Russell Hope

Improved multiplex PCR strategy for rapid assignment of the four major Escherichia coli phylogenetic groups.

ABSTRACT Using data from whole-genome projects, an updated multiplex PCR strategy was developed to assign Escherichia coli isolates rapidly to major phylogenetic groups. This assay accommodates sequence variations detected within target sequences, thereby increasing sensitivity and reliability. It was validated using 185 isolates of known sequence types and showed improved congruence with multilocus sequence typing data.

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

OBJECTIVES 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Non-susceptibility trends among staphylococci from bacteraemias in the UK and Ireland, 2001-06

OBJECTIVES Investigation of the antibiotic susceptibilities and trends for staphylococci collected from bacteraemia cases in the UK and Ireland, from 2001 to 2006, as part of the British Society for Antimicrobial Chemotherapys Bacteraemia Surveillance Programme. METHODS Twenty-five hospitals from the UK and Ireland each collected up to 10 consecutive isolates of both Staphylococcus aureus and coagulase-negative staphylococci (CoNS) per year from 2001 to 2006. MIC determination and identification to species level were carried out centrally. mecA and also mupA alleles were sought by PCR in S. aureus and CoNS from 2005 and 2006, respectively. RESULTS One thousand four hundred and forty-eight S. aureus and 1214 CoNS were collected. The overall prevalence of methicillin resistance was 42% (with </=6% annual fluctuation) for S. aureus and 67% (range 54% to 80%) for CoNS. Resistance to aminoglycosides, macrolides, quinolones and tetracyclines was strongly associated with methicillin resistance in both species groups. Many (20.8%) CoNS and three (0.2%) S. aureus isolates were non-susceptible to teicoplanin, but there was no vancomycin non-susceptibility found in S. aureus and only one vancomycin-intermediate CoNS isolate. There was little evidence of susceptibility trends over time for any antibiotic, with the surveillance period preceding the recent fall in methicillin-resistant S. aureus (MRSA) prevalence indicated by the mandatory surveillance of MRSA bacteraemia in England. The newer antibiotics, ceftobiprole, daptomycin, linezolid, telavancin and tigecycline, all had excellent activity against staphylococci. CONCLUSIONS Multiresistant staphylococci remain abundant in the UK and Ireland but many new antimicrobials are becoming available and these may prove effective alternatives to glycopeptides.

Non-susceptibility trends among Enterobacteriaceae from bacteraemias in the UK and Ireland, 2001–06

BACKGROUND Enterobacteriaceae are common agents of bacteraemia, with Escherichia coli accounting for 20% of the cases. Reflecting this importance, members of the family constitute 4 of the 12 collection groups in the British Society for Antimicrobial Chemotherapy (BSAC) Bacteraemia Surveillance Programme. METHODS MICs from the BSAC surveillance programme were reviewed, along with bacteraemia reports received by the Health Protection Agency (HPA) via its CoSurv/LabBase system. Isolates with unusual resistances were subjected to molecular analysis. RESULTS The BSAC and HPA systems both revealed dramatically increasing resistance to cephalosporins, ciprofloxacin and gentamicin among E. coli and Klebsiella spp., with cephalosporin resistance largely contingent on the spread of CTX-M extended-spectrum beta-lactamases (ESBLs); fluoroquinolone resistance also increased among Proteus mirabilis and ESBL-negative E. coli. Carbapenem resistance remained extremely rare, but two Enterobacter spp., from the same patient in different years, had KPC carbapenemases, while a few isolates had carbapenem resistance contingent upon combinations of beta-lactamase and impermeability, and ertapenem MICs for AmpC-derepressed Enterobacter spp. rose over time. Three new agents-ceftobiprole, doripenem and tigecycline-were tested. Ceftobiprole was broadly active, except against ESBL producers and Klebsiella oxytoca hyperproducing K1 enzyme, and was variable against AmpC-derepressed Enterobacter spp. and against Proteus vulgaris. Doripenem was more potent than imipenem. Tigecycline was almost universally active against E. coli, but low-level non-susceptibility (MIC 2 mg/L) was frequent among Klebsiella spp. CONCLUSIONS E. coli and Klebsiella spp. showed dramatic shifts, with sharply rising non-susceptibility to cephalosporins, ciprofloxacin and gentamicin. The rise in cephalosporin resistance reflected dissemination of CTX-M ESBLs. Carbapenems remain broadly active and new agents offer potential.

