Online-Only Abstract

With the emergence of reduced susceptibility of Clostridium difficile to metronidazole and vancomycin the value of antimicrobial susceptibility testing has increased. The aim of our study was to evaluate disk diffusion for susceptibility testing of C. difficile by comparing disk diffusion results with MICs from gradient tests and to propose zone diameter breakpoint correlates for the EUCAST epidemiological cut-off values (ECOFFs) recently published. We tested 211 clinical isolates of C. difficile, from patients with diarrhoea hospitalized at Aarhus and Odense University Hospitals, Denmark. Furthermore, ten clinical isolates of C. difficile from the Anaerobe Reference Laboratory, University Hospital of Wales, with known reduced susceptibility to either metronidazole or vancomycin, were included. Isolates were tested with Etest gradient strips and disk diffusion towards metronidazole, vancomycin and moxifloxacin on Brucella Blood Agar supplemented with hemin and vitamin K. We found an excellent agreement between inhibition zone diameter and MICs. For each MIC value, the inhibition zones varied from 0 to 8 mm, with 93% of values within 6 mm for metronidazole, 95% of values within 4 mm for vancomycin, and 98% of values within 4 mm for moxifloxacin. With proposed zone diameter breakpoints for metronidazole, vancomycin and moxifloxacin of WT ≥ 23 mm, WT ≥ 19 and WT ≥ 20 mm, respectively, we found no very major errors and only major errors below 2%. In conclusion, we suggest that disk diffusion is an option for antimicrobial susceptibility testing of C. difficile.     

Antimicrobial susceptibility testing of Clostridium difficile using EUCAST epidemiological cut-off values and disk diffusion correlates

With the emergence of reduced susceptibility of Clostridium difficile to metronidazole and vancomycin the value of antimicrobial susceptibility testing has increased. The aim of our study was to evaluate disk diffusion for susceptibility testing of C. difficile by comparing disk diffusion results with MICs from gradient tests and to propose zone diameter breakpoint correlates for the EUCAST epidemiological cut-off values (ECOFFs) recently published. We tested 211 clinical isolates of C. difficile, from patients with diarrhoea hospitalized at Aarhus and Odense University Hospitals, Denmark. Furthermore, ten clinical isolates of C. difficile from the Anaerobe Reference Laboratory, University Hospital of Wales, with known reduced susceptibility to either metronidazole or vancomycin, were included. Isolates were tested with Etest gradient strips and disk diffusion towards metronidazole, vancomycin and moxifloxacin on Brucella Blood Agar supplemented with hemin and vitamin K. We found an excellent agreement between inhibition zone diameter and MICs. For each MIC value, the inhibition zones varied from 0 to 8 mm, with 93% of values within 6 mm for metronidazole, 95% of values within 4 mm for vancomycin, and 98% of values within 4 mm for moxifloxacin. With proposed zone diameter breakpoints for metronidazole, vancomycin and moxifloxacin of WT ≥ 23 mm, WT ≥ 19 and WT ≥ 20 mm, respectively, we found no very major errors and only major errors below 2%. In conclusion, we suggest that disk diffusion is an option for antimicrobial susceptibility testing of C. difficile.     

Interest of the disk diffusion method for screening Clostridium difficile isolates with decreased susceptibility to antibiotics

