Claire Perry

Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study.

Summary Background Gram-negative Enterobacteriaceae with resistance to carbapenem conferred by New Delhi metallo-β-lactamase 1 (NDM-1) are potentially a major global health problem. We investigated the prevalence of NDM-1, in multidrug-resistant Enterobacteriaceae in India, Pakistan, and the UK. Methods Enterobacteriaceae isolates were studied from two major centres in India—Chennai (south India), Haryana (north India)—and those referred to the UKs national reference laboratory. Antibiotic susceptibilities were assessed, and the presence of the carbapenem resistance gene blaNDM-1 was established by PCR. Isolates were typed by pulsed-field gel electrophoresis of XbaI-restricted genomic DNA. Plasmids were analysed by S1 nuclease digestion and PCR typing. Case data for UK patients were reviewed for evidence of travel and recent admission to hospitals in India or Pakistan. Findings We identified 44 isolates with NDM-1 in Chennai, 26 in Haryana, 37 in the UK, and 73 in other sites in India and Pakistan. NDM-1 was mostly found among Escherichia coli (36) and Klebsiella pneumoniae (111), which were highly resistant to all antibiotics except to tigecycline and colistin. K pneumoniae isolates from Haryana were clonal but NDM-1 producers from the UK and Chennai were clonally diverse. Most isolates carried the NDM-1 gene on plasmids: those from UK and Chennai were readily transferable whereas those from Haryana were not conjugative. Many of the UK NDM-1 positive patients had travelled to India or Pakistan within the past year, or had links with these countries. Interpretation The potential of NDM-1 to be a worldwide public health problem is great, and co-ordinated international surveillance is needed. Funding European Union, Wellcome Trust, and Wyeth.

PCR characterization and typing of Klebsiella pneumoniae using capsular type-specific, variable number tandem repeat and virulence gene targets.

A multiplex PCR is described which detects capsular types K1, K2, K5, K54 and K57, which are those most associated with invasive disease or pathogenicity, a further capsular type (K20), two putative virulence factors (rmpA and wcaG) and the 16S-23S internal transcribed spacer unit of Klebsiella pneumoniae, facilitating identification of this organism. wcaG encodes capsular fucose production and was associated with capsular types K1 and K54, but was also found in strains of other capsular types; 18 of the 543 isolates screened were PCR-positive for this gene. An eight-locus variable number tandem repeat (VNTR) scheme was designed, which provided discrimination at a level similar to that afforded by PFGE among a panel of 36 isolates representing 29 PFGE types. All isolates tested of the virulent K1 clone of CC23, associated with pyogenic liver abscesses, shared the same VNTR profile, which may be helpful in identifying this clone; such isolates were also PCR-positive for allS. These methods provide a rapid means of characterizing and typing isolates of this important agent of community-acquired and nosocomial infection.

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.

Clusters of genetically similar isolates of Pseudomonas aeruginosa from multiple hospitals in the UK

Variable number tandem repeat (VNTR) analysis at nine loci of isolates of Pseudomonas aeruginosa submitted to the national reference laboratory from UK hospitals, from over 2000 patients, between June 2010 and June 2012 revealed four widely found types that collectively were received from approximately a fifth of patients, including from those with cystic fibrosis. These types were also prevalent among related submissions from the clinical environment and were received from up to 54 (out of 143) hospitals. Multi-locus sequence typing and blaOXA-50-like sequencing confirmed the clonal relationship within each cluster, and representatives from multiple centres clustered within about 70 % by pulsed-field gel electrophoresis. Illumina sequencing of 12 isolates of cluster A of VNTR profile 8, 3, 4, 5, 2, 3, 5, 2, x (where the repeat number at the last, most discriminatory locus is variable) revealed a large number of variably present targets in the accessory genome and seven of these were sought by PCR among a larger set of isolates. Representatives from patients within a single centre mostly had distinct accessory gene profiles, suggesting that these patients acquired the strain independently, while those with clear epidemiological links shared the same profile. Profiles also varied between representatives from different centres. Epidemiological investigations of widely found types such as these require the use of finer-typing methods, which increasingly will be informed by next generation sequencing.

