Robert Hill

Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study

Summary Background Animals can act as a reservoir and source for the emergence of novel meticillin-resistant Staphylococcus aureus (MRSA) clones in human beings. Here, we report the discovery of a strain of S aureus (LGA251) isolated from bulk milk that was phenotypically resistant to meticillin but tested negative for the mecA gene and a preliminary investigation of the extent to which such strains are present in bovine and human populations. Methods Isolates of bovine MRSA were obtained from the Veterinary Laboratories Agency in the UK, and isolates of human MRSA were obtained from diagnostic or reference laboratories (two in the UK and one in Denmark). From these collections, we searched for mecA PCR-negative bovine and human S aureus isolates showing phenotypic meticillin resistance. We used whole-genome sequencing to establish the genetic basis for the observed antibiotic resistance. Findings A divergent mecA homologue (mecALGA251) was discovered in the LGA251 genome located in a novel staphylococcal cassette chromosome mec element, designated type-XI SCCmec. The mecALGA251 was 70% identical to S aureus mecA homologues and was initially detected in 15 S aureus isolates from dairy cattle in England. These isolates were from three different multilocus sequence type lineages (CC130, CC705, and ST425); spa type t843 (associated with CC130) was identified in 60% of bovine isolates. When human mecA-negative MRSA isolates were tested, the mecALGA251 homologue was identified in 12 of 16 isolates from Scotland, 15 of 26 from England, and 24 of 32 from Denmark. As in cows, t843 was the most common spa type detected in human beings. Interpretation Although routine culture and antimicrobial susceptibility testing will identify S aureus isolates with this novel mecA homologue as meticillin resistant, present confirmatory methods will not identify them as MRSA. New diagnostic guidelines for the detection of MRSA should consider the inclusion of tests for mecALGA251. Funding Department for Environment, Food and Rural Affairs, Higher Education Funding Council for England, Isaac Newton Trust (University of Cambridge), and the Wellcome Trust.

A genomic portrait of the emergence, evolution and global spread of a methicillin resistant Staphylococcus aureus pandemic

The widespread use of antibiotics in association with high-density clinical care has driven the emergence of drug-resistant bacteria that are adapted to thrive in hospitalized patients. Of particular concern are globally disseminated methicillin-resistant Staphylococcus aureus (MRSA) clones that cause outbreaks and epidemics associated with health care. The most rapidly spreading and tenacious health-care-associated clone in Europe currently is EMRSA-15, which was first detected in the UK in the early 1990s and subsequently spread throughout Europe and beyond. Using phylogenomic methods to analyze the genome sequences for 193 S. aureus isolates, we were able to show that the current pandemic population of EMRSA-15 descends from a health-care-associated MRSA epidemic that spread throughout England in the 1980s, which had itself previously emerged from a primarily community-associated methicillin-sensitive population. The emergence of fluoroquinolone resistance in this EMRSA-15 subclone in the English Midlands during the mid-1980s appears to have played a key role in triggering pandemic spread, and occurred shortly after the first clinical trials of this drug. Genome-based coalescence analysis estimated that the population of this subclone over the last 20 yr has grown four times faster than its progenitor. Using comparative genomic analysis we identified the molecular genetic basis of 99.8% of the antimicrobial resistance phenotypes of the isolates, highlighting the potential of pathogen genome sequencing as a diagnostic tool. We document the genetic changes associated with adaptation to the hospital environment and with increasing drug resistance over time, and how MRSA evolution likely has been influenced by country-specific drug use regimens.

