[Show abstract][Hide abstract] ABSTRACT: Whole genome sequencing (WGS) could potentially provide a single platform for extracting all the information required to predict an organism's phenotype. However, its ability to provide accurate predictions has not yet been demonstrated in large independent studies for specific organisms. In this study we aimed to develop a genotypic prediction method for antimicrobial susceptibilities. 501 unrelated S. aureus isolates were whole genome sequenced and the assembled genomes interrogated using BLASTn for a panel of known resistant determinants (chromosomal mutations and genes carried on plasmids). Results were compared with phenotypic susceptibility testing for 12 commonly used antimicrobials (penicillin, methicillin, erythromycin, clindamycin, tetracycline, ciprofloxacin, vancomycin, trimethoprim, gentamicin, fusidic acid, rifampicin, mupirocin) performed by the routine clinical laboratory. We investigated discrepancies by repeat susceptibility testing and manual inspection of the sequences, and used this information to optimise the resistance determinant panel and BLASTn algorithm. We then tested performance of the optimised tool in an independent validation set of 491 unrelated isolates, with phenotypic results obtained in duplicate by automated broth dilution (BD Phoenix) and disc diffusion.In the validation set, overall sensitivity and specificity of the genomic prediction method were 0.97 (95% CI 0.95-0.98) and 0.99 (95% CI 0.99-1) respectively when compared to standard susceptibility testing methods. The very major error rate was 0.5% and the major error rate was 0.7%WGS was as sensitive and specific as routine antimicrobial susceptibility testing methods, and is a promising alternative to culture methods for resistance prediction in S. aureus and ultimately other major bacterial pathogens.
Journal of clinical microbiology 02/2014; · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Methicillin resistance in Staphylococcus spp. results from the expression of an alternative penicillin-binding protein 2a (encoded by mecA) with a low affinity for β-lactam antibiotics. Recently, a novel variant of mecA known as mecC (formerly mecALGA251) was identified in Staphylococcus aureus isolates from both humans and animals. In this study, we identified two Staphylococcus sciuri subsp. carnaticus isolates from bovine infections that harbour three different mecA homologues: mecA, mecA1 and mecC.
We subjected the two isolates to whole-genome sequencing to further understand the genetic context of the mec-containing region. We also used PCR and RT-PCR to investigate the excision and expression of the SCCmec element and mec genes, respectively.
Whole-genome sequencing revealed a novel hybrid SCCmec region at the orfX locus consisting of a class E mec complex (mecI-mecR1-mecC1-blaZ) located immediately downstream of a staphylococcal cassette chromosome mec (SCCmec) type VII element. A second SCCmec attL site (attL2), which was imperfect, was present downstream of the mecC region. PCR analysis of stationary-phase cultures showed that both the SCCmec type VII element and a hybrid SCCmec-mecC element were capable of excision from the genome and forming a circular intermediate. Transcriptional analysis showed that mecC and mecA, but not mecA1, were both expressed in liquid culture supplemented with oxacillin.
Overall, this study further highlights that a range of staphylococcal species harbour the mecC gene and furthers the view that coagulase-negative staphylococci associated with animals may act as reservoirs of antibiotic resistance genes for more pathogenic staphylococcal species.
Journal of Antimicrobial Chemotherapy 12/2013; · 5.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the reliability of cefoxitin and oxacillin for the detection of mecC-positive Staphylococcus aureus.
The susceptibility to cefoxitin and oxacillin of 62 mecC-positive S. aureus isolates was investigated using broth microdilution, agar dilution, Etest and disc diffusion on different types of media. The data were interpreted for the utility of cefoxitin and oxacillin in conjunction with the stated methodologies for the detection of mecC-positive isolates.
Cefoxitin with Mueller-Hinton media from Becton Dickinson and Oxoid detected all mecC-positive isolates when tested by broth microdilution, agar dilution and disc diffusion. By Etest, one isolate was falsely susceptible. Mueller-Hinton agar from bioMérieux was substantially less able to detect these isolates. One isolate was falsely susceptible by agar dilution when using Iso-Sensitest and Columbia agar. Disc diffusion using cefoxitin on Iso-Sensitest agar missed 29% of the isolates. For oxacillin, only agar dilution on Columbia agar + 2% NaCl was able to detect all mecC-positive isolates successfully.
Cefoxitin used with EUCAST methodology and oxacillin used with agar dilution on Columbia agar + 2% NaCl detected all mecC-positive isolates. These methods with their concomitant agars should be preferred over Iso-Sensitest, which is recommended by the BSAC. It should be noted that for disc diffusion Mueller-Hinton media from bioMérieux performed poorly, with 26%-47% of mecC isolates being falsely susceptible.
Journal of Antimicrobial Chemotherapy 09/2013; · 5.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Methods 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.
Results 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.
Conclusions 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.
Journal of Antimicrobial Chemotherapy 06/2013; · 5.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The emergence of mecC MRSA poses a diagnostic challenge for clinical microbiology laboratories. Using the VITEK 2 system, we tested a panel of 896 Staphylococcus aureus isolates, and found that an oxacillin sensitive / cefoxitin resistant profile had a sensitivity of 88.7% and a specificity of 99.5% for the identification of mecC MRSA isolates. The presence of the mecC gene, determined by bacterial whole-genome sequencing, was used as the gold standard. This profile could provide a zero-cost screening method for identification of mecC positive MRSA strains.
Journal of clinical microbiology 05/2013; · 4.16 Impact Factor