BSAC standardized disc susceptibility testing method (version 11).

This article highlights key amendments incorporated into version 11 of the BSAC standardized disc susceptibility testing method, available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/) and on the BSAC web site (http://bsac.org.uk/susceptibility/guidelines-standardized-disc-susceptibility-testing-method/). The basic disc susceptibility testing method remains unchanged, but there have been a number of alterations to the interpretive criteria for certain organism/drug combinations due to continuing harmonization with the EUCAST MIC breakpoints and constant efforts to improve the reliability and clinical applicability of the guidance.

BSAC standardized disc susceptibility testing method (version 10).

The BSAC standardized disc susceptibility testing method remains unchanged, but there are considerable changes to the interpretative criteria due to continuing harmonization with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) MIC breakpoints. There are a number of agents for which interpretative criteria have been removed. These MIC and/or zone diameter breakpoints will be published on the BSAC web site as a 'Legacy' table; they may be used for research or comparative purposes, but are not recommended for clinical management. Notably, testing of staphylococci for susceptibility to glycopeptides by disc diffusion has been removed because this method has been found to be unreliable, particularly for the detection of low-level resistance; low-level vancomycin resistance in staphylococci is increasingly deemed to be of clinical relevance. The tables for anaerobes have been expanded to include MIC breakpoints that have been determined by EUCAST. There are currently no zone diameter breakpoints for these organisms and an MIC method is recommended if susceptibility testing is required.

Emergence of high-level azithromycin resistance in Neisseria gonorrhoeae in England and Wales.

OBJECTIVES: This study aimed to investigate the origin of high-level azithromycin resistance that emerged in isolates of Neisseria gonorrhoeae in England and Wales in 2007, and to establish methods for identifying high-level azithromycin resistance. METHODS: The Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP) data from 2001-07 were examined for emerging trends in azithromycin susceptibility. Further to the identification of six high-level azithromycin-resistant isolates in GRASP 2007, an additional 102 isolates were selected on the basis of azithromycin susceptibility and geographic origin from the GRASP 2006 and 2007 collections. Susceptibility testing by Etest and disc diffusion was performed on all 108 isolates and 75 of these were typed by N. gonorrhoeae multiantigen sequence typing. RESULTS: A slight drift towards higher MICs of azithromycin was observed in the gonococcal population since 2001. Of greater concern was the first example of a shift to high-level resistance observed in six isolates in 2007. All six isolates were sequence type 649, which was not observed in any of the lower-level azithromycin-resistant isolates from 2007 or in any isolates tested from the same geographical locations. Contact tracing data for one patient suggested a link with Scotland. Disc diffusion testing of all 108 isolates showed that azithromycin, but not erythromycin, discs can differentiate between low-level and high-level resistance. CONCLUSIONS: High-level azithromycin resistance has emerged in England and Wales. Contact tracing and typing data suggest this may have originated from Scotland. Surveillance of azithromycin resistance will be key in controlling its further dissemination.

Genetic & virulence profiling of ESBL-positive E. coli from nosocomial & veterinary sources.

CTX-M genes are the most prevalent ESBL globally, infiltrating nosocomial, community and environmental settings. Wild and domesticated animals may act as effective vectors for the dissemination of CTX-producing Enterobacteriaceae. This study aimed to contextualise blaCTX-M-14-positive, cephalosporin-resistant Enterobacteriaceae human infections and compared resistance and pathogenicity markers with veterinary isolates. Epidemiologically related human (n=18) and veterinary (n=4) blaCTX-M-14-positive E. coli were fully characterised. All were typed by XbaI pulsed field gel electrophoresis and ST. Chromosomal/plasmidic locations of blaCTX-M-14 were deduced by S1-nuclease digestion, and association with ISEcp1 was investigated by sequencing. Conjugation experiments assessed transmissibility of plasmids carrying blaCTX-M-14. Presence of virulence determinants was screened by PCR assay and pathogenicity potential was determined by in vitro Galleria mellonella infection models. 84% of clinical E. coli originated from community patients. blaCTX-M-14 was found ubiquitously downstream of ISEcp1 upon conjugative plasmids (25-150 kb). blaCTX-M-14 was also found upon the chromosome of eight E. coli isolates. CTX-M-14-producing E. coli were found at multiple hospital sites. Clonal commonality between patient, hospitals and livestock microbial populations was found. In vivo model survival rates from clinical isolates (30%) and veterinary isolates (0%) were significantly different (p<0.05). Co-transfer of blaCTX-M-14 and virulence determinants was demonstrated. There is evidence of clonal spread of blaCTX-M-14-positive E. coli involving community patients and farm livestock. blaCTX-M-14 positive human clinical isolates carry a lower intrinsic pathogenic potential than veterinary E. coli highlighting the need for greater veterinary practices in preventing dissemination of MDR E. coli among livestock.

Documentation of blood culture results.