Development of a real-time quadruplex PCR assay for simultaneous detection of nuc, Panton–Valentine leucocidin (PVL), mecA and homologue mecALGA251

BACKGROUNDnThe recent discovery of a mecA homologue (mecA(LGA251)) with a high level of variability between the two gene variants suggested that Staphylococcus aureus harbouring mecA(LGA251) could be wrongly identified as methicillin-susceptible S. aureus (MSSA), in the absence of antimicrobial susceptibility testing.nnnMETHODSnIn this context we designed a real-time quadruplex PCR assay to distinguish unequivocally between mecA and mecA(LGA251), alongside the nuc gene (a species-specific marker) and detection of the lukS-PV gene [encoding the Panton-Valentine leucocidin (PVL) toxin].nnnRESULTS AND DISCUSSIONnThe assay was validated using a collection of (i) PVL-positive and PVL-negative MSSA and methicillin-resistant S. aureus (MRSA) and (ii) known MRSA harbouring mecA(LGA251) from the UK, Denmark and France. When applied to a retrospective collection of oxacillin-non-susceptible, mecA-negative human isolates, three were found to encode mecA(LGA251), including one from blood, representing the first hitherto recognized case of bacteraemia due to S. aureus possessing the mecA(LGA251) in England. Finally, the assay was introduced into the routine Staphylococcus Reference Unit (HPA Microbiology Services, London, UK) workflow in August 2011, and, during the first 5 months of use, 10 isolates harbouring the mecA homologue were identified out of 2263 S. aureus tested, suggesting a low but continuous circulation within the human population in England.

Distinct Bacteriophages Encoding Panton-Valentine Leukocidin (PVL) among International Methicillin-Resistant Staphylococcus aureus Clones Harboring PVL

ABSTRACT Genetically diverse community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) can harbor a bacteriophage encoding Panton-Valentine leukocidin (PVL) lysogenized into its chromosome (prophage). Six PVL phages (ΦPVL, Φ108PVL, ΦSLT, ΦSa2MW, ΦSa2USA, and ΦSa2958) are known, and single-nucleotide polymorphisms (SNPs) in the PVL genes have been reported. We sought to determine the distribution of lysogenized PVL phages among MRSA strains with PVL (PVL-MRSA strains), the PVL gene sequences, and the chromosomal phage insertion sites in 114 isolates comprising nine clones of PVL-MRSA that were selected for maximal underlying genetic diversity. The six PVL phages were identified by PCR; ΦSa2USA was present in the highest number of different lineages (multilocus sequence type clonal complex 1 [CC1], CC5, CC8, and sequence type 93 [ST93]) (n = 37 isolates). Analysis of 92 isolates confirmed that PVL phages inserted into the same chromosomal insertion locus in CC22, -30, and -80 but in a different locus in isolates of CC1, -5, -8, -59, and -88 and ST93 (and CC22 in two isolates). Within the two different loci, specific attachment motifs were found in all cases, although some limited inter- and intralineage sequence variation occurred. Overall, lineage-specific relationships between the PVL phage, the genes that encode the toxin, and the position at which the phage inserts into the host chromosome were identified. These analyses provide important insights into the microepidemiology of PVL-MRSA, will prove a valuable adjunct in outbreak investigation, and may help predict the emergence of new strains.

Linezolid-resistant ST36 methicillin-resistant Staphylococcus aureus associated with prolonged linezolid treatment in two paediatric cystic fibrosis patients

OBJECTIVESnTo describe the emergence of linezolid-resistant methicillin-resistant Staphylococcus aureus (MRSA) of sequence type (ST)36 lineage in two paediatric patients with cystic fibrosis, after long-term low-dose linezolid treatment.nnnMETHODSnTwo paediatric males with cystic fibrosis had sputum samples quantitatively cultured during hospitalization. After the isolation of MRSA from both patients, oral treatment with 300 mg linezolid twice daily was initiated for periods of 1-2 months separated by up to 6 months. Isolates cultured 9 months after the start of treatment were tested for resistance to linezolid by agar dilution (BSAC). Resistant isolates were examined for 23S rDNA mutations, and typed by phage and macrorestriction with SmaI. Isolates from follow-up sputum samples were obtained until 44-51 months after treatment with linezolid.nnnRESULTSnColonization with MRSA was at a density of approximately 10(6) cfu/mL sputum for both subjects. Initial isolates were susceptible to linezolid, but, 9 months later, isolates from both patients were resistant (MICs > 16 mg/L). Both isolates were epidemic MRSA-16 variant A1 (ST36-MRSA-II), which is widespread in UK hospitals. Both isolates were heterozygous for a G2576T mutation in their 23S rDNA genes, but one was resistant to fusidic acid and tetracycline. In follow-up sampling, the younger patient yielded linezolid-resistant EMRSA-16 for a further 42 months, whilst the other lost the linezolid-resistant MRSA and had alternately Pseudomonas aeruginosa or linezolid-susceptible EMRSA-16 variant A1 isolated over 35 further months.nnnCONCLUSIONSnLinezolid resistance emerged in two isolates of ST36 MRSA colonizing the lungs of two paediatric cystic fibrosis patients. Subtherapeutic levels of linezolid may have facilitated the selection of resistance.

