Michel Doumith

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.

Molecular mechanisms disrupting porin expression in ertapenem-resistant Klebsiella and Enterobacter spp. clinical isolates from the UK

OBJECTIVES The aim of this study was to investigate relatedness and molecular mechanism(s) of ertapenem resistance in clinical isolates of Klebsiella spp. (n = 28) and Enterobacter spp. (n = 27) referred from multiple hospitals to the UK national reference laboratory. METHODS Investigations included genotyping by PFGE, resistance gene analysis by PCR and antimicrobial susceptibility testing with and without inhibitors of efflux and beta-lactamase activity. Outer membrane proteins (OMPs) were profiled by SDS-PAGE; porin genes were sequenced and their expression was examined by RT-PCR. The contribution of porin deficiency to resistance was investigated by restoring functional porin genes on plasmids. RESULTS PFGE showed significant clonal diversity among ertapenem-resistant isolates, with only small clusters identified. SHV- and CTX-M-type extended-spectrum beta-lactamases were identified in the Klebsiella spp. isolates, whereas AmpC overexpression or KPC carbapenemase was detected in the Enterobacter cloacae isolates. SDS-PAGE showed that Klebsiella pneumoniae and Enterobacter aerogenes with high-level ertapenem resistance (MICs > or = 16 mg/L) consistently lacked both of the two major non-specific porins, whereas variable patterns of OmpC and OmpF were seen in E. cloacae with lower-level ertapenem resistance. Various point mutations or insertion sequences were identified as disrupting the porin-coding sequences, as well as mutations in the promoter region. Functional restoration of OmpK35 or OmpK36 in Klebsiella and OmpC or OmpF in Enterobacter spp. isolates significantly decreased the MICs of all carbapenems, but particularly of ertapenem. We found no evidence of efflux contributing to resistance. CONCLUSIONS Ertapenem resistance was exclusively due to combinations of beta-lactamases with impermeability caused by loss of OMPs. Efflux was not implicated and there was no national spread of resistant clones.

Phosphoethanolamine Modification of Lipid A in Colistin-Resistant Variants of Acinetobacter baumannii Mediated by the pmrAB Two-Component Regulatory System

ABSTRACT Colistin resistance is rare in Acinetobacter baumannii, and little is known about its mechanism. We investigated the role of PmrCAB in this trait, using (i) resistant and susceptible clinical strains, (ii) laboratory-selected mutants of the type strain ATCC 19606 and of the clinical isolate ABRIM, and (iii) a susceptible/resistant pair of isogenic clinical isolates, Ab15/133 and Ab15/132, isolated from the same patient. pmrAB sequences in all the colistin-susceptible isolates were identical to reference sequences, whereas resistant clinical isolates harbored one or two amino acid replacements variously located in PmrB. Single substitutions in PmrB were also found in resistant mutants of strains ATCC 19606 and ABRIM and in the resistant clinical isolate Ab15/132. No mutations in PmrA or PmrC were found. Reverse transcriptase (RT)-PCR identified increased expression of pmrA (4- to 13-fold), pmrB (2- to 7-fold), and pmrC (1- to 3-fold) in resistant versus susceptible organisms. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry showed the addition of phosphoethanolamine to the hepta-acylated form of lipid A in the resistant variants and in strain ATCC 19606 grown under low-Mg2+ induction conditions. pmrB gene knockout mutants of the colistin-resistant ATCC 19606 derivative showed >100-fold increased susceptibility to colistin and 5-fold decreased expression of pmrC; they also lacked the addition of phosphoethanolamine to lipid A. We conclude that the development of a moderate level of colistin resistance in A. baumannii requires distinct genetic events, including (i) at least one point mutation in pmrB, (ii) upregulation of pmrAB, and (iii) expression of pmrC, which lead to addition of phosphoethanolamine to lipid A.

Activities of NXL104 Combinations with Ceftazidime and Aztreonam against Carbapenemase-Producing Enterobacteriaceae

ABSTRACT Combinations of NXL104 with ceftazidime and aztreonam were tested against carbapenem-resistant members of the Enterobacteriaceae. Ceftazidime-NXL104 was active against strains with the OXA-48 enzyme or with combinations of impermeability and an extended-spectrum β-lactamase (ESBL) or AmpC enzyme and also against most Klebsiella spp. with the KPC enzyme, but metallo-β-lactamase producers were resistant. Aztreonam-NXL104 was active against all carbapenemase producers at 4 and 4 μg/ml, including those with metallo-β-lactamases.

