Detection by PCR of the nim genes encoding 5-nitroimidazole resistance in Bacteroides spp.

A PCR method was developed for detection of the nim genes encoding 5-nitrolmidazole resistance in Bacteroides spp. Two PCR primers specific for nim genes were designed. They allowed amplification of a 458-bp fragment from all characterized plasmid- and chromosome-borne metronidazole resistance genes. The specificity of the method was tested with DNA from metronidazole-sensitive Bacteroides spp. strains and from other strains of unrelated species. Each DNA preparation was analyzed with and without an internal positive control to verify that the absence of PCR amplification product was not due to inhibition of the Taq polymerase inhibitors. By this technique, two newly discovered metronidazole-resistant clinical strains of Bacteroides fragilis were shown to harbor resistance genes undetectable by Southern blotting. In spite of the sequence divergence of the nim genes, the PCR method is thus suitable for epidemiological investigations. The amplification method also revealed that nim-related resistance genes were not present in either Streptomyces strain S6670, a natural producer of 2-nitroimidazole, or in Enterococcus faecalis strains, which have been suggested to possess metronidazole-inactivating enzyme.     

Antimicrobial susceptibility of Bacteroides fragilis group isolates in Europe

Objective  To evaluate the activity of old and newer antianaerobic drugs against clinical isolates of Bacteroides fragilis group strains from different parts of Europe.
Methods  Bacteroides fragilis group isolates from 37 laboratories in 19 countries were biochemically characterized. The MICs of seven antimicrobial agents were determined by the agar dilution method as recommended by the NCCLS. Production of beta-lactamase was detected by nitrocefin.
Results  There were 1284 B. fragilis group isolates included in the study. Abdominal infections and wounds were the most common sources of isolation and B. fragilis was the dominating species. Ninety-nine percent of the strains were resistant to ampicillin (breakpoint 2 mg/L), 6% to cefoxitin (64 mg/L), 15% to clindamycin (8 mg/L) and 9% to moxifloxacin (8 mg/L). Less than 1% were resistant to imipenem (16 mg/L), piperacillin-tazobactam (128 mg/L) and metronidazole (32 mg/L). Ninety-six percent of the isolates were beta-lactamase producers.
Conclusions  Antimicrobial resistance among the B. fragilis group is increasing.     

BmeRABC5 is a multidrug efflux system that can confer metronidazole resistance in Bacteroides fragilis

The RND-family efflux pump gene bmeB5 was previously shown to be overexpressed in metronidazole-resistant laboratory mutants of Bacteroides fragilis. In the present study, we characterized the bmeABC5 genes and an upstream putative TetR-family regulator gene (bmeR5). bmeR5 (645 bp) was located 51 bp upstream of bmeA5 and encoded a 24.9-kDa protein. Deletant strains lacking bmeB5 or bmeR5 were constructed from a wild-type B. fragilis strain ADB77. Strain antimicrobial susceptibility was determined and gene expression was quantified. bmeR5 was overexpressed in Escherichia coli using a 6x-His tag system; BmeR5-His6 was isolated from inclusion bodies and its binding to bmeABC5 promoter regions was determined. BmeR5-His6 bound specifically to the bmeR5-bmeC5 intergenic region (IT1). Deletion of bmeR5 (ADB77DeltabmeR5) resulted in a significant (p < 0.05) increase in expression of bmeA5, bmeB5, and bmeC5, and > two-fold increase in minimum inhibitory concentrations (MICs) of ampicillin, cefoxitin, cefoperazone, ciprofloxacin, imipenem, metronidazole, ethidium bromide, and sodium dodecyl sulfate (SDS). MICs were reduced by the efflux pump inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The MICs of ampicillin, cefoperazone, metronidazole, and SDS were reduced by approximately two-fold in ADB77DeltabmeB5. A multidrug (metronidazole)-resistant, nim-negative B. fragilis clinical isolate overexpressed bmeABC5 genes, had a G-->T point mutation in IT1, and significantly reduced binding to BmeR5-His6. These data demonstrate that BmeR5 is a local repressor of bmeABC5 expression and that mutations in IT1 can lead to a derepression and resistance to multiple antimicrobial agents, including metronidazole.     