Use of the Accessory Genome for Characterization and Typing of Acinetobacter baumannii

ABSTRACT Outbreak strains of Acinetobacter baumannii are highly clonal, and cross-infection investigations can be difficult. We sought targets based on AbaR resistance islands and on other genes found in some, but not all, sequenced isolates of A. baumannii among a set of clinical isolates (n = 70) that included multiple representatives of a number of pulsed-field gel electrophoresis (PFGE)-defined types. These included representatives that varied in their profiles at two variable-number tandem repeat (VNTR) loci, which can provide discrimination within a PFGE cluster. Detection, or not, of each element sought provided some degree of discrimination among the set, with the presence or absence of genes coding for a phage terminase (ACICU_02185), a sialic acid synthase (ACICU_00080), a polysaccharide biosynthesis protein (AB57_0094), aphA1, bla TEM, and integron-associated orfX (Kyoto Encyclopedia of Genes and Genomes [KEGG] no. K03830) proving the most helpful in discriminating between closely related isolates in our panel. The results support VNTR data in describing distinct populations of highly similar isolates. Such analysis, in combination with other typing methods, can inform epidemiological investigations and provide additional characterization of isolates. Most genotypes carrying bla OXA-23-like were PCR positive for a yeeA-bla OXA-23 fragment found in an AbaR4-type island, suggesting that this is widespread.

Breakthrough bacteraemia due to tigecycline-resistant Escherichia coli with New Delhi metallo-β-lactamase (NDM)-1 successfully treated with colistin in a patient with calciphylaxis

Sir, We present the case of a patient with calciphylaxis and co-infection with New Delhi metallo-b-lactamase (NDM)-1producing Escherichia coli and Klebsiella pneumoniae, both susceptible to tigecycline and colistin. While receiving tigecycline, the patient developed a bacteraemia due to the NDM-1-positive E. coli, now showing tigecycline resistance. The infection was successfully treated with intravenous colistin. NDMs confer resistance to carbapenems. The NDM-1 enzyme was first recognized in 2008 in K. pneumoniae and E. coli isolates from a patient in Sweden who had been hospitalized in New Delhi. Bacteria with NDM enzymes have since been reported worldwide, often related to travel or hospitalization in the Indian subcontinent. NDM-1 represents an emerging therapeutic challenge; nevertheless, there are few descriptions of treatment experience. Calciphylaxis is a rare disorder, characterized by calcification of arterioles, leading to tissue ischaemia and necrosis. A 59-year-old patient with a history of type II diabetes mellitus presented to our hospital with a 2 month history of bilateral thigh ulcerations. The patient had visited Kenya and India in the year prior to admission; however, there was no reported contact with healthcare facilities during either visit, both for the patient and for close contacts. On admission, the lesions were clinically infected. Empirical treatment was with intravenous (iv) flucloxacillin and benzylpenicillin, then piperacillin/tazobactam, without clinical response. Biopsy of the lesions led to a histological diagnosis of calciphylaxis. A biopsy sample was cultured on standard media, with E. coli, K. pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa identified using the BD Phoenix automated identification system (Oxford, UK). The E. coli and K. pneumoniae were resistant to meropenem (confirmed by Etest) and ertapenem, and to penicillins, oxyimino-cephalosporins and aminoglycosides. They remained susceptible to tigecycline and colistin. Accordingly, antimicrobial therapy was altered to 50 mg of tigecycline twice daily iv (after a 100 mg loading dose) and 500 mg of ciprofloxacin twice daily orally to cover the P. aeruginosa. Screening cultures from stool were negative for carbapenem-resistant Enterobacteriaceae. The K. pneumoniae and E. coli isolates were referred to the Antibiotic Resistance Monitoring & Reference Laboratory of the HPA for MIC determination by BSAC agar dilution and molecular investigation. Both were multiresistant, with susceptibility confirmed only to tigecycline and colistin (MICs both ≤0.5 mg/L); PCR and sequencing confirmed the blaNDM-1 gene in both isolates, carried by plasmids belonging to the A/C rep type, which gave closely similar profiles after digestion with the SacI enzyme. The 16S rRNA methylase gene rmtC was also detected in both isolates by PCR, conferring resistance to all clinically used aminoglycosides. The patient required prolonged hospitalization, interrupted only by 4 weeks at home. Four months after initial presentation, while being treated for ongoing infection of calciphylactic lesions, the patient developed fever. Blood cultures were taken and grew NDM-1-positive E. coli identical by PFGE to that isolated from the original lesions. Susceptibility to colistin remained, but Table 1. MICs in mg/L for NDM-1-producing E. coli isolated from the patient; the second was isolated from blood 4 months after the original was isolated from a calciphylactic skin lesion