AIMS: To evaluate the adequacy of documentation of blood culture results in patients' medical notes. METHODS: A pro-forma was completed following review of medical notes at 24 and 48 hours after a blood culture had been reported as positive. The study was performed on blood cultures received at the Department of Microbiology, Royal Hallamshire Hospital, Sheffield, from two local hospitals. Two periods were studied: (A) May to June 1993 and (B) September to October 1993. RESULTS: There were 43 results studied in period A and 79 in period B, giving a total of 122 results studied. Overall, 72 (59%) of 122 results were recorded in the medical notes at 24 hours. Of those results deemed highly significant, 40 (63%) of 63 were recorded. There was no significant difference in the documentation of results if the result was given personally or via the telephone. Nor was there any difference in documentation between different medical grades. Throughout the study there were six inaccurate records. The cumulative documentation over 48 hours of positive results was 54 (86%) of 63 of highly significant, 27 (69%) of 39 of uncertain significance, and 11 (55%) of 20 probable contaminant results. CONCLUSIONS: Documentation of blood culture results is currently suboptimal.

Cotrimoxazole. Rationale for re-examining its indications for use.

Trimethoprim was specifically developed in the late 1960s as a sulphonamide potentiator and was launched in combination with sulfamethoxazole as cotrimoxazole. Laboratory data showed synergy of antimicrobial action for the combination and suggested that the use of both agents would delay the emergence of resistance. However, the tissue distribution of trimethoprim and sulfamethoxazole does not favour synergy, and resistance among common pathogens to sulfamethoxazole is high. Clinical studies comparing trimethoprim alone with cotrimoxazole for the treatment of respiratory tract and urinary tract infections have failed to show any benefit from the combination. The development of delayed resistance by use of the combination has not been substantiated. The common adverse effects seen with cotrimoxazole are gastrointestinal disturbances and skin rashes which are well described adverse effects of sulphonamides. Comparative studies suggest that these are less common with trimethoprim alone. Serious adverse effects such as liver disorders and Stevens-Johnson syndrome appear more common with cotrimoxazole. Where there is little evidence for benefit from the use of the combination, the exposure of patients to the additional risk from the adverse effects and drug interactions of 2 drugs cannot be justified. Therefore use of cotrimoxazole should be restricted to those situations such as Pneumocystis carnii pneumonia where the combination has been shown to be beneficial.

Campylobacter fetus subspecies fetus septicaemia: SDS-PAGE as an aid to speciation.

We describe a case of bacteraemia due to an atypical strain of Campylobacter fetus ssp. fetus. Conventional biochemical and phenotypic characterisation was unhelpful but whole cell protein profiles obtained by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) allowed us to identify this strain.

Susceptibility testing of Stenotrophomonas maltophilia to carbapenems.

The susceptibility of 20 clinical isolates of Stenotrophomonas maltophilia to the carbapenems imipenem and meropenem was investigated by various methods. S. maltophilia appeared sensitive to meropenem but resistant to imipenem by disc testing in Iso-sensitest agar. Agar dilution MICs were performed using Iso-sensitest agar and with incubation under three sets of atmospheric conditions. MICs of meropenem were considerably lower than those of imipenem; this effect was maximal after incubation in 5% CO2 when the MIC of meropenem was approximately 64 times less than that of imipenem. Induction experiments showed that both carbapenems could induce production of the L1 carbapenemase. However, disc approximation tests showed that imipenem could induced resistance to meropenem. Partially stably derepressed mutants were readily selected in vitro. We conclude that, although S. maltophilia may give large zones of inhibition to meropenem on disc testing, resistant mutants are readily selected and therefore standard sensitivity tests may be poorly predictive of clinical outcome of treatment of S. maltophilia infections with meropenem.

Interactions between methicillin and vancomycin in methicillin-resistant Staphylococcus aureus strains displaying different phenotypes of vancomycin susceptibility.

Vancomycin-sensitive, -intermediate, and -heterointermediate methicillin-resistant Staphylococcus aureus isolates were tested by using E-tests to explore the interaction of methicillin and vancomycin. For the vancomycin-intermediate and -heterointermediate strains both drugs showed antagonism at concentrations below their MICs but synergy at methicillin concentrations near the MIC. This property could be used to screen for heterointermediate S. aureus strains.

Expression and detection of hetero-vancomycin resistance in Staphylococcus aureus.

Isolates of Staphylococcus aureus resistant to vancomycin have been reported but appear to be extremely rare. However, isolates displaying hetero-resistance to vancomycin (hVRSA) are reportedly common in parts of Japan (9.3% of MRSA isolated from a group of university hospitals). We have investigated the reliability of the proposed method for detection of hetero-resistant isolates and the ability of clinical S. aureus isolates to express vancomycin resistance. The original method for identification of hVRSA was found to have poor reproducibility and may select for, rather than detect, vancomycin resistance. There appears to be a spectrum of heterogeneity in the expression of resistance to vancomycin among S. aureus. Until there is a clearer understanding of the mechanism and control of vancomycin resistance in S. aureus, and reliable tests are devised, the clinical relevance of different degrees of hetero-resistance cannot be assessed.

