BSAC Methods Research Articles

Vancomycin MIC as a predictor of outcome in MRSA bacteraemia in the UK context

Received 29 November 2012; returned 20 February 2013; revised 16 May 2013; accepted 18 May 2013 OBJECTIVES: Raised vancomycin MICs have been associated with poor outcomes for methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia in the USA and mainland Europe. We investigated if this also applies in the UK, where EMRSA-15 (clonal complex 22) dominates. Isolates from UK patients receiving vancomycin therapy for MRSA bacteraemia in 2008-10 were collected, along with clinical details. Outcomes (i.e. patient survival or bacteraemia resolution) were reported 28 days after vancomycin therapy ended. The relationship between clinical outcome and MIC--as determined by CLSI and BSAC agar dilution methods--was assessed. Among 228 MRSA bacteraemias, 82% were caused by EMRSA-15; 65% of the patients were male and the median age was 70.5 years. MICs correlated between methods, but CLSI agar dilution testing gave a mode at 1 mg/L with only 12% of results either side, whereas the BSAC method gave a mode straddling 0.7-1 mg/L with <4% outliers. Twenty-three percent of patients died, with MRSA contributory in half; another 17% had unresolved bacteraemia at 28 days. Neither death nor unresolved bacteraemia was significantly associated with higher vancomycin MICs by either method. Rifampicin co-therapy had no quantifiable effect on outcome. The patient's age was the only significant correlate of patient outcome (P < 0.01); the underlying medical condition of the patient was important for the resolution of bacteraemia (P < 0.01), though not for overall mortality. Subtle vancomycin MIC differences did not correlate with worse outcomes for vancomycin monotherapy or for vancomycin/rifampicin co-therapy in MRSA bacteraemia. Regardless of the exact MIC-outcome relationship, detecting such small MIC differences seems unlikely to be reliable in routine laboratories.

Proposed disc zone breakpoints for doripenem for use with the BSAC disc susceptibility testing method

Sir, Doripenem is a new carbapenem, recently licensed in the European Union for treatment of nosocomial pneumonia (including ventilator-associated pneumonia), complicated intraabdominal infections and complicated urinary tract infections. A 10 mg doripenem disc has been proposed for routine susceptibility testing. We determined provisional zone breakpoints corresponding to European Committee on Antimicrobial Susceptibility Testing (EUCAST) MIC breakpoints for these 10 mg doripenem discs in the BSAC disc method. Isolates (n1⁄4810) were selected from the BSAC Bacteraemia Resistance Surveillance Project (2005–06), supplemented with additional, more-resistant, isolates from other survey collections and reference submissions held by the UK HPA’s Antibiotic Resistance Monitoring and Reference Laboratory. Care was taken, whenever possible, to select isolates for which the MICs corresponded with or were within one or two dilutions of the breakpoints. Zones and agar dilution MICs were determined by the BSAC methods. Results were reviewed against EUCAST/ European Medicines Agency (EMEA) MIC breakpoints, as also adopted by the BSAC, of: susceptible, ≤1 mg/L and resistant, .1 mg/L for streptococci; and susceptible, ≤1 mg/L and resistant, .4 mg/L for staphylococci, enterococci, Enterobacteriaceae, Acinetobacter spp. and Pseudomonas spp. Error minimization was used to optimize the values. Zone breakpoints corresponding to these MIC breakpoints were as follows:

Decline of EMRSA-16 amongst methicillin-resistant Staphylococcus aureus causing bacteraemias in the UK between 2001 and 2007

Between 1998 and 2000, 95.6% of methicillin-resistant Staphylococcus aureus (MRSA) bacteraemias in the UK were due to two epidemic strains, namely EMRSA-15 or EMRSA-16 (60.2% and 35.4%, respectively). We sought to determine the proportions of these strains before and after the general decline in MRSA bacteraemia that began around 2004. Consecutive MRSA isolates collected in 2001, 2003, 2005 and 2007 by the BSAC Bacteraemia Surveillance Programme were categorized to multilocus sequence typing (MLST) clonal complex and to SCCmec type by PCR. MICs were determined by the BSAC method. Data trends were tested for significance using a generalized linear regression model. Collectively, EMRSA-15 and EMRSA-16 consistently accounted for approximately 95% of MRSA studied between 2001 and 2007, but the proportions of EMRSA-16 declined from 21.4% in 2001 to 9% in 2007 (P < 0.05), whilst the proportion of EMRSA-15 rose commensurately, accounting for 85% of MRSA in 2007. Ciprofloxacin and erythromycin resistance were common amongst both EMRSA-15 and EMRSA-16. EMRSA-15 and EMRSA-16 remain the main MRSA strains in bacteraemia in the UK, but the proportion of EMRSA-16 declined from the late 1990 s, thus preceding the general decline in MRSA bacteraemias that began in the middle of the present decade.

