Corinne Mory

H Teyssier

J P Hallage

G Carlier

A Grollier

Lozniewski

C Alauzet

F Mory

C Teyssier

H Hallage

J P Carlier

G Grollier

A Lozniewski
email: a.lozniewski@chu-nancy.fr.

Metronidazole Resistance in Prevotella spp. and Description of a New nim Gene in Prevotella baroniae ᰔ

Metronidazole Resistance in Prevotella spp.

The genus Prevotella includes strictly anaerobic, gram-negative, moderately saccharolytic, bile-sensitive rods formerly belonging to the genus Bacteroides [START_REF] Shah | Prevotella, a new genus to include Bacteroides melaninogenicus and related species formerly classified in the genus Bacteroides[END_REF]. These bacteria, which are part of the human oral, intestinal, and urogenital floras, may be involved in various infections, including infections of the head and neck, respiratory tract, central nervous system, and abdominal and urogenital tracts, as well as bacteremia [START_REF] Jousimies-Somer | Wadsworth-KTL anaerobic bacteriology manual[END_REF]. Metronidazole is commonly used for the treatment of infections caused by anaerobic organisms. For a long time, it has been considered that acquired resistance to this antibiotic is rare among anaerobes, despite its extensive use. However, recent studies have shown that this resistance is no longer uncommon among these organisms [START_REF] Behra-Miellet | Evaluation of the in vitro activity of ertapenem and nine other comparator agents against 337 anaerobic bacteria[END_REF][START_REF] Hecht | Prevalence of antibiotic resistance in anaerobic bacteria: worrisome developments[END_REF][START_REF] Jamal | Molecular characterization of nitroimidazole resistance in metronidazole-resistant Bacteroides species isolated from hospital patients in Kuwait[END_REF][START_REF] Lo ¨fmark | Inducible metronidazole resistance and nim genes in clinical Bacteroides fragilis group isolates[END_REF][START_REF] Lubbe | Prevalence of nim genes in anaerobic/facultative anaerobic bacteria isolated in South Africa[END_REF][START_REF] Papaparaskevas | Moxifloxacin resistance is prevalent among Bacteroides and Prevotella species in Greece[END_REF][START_REF] Theron | Nitroimidazole resistance genes (nimB) in anaerobic gram-positive cocci (previously Peptostreptococcus spp.)[END_REF]. Reduced susceptibility to 5-nitroimidazole drugs is generally associated with the presence of a nitroimidazole reductase encoded by a nim gene. This enzyme converts 4-or 5-nitroimidazole to 4-or 5-aminoimidazole, thus avoiding the formation of the toxic nitroso radicals that are essential for antimicrobial activity [START_REF] Carlier | Metabolism of a 5-nitroimidazole in susceptible and resistant isogenic strains of Bacteroides fragilis[END_REF]. Currently, seven nim genes, named nimA to nimG, which are either plasmid or chromosomally encoded, have been described (30), while a new nim gene, nimH (GenBank accession number FJ969397), has been described in Bacteroides fragilis.

Prevotella spp. have only rarely been investigated for the presence of nim genes in studies concerning metronidazole susceptibility [START_REF] Katsandri | Dissemination of nim-class genes, encoding nitroimidazole resistance, among different species of gram-negative anaerobic bacteria isolated in Athens, Greece[END_REF][START_REF] Lubbe | Prevalence of nim genes in anaerobic/facultative anaerobic bacteria isolated in South Africa[END_REF][START_REF] Papaparaskevas | Multicentre survey of the in-vitro activity of seven antimicrobial agents, including ertapenem, against recently isolated gram-negative anaerobic bacteria in Greece[END_REF]. This led us to investigate a large panel of clinical Prevotella strains that belong to different species and that were isolated from patients hospitalized in two French tertiary-care teaching hospitals for the presence of nim genes and for the type of nim genes that they carry.