NDM carbapenemases in the United Kingdom: an analysis of the first 250 cases

OBJECTIVESnGram-negative bacteria with diverse carbapenemases, including New Delhi metallo-β-lactamase (NDM) enzymes, have been increasingly recorded in the UK since 2007. We analysed patient data for NDM-positive isolates confirmed by the national reference laboratory from UK laboratories from February 2008 to July 2013.nnnMETHODSnIsolates resistant to carbapenems and with imipenem MICs reduced ≥8-fold by EDTA were tested by PCR for genes encoding acquired class B carbapenemases. MICs were determined by BSAC agar dilution methodology. When requested by the sender, or when they were members of apparent clusters, NDM-positive isolates were typed by variable number tandem repeat (VNTR) analysis or PFGE. Data provided by the sending laboratories were collated and reviewed.nnnRESULTSnFrom February 2008 to July 2013 the reference laboratory confirmed 326 NDM-positive isolates from 250 patients, submitted by 83 laboratories. Most (85%, 213/250) patients were already hospitalized when the NDM-positive bacteria were detected, were male (61%, 152/250) and were aged >60 years (58%, 145/250). Travel history was available for only 40% of patients, but 52% (53/101) of these had documented healthcare contact within or travel to the Indian subcontinent. Most NDM-positive isolates (94%, 306/326) were Enterobacteriaceae with just 6% (20/326) non-fermenters; the predominant hosts were Klebsiella spp. (55%, 180/326) and Escherichia coli (25%, 80/326). Almost all NDM-positive isolates were resistant to multiple antibiotic classes, but 90% remained susceptible to colistin.nnnCONCLUSIONSnGram-negative bacteria with NDM carbapenemases are a growing challenge, especially for elderly hospitalized patients, including those with healthcare contact in the Indian subcontinent, and leave few therapeutic options. UK outbreaks remain rare and contained.

In vitro activity of temocillin against multidrug-resistant clinical isolates of Escherichia coli, Klebsiella spp. and Enterobacter spp., and evaluation of high-level temocillin resistance as a diagnostic marker for OXA-48 carbapenemase

