Christopher Teale

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.

Rapid detection, differentiation and typing of methicillin-resistant Staphylococcus aureus harbouring either mecA or the new mecA homologue mecALGA251

The recent finding of a new mecA homologue, mecA(LGA251) , with only 70% nucleotide homology to the conventional mecA gene has brought the routine testing for mecA as a confirmatory test for methicillin-resistant Staphylococcus aureus (MRSA) into question. A multiplex PCR was designed to differentiate mecA(LGA251) from the known mecA together with detection of lukF-PV and the spa gene fragments, enabling direct spa typing by sequencing of the PCR amplicons. The PCR analysis and subsequent spa typing were validated on a large collection (n=185) of contemporary MRSA and methicillin-sensitive S. aureus isolates, including 127 isolates carrying mecA(LGA251) . The mecA(LGA251) gene was situated in staphylococcal cassette chromosome mec type XI elements, and sequence variation within a 631-bp fragment of mecA(LGA251) in 79 isolates indicated a very conserved gene sequence. Following a successful validation, the multiplex PCR strategy was implemented in the routine testing of MRSA for national surveillance. Over a 2-month period, among 203 samples tested, 12 new MRSA cases caused by isolates carrying mecA(LGA251) were identified, emphasizing the clinical importance of testing for these new MRSA isolates.

Carbapenemase-producing Enterobacteriaceae and non-Enterobacteriaceae from animals and the environment: an emerging public health risk of our own making?

Acquired carbapenemases pose one of the most pressing public health threats relating to antibiotic resistance. In most countries, the number of carbapenemase-producing bacteria from human clinical specimens is rising, and the epidemiological status of these multiresistant bacteria is progressively worsening. Furthermore, there is a growing number of reports of carbapenemases found either in bacteria isolated from non-human sources or in Salmonella enterica subsp. enterica, a zoonotic species. However, carbapenemases are not yet systematically sought in bacteria from non-human sources, reports of them are largely observational, and there is limited investigation of carbapenemase-positive bacteria in animals and possible links with people who may have acted as potential sources. Active surveillance and monitoring for carbapenem-resistant bacteria in the food chain and other non-human sources is urgently needed, with an enhanced and rigorous follow-up of all positive results. The carbapenems are currently our last good defence against multiresistant Gram-negative bacteria. Our ability to limit the rise and spread of carbapenemase producers, which occur only at basal levels in many countries at present, should serve as a key performance indicator for the success or failure of the efforts that have been called for by international organizations and governments to reduce the impact of antibiotic resistance.

Use of colistin-containing products within the European Union and European Economic Area (EU/EEA): development of resistance in animals and possible impact on human and animal health.

Since its introduction in the 1950s, colistin has been used mainly as a topical treatment in human medicine owing to its toxicity when given systemically. Sixty years later, colistin is being used as a last-resort drug to treat infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa, Acinetobacter baumannii and Enterobacteriaceae (e.g., Escherichia coli, Klebsiella pneumoniae), for which mortality can be high. In veterinary medicine, colistin has been used for decades for the treatment and prevention of infectious diseases. Colistin has been administered frequently as a group treatment for animal gastrointestinal infections caused by Gram-negative bacteria within intensive husbandry systems. Given the ever-growing need to retain the efficacy of antimicrobials used to treat MDR infections in humans, the use of colistin in veterinary medicine is being re-evaluated. Despite extensive use in veterinary medicine, there is limited evidence for the development of resistance to colistin and no evidence has been found for the transmission of resistance in bacteria that have been spread from animals to humans. Since surveillance for colistin resistance in animals is limited and the potential for such transmission exists, there is a clear need to reinforce systematic monitoring of bacteria from food-producing animals for resistance to colistin (polymyxins). Furthermore, colistin should only be used for treatment of clinically affected animals and no longer for prophylaxis of diseases, in line with current principles of responsible use of antibiotics.

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

BACKGROUND The 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. METHODS In 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]. RESULTS AND DISCUSSION The 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.

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

OBJECTIVES To investigate the reliability of cefoxitin and oxacillin for the detection of mecC-positive Staphylococcus aureus. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Pleuromutilins: use in food-producing animals in the European Union, development of resistance and impact on human and animal health

Pleuromutilins (tiamulin and valnemulin) are antimicrobial agents that are used mainly in veterinary medicine, especially for swine and to a lesser extent for poultry and rabbits. In pigs, tiamulin and valnemulin are used to treat swine dysentery, spirochaete-associated diarrhoea, porcine proliferative enteropathy, enzootic pneumonia and other infections where Mycoplasma is involved. There are concerns about the reported increases in the MICs of tiamulin and valnemulin for porcine Brachyspira hyodysenteriae isolates from different European countries, as only a limited number of antimicrobials are available for the treatment of swine dysentery where resistance to these antimicrobials is already common and widespread. The loss of pleuromutilins as effective tools to treat swine dysentery because of further increases in resistance or as a consequence of restrictions would present a considerable threat to pig health, welfare and productivity. In humans, only one product containing pleuromutilins (retapamulin) is authorized currently for topical use; however, products for oral and intravenous administration to humans with serious multidrug-resistant skin infections and respiratory infections, including those caused by methicillin-resistant Staphylococcus aureus (MRSA), are being developed. The objective of this review is to summarize the current knowledge on the usage of pleuromutilins, resistance development and the potential impact of this resistance on animal and human health.