AimIn vitro determination of Clostridium difficile susceptibility to antibiotics is not routinely performed. The aim of this study was to evaluate the performance of antibiotic susceptibility determination with the disk diffusion method for screening C. difficile isolates with decreased susceptibility to antibiotics.MethodsThirty-six C. difficile isolates (toxigenic or not) isolated in 2005 and 2006 from three hospitals Assistance publique–Hôpitaux de Paris (Jean-Verdier, René-Muret, Beaujon) were studied by disk diffusion method with 14 antibiotics. Mueller-Hinton agar supplemented with sheep blood (Bio-Rad*) were swabbed with a C. difficile suspension at 1 McFarland. To check the results obtained with the disk diffusion method, Minimal Inhibitory Concentration (MIC) were performed respectively with E-test for glycopeptides and metronidazole and with the agar dilution reference method and E-test for new molecules with a potential activity against anaerobes: imipenem, ertapenem, linezolid and moxifloxacin.ResultsThe decreased susceptibility (resistant and intermediate) observed was 40% for amoxicillin–clavulanate, 60% for piperacillin–tazobactam, 100% for ceftriaxone, 81% for imipenem, 61% for ertapenem, 2% for chloramphenicol, 34% for erythromycin, 90% for lincomycin, 2% for linezolid, 98% for levofloxacin, 17% for moxifloxacin and 0% for vancomycin, teicoplanin and metronidazole. The results obtained with the disk diffusion method were compared to MICs obtained with E-test and reference method.ConclusionThe disk diffusion method seems to be a good method to detect isolates suspected to have a decreased susceptibility and consequently to reduce MIC determinations.     

Fosfomycin Susceptibility in Carbapenem-Resistant Enterobacteriaceae from Germany

Due to the increase in multidrug-resistant Enterobacteriaceae, the interest in older antimicrobial agents, like fosfomycin, has increased. In this study, we used agar dilution for testing susceptibilities to fosfomycin in a collection of 107 carbapenem-nonsusceptible Enterobacteriaceae isolates, of which 80 produced various types of carbapenemases, including KPC, VIM, NDM, and OXA-48. Overall, 78% of the strains had fosfomycin MICs of ≤32 mg/liter and were thus considered to be susceptible according to the current EUCAST breakpoint. The MIC₅₀ and MIC₉₀ were 8 mg/liter and 512 mg/liter, respectively. Escherichia coli strains had significantly lower fosfomycin MICs than the Klebsiella pneumoniae and Enterobacter cloacae strains. Furthermore, comparisons of the susceptibility testing methods, like Etest and disk diffusion, were performed against agar dilution as the reference method. Essential agreement between Etest and agar dilution was 78.9%, and categorical agreement between the two methods was 92.5%, with 20% very major errors and 2.6% major errors. Disk diffusion was studied with 50-μg and 200-μg fosfomycin disks, but no inhibition zone breakpoint that reduced very major and major errors to an acceptable level was found. Etest and disk diffusion showed poor agreement with fosfomycin agar dilution.     

Prevalence of antimicrobial resistance genes in Bacteroides spp. and Prevotella spp. Dutch clinical isolates

ObjectivesThe prevalence of resistance genes in two important anaerobic genera, Bacteroides and Prevotella, was assessed by applying PCR specifically directed to genes of interest.MethodsA total of 101 Bacteroides spp. and 99 Prevotella spp. human clinical isolates were identified using MALDI-TOF MS. The presence of the resistance genes cfxA, cepA, cfiA, tetQ, ermF and nim, was assessed. Prevalence of resistance genes was compared with the phenotypic resistance against amoxicillin, clindamycin, meropenem and metronidazole.ResultsEven though the majority of the Bacteroides isolates (95.0%) showed resistance towards amoxicillin, only 52/101 of the isolates harboured one of the resistance genes, accounting for this resistance. Within the genus Prevotella the presence of cfxA (50/99) almost perfectly matched the amoxicillin resistance (48/99). No difference in prevalence of the ermF gene (16/101 and 9/99) and clindamycin resistance (16/101 and 10/99) was observed within Bacteroides and Prevotella, respectively. Two isolates of Prevotella were resistant to metronidazole. One harboured the nim gene. One metronidazole-susceptible isolate of Bacteroides harboured a nim gene. Within the Bacteroides and Prevotella genera, 6/101 strains and 5/99 isolates harboured three different resistance genes, respectively, among them tetQ. TetQ is often located on a conjugative transposon, increasing the chance of horizontal gene transfer between isolates.ConclusionsAn unknown mechanism in Bacteroides non-fragilis isolates causes resistance to β-lactam antibiotics. The fact that the prevalence of the tetQ gene among Prevotella is increasing and the existence of isolates harbouring three resistance genes are worrisome developments.     