Sir, Temocillin is a narrow-spectrum penicillin active primarily against Enterobacteriaceae and resistant to hydrolysis by penicillinases, extended-spectrum b-lactamases (ESBLs), and AmpC enzymes. It has been shown also to retain some activity in vitro against Enterobacteriaceae with KPC-type carbapenemases, although not against bacteria with OXA-48-like non-metalloenzymes or the metalloenzymes (including IMP, NDM and VIM types). The emergence and spread of bacteria with acquired carbapenemases raises public health concerns, and there is a clear need for reliable diagnostic tests in routine bacteriology laboratories. Simple phenotypic tests with b-lactam/b-lactamase inhibitor combinations can aid the identification of isolates with KPC-type or metallo-carbapenemases, but cannot detect OXA-48 carbapenemases, for which there is currently no good inhibitor. Molecular tests could overcome this shortcoming but are not an option in many laboratories. The Carba NP acido/colorimetric phenotypic test detects carbapenem hydrolysis per se, but its sensitivity is reportedly lower for OXA-48 producers than for isolates with other carbapenemases. High-level temocillin resistance is a characteristic of many OXA-48 producers and has been included in algorithms to aid their recognition. – 8 We investigated the in vitro activity of temocillin against 2280 clinical isolates of Escherichia coli, Klebsiella spp. and Enterobacter spp. submitted to Public Health England’s Antimicrobial Resistance and Healthcare Associated Infections (AMRHAI) Reference Unit for the investigation of unusual resistance between January 2012 and April 2013. They included 1029 producers of KPC, OXA-48 or IMP, NDM and VIM carbapenemases. We also evaluated the diagnostic potential of high-level temocillin resistance (MIC ≥128 mg/L) to predict the production of an OXA-48 carbapenemase. Temocillin (Eumedica, Brussels, Belgium) was tested in the range 1–128 mg/L using agar dilution methodology; MICs were interpreted using BSAC breakpoints (susceptibility: systemic infections, MICs≤8 mg/L; urinary infections, MICs≤32 mg/L). Isolates were also tested against AMRHAI’s standard Gram-negative antibiotic panel, which includes ertapenem, imipenem (tested+EDTA to indicate likely metallo-carbapenemase producers) and meropenem. Carbapenemase production was sought using in-house PCRs and/or a commercial microarray (Check-MDR CT102; Check-Points, Wageningen, The Netherlands). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of temocillin MIC ≥128 mg/L as an indicator of OXA-48 carbapenemase production were determined using an online tool (http://www. medcalc.org/calc/diagnostic_test.php). Temocillin MIC distributions for the 2280 isolates are shown in Table 1. These were submissions to a reference laboratory so were biased towards multiresistance, with proportions of ‘unusual’ isolates far higher than would be observed in most diagnostic laboratories. Allowing for the fact that a larger proportion of Klebsiella spp. isolates produced carbapenemases (760/1133; 67.1%) than did isolates of E. coli (206/701; 29.4%) or Enterobacter spp. (63/446; 14.1%), no major differences were observed in the MIC distributions of temocillin for the three genera. Overall, 920 (40.4%) isolates were susceptible to temocillin at the systemic breakpoint (MICs ≤8 mg/L); those considered resistant included 329/669 (49.2%) KPC producers, 671/1251 (53.6%) isolates without carbapenemases, and all 360 (100%) isolates with other types of carbapenemase (OXA-48, IMP, NDM or VIM). At the higher, urinary breakpoint, 1758 (77.1%) isolates were susceptible to temocillin (MICs ≤32 mg/L), including 628 (93.4%) KPC producers and 1098 (87.8%) isolates without carbapenemases. However, 328 (91.4%) isolates with other carbapenemases (OXA-48, IMP, NDM or VIM) remained resistant with MICs .32 mg/L. Temocillin MICs ≥128 mg/L were observed for 355 (15.6%) isolates overall, most (292; 82.3%) of which had a carbapenemase. However, 63 isolates (17.7%; 30 E. coli, 22 Klebsiella spp. and 11 Enterobacter spp.) lacked these mechanisms and the nature of their high-level temocillin resistance warrants further investigation. Many of them had complex b-lactam antibiograms—for example, 57 were also non-susceptible to ertapenem (MICs .0.5 mg/L)—consistent with ESBL/AmpC production combined with impermeability (data not shown). High-level temocillin resistance (MICs ≥128 mg/L) was recorded for 98/108 (90.7%) OXA-48 producers compared with 257/2172 (11.8%) isolates lacking OXA-48 or with 72/1920 (3.8%) isolates lacking a metallo-carbapenemase. Based on these proportions, the detection of high-level resistance was not a sufficiently robust marker when used as the sole criterion to predict the presence of an OXA-48-like carbapenemase; the PPV of the test was only 27.6% (Table 2). However, if combined with an absence of imipenem/EDTA synergy (i.e. when there was no phenotypic evidence of metallo-carbapenemase production), the PPV of high-level temocillin resistance predicting OXA-48

Incidence, management and outcomes of the first cfr-mediated linezolid-resistant Staphylococcus epidermidis outbreak in a tertiary referral centre in the Republic of Ireland.