Postantibiotic effects with Bacteroides fragilis determined by viable counts and CO2 generation

Objective: To study the postantibiotic effect (PAE) for Bacteroides fragilis after exposure to common anaerobic antimicrobials with two different methods, by viable counting and by measuring CO₂ generation in a BACTEC® blood culture system.
Method: Four strains of B. fragilis were exposed for 1,2 and 4 h to cefoxitin, chloramphenicol, clindamycin, imipenem or metronidazole at concentrations from 1 to 16 X MIC. The drugs were removed by dilution into BACTEC 7A® vials and growth determined with viability counts and CO₂ production.
Results: The durations of the PAEs determined by the two methods correlated well ( r =0.913, p <0.005). PAEs of up to 4–5 h were induced by imipenem and metronidazole with achievable concentrations and exposure durations. Chloramphenicol induced short or no PAEs, but cefoxitin and clindamycin induced PAEs up to 2 h with high AUC values. The imipenem PAEs and the short cefoxitin and clindamycin PAEs were dependent on AUC.
Conclusions: Significant PAEs against B. fragilis were induced by imipenem and metronidazole. Determining PAE by measuring CO₂ production is an accurate and less time-consuming alternative to the conventional method of viable counts.     

Susceptibility Testing of Anaerobic Bacteria: Evaluation of the Redesigned (Version 96) bioMérieux ATB ANA Device

We compared the susceptibility results for 200 clinical anaerobes with nine antibiotics obtained by using a new ATB ANA (bioMérieux) device against those obtained by the National Committee for Clinical Laboratory Standards (NCCLS) standard agar dilution method. For better evaluation of the device, we added some resistant Bacteroides fragilis group strains from our own collection: 3, 6, and 12 strains that were resistant to imipenem, ticarcillin plus clavulanic acid, and co-amoxiclav, respectively, and 2 other strains with decreased susceptibility to metronidazole. For some strains that did not grow on ATB S medium, tests were performed by using West-Wilkins medium supplemented with 1.5% agar. The new ATB ANA device made clinical categorization of the investigated strains possible, according to French (Committee of the Antibiogram of the French Society of Microbiology) or U.S. (NCCLS) breakpoints, with the following respective results: category agreement, 94.3 and 94.9%; minor errors, 4.8 and 3.8%; major errors, 0.4 and 0.8%; and very major errors 4.6 and 4.2%. The ATB ANA device was able to detect low-level metronidazole-resistant B. fragilis strains according to the French breakpoints but not the NCCLS ones. For B. fragilis and beta-lactamase-positive Prevotella strains, the clustering effect of amoxicillin MICs around the French breakpoints led to more frequent minor errors. ATB ANA is a very convenient method to determine the antibiotic susceptibilities of anaerobes. Results obtained by ATB ANA correlated well with those obtained by the reference method.     

Metronidazole resistance in Clostridium difficile is heterogeneous

At our institution, the prevalence of clinical isolates of Clostridium difficile with resistance to metronidazole is 6.3%. We observed that initial metronidazole MICs of 16 to 64 mg/liter against toxigenic, primary fresh C. difficile isolates, as determined by agar dilution, decreased to 0.125 mg/liter after the isolates were thawed. In this study, we examined the possibility of heterogeneous or inducible resistance. Totals of 14 metronidazole-resistant and 10 metronidazole-susceptible clinical isolates of toxigenic C. difficile were studied. The isolates were investigated for the presence of nim genes by PCR. After the isolates were thawed, susceptibility testing was done by agar dilution, by disc diffusion using a 5-μg metronidazole disc, and by the Etest method. An experiment for determining the effect of prolonged exposure to metronidazole was applied to all resistant isolates and to susceptible control strains. None of the isolates presented the nim genes. All initially metronidazole-resistant C. difficile isolates became susceptible after thawing; however, they presented slow-growing subpopulations within the inhibition zones of both the disk and the Etest strip. All metronidazole-susceptible isolates remained homogeneously susceptible by both methods. After prolonged exposure in vitro to metronidazole, no zone of inhibition was found around the 5-μg disk in any of the metronidazole-resistant isolates, and the MICs as determined by the Etest method ranged from 0.125 to >256 mg/liter, with colonies growing inside the inhibition zone. Our results indicate that (i) resistance to metronidazole was not due to the presence of nim genes, (ii) resistance to metronidazole in toxigenic C. difficile isolates is heterogeneous, and (iii) prolonged exposure to metronidazole can select for in vitro resistance. We recommend routine performance of the disk diffusion method (5-μg metronidazole disk) with primary fresh C. difficile isolates in order to ensure that metronidazole-heteroresistant populations do not go undetected.     