Evaluation of CHROMagar CTX, a novel medium for isolating CTX-M-ESBL-positive Enterobacteriaceae while inhibiting AmpC-producing strains

OBJECTIVES The aim of this study was to evaluate CHROMagar CTX (CHROMagar France), a novel agar for the selective isolation of Enterobacteriaceae expressing the bla(CTX-M) gene in the presence of enteric bacteria expressing AmpC enzymes. METHODS A panel of 150 Gram-negative bacteria (mainly Escherichia coli, Enterobacter, Klebsiella, Pseudomonas and Salmonella) isolated from humans and animals were assembled for the purpose of evaluating CHROMagar CTX and comparing it with CHROMagar ECC with the addition of 1, 2, 4 and 8 mg/L cefotaxime or ceftazidime and with bioMérieux extended-spectrum beta-lactamase (ESBL)-Bx agar. CHROMagar CTX was also assessed for its ability to isolate bla(CTX-M) strains from farm animal faeces (n = 342). RESULTS The panel contained CTX-M-positive (n = 70) strains (CTX-M types 1, 9, 14 and 15), ESBLs (n = 31) belonging to other families (OXA, PER, SHV, TEM, VEB), strains positive for ampC genes (n = 31), strains that overexpressed ampC (n = 6), non-ESBL/AmpC strains (n = 11) and Klebsiella oxytoca (n = 1). CHROMagar CTX was superior to other agars tested for selective isolation of Enterobacteriaceae expressing the bla(CTX-M) gene with 100% sensitivity and 64.2% specificity for CTX-M strains in the panel and 90.1% of the colonies from animal faeces plated on CHROMagar CTX were CTX-M strains. CONCLUSIONS CHROMagar CTX is a valuable agar in situations where it is important to isolate bla(CTX-M) strains in the presence of AmpC strains. The agar may be particularly useful in veterinary studies, where AmpC-producing commensal E. coli can be encountered reasonably frequently in the enteric flora of some animal species and may also be useful, following further evaluation, for samples from humans.

Public health risk of antimicrobial resistance transfer from companion animals.

Antimicrobials are important tools for the therapy of infectious bacterial diseases in companion animals. Loss of efficacy of antimicrobial substances can seriously compromise animal health and welfare. A need for the development of new antimicrobials for the therapy of multiresistant infections, particularly those caused by Gram-negative bacteria, has been acknowledged in human medicine and a future corresponding need in veterinary medicine is expected. A unique aspect related to antimicrobial resistance and risk of resistance transfer in companion animals is their close contact with humans. This creates opportunities for interspecies transmission of resistant bacteria. Yet, the current knowledge of this field is limited and no risk assessment is performed when approving new veterinary antimicrobials. The objective of this review is to summarize the current knowledge on the use and indications for antimicrobials in companion animals, drug-resistant bacteria of concern among companion animals, risk factors for colonization of companion animals with resistant bacteria and transmission of antimicrobial resistance (bacteria and/or resistance determinants) between animals and humans. The major antimicrobial resistance microbiological hazards originating from companion animals that directly or indirectly may cause adverse health effects in humans are MRSA, methicillin-resistant Staphylococcus pseudintermedius, VRE, ESBL- or carbapenemase-producing Enterobacteriaceae and Gram-negative bacteria. In the face of the previously recognized microbiological hazards, a risk assessment tool could be applied in applications for marketing authorization for medicinal products for companion animals. This would allow the approval of new veterinary medicinal antimicrobials for which risk levels are estimated as acceptable for public health.

Prevalence of extended-spectrum-β-lactamase-producing Escherichia coli from pigs at slaughter in the UK in 2013

OBJECTIVES To determine the prevalence and types of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli occurring in pigs at slaughter in the UK in 2013. METHODS Caecal samples from 637 pigs, sampled via a UK-wide monitoring programme in 2013, were enriched overnight in buffered peptone water, before plating to CHROMagar CTX and Oxoid Brilliance ESBL agar. Presumptive ESBL-producing E. coli from both media were tested for ESBL phenotype using MAST ESβL ID discs. Isolates with an ESBL phenotype were examined for the presence of blaCTX-M, blaOXA, blaSHV and blaTEM genes using a multiplex PCR. All blaCTX-M and blaSHV genes identified by PCR were sequenced. RESULTS A total of 23.4% (95% CI 19.2-27.6) of pigs were positive for ESBL-producing E. coli; 22% (95% CI 17.8-26.1) of the pigs carried E. coli producing CTX-M enzymes [comprising enzyme types 1 (18.7% of pigs), 3 (0.2%), 14 (0.5%), 15 (1.4%), 27 (0.5%), 32 (0.5%) and 55 (0.3%)] and 2.2% (95% CI 0.8-3.6) of the pigs carried E. coli producing SHV-12. Five pigs carried both CTX-M- and SHV-12-producing E. coli as different isolates. There were no statistically significant differences observed between the two medium types in terms of the proportions of each CTX-M enzyme type isolated. CONCLUSIONS In this UK study, 23.4% of pigs were found to be positive for ESBL-producing E. coli using selective culture media. The use of two different commercially available ESBL isolation media was found to improve the detection of ESBL-producing E. coli.