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.     

Multiplex PCR targeting tpi (triose phosphate isomerase), tcdA (Toxin A), and tcdB (Toxin B) genes for toxigenic culture of Clostridium difficile

A multiplex PCR toxigenic culture approach was designed for simultaneous identification and toxigenic type characterization of Clostridium difficile isolates. Three pairs of primers were designed for the amplification of (i) a species-specific internal fragment of the tpi (triose phosphate isomerase) gene, (ii) an internal fragment of the tcdB (toxin B) gene, and (iii) an internal fragment of the tcdA (toxin A) gene allowing distinction between toxin A-positive, toxin B-positive (A+B+) strains and toxin A-negative, toxin B-positive (A−B+) variant strains. The reliability of the multiplex PCR was established by using a panel of 72 C. difficile strains including A+B+, A−B−, and A−B+ toxigenic types and 11 other Clostridium species type strains. The multiplex PCR assay was then included in a toxigenic culture approach for the detection, identification, and toxigenic type characterization of C. difficile in 1,343 consecutive human and animal stool samples. Overall, 111 (15.4%) of 721 human samples were positive for C. difficile; 67 (60.4%) of these samples contained A+B+ toxigenic isolates, and none of them contained A−B+ variant strains. Fifty (8%) of 622 animal samples contained C. difficile strains, which were toxigenic in 27 (54%) cases, including 1 A−B+ variant isolate. Eighty of the 721 human stool samples (37 positive and 43 negative for C. difficile culture) were comparatively tested by Premier Toxins A&B (Meridian Bioscience) and Triage C. difficile Panel (Biosite) immunoassays, the results of which were found concordant with toxigenic culture for 82.5 and 92.5% of the samples, respectively. The multiplex PCR toxigenic culture scheme described here allows combined diagnosis and toxigenic type characterization for human and animal C. difficile intestinal infections.     

Effects of Treated versus Untreated Polystyrene on Caspofungin In Vitro Activity against Candida Species

Significant interlaboratory variability is observed in testing the caspofungin susceptibility of Candida species by both the CLSI and EUCAST broth microdilution methodologies. We evaluated the influence of treated versus untreated polystyrene microtiter trays on caspofungin MICs using 209 isolates of four Candida species, including 16 C. albicans and 11 C. glabrata isolates with defined FKS mutations. Caspofungin MICs were also determined using the commercially available YeastOne and Etest assays and 102 isolates. All C. glabrata isolates had caspofungin MICs of ≥0.5 μg/ml, the clinical breakpoint for caspofungin resistance in this species, measured using trays made of treated polystyrene, regardless of the FKS status. In contrast, susceptible isolates could readily be distinguished from resistant/non-wild-type isolates when caspofungin MICs were measured using untreated polystyrene trays and both the YeastOne and Etest assays. Similar results were also observed for C. krusei isolates, as all isolates had caspofungin MICs above the threshold for resistance measured using treated polystyrene trays. In contrast, C. albicans isolates could be correctly identified as susceptible or resistant when caspofungin MICs were measured with treated or untreated trays and with the YeastOne and Etest assays. MICs falsely elevated above the resistance breakpoint were also not observed for C. tropicalis isolates. These results demonstrated that the use of treated polystyrene may be one factor that leads to falsely elevated caspofungin in vitro susceptibility results and that this may also be a greater issue for some Candida species than for others.     