AIMnTo report the first Irish outbreak of cfr-mediated linezolid-resistant Staphylococcus epidermidis.nnnMETHODSnLinezolid-resistant S. epidermidis isolated at University Hospital Limerick from four blood cultures, one wound and four screening swabs (from nine patients) between April and June 2013 were characterized by pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST) and staphylococcal cassette chromosome (SCCmec) typing. Antibiotic susceptibilities were determined according to the guidelines of the British Society for Antimicrobial Chemotherapy. The outbreak was controlled through prohibiting prescription and use of linezolid, adherence to infection prevention and control practices, enhanced environmental cleaning, isolation of affected patients, and hospital-wide education programmes.nnnFINDINGSnPFGE showed that all nine isolates represented a single clonal strain. MLST showed that they belonged to ST2, and SCCmec typing showed that they encoded a variant of SCCmecIII. All nine isolates were cfr positive, and eight isolates were positive for the G2576T 23S rRNA mutation commonly associated with linezolid resistance. Isolates exhibited multiple antibiotic resistances (i.e. linezolid, gentamicin, methicillin, clindamycin, ciprofloxacin, fusidic acid and rifampicin). The adopted infection prevention intervention was effective, and the outbreak was limited to the affected intensive care unit.nnnCONCLUSIONSnThis is the first documented outbreak of cfr-mediated linezolid-resistant S. epidermidis in the Republic of Ireland. Despite this, and due to existing outbreak management protocols, the responsible micro-organism and source were identified efficiently. However, it became apparent that staff knowledge of antimicrobial susceptibilities and appropriate hygiene practices were suboptimal at the time of the outbreak, and that educational interventions (and re-inforcement) are necessary to avoid occurrence of antimicrobial resistance and outbreaks such as reported here.

Molecular diversity within clonal complex 22 methicillin-resistant Staphylococcus aureus encoding Panton-Valentine leukocidin in England and Wales.

Panton-Valentine leukocidin (PVL)-positive methicillin-resistant Staphylococcus aureus (MRSA) that are multi-locus sequence type clonal complex 22 (CC22) comprise a significant public health problem in the UK. In the present study we sought to determine the genetic diversity, and the respective patient demographics, among 47 PVL-MRSA with a CC22 pulsotype that occurred sporadically or in clusters in community and healthcare settings in eight of nine geographic regions in England and Wales between January 2005 and September 2007. Patient demographics and disease presentations were typical for PVL-S. aureus infections (mostly skin and soft tissue infections in individuals <40 years old); one patient with community-acquired pneumonia died. Although the isolates were closely genotypically related by spa typing and pulsed field gel electrophoresis, at least two variant groups were suggested. PCR detections demonstrated that the majority of the CC22 PVL-MRSA identified (n = 42; 89%) harboured SCCmecIVc, three had SCCmecIVd, one had SCCmecIV but was non-subtypeable, and one isolate harboured SCCmecV. At least three different PVL-encoding phages were detected: ФPVL, Ф108PVL and an unidentified icosahedral phage. Agar dilution MIC determinations showed that the CC22 PVL-MRSA identified were typically resistant to gentamicin and trimethoprim (43 of 47 isolates) and ciprofloxacin resistance was also noted in six isolates. In conclusion, the CC22 PVL-MRSA tested were geographically disseminated but highly genetically related. The observed variances in acquired elements (most notably SCCmec and PVL-encoding phages) suggested that CC22 PVL-MRSA in England and Wales have evolved on multiple occasions.

Phenotypic detection of mecC-MRSA: cefoxitin is more reliable than oxacillin.

OBJECTIVESnTo investigate the reliability of cefoxitin and oxacillin for the detection of mecC-positive Staphylococcus aureus.nnnMETHODSnThe 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.nnnRESULTSnCefoxitin 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.nnnCONCLUSIONSnCefoxitin 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.