Associated antimicrobial resistance in Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus pyogenes

AbstractAssociated resistance to four to six related and unrelated antimicrobial agents was investigated in consecutive non-duplicate isolates of Escherichia coli (n = 39 425), Pseudomonas aeruginosa (n = 1070), Staphylococcus aureus (n = 7489), Streptococcus pneumoniae (n = 1604) and Streptococcus pyogenes (n = 2531). In all species, high proportions (76.5–88.9%) of isolates were susceptible to all the drugs investigated. Irrespective of species, isolates resistant to one drug were more likely to be resistant to any of the other drugs than were susceptible isolates. Thus, trimethoprim resistance in E. coli was 38.4% among ampicillin-resistant vs. 3.9% among ampicillin-susceptible isolates, and erythromycin resistance in Strep. pneumoniae was 41% among doxycycline-resistant vs. 1% among doxycycline-susceptible isolates. In all five species investigated, there was also significant associated resistance among unrelated drugs, highlighting the fact that resistance development occurs primarily among bacteria already resistant to one or more antimicrobial agents. For the clinician, pronounced resistance associations mean that when empirical therapy fails because of resistance, there is a reduced chance of choosing an alternative successful empirical agent. For the epidemiologist, who uses routine clinical susceptibility data to describe resistance development, resistance associations mean that if the dataset contains results for isolates selected on the basis of their susceptibility to another drug, structurally related or not, a bias of false resistance is introduced

The need for European professional standards and the challenges facing clinical microbiology.

Microorganisms spread across national boundaries and the professional activities of clinical (medical) microbiologists are critical in minimising their impact. Clinical microbiologists participate in many activities, e.g. diagnosis, antibiotic therapy, and there is a need for a set of professional standards for Europe with a common curriculum, to build upon the current strengths of the specialty and to facilitate the free movement of specialists within the European Union. Such standards will also better highlight the important contribution of clinical microbiologists to healthcare. There is a move to larger centralised microbiology laboratories often located off-site from an acute hospital, driven by the concentration of resources, amalgamation of services, outsourcing of diagnostics, automation, an explosion in the range of staff competencies and accreditation. Large off-site centralised microbiology laboratories are often distant to the patient and may not facilitate the early detection of microbial spread. Ultimately, the needs of patients and the public are paramount in deciding on the future direction of clinical microbiology. Potential conflicts between integration on an acute hospital site and centralisation can be resolved by a common set of professional standards and a team-based approach that puts patients first

A new method for normalized interpretation of antimicrobial resistance from disk test results for comparative purposes.

Objective To evaluate a calibration method for disk diffusion antibiotic susceptibility tests, using zone diameter values generated in the individual laboratory as the internal calibrator for combinations of antibiotic and bacterial species. Methods The high-zone side of zone histogram distributions was first analyzed by moving averages to determine the peak position of the susceptible population. The accumulated percentages of isolates for the high zone diameter values were calculated and converted into probit values. The normal distribution of the ideal population of susceptible strains was then determined by using the least-squares method for probit values against zone diameters, and the ideal population was thereby defined, including mean and standard deviation. Zone diameter values were obtained from laboratories at the Karolinska Hospital (KS) and Växjö Hospital (VX), and from two laboratories (LabA, LabB) in Argentina. The method relies on well standardized disk tests, but is independent of differences in MIC limits and zone breakpoints, and does not require the use of reference strains. Resistance was tentatively set at below 3 SD from the calculated, ideal mean zone diameter of the susceptible population. Results The method, called normalized interpretation of antimicrobial resistance, was tested on results from the KS and VX clinical microbiology laboratories, using the disk diffusion method for antimicrobial susceptibility tests, and for two bacterial species, Staphylococcus aureus and Escherichia coli. In total, 114 217 test results were included for the clinical isolates, and 3582 test results for control strains. The methodology at KS and VX followed the standard of the Swedish Reference Group for Antibiotics (SRGA). Zone diameter histograms for control strains were first analyzed to validate the procedure, and a comparison of actual means with the calculated means showed a correlation coefficient of r = 0.998. Results for clinical isolates at the two laboratories showed an excellent agreement for 54 of 57 combinations of antibiotic and bacterial species between normalized interpretations and the interpretations given by the laboratories. There were difficulties with E. coli and mecillinam, and S. aureus and tetracycline and rifampicin. The method was also tested on results from two laboratories using the NCCLS standard, and preliminary results showed very good agreement with quality-controlled laboratory interpretations. Conclusions The normalized resistance interpretation offers a new approach to comparative surveillance studies whereby the inhibition zone diameter results from disk tests in clinical laboratories can be used for calibration of the test

Little difference between minimum inhibitory concentrations of Mycobacterium tuberculosis wild-type organisms determined with BACTEC MGIT 960 and Middlebrook 7H10

AbstractThe MIC wild-type (WT) distribution for Mycobacterium tuberculosis in BACTEC 960 MGIT is not defined, which may result in poor reproducibility for drug susceptibility testing (DST), as several DST methods with different breakpoints are in use. In a comparison between MGIT and Middlebrook 7H10 medium of seven first- and second-line drugs, including 133 MIC determinations of 15 WT isolates, we found an agreement of 91.7% within ± one MIC dilution step. The results confirm the agreement in MIC testing between 7H10 and MGIT and indicate that breakpoints could be harmonized in order to avoid misclassification

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

AbstractWith 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 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

EUCAST rapid antimicrobial susceptibility testing (RAST) in blood cultures: Validation in 55 european laboratories

Objectives: When bloodstream infections are caused by resistant bacteria, rapid antimicrobial susceptibility testing (RAST) is important for adjustment of therapy. The EUCAST RAST method, directly from positive blood cultures, was validated in a multi-laboratory study in Europe.Methods: RAST was performed in 40 laboratories in northern Europe (NE) and 15 in southern Europe (SE) from clinical blood cultures positive for Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus or Streptococcus pneumoniae. Categorical results at 4, 6 and 8 h of incubation were compared with results for EUCAST standard 16-20 h disc diffusion. The method, preliminary breakpoints and the performance of the laboratories were evaluated.Results: The total number of isolates was 833/318 in NE/SE. The number of zone diameters that could be read (88%, 96% and 99%) and interpreted (70%, 81% and 85%) increased with incubation time (4, 6 and 8 h). The categorical agreement was acceptable, with total error rates in NE/SE of 2.4%/4.9% at 4 h, 1.1%/3.5% at 6 h and 1.1%/3.3% at 8 h. False susceptibility at 4, 6 and 8 h of incubation was below 0.3% and 1.1% in NE and SE, respectively, and the corresponding percentages for false resistance were below 1.9% and 2.8%. After fine-tuning breakpoints, more zones could be interpreted (73%, 89% and 93%), with only marginally affected error rates.Conclusions: The EUCAST RAST method can be implemented in routine laboratories without major investments. It provides reliable antimicrobial susceptibility testing results for relevant bloodstream infection pathogens after 4-6 h of incubation.</p

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