MATERIALS AND METHODS

Bacterial isolates, identification, and culture. One hundred eighty-eight nonduplicate Prevotella isolates recovered from patients hospitalized between 2003 and 2006 in two French tertiary-care teaching hospitals (university hospital center of Nancy, n ϭ 157; university hospital center of Poitiers, n ϭ 31) were investigated. These strains were isolated from patients with various clinically significant infections (head and neck, n ϭ 49; skin and soft tissues, n ϭ 36; intra-abdominal, n ϭ 30; pleuropulmonary, n ϭ 25; urogenital, n ϭ 20; osteoarticular, n ϭ 13; bacteremia, n ϭ 11; other, n ϭ 4). Isolates were identified by phenotypic methods as well as by 16S rRNA gene sequence analysis, if necessary (Prevotella bivia, n ϭ 41; Prevotella buccae, n ϭ 35; Prevotella denticola, n ϭ 28; Prevotella melaninogenica, n ϭ 17; Prevotella disiens, n ϭ 10; Prevotella nanceiensis, n ϭ 10; Prevotella oris, n ϭ 9; Prevotella baroniae, n ϭ 7; Prevotella nigrescens, n ϭ 5; Prevotella oralis, n ϭ 5; Prevotella spp., n ϭ 5; Prevotella loescheii, n ϭ 3; Prevotella salivae, n ϭ 3; Prevotella veroralis, n ϭ 3; Prevotella bergensis, n ϭ 2; Prevotella intermedia, n ϭ 2; Prevotella corporis, n ϭ 1; Prevotella heparinolytica, n ϭ 1; Prevotella multiformis, n ϭ 1) [START_REF] Alauzet | Prevotella nanceiensis sp. nov., isolated from human clinical samples[END_REF][START_REF] Holdeman | Anaerobe laboratory manual[END_REF][START_REF] Jousimies-Somer | Wadsworth-KTL anaerobic bacteriology manual[END_REF]. The strains were stored in brucella broth containing 15% (wt/vol) glycerol at Ϫ80°C prior to the assays. For all experiments, the strains were grown at 37°C on brucella agar supplemented with 5% sheep blood, hemin, and vitamin K 1 (BBA) under anaerobic conditions.

Metronidazole susceptibility testing. The MICs of metronidazole were determined on BBA by the agar dilution method, according to CLSI standards (document M11-A7) [START_REF]Methods for antimicrobial susceptibility testing of anaerobic bacteria[END_REF], and by the Etest method (AB Biodisk, Solna, Sweden), according to the manufacturer's instructions. Readings were performed after incubation for 48 h at 35°C under anaerobic conditions. The MIC results were interpreted in accordance with the guidelines of the European Committee on Antimicrobial Susceptibility Testing (susceptibility, MIC Յ 4 g/ml; resistance, MIC Ն 8 g/ml) [START_REF]Clinical breakpoints and epidemiological cut-off values: metronidazole-EUCAST clinical MIC breakpoints 2008-06-19[END_REF]. Bacteroides fragilis ATCC 25285 T and Bacteroides thetaiotaomicron ATCC 29741 were included as controls. As it has previously been shown that metronidazole resistance may be detected only after prolonged in-cubation [START_REF] Dublanchet | Bacteroides de sensibilite ´re ´duite au me ´tronidazole[END_REF][START_REF] Gal | Metronidazole resistance in Bacteroides spp. carrying nim genes and the selection of slow-growing metronidazole-resistant mutants[END_REF][START_REF] Mory | Bacteremia caused by a metronidazole-resistant Prevotella sp. strain[END_REF], a second reading of all tests was performed after an additional 72 h. All assays were performed in duplicate.

