Antimicrobial susceptibility testing of Bacteroides species by disk diffusion: The NordicAST Bacteroides study

Objectives - Antimicrobial susceptibility testing (AST) of anaerobic bacteria has until recently been done by MIC methods. We have carried out a multi-centre evaluation of the newly validated EUCAST disk diffusion method for AST of Bacteroides spp. Methods - A panel of 30 Bacteroides strains was assembled based on reference agar dilution MICs, resistance gene detection and quantification of cfiA carbapenemase gene expression. Nordic clinical microbiology laboratories (n = 45) performed disk diffusion on Fastidious Anaerobe Agar with 5% mechanically defibrinated horse blood (FAA-HB) for piperacillin-tazobactam, meropenem and metronidazole. Results - A total of 43/45 (95.6%) laboratories carried out disk diffusion per protocol. Intraclass correlation coefficients were 0.87 (0.80–0.93) for piperacillin-tazobactam, 0.95 (0.91–0.97) for meropenem and 0.89 (0.83–0.94) for metronidazole. For metronidazole, one media lot yielded smaller zones and higher variability than another. Piperacillin-tazobactam and meropenem zone diameters correlated negatively with cfiA expression. A meropenem zone diameter of Conclusions - Inter-laboratory agreement by disk diffusion was good or very good. The main challenges were media-related variability for metronidazole and categorical disagreement with the reference method for piperacillin-tazobactam in some cfiA-positive strains. An area of technical uncertainty specific for such strains may be warranted

Molecular Characterisation of Trimethoprim Resistance in Escherichia coli and Klebsiella pneumoniae during a Two Year Intervention on Trimethoprim Use

BACKGROUND: Trimethoprim resistance is increasing in Enterobacteriaceae. In 2004-2006 an intervention on trimethoprim use was conducted in Kronoberg County, Sweden, resulting in 85% reduction in trimethoprim prescriptions. We investigated the distribution of dihydrofolate reductase (dfr)-genes and integrons in Escherichia coli and Klebsiella pneumoniae and the effect of the intervention on this distribution. METHODOLOGY/PRINCIPAL FINDINGS: Consecutively isolated E. coli (n = 320) and K. pneumoniae (n = 54) isolates phenotypically resistant to trimethoprim were studied. All were investigated for the presence of dfrA1, dfrA5, dfrA7, dfrA8, dfrA12, dfrA14, dfrA17 and integrons class I and II. Isolates negative for the seven dfr-genes (n = 12) were also screened for dfr2d, dfrA3, dfrA9, dfrA10, dfrA24 and dfrA26. These genes accounted for 96% of trimethoprim resistance in E. coli and 69% in K. pneumoniae. The most prevalent was dfrA1 in both species. This was followed by dfrA17 in E. coli which was only found in one K. pneumoniae isolate. Class I and II Integrons were more common in E. coli (85%) than in K. pneumoniae (57%). The distribution of dfr-genes did not change during the course of the 2-year intervention. CONCLUSIONS/SIGNIFICANCE: The differences observed between the studied species in terms of dfr-gene and integron prevalence indicated a low rate of dfr-gene transfer between these two species and highlighted the possible role of narrow host range plasmids in the spread of trimethoprim resistance. The stability of dfr-genes, despite large changes in the selective pressure, indirectly suggests a low fitness cost of dfr-gene carriage

How to: interpret MICs of antifungal compounds according to the revised clinical breakpoints v. 10.0 European committee on antimicrobial susceptibility testing (EUCAST)

BACKGROUND: EUCAST has revised the definition of the susceptibility category "I" from "Intermediate" to "Susceptible, Increased exposure". This implies that "I" can be used where the drug-concentration at the site of infection is high, either because of dose escalation or through other means to ensure efficacy. Consequently, "I" is no longer used as a buffer-zone to prevent technical fact