Klebsiella pneumoniae producing KPC carbapenemase in a district general hospital in the UK

We report two patients with multidrug-resistant KPC-carbapenemase-producing Klebsiella pneumoniae urinary tract infections. A bla(KPC-2) gene was detected in both of the isolates by polymerase chain reaction and sequencing. The isolates had identical pulsed-field gel electrophoresis patterns and belonged to sequence type ST11. The index patient probably acquired the KPC-producing strain while in hospital in Curaçao, with subsequent nosocomial transmission to the second patient occurring in our hospital. We describe the interventions that were taken to prevent its further spread within the acute Trust and the community.

Clonal expansion of Escherichia coli ST38 carrying a chromosomally integrated OXA-48 carbapenemase gene

Many isolates of Escherichia coli carrying blaOXA-48 referred to Public Health Englands national reference laboratory during 2014 and 2015 shared similar pulsed-field gel electrophoresis (PFGE) profiles, despite coming from patients in multiple different hospitals and regions. Whole genome sequencing on an Illumina platform revealed that these belonged to sequence type (ST) 38. The OXA-48 gene is usually carried on a 62 kb IncL/M plasmid (pOXA48a), but those belonging to this ST appeared either to lack plasmid elements or to have only a partial complement. Two isolates, one belonging to a main cluster sharing identical PFGE profiles and the other having a distinct profile, were further sequenced on a minION. The long reads provided by the nanopore sequencing technology facilitated assembly of a much larger contig around the blaOXA-48 region, showing that both isolates shared a similar arrangement, with a plasmid fragment containing blaOXA-48 flanked by IS1R elements integrated into the chromosome, although the length of the plasmid fragment and the insertion site differed between the two isolates. That belonging to the main cluster contained a 21.9 kb Tn6237 insert, as previously described in E. coli EC-15 from Lebanon, but in a different insertion site. PCR mapping indicated that a further 14/31 representatives of this cluster also contained this insert in the same insertion site, with most of the remainder differing only by having additional E. coli sequence on one side of the insertion. This sub-cluster of ST38 was found from 25 different hospital laboratories, suggesting widespread distribution of a successful type.

Use of nrdA gene sequence clustering to estimate the prevalence of different Achromobacter species among Cystic Fibrosis patients in the UK

BACKGROUND We aimed to estimate the prevalence of different Achromobacter species among UK Cystic Fibrosis (CF) patients. METHODS nrdA sequence clustering was used to identify 147 Achromobacter isolates from 96 patients from 27 hospitals to species level. Potential cross-infection was investigated by MLST, pulsed-field gel electrophoresis and whole genome sequencing (WGS). RESULTS Achromobacter xylosoxidans was the most prevalent species affecting 59 of 96 (61%) patients, followed by Achromobacter insuavis and Achromobacter dolens (12.4% and 8%, respectively). Three novel nrdA clusters were identified. One was further characterised by sequencing the intrinsic blaOXA gene, revealing novel variants. WGS of A. insuavis 2a isolates from four patients attending the same paediatric unit revealed that three were ST144, but differed from one another by a minimum of 385 SNPs, suggesting cross-infection was unlikely. CONCLUSIONS nrdA sequence clustering permitted an estimation of UK Achromobacter species prevalence, highlighted additional novel species, and aided cross-infection investigations.

Emergence of carbapenem resistance due to porin loss in an extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae strain during meropenem therapy.

Here we report the emergence of carbapenem resistance in an extended-spectrum β-lactamase-producing strain of Klebsiella pneumoniae (ESBL-KP) owing to loss of outer membrane proteins (OMPs)