Phenotypic detection of methicillin resistance in Staphylococcus aureus by disk diffusion testing and Etest on Mueller-Hinton agar

Cefoxitin is increasingly recommended for detection of methicillin resistance in Staphylococcus aureus (MRSA) when using disk diffusion testing. In this study, 95 mecA-negative S. aureus isolates and a highly genetically diverse collection of mecA-positive S. aureus types (n=50) were used to investigate the influence of technical factors such as disk potency, incubation time, and temperature on Mueller-Hinton agar. The use of cefoxitin MIC testing by Etest for the same purpose was investigated under similar conditions. For disk diffusion, the accuracy was high at both 35 degrees C and 36 degrees C using overnight incubation, while incubation at 30 degrees C or 37 degrees C was associated with slightly lower accuracy. Increasing incubation times from 18 to 24 h did not improve accuracy at either temperature. Cefoxitin Etest MICs for mecA-positive strains were 6 mg/liter or higher, while cefoxitin Etest MICs for mecA-negative strains were 4 mg/liter, corresponding to S>or=22 mm and Ror=17 mm and R相似文献   

Evaluation of Mannitol Salt Agar for Detection of Oxacillin Resistance in Staphylococcus aureus by Disk Diffusion and Agar Screening

Mannitol salt agar was evaluated for detection of oxacillin resistance in 136 Staphylococcus aureus isolates. All mecA-positive isolates (n = 54) were correctly categorized as oxacillin resistant by the disk diffusion test (1-μg disk; zone diameter, <16 mm); the specificity was 97.6%. Agar screening (2 μg of oxacillin per ml) revealed a sensitivity of 98.1% and a specificity of 95.1%.     

The ClosER study: results from a three-year pan-European longitudinal surveillance of antibiotic resistance among prevalent Clostridium difficile ribotypes, 2011–2014

ObjectivesUntil the introduction of fidaxomicin, antimicrobial treatment for Clostridium difficile infection (CDI) was limited to metronidazole and vancomycin. The changing epidemiology of CDI and the emergence of epidemic C. difficile PCR ribotype 027 necessitate continued surveillance to identify shifts in antibiotic susceptibility. ClosER, currently the largest pan-European epidemiological study of C. difficile ribotype distribution and antibiotic susceptibility, aimed to undertake antimicrobial resistance surveillance pre- and post-introduction of fidaxomicin.MethodsBetween July 2011 and July 2014, 39 sites across 22 European countries submitted 2830 C. difficile isolates for ribotyping, toxin testing and susceptibility testing to metronidazole, vancomycin, fidaxomicin, rifampicin, moxifloxacin, clindamycin, imipenem, chloramphenicol and tigecycline.ResultsRibotypes 027, 014, 001, 078, 020, 002, 126, 015 and 005 were most frequently isolated, and emergent ribotypes 198 and 356 were identified in Hungary and Italy, respectively. All isolates were susceptible to fidaxomicin, with scarce resistance to metronidazole (0.2%, 6/2694), vancomycin (0.1%, 2/2694) and tigecycline (0%). Rifampicin, moxifloxacin and clindamycin resistance was evident in multiple ribotypes. Lack of ribotype diversity correlated with greater antimicrobial resistance. Epidemic ribotypes (027/001) were associated with multiple antimicrobial resistance, and ribotypes 017, 018 and 356 with high-level resistance. Additional factors may also influence local ribotype prevalence.ConclusionsFidaxomicin susceptibility was retained post-introduction, and resistance to metronidazole and vancomycin was rare. Continued surveillance is needed, with more accurate classification and clarification of ribotype subtypes to further understand their role in the spread of resistance. Other factors may also influence changes in prevalence of C. difficile ribotypes with reduced antibiotic susceptibility.     