Detection of enterotoxigenic Bacteroides fragilis by PCR.

Strains of enterotoxigenic Bacteroides fragilis (ETBF) are associated with diarrhea in young farm animals and, at least in particular settings, in children. Enterotoxin production by ETBF is currently detected by a tissue culture assay with HT-29 cells. We have developed a PCR assay based on the detection of the enterotoxin gene to identify ETBF in culture and in stool samples. Overall, 113 bacterial strains were examined, including 3 B. fragilis reference strains, 75 B. fragilis isolates (comprising 40 ETBF isolates), 20 Bacteroides spp. other than B. fragilis, and 15 strains belonging to other genera. Complete agreement was found between the results of the tissue culture assay and those of the PCR for our strains. PCR was also used to detect ETBF directly in fecal samples. Stools from two healthy volunteers were spiked with known numbers of ETBF and were processed by three different methods. A culture method, which required inoculation of the stools on selective plates and the collection of the whole bacterial growth ("sweeps"), was found to be the most sensitive. PCR performed with the plate sweeps yielded amplification products with a detection limit of 10(5) to 10(4) CFU/g of feces. By this method 18 samples of diarrheic stools (10 positive and 8 negative for ETBF) were examined. The results of the PCR were in accordance with the culture results in all cases. The proposed PCR assay represents a diagnostic tool for the rapid identification of ETBF in culture as well as in fecal samples.     

PCR-restriction fragment length polymorphism analysis for identification of Bacteroides spp. and characterization of nitroimidazole resistance genes

Bacteroides spp. are opportunist pathogens that cause blood and soft tissue infections and are often resistant to antimicrobial agents. We have developed a combined PCR-restriction fragment length polymorphism (RFLP) technique to characterize the 16S rRNA gene for identification purposes and the nitroimidazole resistance (nim) gene for detection of resistance to the major antimicrobial agent used to treat Bacteroides infections: metronidazole (MTZ). PCR-RFLP analysis of 16S ribosomal (rDNA) with HpaII and TaqI produced profiles that enabled discrimination of type strains and identification of 70 test strains to the species level. The 16S rDNA PCR-RFLP identification results agreed with routine phenotypic testing for 62 of the strains. The discrepancies between phenotypic and PCR-RFLP methods for eight strains were resolved by 16S rDNA sequencing in three cases, but five strains remain unidentified. The presence of nim genes was indicated by PCR in 25 of 28 strains that exhibited reduced sensitivity to MTZ. PCR-RFLP of the nim gene products identified the four reported genes (nimA, -B, -C, and -D) and indicated the presence of a previously unreported nim gene in 5 strains. This novel nim gene exhibited 75% DNA sequence similarity with nimB. These rapid, accurate, and inexpensive methods should enable improved identification of Bacteroides spp. and the detection of MTZ resistance determinants.     

Isolation of metronidazole-resistant Bacteroides fragilis carrying the nimA nitroreductase gene from a patient in Washington State

Members of the Bacteroides fragilis group are among the most common anaerobic bacterial isolates in clinical specimens. Metronidazole, a 5-nitroimidazole, is often used as empirical therapy for anaerobic infections. Susceptibility testing is not routinely performed because of nearly universal susceptibility of Bacteroides spp. to this agent. We report a case of metronidazole-resistant Bacteroides fragilis in the United States and demonstrate the presence of the nimA gene, encoding a nitroreductase previously shown to mediate resistance to 5-nitroimidazole antimicrobial agents in B. fragilis strains from Europe and Africa. Because clinical failures in Bacteroides infections have been associated with the use of inactive antimicrobial agents, clinicians need to be aware of the possibility of metronidazole-resistant B. fragilis strains in the United States and the importance of susceptibility testing in selected situations.     