Detection of nim genes and IS elements. DNA was extracted by using a QIAamp DNA minikit (Qiagen Inc., Hilden, Germany). The strains were investigated for the presence of nim genes by PCR with universal primers NIM-3 and NIM-5, as described previously [START_REF] Mory | Bacteremia caused by a metronidazole-resistant Prevotella sp. strain[END_REF][START_REF] Trinh | Detection by PCR of the nim genes encoding 5-nitroimidazole resistance in Bacteroides spp[END_REF]. For the insertion sequence (IS) elementspecific PCR amplifications, previously published primers were used [START_REF] Haggoud | Nucleotide sequence analysis of two 5-nitroimidazole resistance determinants from Bacteroides strains and of a new insertion sequence upstream of the two genes[END_REF][START_REF] So ´ki | Molecular investigation of genetic elements contributing to metronidazole resistance in Bacteroides strains[END_REF][START_REF] Trinh | Plasmids pIP419 and pIP421 from Bacteroides: 5-nitroimidazole resistance genes and their upstream insertion sequence elements[END_REF][START_REF] Trinh | Detection by PCR of the nim genes encoding 5-nitroimidazole resistance in Bacteroides spp[END_REF]. By using the forward primer specific to the amplified IS element and the reverse NIM-5 primer, further PCR experiments were performed to determine whether the IS was upstream of the nim gene, as described by So ´ki et al. [START_REF] So ´ki | Molecular characterization of imipenem-resistant, cfiA-positive Bacteroides fragilis isolates from the USA, Hungary and Kuwait[END_REF][START_REF] So ´ki | Molecular investigation of genetic elements contributing to metronidazole resistance in Bacteroides strains[END_REF]. The following positive control strains containing nim genes and IS elements were used: B. fragilis 638R(pIP417) (nimA, IS1168), B. fragilis BF8 (nimB, IS1168), B. thetaiotaomicron BT13(pIP419) (nimC, IS1170), B. fragilis 638R(pIP421) (nimD, IS1169), B. fragilis BF388(pBF388c) (nimE, ISBf6), and B. fragilis BF6712 (IS612). B. fragilis ATCC 25285 T was included as a nim-negative control. The IS element and nim gene PCR products were sequenced by using an ABI Prism BigDye Terminator sequencing kit on an ABI Prism 3100 automated sequencer (Applied Biosystems, Les Ulis, France). The nucleotide sequences were analyzed by using SeqScape software (version 2.5; Applied Biosystems). The sequences were compared to those deposited in the GenBank database by using the BLAST program [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF]. The sequences of the Nim proteins were determined by using the TRANSLATE program of the ExPASy proteomics server (www.expasy.org). The protein sequences were analyzed and aligned by using the BioEdit sequence alignment editor program (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). A phylogenetic dendrogram was generated by the neighbor-joining method with MEGA software, version 2.1 [START_REF] Kumar | MEGA2: molecular evolutionary genetics analysis software[END_REF].

Localization of nim genes. Plasmid extraction and Southern hybridization of nim-positive strains were used to determine whether the nim genes were present in either plasmid or chromosomal DNA, as described previously [START_REF] Gal | Metronidazole resistance in Bacteroides spp. carrying nim genes and the selection of slow-growing metronidazole-resistant mutants[END_REF]. Briefly, plasmid DNA was extracted with a plasmid minipreparation kit (Qiagen). Uncut plasmid DNA and EcoRI-digested chromosomal DNA were run on electrophoresis gels and then transferred by capillarity to nylon membranes (Hybond-Nϩ; Amersham Biosciences). Nim-specific digoxigenin-labeled probes were obtained by PCR, as described above, with primer pair NIM-3 and NIM-5 with a deoxynucleoside triphosphate mixture containing 0.1 mM digoxigenin-dUTP (Roche Diagnostics). The hybridization of the probes was detected with a CSPD chemiluminescence system (Roche Diagnostics). B. fragilis 638R(pIP417) and B. fragilis BF8 were used as positive and negative controls for plasmid and chromosomal localization, respectively.