Determination of disc breakpoints and evaluation of Etests for tigecycline susceptibility testing by the BSAC method

Tigecycline has been recently licensed in Europe for intra-abdominal and complicated skin and soft tissue infections. We determined zone breakpoints for use with 15 microg tigecycline discs and evaluated Etests for the routine determination of tigecycline susceptibility by BSAC methods. Disc zones for 2236 isolates and MICs by Etest for 531 isolates were compared with MICs obtained by the BSAC agar dilution method. Based on error minimization, we propose zone breakpoints for 15 microg tigecycline discs as follows: alpha/beta-haemolytic streptococci, S > or = 25 mm, R < or = 19 mm; Acinetobacter spp. and Enterobacteriaceae, S > or = 24 mm, R < or = 19 mm; Enterococcus spp., S > or = 21 mm, R < or = 20 mm; Haemophilus spp., S > or = 28 mm, R < or = 27; Streptococcus pneumoniae, S > or = 24 mm, R < or = 23 mm; and staphylococci, S > or = 26 mm, R < or = 25 mm. These criteria gave overall false resistance rates of < or =0.8% and false susceptibility rates of < or =0.7%. Tigecycline Etests, used on Iso-Sensitest agar, gave MICs within one doubling dilution of those by agar dilution in 97% of cases. Categorization agreement was good for isolates with borderline susceptibility or resistance-a group where Etests are likely to be used in order to verify disc-based results. MICs for highly susceptible alpha-haemolytic streptococci were underestimated by Etest, but this seems unlikely to be significant. Disc breakpoints corresponding to BSAC MIC breakpoints were defined for 15 microg tigecycline discs and have been adopted by the BSAC. Tigecycline Etest gave results in good agreement with agar dilution.

Antimicrobial susceptibility of community-acquired respiratory tract pathogens in the UK during 2002/3 determined locally and centrally by BSAC methods

To determine the antimicrobial susceptibility of Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae causing community-acquired lower respiratory tract infection in the UK during 2002/2003 and to compare susceptibilities determined locally by disc diffusion with agar dilution MICs determined at a central laboratory. H. influenzae, M. catarrhalis and S. pneumoniae were isolated in 30 laboratories and susceptibility determined locally by the BSAC standardized disc diffusion method. At a central laboratory, isolates were re-identified, tested for beta-lactamase production (H. influenzae and M. catarrhalis only) and MICs determined using the BSAC agar dilution method. Five hundred and eighty-one H. influenzae, 269 M. catarrhalis and 519 S. pneumoniae were collected. Over 93% of M. catarrhalis and nearly 15% of H. influenzae were beta-lactamase positive rendering these sub-populations resistant to aminopenicillins. Overall, the antibacterial susceptibility rates for the isolates were high. However, macrolides showed poor activity against H. influenzae (0.86-1.38% susceptible by disc or MIC methods) and, compared with other antimicrobials, against S. pneumoniae (approximately 88% susceptible). Between 84% and 95% of H. influenzae, M. catarrhalis and S. pneumoniae were susceptible to cefuroxime but all isolates were susceptible to cefotaxime. Eighty-five percent of H. influenzae were susceptible to trimethoprim. The fluoroquinolones were very active against the isolates, with moxifloxacin showing lower MICs than levofloxacin against S. pneumoniae. Susceptibility determined locally by disc diffusion was in general agreement with that determined centrally by agar dilution MIC testing. However, there was one inconsistency with H. influenzae where disc diffusion indicated 22.9% and 46.8% resistance to clarithromycin and erythromycin, respectively but by MIC, only 0.9% and 6.9% were resistant, respectively. Rates of resistance within community-acquired respiratory tract isolates were relatively low in the UK, in agreement with other studies. Moxifloxacin was the only antibacterial with over 99% isolates susceptible for each of the three pathogens investigated where breakpoints are available. The comparison between disc susceptibility testing and MIC determination using BSAC methods indicated generally good correlation but has highlighted a methodological problem with macrolides against H. influenzae in particular.

Activity of dalbavancin against staphylococci and streptococci, assessed by BSAC and NCCLS agar dilution methods.