Multicentre survey of the in-vitro activity of seven antimicrobial agents, including ertapenem, against recently isolated Gram-negative anaerobic bacteria in Greece

The in-vitro activities of penicillin, ticarcillin-clavulanic acid, cefoxitin, imipenem, ertapenem, metronidazole and clindamycin were evaluated against 138 Gram-negative anaerobic isolates (82 Bacteroides fragilis group, 17 non-fragilis Bacteroides spp., 31 Prevotella spp., four Fusobacterium spp., two Veillonella spp., one Porphyromonas sp. and one Tissierella praeacuta) collected from six general hospitals in Athens, Greece. Overall rates of non-susceptibility (both resistant and intermediately-resistant) to penicillin and ticarcillin-clavulanic acid were 81.8% and 2.3%, respectively. The rates of non-susceptibility to cefoxitin and clindamycin were 30.3% and 31.1%, respectively, and that for metronidazole was 4.3% (four Prevotella spp. isolates, one Porphyromonas sp. isolate and one B. fragilis isolate). Only the single B. fragilis isolate was nim-positive by PCR. Only one B. fragilis isolate was resistant to both carbapenems tested, while six more Bacteroides spp. isolates were imipenem-susceptible and ertapenem-non-susceptible. The MIC range, MIC(50) and MIC(90) values were comparable for imipenem and ertapenem, although ertapenem MIC(90)s were one or two two-fold dilutions higher.     

Antimicrobial resistance patterns of Bacteroides fragilis group organisms in Korea

Antimicrobial resistance patterns of 913 clinical isolates of Bacteroides fragilis group organisms were monitored during an 8-year period in Korea. In general the resistance rates of the non-fragilis B. fragilis group species were higher than those of B. fragilis for all the drugs tested. The rate of resistance to clindamycin remarkably increased and those to some beta-lactam drugs such as piperacillin and cefotaxime also increased. No isolates were found to be resistant to imipenem, metronidazole, or chloramphenicol. beta-lactam and beta-lactamase inhibitor combinations and cefoxitin were more active than the other beta-lactams. Therefore, these agents may be considered when empirical selection of antimicrobial agents is required to treat severe anaerobic infections.     

Quantitative detection of enterotoxigenic Bacteroides fragilis subtypes isolated from children with and without diarrhea

A rapid real-time PCR (RT-PCR) approach was developed to detect the bft gene subtypes in Bacteroides fragilis isolated from fecal samples. DNA obtained from diarrhea (110) and nondiarrhea (150) samples was evaluated. Subtype 1 was observed in 9 (8.2%) diarrhea and 7 (4.7%) nondiarrhea samples. Subtype 2 was not detected in any DNA samples, and subtype 3 was observed in only 1 diarrhea sample. The presence of the bft-1 gene did not show any statistically significant differences between the groups of children. This technique could be used to evaluate a possible correlation between disease and the presence of B. fragilis enterotoxin.     

Susceptibility of 114 isolates of the Bacteroides fragilis group to imipenem and eight other antimicrobial agents

The minimal inhibitory concentrations (MICs) of the newer beta-lactam antibiotic, imipenem, were compared with those of cefoxitin, cefotetan, penicillin, amoxycillin, ticarcillin and metronidazole against 114 clinical isolates of the Bacteroides fragilis group of anaerobic organisms. The ability of clavulanic acid, a beta-lactamase inhibitor, to potentiate the in vitro activity of amoxycillin and ticarcillin was also studied. Using an agar dilution technique we found imipenem to be the most active beta-lactam antibiotic tested having an MIC50 of 0.25 microgram/ml and inhibiting all isolates at a concentration of 4 micrograms/ml. Metronidazole had comparable activity with a MIC50 of 0.5 microgram/ml and all isolates inhibited by 1 microgram/ml. Cefoxitin and cefotetan showed similar activity both with a MIC50 of 8 micrograms/ml against the B. fragilis group, while penicillin, amoxycillin and ticarcillin all had a MIC50 of 16 micrograms/ml. Clavulanic acid significantly reduced the MIC50 of amoxycillin and ticarcillin to 0.5 micrograms/ml and 0.25 micrograms/ml, respectively.     