Selection and stability of metronidazole resistance. Eleven clinical isolates (five nim-positive strains and six nim-negative strains), as well as Prevotella baroniae DSM 16972 T and B. fragilis 25285 T , were used to test for the induction of metronidazole resistance. For this purpose, bacterial suspensions were inoculated on BBA plates containing metronidazole at increasing concentrations, as previously described by Schaumann et al. [START_REF] Schaumann | Inducible metronidazole resistance in nim-positive and nim-negative Bacteroides fragilis group strains after several passages on metronidazole containing Columbia agar plates[END_REF]. The susceptibility of colonies growing in the presence of the highest concentration was determined by the agar dilution method. The stability of the resistance was evaluated by determining the MIC of metronidazole after three consecutive subcultures on drug-free BBA. As described by Pela ´ez et al. [START_REF] Pela ´ez | Metronidazole resistance in Clostridium difficile is heterogeneous[END_REF], we considered that the resistance was stable when the MIC of metronidazole for Prevotella spp. was maintained (within Ϯ1 dilution) after the passages. The experiments were performed in duplicate.

Nucleotide sequence accession numbers. The 16S rRNA and nim gene sequences of the P. baroniae isolates have been deposited in the GenBank database under accession numbers FJ940875 to FJ940882 and FJ940883 to FJ940890, respectively.

RESULTS

Of the 188 clinical isolates tested, 3 were found to be resistant to metronidazole either by the agar dilution method (isolates LBN 464 and LBN 467) or by the Etest method (isolate LBN 298) after 48 h of incubation (Table 1). For 30 isolates, slowly growing colonies were visualized inside the inhibition zone of the Etest strip after prolonged incubation. Colonies from within the inhibition zone were subcultured without antibiotic and were retested by the agar dilution method to determine whether the MIC had changed from its original value. These subpopulations showed MICs ranging from 8 to 32 g/ml.

PCR amplification with universal primers targeting the nim genes yielded products of about 460 bp for seven clinical isolates. All of these strains were genotypically identified as P. baroniae. The same nim gene was amplified from P. baroniae DSM 16972 T . All P. baroniae strains tested were considered susceptible to metronidazole by the agar dilution method and the Etest method (Table 1). The distance matrix constructed by using the Similarity table program of the PHYLIP package [START_REF] Felsenstein | PHYLIP (phylogeny inference package), version 3.5c. Distributed by the author[END_REF] showed that the P. baroniae strains shared from 97.1 to 100% of their nim gene nucleotide positions. The nim gene detected in the P. baroniae type strain (GenBank accession no. FJ940883) showed the highest degree of identity with nimH (63.8%), followed by nimF (63.2%), nimD (62.8%), nimG (62.5%), nimC (62.1%), nimE (61.7%), nimA (60.1%), and nimB (59.7%). This potentially new nim gene was named nimI. The predicted amino acid sequence alignment of NimI with homologs from other anaerobic bacteria showed that NimI formed a homogeneous group distinct from the other Nim types, with which NimI exhibited 55.9 to 64.4% identity. Insertion elements that have been associated with nim genes (IS1168, IS1169, IS1170, ISBf6, and IS612) [START_REF] So ´ki | Molecular investigation of genetic elements contributing to metronidazole resistance in Bacteroides strains[END_REF][START_REF] Trinh | Plasmids pIP419 and pIP421 from Bacteroides: 5-nitroimidazole resistance genes and their upstream insertion sequence elements[END_REF] were not found in any of the nimI-positive strains except the type strain, in which IS1168 was detected. However, PCR mapping and sequencing showed that this sequence was not localized upstream of the nimI gene.

No plasmid was found in any of the P. baroniae isolates tested or in B. fragilis BF8, whereas a nimA-carrying plasmid of about 7 to 8 kb was detected in B. fragilis 638R(pIP417), as expected [START_REF] Haggoud | Nucleotide sequence analysis of two 5-nitroimidazole resistance determinants from Bacteroides strains and of a new insertion sequence upstream of the two genes[END_REF]. To determine whether the nimI gene was present in chromosomal DNA, hybridizations were performed with nimA, nimB, and nimI gene probes on chromosomal blots with EcoRI-digested DNA. No hybridization with any of the strains tested with the nimA gene probe was found. A positive hybridization was observed for B. fragilis BF8 with the nimB gene probe and for all P. baroniae strains with the nimI gene probe. In all cases, only one copy of the nimI gene was observed.