Isolation of fluoroquinolone-resistant O25b:H4-ST131 Escherichia coli with CTX-M-14 extended-spectrum β-lactamase from UK river water

OBJECTIVES We analysed water sampled from the River Thames in London for Escherichia coli resistant to oxyimino-cephalosporins and/or fluoroquinolones, particularly seeking isolates with CTX-M extended-spectrum β-lactamases (ESBLs) and members of the clinically important O25b:H4-ST131 lineage. METHODS River water was collected from three urban sites on the River Thames by the City of London Port Health Authority on two occasions 1 week apart. Coliforms and E. coli were identified by the Quanti-Tray™ method. Disc susceptibility tests were performed and MICs were determined for E. coli isolates resistant to either ciprofloxacin or cefpodoxime and genetic relatedness was determined by PFGE and real-time PCR. PCR was used for phylogenetic and plasmid typing, to detect antibiotic resistance genes and to detect ISEcp1 upstream of bla(CTX-M) genes. bla(CTX-M) alleles were identified by sequencing. RESULTS The mean E. coli count, as the most probable number, from the first river samples, taken on a falling tide on 23 March 2010, was 4.7 × 10(4)/100 mL and 30 ciprofloxacin-resistant colonies were isolated. Twenty of the 30 colonies belonged to clone ST131; 10 of these had bla(CTX-M-14) whereas the remaining 10 lacked ESBLs. The ST131 isolates represented two different PFGE types. No ciprofloxacin- or cefpodoxime-resistant E. coli were isolated from the second river sample taken at low tide. CTX-M-15, the most common ESBL in clinical E. coli, was not detected in the river samples. CONCLUSIONS Water from the River Thames in West London is contaminated, perhaps transiently, with antibiotic-resistant E. coli belonging to the clinically important O25b:H4-ST131 lineage.

Effects of control interventions on Clostridium difficile infection in England: an observational study

Summary Background The control of Clostridium difficile infections is an international clinical challenge. The incidence of C difficile in England declined by roughly 80% after 2006, following the implementation of national control policies; we tested two hypotheses to investigate their role in this decline. First, if C difficile infection declines in England were driven by reductions in use of particular antibiotics, then incidence of C difficile infections caused by resistant isolates should decline faster than that caused by susceptible isolates across multiple genotypes. Second, if C difficile infection declines were driven by improvements in hospital infection control, then transmitted (secondary) cases should decline regardless of susceptibility. Methods Regional (Oxfordshire and Leeds, UK) and national data for the incidence of C difficile infections and antimicrobial prescribing data (1998–2014) were combined with whole genome sequences from 4045 national and international C difficile isolates. Genotype (multilocus sequence type) and fluoroquinolone susceptibility were determined from whole genome sequences. The incidence of C difficile infections caused by fluoroquinolone-resistant and fluoroquinolone-susceptible isolates was estimated with negative-binomial regression, overall and per genotype. Selection and transmission were investigated with phylogenetic analyses. Findings National fluoroquinolone and cephalosporin prescribing correlated highly with incidence of C difficile infections (cross-correlations >0·88), by contrast with total antibiotic prescribing (cross-correlations <0·59). Regionally, C difficile decline was driven by elimination of fluoroquinolone-resistant isolates (approximately 67% of Oxfordshire infections in September, 2006, falling to approximately 3% in February, 2013; annual incidence rate ratio 0·52, 95% CI 0·48–0·56 vs fluoroquinolone-susceptible isolates: 1·02, 0·97–1·08). C difficile infections caused by fluoroquinolone-resistant isolates declined in four distinct genotypes (p<0·01). The regions of phylogenies containing fluoroquinolone-resistant isolates were short-branched and geographically structured, consistent with selection and rapid transmission. The importance of fluoroquinolone restriction over infection control was shown by significant declines in inferred secondary (transmitted) cases caused by fluoroquinolone-resistant isolates with or without hospital contact (p<0·0001) versus no change in either group of cases caused by fluoroquinolone-susceptible isolates (p>0·2). Interpretation Restricting fluoroquinolone prescribing appears to explain the decline in incidence of C difficile infections, above other measures, in Oxfordshire and Leeds, England. Antimicrobial stewardship should be a central component of C difficile infection control programmes. Funding UK Clinical Research Collaboration (Medical Research Council, Wellcome Trust, National Institute for Health Research); NIHR Oxford Biomedical Research Centre; NIHR Health Protection Research Unit on Healthcare Associated Infection and Antimicrobial Resistance (Oxford University in partnership with Public Health England [PHE]), and on Modelling Methodology (Imperial College, London in partnership with PHE); and the Health Innovation Challenge Fund.