Evaluation of a New Automated Homogeneous PCR Assay,GenomEra C. difficile,for Rapid Detection of Toxigenic Clostridium difficile in Fecal Specimens

We evaluated a new automated homogeneous PCR assay to detect toxigenic Clostridium difficile, the GenomEra C. difficile assay (Abacus Diagnostica, Finland), with 310 diarrheal stool specimens and with a collection of 33 known clostridial and nonclostridial isolates. Results were compared with toxigenic culture results, with discrepancies being resolved by the GeneXpert C. difficile PCR assay (Cepheid). Among the 80 toxigenic culture-positive or GeneXpert C. difficile assay-positive fecal specimens, 79 were also positive with the GenomEra C. difficile assay. Additionally, one specimen was positive with the GenomEra assay but negative with the confirmatory methods. Thus, the sensitivity and specificity were 98.8% and 99.6%, respectively. With the culture collection, no false-positive or -negative results were observed. The analytical sensitivity of the GenomEra C. difficile assay was approximately 5 CFU per PCR test. The short hands-on (<5 min for 1 to 4 samples) and total turnaround (<1 h) times, together with the high positive and negative predictive values (98.8% and 99.6%, respectively), make the GenomEra C. difficile assay an excellent option for toxigenic C. difficile detection in fecal specimens.     

Quantitative Fecal Lactoferrin in Toxin-Positive and Toxin-Negative Clostridium difficile Specimens

Quantitative fecal lactoferrin was measured in 112 patients tested for toxigenic Clostridium difficile using glutamate dehydrogenase (GDH) and toxin immunoassays combined with tcdB PCR. Lactoferrin levels were higher in the GDH-positive/toxin-positive group (79 μg/ml) than in the GDH-positive/toxin-negative/PCR-positive (21 μg/ml) and the GDH-negative groups (13 μg/ml). Differences in fecal lactoferrin levels suggest variable presence or severity of C. difficile infection among toxin-positive and toxin-negative patients.     

Fluconazole Disk Diffusion Susceptibility Testing of Candida Species

We describe a simple procedure for detecting fluconazole-resistant yeasts by a disk diffusion method. Forty clinical Candida sp. isolates were tested on RPMI-glucose agar with either 25- or 50-μg fluconazole disks. With 25-μg disks, zones of inhibition of ≥20 mm at 24 h accurately identified 29 of 29 isolates for which MICs were ≤8 μg/ml, and with 50-μg disks, zones of ≥27 mm identified 28 of 29 such isolates. All 11 isolates for which MICs were >8 μg/ml were identified by using either disk. Disk diffusion may be a useful screening method for clinical microbiology laboratories.     

Multicenter Study To Determine Disk Diffusion and Broth Microdilution Criteria for Prediction of High- and Low-Level Mupirocin Resistance in Staphylococcus aureus