Experiments performed to detect whether metronidazoleresistant subpopulations could be induced or selected from nim-positive (n ϭ 6) or nim-negative (n ϭ 6) Prevotella strains showed that all isolates exhibited significantly enhanced MICs (8ϫ to 256ϫ the original MIC) after several passages on plates containing increasing concentrations of metronidazole (Table 2). The induced resistance was stable in all strains after subculture in the absence of metronidazole and also after storage and freezing. For the nimI-positive strains, no differences in the nim gene sequences were found in strains that converted from metronidazole susceptibility to metronidazole resistance.

DISCUSSION

In the few studies examining the distribution of nim genes in Prevotella spp. [START_REF] Katsandri | Dissemination of nim-class genes, encoding nitroimidazole resistance, among different species of gram-negative anaerobic bacteria isolated in Athens, Greece[END_REF][START_REF] Lubbe | Prevalence of nim genes in anaerobic/facultative anaerobic bacteria isolated in South Africa[END_REF][START_REF] Papaparaskevas | Multicentre survey of the in-vitro activity of seven antimicrobial agents, including ertapenem, against recently isolated gram-negative anaerobic bacteria in Greece[END_REF], nim-positive Prevotella strains have rarely been found. Lubbe et al. [START_REF] Lubbe | Prevalence of nim genes in anaerobic/facultative anaerobic bacteria isolated in South Africa[END_REF] detected one nimA-positive strain (Prevotella bivia) among seven strains screened, while Katsandri et al. [START_REF] Katsandri | Dissemination of nim-class genes, encoding nitroimidazole resistance, among different species of gram-negative anaerobic bacteria isolated in Athens, Greece[END_REF] recovered one nimC-positive strain (Prevotella oralis) and two nimE-positive isolates (P. oralis, Prevotella buccalis) from among 57 isolates tested. In our study, no nim gene was amplified from any of the 30 isolates exhibiting reduced susceptibility to metronidazole after 48 h of incubation and/or isolates that presented slowly growing resistant subpopulations. This suggests that other potential resistance mechanisms may exist for these isolates, such as decreased pyruvate:ferredoxin oxidoreductase activity, overexpression of efflux pumps, or alterations of the rhamnose catabolism pathway [START_REF] Diniz | Differential gene expression in a Bacteroides fragilis metronidazoleresistant mutant[END_REF][START_REF] Edwards | Nitroimidazole drugs-action and resistance mechanisms. II. Mechanisms of resistance[END_REF][START_REF] Patel | Overexpression of the rhamnose catabolism regulatory protein, RhaR: a novel mechanism for metronidazole resistance in Bacteroides thetaiotaomicron[END_REF][START_REF] Pela ´ez | Metronidazole