Cephalosporin resistance mechanisms in Escherichia coli isolated from raw chicken imported into the UK

OBJECTIVES We characterized mechanisms of resistance to oxyimino-cephalosporins in Escherichia coli isolated from raw chicken meat imported into the UK from South America, to ascertain whether this foodstuff contributes to the dissemination in the UK of extended-spectrum β-lactamase (ESBL)-producing E. coli belonging to the international uropathogenic ST131 clone. METHODS Sampling and collection of imported raw chicken meat was performed in accordance with regulatory guidelines by the London Port Health Authority at Tilbury. E. coli strains producing ESBLs were isolated based on growth within the zones of cefpodoxime (10 μg) discs. MICs were determined by agar dilution and interpreted using BSAC/EUCAST breakpoints. PCR was used to determine the phylogenetic groups of E. coli, to detect ESBL genes and to determine the incompatibility groups of plasmids encoding CTX-M enzymes. The molecular environments surrounding bla(CTX-M) were determined by DNA sequencing and PCR mapping. RESULTS A total of 141 oxyimino-cephalosporin-resistant E. coli were isolated from 62 of 210 batches of imported raw chicken sampled. Thirty percent of these isolates produced group 2 CTX-M ESBLs, 27% produced group 8 CTX-M ESBLs, 42% produced CMY-type AmpC enzymes and 1% produced a group 2 CTX-M along with a CMY enzyme; none produced CTX-M-15 ESBL and none belonged to the ST131 clone. In contrast to human clinical ESBL E. coli, >90% of isolates were susceptible to ciprofloxacin and 74% to all aminoglycosides. CONCLUSIONS Raw chicken imported into the UK from South America commonly carries ESBL-producing E. coli, but is not a significant source for the ST131 clone or for the CTX-M-15 ESBL.

Variation in the genetic environments of blaCTX-M-15 in Escherichia coli from the faeces of travellers returning to the United Kingdom

OBJECTIVE The genetic surroundings of bla(CTX-M-15) in Escherichia coli recovered from faeces of travellers returning to the UK from overseas were compared with those among established UK strains to provide further insights into the spread of bla(CTX-M-15) in the UK. METHODS From August 2006 to January 2008, 1031 faecal specimens were collected at the North West London NHS Trust from general practice patients with a clinical history of diarrhoea following recent international travel. Cefuroxime-resistant E. coli were isolated on cystine-lactose-electrolyte deficient agar and those that produced extended-spectrum β-lactamases (ESBLs) were identified by double disc synergy test (DDST). The molecular environments surrounding bla(CTX-M-15) were investigated by PCR, DNA sequencing, gene cloning and northern blotting. RESULTS 182/1031 (18%) E. coli isolated from returning travellers gave a positive DDST, and were confirmed by PCR to produce CTX-M ESBLs; 174 (96%) had bla(CTX-M-15), including 21 belonging to clone ST131. Among these 174 isolates, the environment upstream of bla(CTX-M-15) consisted of either: (i) an intact ISEcp1 (n = 108); (ii) various lengths of truncated ISEcp1 (n = 58); or (iii) a 24 bp remnant of ISEcp1 (n = 8). Two different promoters were found to transcribe bla(CTX-M-15), resulting in different levels of cephalosporin resistance. CONCLUSION E. coli with CTX-M-15 ESBL from returning travellers harboured previously seen UK bla(CTX-M-15) genetic environments (intact or 24 bp remnant of ISEcp1) as well as bla(CTX-M-15) genetic environments previously unseen in the UK (various lengths of truncated ISEcp1), which suggest overseas acquisition and highlight the difficulty of control in a time of population mobility and travel.

