Antibiotic susceptibility of Legionella pneumophila strains isolated in England and Wales 2007-17.

Objectives: Antibiotic susceptibility of Legionella pneumophila is poorly understood, with treatment of Legionnaires' disease often based on empirical choice. The aim of this study was to determine the antibiotic susceptibility of L. pneumophila strains. Methods: Antibiotic susceptibility of 92 L. pneumophila strains isolated in England and Wales between 2007 and 2017 was determined using a microbroth dilution methodology for each agent tested. MICs and MBCs were determined and compared with published intracellular concentrations of each agent tested. Results: The MIC range of erythromycin was 0.06-1 mg/L, the MIC range of rifampicin was 0.0001 mg/L, the MIC range of ciprofloxacin was 0.004-0.25 mg/L and the MIC range of levofloxacin and moxifloxacin was 0.03-0.25 mg/L. The MBC range of erythromycin was 1-32 mg/L, but the MBC range of ciprofloxacin was the same as the MIC range. For levofloxacin and moxifloxacin the MBC range was elevated by one dilution and two dilutions, respectively. Typically, intracellular bronchial secretion concentrations of erythromycin might be expected to reach a suitable level to exceed the MIC range; however, 91 of 92 (98.9%) isolates had an MBC below the expected intracellular concentrations, which indicated erythromycin may have variable efficacy. MIC and MBC values of ciprofloxacin, levofloxacin and moxifloxacin were below achievable intracellular levels within bronchial secretions. Comparison of the MIC/MBC correlation showed very little clustering for erythromycin, but strong clustering for levofloxacin and to a lesser extent ciprofloxacin. Conclusions: Use of the MIC/MBC linkage analysis seems an appropriate way forward for antimicrobial susceptibility testing and supports current guidance recommending levofloxacin for the treatment of Legionnaires' disease.

The use of high-throughput sequencing to investigate an outbreak of glycopeptide-resistant Enterococcus faecium with a novel quinupristin-dalfopristin resistance mechanism.

High-throughput sequencing (HTS) has successfully identified novel resistance genes in enterococci and determined clonal relatedness in outbreak analysis. We report the use of HTS to investigate two concurrent outbreaks of glycopeptide-resistant Enterococcus faecium (GRE) with an uncharacterised resistance mechanism to quinupristin-dalfopristin (QD). Seven QD-resistant and five QD-susceptible GRE isolates from a two-centre outbreak were studied. HTS was performed to identify genes or predicted proteins that were associated with the QD-resistant phenotype. MLST and SNP typing on HTS data was used to determine clonal relatedness. Comparative genomic analysis confirmed this GRE outbreak involved two distinct clones (ST80 and ST192). HTS confirmed the absence of known QD resistance genes, suggesting a novel mechanism was conferring resistance. Genomic analysis identified two significant genetic determinants with explanatory power for the high level of QD resistance in the ST80 QD-resistant clone: an additional 56aa leader sequence at the N-terminus of the lsaE gene and a transposon containing seven genes encoding proteins with possible drug or drug-target modification activities. However, HTS was unable to conclusively determine the QD resistance mechanism and did not reveal any genetic basis for QD resistance in the ST192 clone. This study highlights the usefulness of HTS in deciphering the degree of relatedness in two concurrent GRE outbreaks. Although HTS was able to reveal some genetic candidates for uncharacterised QD resistance, this study demonstrates the limitations of HTS as a tool for identifying putative determinants of resistance to QD.

Detection and molecular characterization of Livestock-Associated MRSA in raw meat on retail sale in North West England.

Limited data are available on the prevalence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in the UK. We tested 124 raw meat samples for MRSA including pork (n = 63), chicken (n = 50) and turkey (n = 11) collected from retail outlets in North West England between March and July 2015. MRSA was recovered from nine (7·3%) samples (four chicken, three pork and two turkey) from different butchers and supermarkets. Four were labelled of UK origin, three were from continental Europe; the origin was not specified for two samples. Whole-genome sequencing (WGS), spa typing and the presence of lineage-specific canonical single nucleotide polymorphisms confirmed that they belonged to the livestock-associated clade of clonal complex (CC) 398. Seven (77·8%) isolates were multi-drug resistant. Phylogenetic analyses showed the isolates were diverse, suggesting multiple silent introductions of LA-MRSA into the UK food chain. Two chicken meat isolates belonged to a sub-clade recently reported from human cases in Europe where poultry meat was the probable source. The low levels of MRSA identified (<20 CFU per g) and absence of enterotoxin genes suggest the risk of acquisition of, or food-poisoning due to, LA-MRSA is low. Nevertheless, the MRSA contamination rate is higher than previously estimated; further evaluation of the public health impacts of LA-MRSA is warranted. SIGNIFICANCE AND IMPACT OF THE STUDY: Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is a public health concern worldwide, but has only been reported sporadically in the UK. In the largest UK study to date, samples of raw meat at retail sale were examined for both the presence and levels of MRSA. We report the first isolations of CC398 LA-MRSA from poultry meat in the UK including representatives of a particular sub-clade associated with cases of human infection/colonization in Europe. Although levels were low (<20 CFU per g), the contamination rate was higher than previous UK studies. Moreover, whole-genome sequencing revealed multiple independent introductions of LA-MRSA into the UK food chain.