resistance in Clostridium difficile is heterogeneous[END_REF][START_REF] Pumbwe | Efflux pump overexpression in multiple-antibiotic-resistant mutants of Bacteroides fragilis[END_REF]. However, a potentially novel nim gene, nimI, was present and silent in 7 of the 158 remaining clinical isolates, for which neither increased initial MICs nor slowly growing resistant populations were observed. For Bacteroides spp., it has been reported that strains harboring silent nim genes may become resistant to metronidazole after prolonged exposure to this antibiotic [START_REF] Gal | Metronidazole resistance in Bacteroides spp. carrying nim genes and the selection of slow-growing metronidazole-resistant mutants[END_REF][START_REF] Lo ¨fmark | Inducible metronidazole resistance and nim genes in clinical Bacteroides fragilis group isolates[END_REF] and that this phenomenon may be a consequence of the activation of the nim gene as a result of point mutations, the insertion of an IS element promoter immediately upstream of the gene, or the formation of a new promoter following insertion [START_REF] Gal | Metronidazole resistance in Bacteroides spp. carrying nim genes and the selection of slow-growing metronidazole-resistant mutants[END_REF][START_REF] Trinh | Plasmids pIP419 and pIP421 from Bacteroides: 5-nitroimidazole resistance genes and their upstream insertion sequence elements[END_REF]. In the present study, metronidazole resistance could be selected after prolonged in vitro exposure to this drug in all nim-positive Prevotella strains tested. The possibility that mutation within the nimI gene might have led to the conversion from silent to constitutive expression was ruled out by nim gene sequencing in the pre-and postinduction states. However, none of the known IS elements that have been associated with nim genes were detected upstream of nimI. These findings are in accordance with those of previous studies that have reported that nim-positive strains may be metronidazole resistant in the absence of known activating IS elements [START_REF] Gal | Metronidazole resistance in Bacteroides spp. carrying nim genes and the selection of slow-growing metronidazole-resistant mutants[END_REF][START_REF] Katsandri | Dissemination of nim-class genes, encoding nitroimidazole resistance, among different species of gram-negative anaerobic bacteria isolated in Athens, Greece[END_REF][START_REF] Lo ¨fmark | Inducible metronidazole resistance and nim genes in clinical Bacteroides fragilis group isolates[END_REF]. This suggests that, as for other nim genes, the expression of nimI may be modulated by mechanisms other than those related to the presence of IS elements, although it cannot be ruled out that new IS elements that are not recognized by the primers used may be involved.