Dalbavancin is a long-half-life (9-12 days) glycopeptide, now in Phase 3 development. Its pharmacokinetics may facilitate home intravenous therapy, early discharge and long prophylaxis. Dalbavancin and comparators were tested in vitro against staphylococci and streptococci to determine (i) activity and (ii) the comparability of agar dilution MICs by the BSAC and the NCCLS methods. The test panels comprised 92-93 isolates each of Staphylococcus aureus, coagulase-negative staphylococci (CoNS) and 'viridans' streptococci, chosen for epidemiological diversity and to over-represent strains resistant to conventional agents, including teicoplanin. Dalbavancin MICs by the BSAC and NCCLS methods generally were identical, or else those by the BSAC method were two-fold lower. In both cases the MIC distributions of dalbavancin within species groups were unimodal, with peaks at 0.25, 0.12/0.25 and 0.12 mg/L for S. aureus, CoNS and viridans streptococci, respectively. MIC differences between the BSAC and NCCLS methods were similarly small for other glycopeptides and, generally, non-glycopeptides. Dalbavancin MICs were mostly two- to 16-fold below those of vancomycin and dalbavancin-like vancomycin-remained highly active against teicoplanin-non-susceptible staphylococci. Dalbavancin has good activity versus streptococci and staphylococci, including teicoplanin-resistant strains. MICs by BSAC and NCCLS agar dilution methods were comparable to each other; slightly lower MIC values, nevertheless, have been recorded by broth microdilution.

Susceptibility of pneumococci to evernimicin: effect of CO2 and different methodologies

Accurate determination of the antibiotic susceptibility of pathogens is essential for both patient management and epidemiologic purposes. Unfortunately, there are differences in methodology (e.g. media, inoculum, incubation conditions) and interpretation (e.g. numerical values of breakpoint concentrations and zone sizes) between recommendations approved by various organizations. Results using different methods have not been correlated for all antibiotics. Such differences appear to be of particular importance in the case of pneumococci, as incubation in the presence of CO2 is recommended by some but not all testing protocols. For example, incubation in CO2 is recommended by the NCCLS and BSAC for agar diffusion [1,2], by the Swedish guidelines [3] and for the E test [4]. On the other hand, incubation in air is suggested by the NCCLS for broth dilution [5], and by BSAC for agar dilution [6]. The European Committee [7] advises the use of CO2 only when ‘necessary’, while French guidelines [8] do not address this problem. CO2 is a well recognized confounding factor in susceptibility testing, as, among other factors, the reduction in pH it engenders may significantly alter the apparent activity of some antibiotics. Thus, ideally it should be used routinely only when absolutely necessary for the growth of the organism being tested (e.g. for gonococci), which, notwithstanding the recommendationsmentioned above, is not the case for pneumococci.We have recently found (unpublished) that only six of 175 freshly isolated strains of pneumococci (3.4%) were strictly capnophilic. We wish to illustrate discrepancies that can arise between results obtained using different methods, giving as an example comparative data recently obtained with evernimicin, a member of the orthosomycin family of polysaccharide antibiotics [9]. MICs determined by plate dilution were compared with results of E tests, using different media and incubation conditions, as recommended by the NCCLS, the BSAC and AB Biodisk (the manufacturers of the E test). Evernimicin has been shown to diffuse too poorly to be tested by the standard disk diffusion method [10]. MICs of evernimicin (SCH 27899, kindly supplied by the Schering Plough Research Institute) against 50 clinical isolates of Streptococcus pneumoniae were determined by four different methods: agar dilution, according to NCCLS and BSAC recommendations, and E test using Mueller–Hinton agar (MHA) and IsoSensitest agar (ISA). In addition, the effect of CO2 on the activity of evernimicin was investigated by testing 20 of the strains on MHA incubated in CO2 (i.e. standard NCCLS conditions) and in air. Results of E tests were read using an endpoint of 80% inhibition, as recommended by Marshall et al [11]. Media were obtained from Unipath Ltd, Basingstoke, UK. The control strain was S. pneumoniae ATCC 49619, as recommended by the NCCLS [5]. Table 1 shows that MICs obtained using the BSAC method were almost double those determined according to the NCCLS protocol. This difference can be explained by the effect of CO2—present in the test conducted following NCCLS guidelines, absent when using the BSACmethod. From Table 2 it can be seen that under otherwise identical conditions, incubation in CO2 causes a two-fold reduction in the MIC of evernimicin. Marshall et al [11] and Jones and Barrett [12] have, respectively, shown that MICs for pneumococci in MH medium incubated in CO2 are virtually identical in broth and agar, and our results (Table 1) agree very closely with those of the latter workers. The results of E tests in the different media (both carried out in the presence of CO2) were almost identical, but consistently lower than the respective results obtained by the dilution method. A partial explanation of this may be that, as recommended, E test endpoints were read at 80% inhibition, whereas the plate MIC was read, as customary, at 100% inhibition. However, there was little or no discrepancy between E test and plate dilution results when staphylococci and enterococci were tested in a similar manner (unpublished results), so this may be a phenomenon peculiar to pneumococci. It is not clear which of theMIC values obtained is ‘correct’ in terms of prediction of therapeutic efficacy. The results here