Increased Mortality in Diabetic Foot Ulcer Patients: The Significance of Ulcer Type.

Diabetic foot ulcer (DFU) patients have a greater than twofold increase in mortality compared with nonulcerated diabetic patients. We investigated (a) cause of death in DFU patients, (b) age at death, and (c) relationship between cause of death and ulcer type. This was an eleven-year retrospective study on DFU patients who attended King's College Hospital Foot Clinic and subsequently died. A control group of nonulcerated diabetic patients was matched for age and type of diabetes mellitus. The cause of death was identified from death certificates (DC) and postmortem (PM) examinations. There were 243 DFU patient deaths during this period. Ischaemic heart disease (IHD) was the major cause of death in 62.5% on PM compared to 45.7% on DC. Mean age at death from IHD on PM was 5 years lower in DFU patients compared to controls (68.2 ± 8.7 years versus 73.1 ± 8.0 years, P = 0.015). IHD as a cause of death at PM was significantly linked to neuropathic foot ulcers (OR 3.064, 95% CI 1.003-9.366, and P = 0.049). Conclusions. IHD is the major cause of premature mortality in DFU patients with the neuropathic foot ulcer patients being at a greater risk.

Emergence of carbapenem-resistant Enterobacteriaceae in a UK paediatric hospital.

BACKGROUND: Carbapenem-resistant Enterobacteriaceae are an emerging global infection threat. However, there are few data describing their clinical importance in children. AIM: This retrospective study reviewed the prevalence and resistance mechanisms of carbapenem-resistant Enterobacteriaceae grown from clinical and surveillance samples in a large tertiary referral children's hospital in the UK. METHODS: Carbapenem-resistant Enterobacteriaceae were sought in specimens submitted for diagnostic and surveillance purposes at Alder Hey Children's NHS Foundation Trust, Liverpool, between September 2011 and August 2012. Mechanisms of resistance were identified using phenotypic and/or molecular methods. Variable number tandem repeat profiling was used to type carbapenemase-producing strains. FINDINGS: During the 12-month study period, carbapenem-resistant Enterobacteriaceae were recovered from 24 patients. Five isolates were from clinical diagnostic specimens whereas 19 of 421 patients had positive rectal surveillance swabs (4.5%). Of the 24 isolates, seven (all Klebsiella spp.) harboured carbapenemases: three had blaKPC and four blaNDM, whereas 17 had resistance due to combinations of AmpC or extended-spectrum ß-lactamase activity plus impermeability. CONCLUSION: Carbapenem-resistant Enterobacteriaceae and, in particular, those with carbapenemases, are an emerging infection problem in a major paediatric hospital in the UK. Active surveillance is required to monitor and control their spread.

The bioavailability of mupirocin in nasal secretions in vitro.

AIM: To determine the bioavailability of mupirocin in human nasal secretions and to assess whether the contents of nasal secretions interact appreciably with this antibiotic. METHODS: The comparative bioavailability of mupirocin and chlorhexidine in nasal secretions was determined by bioassay after one, four, and eight hours of incubation with pooled secretions from three subjects. The interaction of mupirocin with nasal secretions was characterised by matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF). RESULTS: MALDI-TOF analysis showed that mupirocin was not absorbed by the main fraction of pooled nasal secretions and should remain active. In bioassay, mupirocin retained 100% of its antistaphylococcal activity in nasal secretions, whereas chlorhexidine was significantly reduced from 100 mg/litre to 1.5 mg/litre and from 1000 mg/litre to 38.5 mg/litre, irrespective of incubation time. CONCLUSIONS: The high bioavailability of mupirocin in nasal secretions results from the lack of appreciable molecular interactions.

Is neuropathic ulceration the key to understanding increased mortality due to ischaemic heart disease in diabetic foot ulcer patients? A population approach using a proportionate model.

Ischaemic heart disease (IHD) is the commonest cause of death in diabetic foot ulcer patients and non-ulcerated diabetic patients, yet the mortality rate of diabetic foot ulcer patients is over twice that of non-ulcerated patients. As the cause of this increased mortality is not understood, we plotted the ratio of deaths due to ischaemic heart disease (IHDn) to other causes of death (i.e. IHDn:OCDn) against age for 242 diabetic foot ulcer patients and 121 controls (non-ulcerated diabetic patients). The IHDn:OCDn ratio rose above 1.0 from age 40 years onwards for diabetic foot ulcer patients, but from age 70 years onwards for controls, demonstrating differentially increased mortalities due to IHD. A population model involving summation of IHDn:OCDn ratios for neuropathic and neuroischaemic diabetic foot ulcer patients calculated an overall increased mortality rate of 1.8 compared with that of non-ulcerated diabetics. The model predicted that a 25% reduction in neuropathic diabetic foot ulcer patients dying from IHD would eliminate the increased mortality, demonstrating that neuropathic rather than ischaemic ulceration defines the cause of increased mortality among diabetic foot ulcer patients.