In Bacteroides spp., it has been shown that exposure to metronidazole can select for resistant subpopulations exhibiting either a nonstable phenotype that reverted to susceptibility during growth in the absence of the antibiotic or a constitutive phenotype that remained resistant after removal of the drug [START_REF] Gal | Metronidazole resistance in Bacteroides spp. carrying nim genes and the selection of slow-growing metronidazole-resistant mutants[END_REF][START_REF] Lo ¨fmark | Inducible metronidazole resistance and nim genes in clinical Bacteroides fragilis group isolates[END_REF][START_REF] Schaumann | Inducible metronidazole resistance in nim-positive and nim-negative Bacteroides fragilis group strains after several passages on metronidazole containing Columbia agar plates[END_REF]. In the present study, metronidazole resistance could be selected and remained stable in both the nim-positive and the nim-negative Prevotella strains tested, as well as in B. fragilis ATCC 25285 T . Other authors have also shown that metronidazole-resistant mutants may be selected from nimnegative Bacteroides sp. isolates, including the B. fragilis type strain [START_REF] Diniz | Differential gene expression in a Bacteroides fragilis metronidazoleresistant mutant[END_REF][START_REF] Schaumann | Inducible metronidazole resistance in nim-positive and nim-negative Bacteroides fragilis group strains after several passages on metronidazole containing Columbia agar plates[END_REF]. In contrast, Gal and Brazier (14) as well as Lo ¨fmark et al. [START_REF] Lo ¨fmark | Inducible metronidazole resistance and nim genes in clinical Bacteroides fragilis group isolates[END_REF] did not observe any selection of resistant mutants when nim-negative Bacteroides isolates were tested. Possible reasons for the discrepancies between the findings of those studies can be attributed, at least partially, to the use of different protocols for the selection of metronidazole-resistant subpopulations. It has been reported that the selection of microbial subpopulations that are resistant to the therapy administered may lead to the failure of treatment for infections caused by different bacterial species, including Prevotella spp. [START_REF] Falagas | Heteroresistance: a concern of increasing clinical significance?[END_REF][START_REF] Moore | Vancomycin treatment failure associated with heterogeneous vancomycin-intermediate Staphylococcus aureus in a patient with endocarditis and in the rabbit model of endocarditis[END_REF][START_REF] Mory | Bacteremia caused by a metronidazole-resistant Prevotella sp. strain[END_REF][START_REF] Pela ´ez | Metronidazole resistance in Clostridium difficile is heterogeneous[END_REF]. Furthermore, Diniz et al. have also shown that the in vivo selection of resistant Bacteroides isolates by low doses of metronidazole may lead to enhanced pathogenicity [START_REF] Diniz | Enhanced pathogenicity of susceptible strains of the Bacteroides fragilis group subjected to low doses of metronidazole[END_REF]. In the present study, the review of the charts of patients with P. baroniae infections revealed that treatment failure was not observed in the only patient who was treated with metronidazole. However, no conclusion can be drawn from this observation since that patient also received another active antibiotic. The failure of metronidazole treatment was observed in two patients infected with nim-negative Prevotella isolates, isolates LBN 293b (Prevotella nanceiensis) and LBN 465 (P. buccae), both of which were initially considered susceptible to metronidazole. High-level metronidazole resistance could be induced in both strains, while slowly growing subpopulations within the inhibition zone of the Etest strip were observed only for strain LBN 293b. That strain was isolated from blood cultures (25), while strain LBN 465 was recovered from pancreatic necrotic tissue in a patient with acute pancreatitis. For that patient, who remained febrile during treatment with metronidazole and who was operated on again, a metronidazole-resistant P. buccae isolate (MIC, 32 g/ml) was obtained from a surgical drainage sample. That strain exhibited the same pulsed-field gel electrophoresis pattern as strain LBN 465 after DNA digestion was performed by using XbaI (data not shown). The patient became apyretic after treatment with metronidazole was changed to treatment with imipenem. Thus, the in vivo selection of a resistant subpopulation, which was responsible for treatment failure, was demonstrated and corroborated the previous observation that the initial isolate became resistant after prolonged in vitro exposure to metronidazole. It is noteworthy that nimI was found in all P. baroniae isolates tested, including the type strain originating from the United Kingdom (8) and clinical isolates obtained in two geographically distant sites in France. NimI was also recently detected in two other P. baroniae clinical strains isolated in our laboratory, whereas this gene was not detected in 33 type strains belonging to other Prevotella species. Moreover, a phylogenetic tree based on Nim amino acid sequences showed that NimI formed a new homogeneous group distant from the other Nim types involved in metronidazole resistance in anaerobic bacteria (data not shown). These results, associated with the fact that nimI had a chromosomal localization, suggest that this gene might be intrinsic in P. baroniae. However, further studies with a larger number of strains are needed to confirm this hypothesis. It is interesting to note that 16S rRNA gene sequencing was necessary to unambiguously identify all P. baroniae strains, suggesting that the clinical implication of this species might be underestimated, since molecular identification is not widely used in routine clinical laboratories.