PCR ribotyping and arbitrarily primed PCR for the comparison of enterotoxigenic Bacteroides fragilis strains from two Polish university hospitals

Objective: To study the clinical incidence and possible clonal relatedness of enterotoxigenic strains of Bacteroides fragilis among pediatric and adult patients in two Polish university hospitals.
Method: Fecal samples from 201 adults and 131 infants (with or without diarrhea) and vaginal samples from 100 pregnant women nursed in two Polish university hospitals were analyzed with respect to carriage of enterotoxin-producing Bacteroides fragilis (ETBF). This putative pathogen was identified by cultivation and subsequent cytopathogenicity testing of culture supernatants on HT/29 C1 cells.
Results and discussion: Two ETBF strains were isolated from childrens' feces; two additional strains were isolated from adults, and from the vaginal samples only a single strain was isolated. One strain (W2) was isolated from a child with diarrhea. These incidence figures, the fact that all ETBF isolates were shown to produce strongly differing amounts of the cytotoxin, and the genetic unrelatedness of the strains as demonstrated by two different PCR-mediated DNA typing procedures, indicates that clonal spread of ETBF is presently not a clinical problem in these hospitals. It was shown that PCR-mediated ribotyping and arbitrarily primed PCR can be applied with success to study the epidemiology of ETBF.     

Clonal dissemination of a toxin-A-negative/toxin-B-positive Clostridium difficile strain from patients with antibiotic-associated diarrhea in Poland

Objective To determine the incidence of toxin-A-negative/toxin-B-positive Clostridium difficile strains and their genetic relatedness in the feces of patients suffering from antibiotic-associated diarrhea (AAD) in Polish hospitals.
Methods C. difficile strains were cultured from patients' stool samples. The present study characterises these strains with respect to their cytopathogenicity on McCoy cells and the absence of toxin A despite a functional toxin B as determined with commercial test kits (Culturette Brand Toxin CD-TCD toxin A test and C. difficile Tox A/B test). In addition, PCR using different primer pairs aiming at non-repeating or repeating regions of the toxin A and B genes were used to confirm the findings. All toxin A^–B⁺ strains were genetically characterised by random amplification of polymorphic DNA (RAPD) analysis, PCR ribotyping and, in part, pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments.
Results We here present the presence of 17 toxin A^–B⁺ strains among 159 C. difficile strains (11%) isolated from fecal samples from 413 patients with antibiotic-associated diarrhea. All 17 strains possessed the toxin B gene, demonstrated a cytopathogenic effect on the McCoy cells, and were positive in the Tox A/B test. Molecular typing of these 17 C. difficile strains revealed that 7 of 17 (41%) toxin A^–/B⁺ C. difficile strains could not be discriminated. It appeared that these strains had a genotype that could not be distinguished from that of a Japanese control strain.
Conclusion Our observations imply that a particular genotype of toxin A^–B⁺ C. difficile has spread extensively, not only in Poland but possibly even worldwide.     

Metronidazole resistance test in vitro in strains of Trichomonas vaginalis isolated from clinical material. 2. Proposal for a standard tests]

The purpose of this paper is to recommend a dependable susceptibility assays for detection of resistance to metronidazole in Trichomonas vaginalis, suitable for routine use in clinical and public health care laboratories. Two different assays, the microtitre plate test based on Meingassner's technique and an alternative tube-assay, were scrutinized and compared using 10 metronidazole-resistant isolates from Czechoslovakia, other European countries and USA and 10 drug-susceptible strains isolated in Czechoslovakia. The minimal lethal concentrations of metronidazole determined for the resistant strains ranged from 25 to 317.5 micrograms.ml-1 while those of drug-susceptible from 3.1-12.5 micrograms.ml-1 metronidazole. Results obtained by both assays were highly reliable and mutually comparable. The authors recommend the microtitre plate test as a method of choice.     