Mupirocin susceptibility testing of Staphylococcus aureus has become more important as mupirocin is used more widely to suppress or eliminate S. aureus colonization and prevent subsequent health care- and community-associated infections. The present multicenter study evaluated two susceptibility testing screening methods to detect mupirocin high-level resistance (HLR), broth microdilution (BMD) MICs of ≥512 μg/ml, and a 6-mm zone diameter for a disk diffusion (DD) test with a 200-μg disk. Initial testing indicated that with Clinical and Laboratory Standards Institute methods for BMD and DD testing, the optimal conditions for the detection of mupirocin HLR were 24 h of incubation and reading of the DD zone diameters with transmitted light. Using the presence or absence of mupA as the “gold standard” for HLR, the sensitivity and specificity of a single-well 256 μg/ml BMD test were 97 and 99%, respectively, and those for the 200-μg disk test were 98 and 99%, respectively. Testing with two disks, 200 μg and 5 μg, was evaluated for its ability to distinguish HLR isolates (MICs ≥ 512 μg/ml), low-level-resistant (LLR) isolates (MICs = 8 to 256 μg/ml), and susceptible isolates (MICs ≤ 4 μg/ml). Using no zone with both disks as an indication of HLR and no zone with the 5-μg disk plus any zone with the 200-μg disk as LLR, only 3 of the 340 isolates were misclassified, with 3 susceptible isolates being classified as LLR. Use of standardized MIC or disk tests could enable the detection of emerging high- and low-level mupirocin resistance in S. aureus.Mupirocin is a topical antibacterial agent that is used both for the treatment of skin infections and for the suppression or elimination of nasal carriage of Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA) (8). The recommendations of the Healthcare Infection Control Practices Advisory Committee suggest the use of a tiered approach to the prevention and control of infections with multidrug-resistant organisms, including MRSA, in acute-care settings (20). In their recommendations, decolonization is presented as one intervention that may be considered when intensified MRSA control measures are needed; if decolonization is used, susceptibility testing and monitoring for the emergence of resistance to the decolonization agent are recommended in one study (21).There are two levels of resistance to mupirocin: low-level resistance (LLR), for which the MICs are 8 to 256 μg/ml, and high-level resistance (HLR), for which the MICs are ≥512 μg/ml (11). The mupirocin MICs of strains susceptible to mupirocin are MICs ≤4 μg/ml. HLR is associated with the presence of the plasmid-mediated mupA gene, which encodes a mupirocin-resistant isoleucyl-tRNA synthetase, although S. aureus strains with HLR that lack mupA have occurred (this study) and can also be created in the laboratory (23). LLR results from mutation of the native, chromosomal isoleucyl-tRNA synthetase ileS gene (1). Studies suggest that S. aureus strains with HLR to mupirocin cannot be successfully eliminated with mupirocin and that the occurrence of HLR is increasing (22). It has been suggested that S. aureus strains demonstrating LLR could be eliminated by topical application of mupirocin because of the high concentrations achieved locally, but this has not been demonstrated definitively (11, 21).Until recently, methods for testing topical agents have not been included in susceptibility testing documents published by the Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS), although guidelines for testing by various methods have been suggested by others (9, 10, 12, 13, 16, 17). The British Society for Antimicrobial Chemotherapy has formal recommendations for the testing of mupirocin (www.bsac.org.uk) that include testing of a 5-μg and a 20-μg mupirocin disk. Their recommendations require MIC testing to determine the level of resistance if a 5-μg disk is used alone. An initial investigation at the Centers for Disease Control and Prevention (CDC), Atlanta, GA, showed that a 200-μg mupirocin disk was able to differentiate isolates with LLR from those with HLR (15). We undertook the study described here to determine the MIC and disk diffusion criteria for the detection of S. aureus strains with high- or low-level mupirocin resistance and to validate quality control tests. Using data from this study, a screen test for prediction of high-level mupirocin resistance is now included in CLSI susceptibility testing documents (3, 6, 7).     

Comparing Etest and Broth Microdilution for Antifungal Susceptibility Testing of the Most-Relevant Pathogenic Molds

Invasive mold infections are life-threatening diseases for which appropriate antifungal therapy is crucial. Their epidemiology is evolving, with the emergence of triazole-resistant Aspergillus spp. and multidrug-resistant non-Aspergillus molds. Despite the lack of interpretive criteria, antifungal susceptibility testing of molds may be useful in guiding antifungal therapy. The standard broth microdilution method (BMD) is demanding and requires expertise. We assessed the performance of a commercialized gradient diffusion method (Etest method) as an alternative to BMD. The MICs or minimal effective concentrations (MECs) of amphotericin B, voriconazole, posaconazole, caspofungin, and micafungin were assessed for 290 clinical isolates of the most representative pathogenic molds (154 Aspergillus and 136 non-Aspergillus isolates) with the BMD and Etest methods. Essential agreements (EAs) within ±2 dilutions of ≥90% between the two methods were considered acceptable. EAs for amphotericin B and voriconazole were >90% for most potentially susceptible species. For posaconazole, the correlation was acceptable for Mucoromycotina but Etest MIC values were consistently lower for Aspergillus spp. (EAs of <90%). Excellent EAs were found for echinocandins with highly susceptible (MECs of <0.015 μg/ml) or intrinsically resistant (MECs of >16 μg/ml) strains. However, MEC determinations lacked consistency between methods for strains exhibiting mid-range MECs for echinocandins. We concluded that the Etest method is an appropriate alternative to BMD for antifungal susceptibility testing of molds under specific circumstances, including testing with amphotericin B or triazoles for non-Aspergillus molds (Mucoromycotina and Fusarium spp.). Additional study of molecularly characterized triazole-resistant Aspergillus isolates is required to confirm the ability of the Etest method to detect voriconazole and posaconazole resistance among Aspergillus spp.     