Performance of the EUCAST disc diffusion method and two MIC methods in detection of Enterobacteriaceae with reduced susceptibility to meropenem: the NordicAST CPE study.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked DownloadTo examine performance of EUCAST disc diffusion and supplementary MIC methods for detection of Enterobacteriaceae with reduced susceptibility to meropenem using EUCAST screening recommendations. Sixty-one Nordic laboratories delivered data on EUCAST disc diffusion (n = 61), semi-automated meropenem MIC (n = 23; VITEK2, n = 20 and Phoenix, n = 3) and gradient meropenem MIC (n = 58) methods. The strains (n = 27) included the major carbapenemase classes (A, n = 4; B, n = 9; D, n = 6) involved in the global spread of carbapenemase-producing Enterobacteriaceae (CPE) and non-CPE strains (n = 8) covering a range of broth microdilution (BMD) meropenem MICs. A triplicate Klebsiella variicola (meropenem MIC 0.5 mg/L) harbouring OXA-48 and Escherichia coli ATCC 25922 showed an overall good precision. Meropenem zone diameters below the EUCAST screening cut-off (<27 mm) were reported for strains with MIC ≥1 mg/L (n = 21), irrespective of resistance mechanism. For three strains (MIC = 0.5 mg/L) with OXA-48/-181, eight laboratories provided meropenem zone diameters above the screening cut-off. Very major errors (VMEs) were not observed. The overall distributions of major errors (MEs) and minor errors (mEs) were 9% and 36% (disc diffusion), 26% and 18% (VITEK2) and 7% and 20% (gradient MIC), respectively. Differences in ME and mE distributions between disc diffusion and MIC gradient tests compared with semi-automated methods were significant (P < 0.0001), using BMD MICs as a reference for categorization. The EUCAST disc diffusion method is a robust method to screen for CPE but isolates with meropenem MICs <1 mg/L pose challenges. The high ME rate in semi-automated methods might deter appropriate use of carbapenems in CPE infections with limited therapeutic options.Northern Norway Regional Health Authority (Helse Nord RHF) Norwegian Directorate of Healt

Biological Costs and Mechanisms of Fosfomycin Resistance in Escherichia coli

Fosfomycin is a cell wall inhibitor used mainly for the treatment of uncomplicated lower urinary tract infections. As shown here, resistance to fosfomycin develops rapidly in Escherichia coli under experimental conditions, but in spite of the relatively high mutation rate in vitro, resistance in clinical isolates is rare. To examine this apparent contradiction, we mathematically modeled the probability of resistance development in the bladder during treatment. The modeling showed that during a typical episode of urinary tract infection, the probability of resistance development was high (>10(−2)). However, if resistance was associated with a reduction in growth rate, the probability of resistance development rapidly decreased. To examine if fosfomycin resistance causes a reduced growth rate, we isolated in vitro and in vivo a set of resistant strains. We determined their resistance mechanisms and examined the effect of the different resistance mutations on bacterial growth in the absence and presence of fosfomycin. The types of mutations found in vitro and in vivo were partly different. Resistance in the mutants isolated in vitro was caused by ptsI, cyaA, glpT, uhpA/T, and unknown mutations, whereas no cyaA or ptsI mutants could be found in vivo. All mutations caused a decreased growth rate both in laboratory medium and in urine, irrespective of the absence or presence of fosfomycin. According to the mathematical model, the reduced growth rate of the resistant strains will prevent them from establishing in the bladder, which could explain why fosfomycin resistance remains rare in clinical isolates

Invasive pneumococcal disease in patients with an underlying pulmonary disorder.

Chronic pulmonary disease is a recognized risk factor for invasive pneumococcal disease (IPD). However, previous studies have often not been large enough to allow detailed analyses of less prevalent pulmonary diseases, and findings regarding case fatality have been inconsistent. We examined the associations between an underlying pulmonary disease and IPD, and the impact of these diseases on the case fatality rate. Patients with IPD ≥18 years of age, between 1990 and 2008, were identified in microbiological databases. The associations between IPD and the pulmonary diseases were assessed using conditional logistic regression, comparing IPD cases to ten control subjects per case, randomly selected from the general population (matched for gender, year of birth and county of residence). Adjustments were made for other co-morbidities, level of education and socio-economic status, 4085 cases of IPD and 40 353 controls were identified. A more than four-fold increased risk of IPD was seen in chronic obstructive pulmonary disease, a doubled risk in asthma and a five-fold increased risk in subjects with pulmonary fibrosis. In univariate analysis, sarcoidosis and bronchiectasis were associated with a two-fold to seven-fold increase in the risk of IPD, but there was no statistical support for the associations when adjustments for confounders were made. No increased risk was seen in subjects with a history of pneumoconiosis or allergic alveolitis. The mortality following IPD was not increased in patients with chronic obstructive pulmonary disease, asthma, pulmonary fibrosis or bronchiectasis. Several chronic pulmonary diseases increase the risk of IPD but mortality following IPD seems not to be affected