Alterations in rdxA and frxA genes and their upstream regions in metronidazole-resistant Helicobacter pylori isolates

Metronidazole resistance among Helicobacter pylori strains has been related to alterations in gene products having metronidazole nitroreductase activities. RdxA and FrxA proteins are the two major contributing factors. In this investigation, the rdxA and frxA genes and their upstream regions were analyzed in 19 H. pylori isolates, 8 of which were metronidazole-sensitive (MIC < or = 8 microg/mL) and 11 of which were metronidazole-resistant (MIC > or = 8 microg/mL), as determined by the E-test. Among the metronidazole-resistant isolates, three contained both RdxA and FrxA proteins with premature truncation caused by gene nonsense mutations or frameshift mutations, while three contained only stop mutations in FrxA and two only in RdxA. Substitutions of amino acids occurred in other RdxA (5/6) and FrxA (4/5) proteins from metronidazole resistant isolates as compared with those from metronidazole-sensitive ones. All metronidazole-resistant isolates had alterations in RdxA and/or FrxA proteins. Moreover, the upstream regions (-1 to -35) of rdxA and frxA genes in some metronidazole-resistant isolates varied by nucleotide insertion and/or deletion or substitution. The patterns of variation in both genes and their upstream regions were highly diversified. Alterations in rdxA and frxA genes and their upstream regions may be involved in the development of metronidazole resistance in H. pylori.     

Molecular epidemiology of Korean porcine sapeloviruses

To evaluate the prevalence and genetic diversity of porcine sapeloviruses (PSVs) in Korea, a total of 100 diarrhea fecal samples from pigs were analyzed by RT-PCR and nested PCR assays with primer pairs specific for the VP1 gene. Overall, 34 % of the diarrhea samples tested positive for PSV, and a high proportion of infections occurred along with a variety of other enteric viruses and bacteria. Genomic and phylogenetic analysis of the VP1 genes revealed pronounced genetic diversities between PSVs from Korean and elsewhere. Our results indicate that PSV infections are very common in Korean pigs with diarrhea. The infecting strains are genetically diverse.     

汕头地区耐亚胺培南铜绿假单胞菌耐药分析

目的：了解汕头地区对亚胺培南耐药的铜绿假单胞菌（PA）的耐药情况及耐药机制。方法：收集临床分离耐亚胺培南的PA共141株，双纸片协同实验检测金属酶表型，PCR法检测外膜孔蛋白OprD2和金属β内酰胺酶（IMP、VIM、SPM）基因。结果：耐亚胺培南的铜绿假单胞菌均为多重耐药茵，对头孢哌酮／舒巴坦的耐药率较低，未发现产金属酶菌株，仅22株菌株扩增出OprD2基因。结论：头孢哌酮／舒巴坦可作为本地区临床治疗耐亚胺培南铜绿假单胞茵所致感染的首选经验用药，OprDa表达减少或不表达可能是临床分离铜绿假单胞茵对亚胺培南耐药的主要机制。     

Down-regulation of flavin reductase and alcohol dehydrogenase-1 (ADH1) in metronidazole-resistant isolates of Trichomonas vaginalis

The microaerophilic parasite Trichomonas vaginalis is a causative agent of painful vaginitis or urethritis, termed trichomoniasis, and can also cause preterm delivery or stillbirth. Treatment of trichomoniasis is almost exclusively based on the nitroimidazole drugs metronidazole and tinidazole. Metronidazole resistance in T. vaginalis does occur and is often associated with treatment failure. In most cases, metronidazole-resistant isolates remain susceptible to tinidazole, but cross resistance between the two closely related drugs can be a problem. In this study we measured activities of thioredoxin reductase and flavin reductase in four metronidazole-susceptible and five metronidazole-resistant isolates. These enzyme activities had been previously found to be downregulated in T. vaginalis with high-level metronidazole resistance induced in the laboratory. Further, we aimed at identifying factors causing metronidazole resistance and compared the protein expression profiles of all nine isolates by application of two-dimensional gel electrophoresis (2DE). Thioredoxin reductase activity was nearly equal in all strains assayed but flavin reductase activity was clearly down-regulated, or even absent, in metronidazole-resistant strains. Since flavin reductase has been shown to reduce oxygen to hydrogen peroxide, its down-regulation could significantly contribute to the impairment of oxygen scavenging as reported by others for metronidazole-resistant strains. Analysis by 2DE revealed down-regulation of alcohol dehydrogenase 1 (ADH1) in strains with reduced sensitivity to metronidazole, an enzyme that could be involved in detoxification of intracellular acetaldehyde.