Carbapenem Heteroresistance in VIM-1-Producing Klebsiella pneumoniae Isolates Belonging to the Same Clone: Consequences for Routine Susceptibility Testing

Susceptibility results with low reproducibility by the same or different methods have been observed for metallo-beta-lactamase (MBL)-producing Enterobacteriaceae. Eighteen VIM-1-producing Klebsiella pneumoniae isolates (one per patient) belonging to a single epidemic clone in our hospital (2005 to 2008) but with different susceptibilities to carbapenems were studied. Imipenem MICs ranged from 8 to >128 mg/liter by standard CLSI microdilution, from ≤1 to >8 mg/liter by the semiautomatic Wider system, and from 0.75 to >32 mg/liter by Etest. Meropenem MICs ranged from 0.5 to 128, ≤1 to >8, and 0.38 to >32 mg/liter, respectively. Ertapenem MICs by CLSI microdilution and Etest ranged from 1 to 64 and 0.75 to >32 mg/liter, respectively. The rates of essential agreement (±1 log₂ dilution) for imipenem and meropenem MICs between the Wider system and the reference microdilution method were 45% and 49%, respectively. Those between Etest and the reference microdilution method for imipenem, meropenem, and ertapenem MICs were 33%, 67%, and 84%. The rates of very major errors for the Wider system and Etest were 33% and 28% for imipenem and 25% and 75% for meropenem, respectively. Low MIC reproducibility was observed even when the same inoculum was used (differences up to 4-fold dilutions). Heteroresistance was suspected due to the presence of colonies in the Etest inhibition zone. It was confirmed by population analysis profiles of 4 isolates displaying different imipenem MICs, with the exception of an OmpK36-porin-deficient isolate that homogeneously expressed carbapenem resistance (MIC, >128 mg/liter). Low carbapenem MIC reproducibility could be due to the presence of resistant subpopulations and variable expression of the resistance mechanisms. Since carbapenem MICs are not good markers of MBL production, reliable and reproducible phenotypic methods are needed to detect the presence of this mechanism.Resistance to carbapenems due to metallo-beta-lactamases (MBLs) in Enterobacteriaceae is increasingly recognized, and different levels of resistance to carbapenems have been observed in these isolates (18). The detection of MBL-carrying isolates in clinical laboratories presents practical difficulties when routine susceptibility testing is performed (10, 28). Automated systems do not accurately detect enzyme-mediated carbapenem resistance when it is expressed at low levels, and MBL-carrying organisms can even appear to be fully susceptible to these compounds (9, 28). Additionally, reproducible results within the same method or among different methods, such as disk diffusion or Etest, are not always obtained, and discrepancies in the susceptibility to imipenem and meropenem of carbapenemase (KPC or VIM types)-producing Klebsiella pneumoniae isolates have already been reported (10, 28).In addition, some studies describing heteroresistance to carbapenems in Acinetobacter baumannii isolates demonstrate its presence by the growth of colonies in the inhibition zone of the Etest strip (12, 19). This phenomenon has also been reported for Klebsiella pneumoniae (28), Pseudomonas aeruginosa (20), and, more recently, Enterobacter aerogenes isolates (11).The objective of this work was to analyze the carbapenem susceptibility profiles of VIM-1-producing K. pneumoniae isolates by different susceptibility testing methods. These isolates belong to a single clone that was involved in an epidemic in our hospital (2005 to 2008) affecting 18 patients in different wards (25).     