Identifying bacteria in human urine: current practice and the potential for rapid, near-patient diagnosis by sensing volatile organic compounds.

Urinary tract infection (UTI) represents a significant burden for the National Health Service. Extensive research has been directed towards rapid detection of UTI in the last thirty years. A wide range of microbiological and chemical techniques are now available to identify and quantify bacteria in urine. However, there is a clear and present need for near, rapid, sensitive, reliable analytical methods, preferably with low-running costs, that could allow early detection of UTI and other diseases in urine. Here we review the "state of the art" of current practice for the detection of bacteria in urine and describe the advantages of the recent "e-nose" technology as a potential tool for rapid, near-patient diagnosis of UTI, by sensing volatile organic compounds (VOCs).

A modified population analysis profile (PAP) method to detect hetero-resistance to vancomycin in Staphylococcus aureus in a UK hospital.

One hundred methicillin-resistant Staphylococcus aureus (MRSA) strains, isolated between 1983 and 1999, were tested alongside the vancomycin hetero-resistant S. aureus (hVRSA) strain Mu 3, and vancomycin-resistant S. aureus (VRSA) strain Mu 50, for their resistance to vancomycin. This was achieved using the screening method described by Hiramatsu, gradient plates, agar incorporation, standard Etest, macrodilution Etest and a modified population analysis. Using Hiramatsu's screening method, 5% of the 100 MRSA were identified as VRSA and 5% identified as hVRSA, the gradient plates identified 7% hVRSA, and the standard and macrodilution Etests identified no hVRSA. Mu 3 appeared to be vancomycin-susceptible using both the agar incorporation and standard Etest methods, but was classified as hVRSA using the macrodilution Etest. The modified population analysis reliably detected vancomycin hetero-resistance in Mu 3 and identified no hVRSAs within the 100 MRSA sample.

The activity of vancomycin against heterogeneous vancomycin-intermediate methicillin-resistant Staphylococcus aureus explored using an in vitro pharmacokinetic model.

Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) may account for treatment failure with vancomycin and act as a precursor of vancomycin-intermediate or -resistant S. aureus. The activity of vancomycin was assessed against vancomycinsusceptible, hVISA and VISA strains in a dilutional pharmacokinetic model. Over a 48 h period, total bacteria and cells with a vancomycin-intermediate phenotype were quantified. Total counts of hVISA were reduced by vancomycin in a similar way to a vancomycin-susceptible control. The vancomycin-intermediate sub-population was eradicated from the model within one dose interval. Exposure to low vancomycin concentrations did not result in an increase in the proportion of cells which were vancomycin intermediate. Short-term exposure of hVISA to vancomycin at gradient concentrations did not increase the proportion of cells with vancomycin-intermediate phenotype.

Evaluation of current methods for detection of staphylococci with reduced susceptibility to glycopeptides.

The sensitivity and specificity of seven methods (agar dilution, broth microdilution, Etest at 0.5 and 2.0 McFarland (McF) inocula, two agar screening methods, and population studies [PS]) were evaluated in a double-blind study involving 284 methicillin-resistant Staphylococcus aureus (MRSA) strains and 45 Staphylococcus strains with reduced susceptibilities to vancomycin (SRSV). The results were compared to the population analysis profile-area under the curve ratio method (PAP-AUC ratio compared to that of Mu3) as described by Wootton et al. The agar screening method using brain heart infusion agar (6 microg of vancomycin per ml) gave a sensitivity of 22% and a specificity of 97%. A similar method using Mueller-Hinton agar (5 microg of vancomycin per ml) gave a sensitivity of 20% and a specificity of 99%. The PS method detected 34 false positives (12%) and gave a sensitivity of 71% and a specificity of 88%. Etest using 0.5 and 2.0 McF inocula gave sensitivities and specificities of 82 and 93% and of 96 and 97%, respectively. The best Etest interpretative criteria for the 2.0 McF inoculum was > or =8 mg of vancomycin per liter and > or =8 microg teicoplanin per ml or > or =12 microg of teicoplanin per ml. The direct colony suspension inoculum for this method was found to be equally accurate in detecting (hetero-)glycopeptide-intermediate S. aureus compared to the overnight broth inoculum preparation method. Agar dilution and broth microdilution using the NCCLS breakpoint criteria for vancomycin gave sensitivities and specificities of 20 and 100% and of 11 and 100%, respectively. Using the Etest with a 2.0 McF inoculum, six different media were assessed against a selection of SRSV (n = 48) and MRSA (n = 12). Brain heart infusion agar yielded the highest sensitivity and specificity values: 88 and 88%, respectively.