In conclusion, this study led to the description of a new nim gene which seems to be intrinsic to the species P. baroniae. This finding underscores the importance of a correct identification at the species level, at least for isolates responsible for severe infections. Moreover, we showed for the first time that subpopulations exhibiting high-level resistance to metronidazole can be selected from both silent nimI-positive and nim-nega-tive Prevotella sp. isolates after prolonged exposure to this antibiotic. These findings not only confirm that nim genes are not the only factors involved in the decreased susceptibility of Prevotella spp. to metronidazole but also emphasize the importance of careful susceptibility testing of anaerobes and the usefulness of 16S rRNA gene-based identification methods, especially in cases of severe infections or treatment failures.

TABLE 1 .

 1 MICs of metronidazole for nimI-positive strains and nim-negative strains exhibiting reduced susceptibility to metronidazole

			MIC determined by
	Strain a	Presence of nim gene	agar dilution (g/ml)
			Original	SGC b
	P. baroniae
	DSM 16972 T	ϩ	2	N A c
	LBN 427	ϩ	4	N A
	LBN 430	ϩ	4	N A
	LBN 432	ϩ	2	N A
	LBN 466	ϩ	2	N A
	LBN 475	ϩ	4	N A
	LBP 9	ϩ	2	N A
	LBP 19	ϩ	1	N A
	P. bivia
	LBN 330	Ϫ	2	1 6
	LBN 331	Ϫ	2	1 6
	LBN 332	Ϫ	4	3 2
	LBN 333	Ϫ	2	3 2
	LBN 334	Ϫ	2	1 6
	LBN 336	Ϫ	2	3 2
	LBN 339	Ϫ	2	1 6
	LBN 343	Ϫ	1	1 6
	LBN 346	Ϫ	1	1 6
	LBN 350	Ϫ	2	1 6
	LBN 361	Ϫ	2	3 2
	LBN 365	Ϫ	4	8
	LBN 371	Ϫ	4	3 2
	LBN 374	Ϫ	2	8
	LBN 445	Ϫ	0.5	16
	LBN 464	Ϫ	8	1 6
	LBN 467	Ϫ	16	32
	LBN 478	Ϫ	4	8
	LBP 4	Ϫ	2	3 2
	LBP 38	Ϫ	2	8
	LBP 51	Ϫ	2	3 2
	LBP 52	Ϫ	4	1 6
	LBP 65	Ϫ	4	3 2
	LBP 69	Ϫ	2	8
	LBP 78	Ϫ	4	8
	P. nanceiensis
	LBN 293b	Ϫ	2	3 2
	LBN 297	Ϫ	2	1 6
	LBN 298 d	Ϫ	4	3 2
	LBN 410	Ϫ	1	3 2
	LBP 66	Ϫ	2	8

a LBN, Laboratoire de Bactériologie de Nancy; LBP, Laboratoire de Bactériologie de Poitiers.

b SGC, slowly growing colonies within the inhibition zone of the Etest strip. c NA, not applicable (absence of slowly growing colonies).

d Strain with reduced susceptibility to metronidazole (MIC, 16 g/ml), as determined at 48 h by the Etest method.

TABLE 2 .

 2 Induction and stability of resistance to metronidazole in 11 clinical Prevotella isolates, P. baroniae DSM 16972 T , and B.

	62	ALAUZET ET AL.			ANTIMICROB. AGENTS CHEMOTHER.
		fragilis ATCC 25285 T
		Strain (nimI gene a )		MIC (g/ml)
			Original	Postinduction	Poststability
	P. baroniae DSM 16972 T (ϩ)	2	1 6	1 6
	P. baroniae LBN 427 (ϩ)	4	128	128
	P. baroniae LBN 430 (ϩ)	4	128	128
	P. baroniae LBN 432 (ϩ)	2	64	64
	P. baroniae LBN 475 (ϩ)	4	256	256
	P. baroniae LBP19 (ϩ)	1	128	128
	P. bivia LBN 332 (Ϫ)	4	Ͼ256	Ͼ256
	P. bivia LBN 371 (Ϫ)	4	Ͼ256	Ͼ256
	P. bivia LBN 467 (Ϫ)	1 6	Ͼ256	Ͼ256
	P. buccae LBN 465 (Ϫ)	0.5	128	64
	P. nanceiensis LBN 293b (Ϫ)	2	256	256
	P. nanceiensis LBN 410 (Ϫ)	1	256	256
	B. fragilis ATCC 25285 T (Ϫ)	0.5	256	128
	a ϩ, positive; Ϫ, negative.

ACKNOWLEDGMENTS

We are very grateful to J. So ´ki for kindly supplying the strains used as positive controls: B. thetaiotaomicron BT13 (nimC, IS1170), B. fragilis BF388 (nimE, ISBf6), and B. fragilis BF6712 (IS612). We thank I. Scholtus, M. R. Balland, C. Nicolas, and A. M. Carpentier for technical assistance.