The emergence of Clostridium difficile PCR ribotype 078 in piglets in the Czech Republic clusters with Clostridium difficile PCR ribotype 078 isolates from Germany,Japan and Taiwan

Clostridium difficile is a major nosocomial pathogen in humans with an increasing incidence in the community. The “one-health” approach of research is needed to investigate possible reservoirs of C. difficile and route of its transmission. The objective of this study is to investigate the occurrence of C. difficile in pigs in the Czech Republic with characterisation of the isolates to determine their genetic relatedness to C. difficile isolates from European and Asian pigs. A total of 198 pig faeces samples from 23 farms were investigated and of those 57 samples (55 piglets, 2 sows) from 11 farms were confirmed as C. difficile positive. The majority of C. difficile isolates belonged to the sequence type 11 and clade 5. The predominant ribotypes were 078 (n = 23), 078-variant (n = 5), 033 (n = 10) followed by RTs 150 (n = 7), 011 (n = 5), 045 (n = 4), 126, 014, 002 (n = 1, each). All isolates were susceptible to metronidazole, vancomycin and tetracycline. Isolates of RTs 150 and 078-variant were moxifloxacin resistant (MIC≥32 mg/L) and carried the amino acid substitution Thr82Ile in the GyrA. A multi-locus variable number tandem-repeats analysis (MLVA) revealed a clonal relatedness of isolates within individual farms and in C. difficile RT078 isolates between two Czech farms. Czech C. difficile RT078 isolates clustered with German C. difficile RT078 isolates and Czech C. difficile 078-variant isolates clustered with C. difficile RT078 isolates from Japan and Taiwan. This study found an emergence of C. difficile RT078 in Czech piglets that was related genetically to C. difficile RT078 isolates from Germany, Japan and Taiwan.     

Frequency of Resistance in Obligate Anaerobic Bacteria Isolated from Dogs,Cats, and Horses to Antimicrobial Agents

Clinical specimens from dogs, cats, and horses were examined for the presence of obligate anaerobic bacteria. Of 4,018 specimens cultured, 368 yielded 606 isolates of obligate anaerobic bacteria (248 from dogs, 50 from cats, and 308 from horses). There were 100 specimens from 94 animals from which only anaerobes were isolated (25 dogs, 8 cats, and 61 horses). The most common sites tested were abdominal fluid (dogs and cats) and intestinal contents (horses). The most common microorganism isolated from dogs, cats, and horses was Clostridium perfringens (75, 13, and101 isolates, respectively). The MICs of amoxicillin with clavulanate, ampicillin, chloramphenicol, metronidazole, and penicillin were determined using a gradient endpoint method for anaerobes. Isolates collected at necropsy were not tested for antimicrobial susceptibility unless so requested by the clinician. There were 1/145 isolates tested that were resistant to amoxicillin-clavulanate (resistance breakpoint ≥ 16/8 μg/ml), 7/77 isolates tested were resistant to ampicillin (resistance breakpoint ≥ 2 μg/ml), 4/242 isolates tested were resistant to chloramphenicol (resistance breakpoint ≥ 32 μg/ml), 12/158 isolates tested were resistant to clindamycin (resistance breakpoint ≥ 8 μg/ml), 10/247 isolates tested were resistant to metronidazole (resistance breakpoint ≥ 32 μg/ml), and 54/243 isolates tested were resistant to penicillin (resistance breakpoint ≥ 2 μg/ml). These data suggest that anaerobes are generally susceptible to antimicrobial drugs in vitro.     

High in vitro activity of fidaxomicin against Clostridium difficile isolates from a university teaching hospital in China

Background

Clostridium difficile infection (CDI) is a significant cause of morbidity and mortality in both the acute care setting and the wider healthcare system. The purpose of this study was to evaluate the in vitro activity of fidaxomicin against C. difficile isolates from a university teaching hospital in China.