Declining cephalosporin and fluoroquinolone non-susceptibility among bloodstream Enterobacteriaceae from the UK: links to prescribing change?

OBJECTIVES The UK saw major increases in cephalosporin and quinolone resistance amongst Enterobacteriaceae from 2001 to 2006, with cephalosporin resistance largely reflecting dissemination of CTX-M extended-spectrum β-lactamases (ESBLs). We review subsequent trends. METHODS Data were extracted from Public Health Englands national database (LabBase), which collects susceptibility results for bloodstream isolates from hospital microbiology laboratories in England, Wales and Northern Ireland, and from the BSAC Bacteraemia Resistance Surveillance System, which centrally tests bloodstream isolates from 25-40 sentinel UK and Irish laboratories. Reference laboratory submissions were also reviewed. RESULTS LabBase and BSAC data showed that rates of non-susceptibility to cephalosporins and quinolones rose amongst Escherichia coli and Klebsiella spp. until mid-decade (2004-07) before plateauing or falling; similar falls in non-susceptibility began slightly earlier in Enterobacter spp. These reversals in trend occurred whilst the incidence of E. coli bacteraemias was rising, the incidence of Klebsiella bacteraemias was stable and the incidence of Enterobacter bacteraemias was falling; they were not paralleled in EARS-Net data for continental Europe and did not reflect the displacement of single mechanisms. They coincided with large reductions in hospital cephalosporin and quinolone use, owing to concern about Clostridium difficile, with replacement by penicillin/β-lactamase inhibitor combinations, which have borderline activity against ESBL producers, but consistently lack activity against carbapenemase producers. CONCLUSIONS Non-susceptibility to cephalosporins and quinolones has declined among bloodstream Enterobacteriaceae in the UK, probably reflecting prescribing shifts. The penicillin/β-lactamase inhibitor combinations that have largely replaced cephalosporins and quinolones may add to selection for carbapenemase producers.

Molecular epidemiology of fluoroquinolone-resistant ST131 Escherichia coli producing CTX-M extended-spectrum β-lactamases in nursing homes in Belfast, UK

OBJECTIVES Between January 2004 and May 2006 Escherichia coli producing extended-spectrum β-lactamases (ESBLs) were isolated from the faeces of 118/294 residents from 16 nursing homes in Belfast. Of these, 58 isolates belonged to UK strain A, a variant of the international ST131 clone. Here we investigated the remaining 60 ESBL producers. METHODS MICs were determined and interpreted using BSAC methodology. Isolates were characterized by phylogenetic typing, real-time PCR and PFGE. Plasmids were rep typed by PCR and their similarity to IncI1 reference plasmid pEK204 was investigated by restriction fragment length polymorphism analysis. The molecular environments surrounding bla(CTX-M) were determined by DNA sequencing and PCR. RESULTS Fifty-nine of 60 isolates belonged to the B2, ST131 lineage; of these 28 belonged to the previously defined UK strain C, while the other 31 were clustered into five groups by PFGE. Forty-nine isolates harboured bla(CTX-M-3) on plasmids of five different rep types (I1, FIA, FIA-FIB, N and Y) and 11 harboured bla(CTX-M-15) on F-type plasmids (FIA and FIA-FIB). All CTX-M-3 ESBL producers and three with CTX-M-15 ESBL had an intact copy of ISEcp1 immediately upstream of bla(CTX-M); the remaining eight with CTX-M-15 ESBL had a truncated ISEcp1. CONCLUSIONS Gut colonization among nursing home residents in Belfast with ciprofloxacin-resistant E. coli producing ESBLs almost entirely involves clonal spread of ST131 variants, with similar genetic environments for bla(CTX-M-3) or bla(CTX-M-15) as in pEK204 and pEK499. Such diversity indicates dissemination of both plasmids and ESBL genes among a single commonly multiresistant clone.