistant Bacteroides spp. are being regularly referred to the ARL and some of these isolates show multiple resistance to other commonly used antibiotics such as co-amoxiclav, erythromycin and tetracycline. It is possible that metronidazole resistance in other anaerobes is being under-reported as, routinely, in the UK it is often assumed that on primary culture colonies that are inhibited by a 5 µg metronidazole disc placed on the streaked-out inoculum are obligate anaerobes and any colonies within the inhibition zone are assumed to be facultative species.
The development of resistance to metronidazole leading to treatment failure during the course of metronidazole therapy in a patient has been reported9 and a recent metronidazole-resistant B. fragilis referred to the ARL mentioned failure of the patient to respond to metronidazole therapy (unpublished data). Furthermore, Bacteroides isolates for which the metronidazole MIC is increased have been recovered from patients with Crohn's disease on long-term treatment with metronidazole.20 It has also been observed that metronidazole-resistant and -susceptible B. fragilis strains became more virulent following exposure to low doses of metronidazole.32,33 It is possible that exposure of Bacteroides spp. to sub-inhibitory concentrations of metronidazole could lead to increased pathogenicity and metronidazole resistance with the development of either non-stable phenotypes or permanently resistant mutants.
In conclusion, some nim-gene-positive Bacteroides spp. with low-level metronidazole resistance can be selected for in vitro and sub-populations of these may convert to stable high-level resistance. Therefore, given that there could be a correlation between the events occurring in vivo and in vitro, screening for these silent nim genes may need to become a more regular investigation. Since 2001, Bacteroides isolates referred to the ARL have been screened by PCR for nim genes in addition to metronidazole disc susceptibility testing. During this time period this regimen has increased the number of Bacteroides spp. detected with the potential for metronidazole resistance from 5% to >15%. This has implications for the continued surveillance of putative inducible resistance to this important agent within the genus Bacteroides and, ultimately, perhaps for clinical treatment.
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Acknowledgements |
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Footnotes |
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References |
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2
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Brazier, J. S., Stubbs, S. & Duerden, B. I. (1999). Metronidazole resistance among clinical isolates belonging to the Bacteroides fragilis group: time to be concerned? Journal of Antimicrobial Chemotherapy 44, 5801.
3 . Breuill, J., Burnat, C., Patey, O. et al. (1989). Survey of Bacteroides fragilis susceptibility patterns in France. Journal of Antimicrobial Chemotherapy 24, 6975.[Abstract]
4 . Chaudry, R., Mathur, P., Dhawan, B. et al. (2001). Emergence of metronidazole-resistant Bacteroides fragilis, India. Emerging Infectious Disease 7, 4856.[ISI]
5 . Haggoud, A., M'Hand, R. A., Reysset, G. et al. (2001). Prevalence and characteristics of nim genes encoding 5-nitroimidazole resistance among Bacteroides strains isolated in Morocco. Microbial Drug Resistance 7, 17781.[CrossRef][ISI][Medline]
6 . Lubbe, M. M., Stanley, K., Chalkley, L. J. et al. (1999). Prevalence of nim genes in anaerobic/facultative anaerobic bacteria isolated in South Africa. FEMS Microbiology Letters 172, 7983.[CrossRef][ISI][Medline]
7 . Merad, A. S., Ghemati, M., Faid, N. et al. (1998). Antibiotic sensitivity of Bacteroides fragilis group in Algeria. Archives de l'Institute Pasteur d'Algerie 62, 91110.
8 . Nagy, E., Soki, J., Urban, E. et al. (2001). Occurrence of metronidazole and imipenem resistance among Bacteroides fragilis group isolates in Hungary. Acta Biologica Hungarica 52, 27180.[ISI][Medline]
9 . Rotimi, V. O., Khourshed, J. S., Brazier, J. S. et al. (1999). Bacteroides species highly resistant to metronidazole: an emerging clinical problem? Clinical Microbiology and Infection 5, 1669.[Medline]
10
.
Teng, L., Hsue, P., Tsai, J. et al. (2002). High incidence of cefoxitin and clindamycin resistance among anaerobes in Taiwan. Antimicrobial Agents and Chemotherapy 46, 290813.
11 . Urban, E., Soki, J., Brazier, J. S. et al. (2002). Prevalence and characterization of nim genes of Bacteroides spp. isolated in Hungary. Anaerobe 8, 1759.[CrossRef][ISI]
12 . Wojcik-Stojek, B., Bulanda, M., Martirosian, M. et al. (2000). In vitro susceptibility of Bacteroides fragilis strains isolated from excised appendix of patients with phlegmonous or gangrenous appendicitis. Acta Microbiologica Polonica 44, 1715.
13
.
Stubbs, S. L. J., Brazier, J. S., Talbot, P. R. et al. (2000). PCR-restriction fragment length polymorphism analysis for identification of Bacteroides spp. and characterization of nitroimidazole resistance genes. Journal of Clinical Microbiology 38, 320913.