Performance of the EUCAST disk diffusion method, the CLSI agar screen method, and the Vitek 2 automated antimicrobial susceptibility testing system for detection of clinical isolates of enterococci with low- and medium-level VanB-type vancomycin resistance: A multicenter study

Different antimicrobial susceptibility testing methods to detect low-level vancomycin resistance in enterococci were evaluated in a Scandinavian multicenter study (n = 28). A phenotypically and genotypically well-characterized diverse collection of Enterococcus faecalis (n = 12) and Enterococcus faecium (n = 18) strains with and without nonsusceptibility to vancomycin was examined blindly in Danish (n = 5), Norwegian (n = 13), and Swedish (n = 10) laboratories using the EUCAST disk diffusion method (n = 28) and the CLSI agar screen (n = 18) or the Vitek 2 system (bioMérieux) (n = 5). The EUCAST disk diffusion method (very major error [VME] rate, 7.0%; sensitivity, 0.93; major error [ME] rate, 2.4%; specificity, 0.98) and CLSI agar screen (VME rate, 6.6%; sensitivity, 0.93; ME rate, 5.6%; specificity, 0.94) performed significantly better (P = 0.02) than the Vitek 2 system (VME rate, 13%; sensitivity, 0.87; ME rate, 0%; specificity, 1). The performance of the EUCAST disk diffusion method was challenged by differences in vancomycin inhibition zone sizes as well as the experience of the personnel in interpreting fuzzy zone edges as an indication of vancomycin resistance. Laboratories using Oxoid agar (P < 0.0001) or Merck Mueller-Hinton (MH) agar (P = 0.027) for the disk diffusion assay performed significantly better than did laboratories using BBL MH II medium. Laboratories using Difco brain heart infusion (BHI) agar for the CLSI agar screen performed significantly better (P = 0.017) than did those using Oxoid BHI agar. In conclusion, both the EUCAST disk diffusion and CLSI agar screening methods performed acceptably (sensitivity, 0.93; specificity, 0.94 to 0.98) in the detection of VanB-type vancomycin-resistant enterococci with low-level resistance. Importantly, use of the CLSI agar screen requires careful monitoring of the vancomycin concentration in the plates. Moreover, disk diffusion methodology requires that personnel be trained in interpreting zone edges

Evaluation of wild-type MIC distributions as a tool for determination of clinical breakpoints for Mycobacterium tuberculosis

Objectives: The aim of this study was to establish wild-type MIC distributions of first-line drugs for Mycobacterium tuberculosis, as well as to explore the usefulness of such distributions when setting clinical breakpoints. Methods: We determined the MICs of rifampicin, isonlazid and ethambutol for M. tuberculosis using a Middlebrook 7H10 dilution method for 90 consecutive clinical isolates, 8 resistant strains and 16 isolates from the WHO proficiency test panel. M. tuberculosis H37Rv was used for quality control and susceptibility results using 7H10 were compared with the results obtained with BACTEC460. Results: The agreement with BACTEC460 was very high for isonlazid (99.1%) and rifampicin (99.1%) but lower for ethambutol (94.7%). Intra- and inter-assay variation was below one MIC dilution. The MIC distributions for isoniazid and rifampicin provided a clear separation between susceptible and resistant strains. Regarding ethambutol, the current breakpoint for 7H10 (5 mg/L) is close to the wild-type and all strains (n=6) showing a disagreement between BACTEC460 and 7H10 were distributed very close to the breakpoint (MIC 4-8 mg/L). This problematic relation was confirmed by investigating isolates from the WHO panel with an agreement <95% (64%-88% among 26 laboratories, n=4) for which the MICs were 4-8 mg/L . Conclusions: Utilizing the wild-type MIC distribution was found to be as useful in M. tuberculosis as in other bacteria when setting clinical breakpoints. We suggest that the present clinical breakpoints should be re-evaluated, taking into account wild-type MIC distributions and available pharmacokinetic data