14 . Haggoud, A., Reysset, G., Azedoug, H. et al. (1994). Nucleotide sequence analysis of two 5-nitroimidazole resistance determinants from Bacteroides strains and of a new insertion sequence upstream of the two genes. Antimicrobial Agents and Chemotherapy 38, 104751.[Abstract]
15 . Reysset, G. (1996). Genetics of 5-nitroimidazole resistance in Bacteroides species. Anaerobe 2, 5969.[CrossRef][ISI]
16 . Reysset, G., Haggoud, A., Su, W. J. et al. (1992). Genetic and molecular analysis of pIP417 and pIP419: Bacteroides plasmids encoding 5-nitroimidazole resistance. Plasmid 27, 18190.[ISI][Medline]
17 . Carlier, J. P., Sellier, N., Ranger, M. N. et al. (1997). Metabolism of a 5-nitroimidazole in susceptible and resistant isogenic strains of Bacteroides fragilis. Antimicrobial Agents and Chemotherapy 41, 14959.[Abstract]
18 . Trinh, S., Haggoud, A., Reysset, G. et al. (1995). Plasmids pIP417 and pIP419 from Bacteroides: 5-nitroimidazole resistance genes and their upstream insertion sequence elements. Microbiology 141, 92735.[Abstract]
19 . Ingham, H. R., Eaton, S., Venables, C. W. et al. (1978). Bacteroides fragilis resistant to metronidazole after long-term therapy. Lancet 1, 214.
20 . Krook, A., Kjellander, J. & Danielsson, D. (1982). Susceptibility of Bacteroides species during treatment of patients with Crohn's disease and healthy individuals. Scandinavian Journal of Infectious Diseases 14, 458.[ISI][Medline]
21 . Holdeman, L. V., Cato, E. P. & Moore, W. E. C. (1977). Anaerobe Laboratory Manual, 4th edn. Virginia Polytechnic Institute and State University, Blacksburg, VA, USA.
22 . Podglajen, I., Breuil, J. & Collatz, E. (1994). Insertion of a novel DNA sequence, IS1186, upstream of the silent carbapenamase gene cfiA, promotes expression of carbapenamase resistance in clinical isolates of Bacteroides fragilis. Molecular Microbiology 12, 10514.[ISI][Medline]
23 . Podglajen, I., Breuil, J., Borden, F. et al. (1992). A silent carbapenamase gene in strains of Bacteroides fragilis can be expressed after a one-step mutation. FEMS Microbiology Letters 91, 2130.[CrossRef][ISI]
24 . Trinh, S. & Reysset, G. (1996). Detection by PCR of the nim genes encoding 5-nitroimidazole resistance in Bacteroides spp. Journal of Clinical Microbiology 34, 207884.[Abstract]
25 . Rasmussen, B. A. & Kovacs, E. (1991). Identification and DNA sequence of a new Bacteroides fragilis insertion sequence-like element. Plasmid 25, 1414.[ISI][Medline]
26
.
Altschul, S. F., Madden, T. L., Schäffer, A. A. et al. (1997). GappedBLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389402.
27 . Reig, M. & Baquero, F. (1989). Selection of constitutively resistant mutants of inducible clindamycin-resistant Bacteroides vulgatus. European Journal of Clinical Microbiology and Infectious Diseases 8, 7115.[ISI][Medline]
28 . Edwards, D. I. (1993). Nitroimidazole drugsaction and resistance mechanisms. 1. Mechanisms of action. Journal of Antimicrobial Chemotherapy 31, 920.[ISI][Medline]
29
.
Edwards, R., Hawkyard, C. V., Garvey, M. T. et al. (1999). Prevalence and degree of expression of the carbapenamase gene (cfiA) among clinical isolates of Bacteroides fragilis in Nottingham, UK. Journal of Antimicrobial Chemotherapy 43, 2736.
30
.
Edwards, R. & Read, P. N. (2000). Expression of the carbapenamase gene (cfiA) in Bacteroides fragilis. Journal of Antimicrobial Chemotherapy 46, 100912.
31
.
Podglajen, I., Breuil, J., Rohaut, A. et al. (2001). Multiple mobile promoter regions for the rare carbapenem resistance gene of Bacteroides fragilis. Journal of Bacteriology 183, 35315.
32 . Diniz, C. G., Arantes, R. M., Cara, D. C. et al. (2003). Enhanced pathogenicity of susceptible strains of the Bacteroides fragilis group subjected to low doses of metronidazole. Microbes and Infection 5, 1926.[CrossRef][ISI][Medline]
33
.
Diniz, C. G., Cara, D. C., Nicoli, J. R. et al. (2000). Effect of metronidazole on the pathogenicity of resistant Bacteroides strains in gnotobiotic mice. Antimicrobial Agents and Chemotherapy 44, 241923.