Activity of aminoglycosides, including ACHN-490, against carbapenem-resistant Enterobacteriaceae isolates

BACKGROUND the emergence of carbapenemases in Enterobacteriaceae is driving a search for therapeutic alternatives. We tested ACHN-490, a sisomicin derivative that evades all plasmid-mediated aminoglycoside-modifying enzymes, against 82 carbapenem-resistant Enterobacteriaceae isolates. Comparators included internationally and locally available aminoglycosides. Methods The isolates variously had KPC (n = 12), SME-1 (n = 1), IMP (n = 13), VIM (n = 5), NDM (n = 17) or OXA-48 (n = 19) carbapenemases, or had combinations of impermeability with AmpC (n = 5) or extended-spectrum β-lactamases (n = 10). They included 53 Klebsiella spp., 19 Enterobacter spp., 6 Escherichia coli and 4 others; most were multiresistant. Genes were identified by PCR and sequencing; MICs were measured by CLSI agar dilution. RESULTS ACHN-490 was active at ≤ 2 mg/L against all 65 isolates with carbapenem resistance mechanisms other than NDM enzyme, mostly with MICs of 0.12-0.5 mg/L; isepamicin was active against 63/65 at ≤ 8 mg/L. In contrast, 35% were resistant to gentamicin at 4 mg/L, 61% to tobramycin at 4 mg/L and 20% to amikacin at 16 mg/L. However, 16 of the 17 isolates with NDM-1 enzyme were resistant to ACHN-490, with MICs ≥ 64 mg/L, and these were cross-resistant to all other human-use aminoglycosides tested. Their behaviour was associated with ArmA and RmtC 16S rRNA methylases. Apramycin (a veterinary aminoglycoside) retained its full activity, with MICs of 4-8 mg/L versus strains with armA or rmtC, though resistance was seen in one Klebsiella pneumoniae with AAC(3)-IV (MIC ≥ 256 mg/L). CONCLUSIONS ACHN-490 has potent activity versus carbapenem-resistant isolates, except those also harbouring 16S rRNA methylases; isepamicin is also widely active, though less potent than ACHN-490. Evasion of 16S rRNA methylases by apramycin is noteworthy and may provide a starting point for future aminoglycoside development.

Phylogenetic diversity of Escherichia coli strains producing NDM-type carbapenemases

BACKGROUND The global accumulation of Escherichia coli with CTX-M extended-spectrum β-lactamases partly reflects the dissemination of clonal lineages, notably ST131 and ST405. More recently, E. coli have emerged that produce NDM carbapenemase. We sought to determine the clonal diversity of E. coli with this enzyme from English hospitals, and to compare them with isolates from Pakistan and India. METHODS The 18 NDM-positive E. coli were from hospitals in England (n = 10), Pakistan (n = 7) and India (n = 1). Isolates were compared by phylogenetic grouping, multilocus sequence typing and PFGE of XbaI-digested DNA. Isolates were screened by PCR for acquired AmpC genes, bla(CTX-M), and the 16S rRNA methylase genes armA and rmtC. RESULTS Most of the isolates belonged to phylogenetic groups B1 (n = 9) or D (n = 7); two were group A and none was group B2. Nine isolates from England and Pakistan belonged to the B1 lineage ST101, with seven of these clustering at >77% similarity by PFGE. Other lineages included ST405 (n = 3, group D), ST648 (n = 3, group D), the ST23 complex (one each of ST90 and ST410, both group A) and ST156 (n = 1, group D). Sixteen of 18 isolates had a group 1 CTX-M gene, 13 had a CIT-type acquired AmpC, and 16 had either or both of armA and rmtC. CONCLUSIONS The E. coli isolates producing NDM-1 carbapenemase belonged to six sequence types and included diverse clonal lineages. Nevertheless, isolates of B1-ST101 accounted for half the collection, and included isolates from both England and Pakistan. None of the isolates belonged to ST131 or to phylogroup B2.

Detection of the plasmid-mediated mcr-1 gene conferring colistin resistance in human and food isolates of Salmonella enterica and Escherichia coli in England and Wales

OBJECTIVES In response to the first report of transmissible colistin resistance mediated by the mcr-1 gene in Escherichia coli and Klebsiella spp. from animals and humans in China, we sought to determine its presence in Enterobacteriaceae isolated in the UK. METHODS The PHE archive of whole-genome sequences of isolates from surveillance collections, submissions to reference services and research projects was retrospectively analysed for the presence of mcr-1 using Genefinder. The genetic environment of the gene was also analysed. RESULTS Rapid screening of the genomes of ∼24 000 Salmonella enterica, E. coli, Klebsiella spp., Enterobacter spp., Campylobacter spp. and Shigella spp. isolated from food or humans identified 15 mcr-1-positive isolates. These comprised: 10 human S. enterica isolates submitted between 2012 and 2015 (8 Salmonella Typhimurium, 1 Salmonella Paratyphi B var Java and 1 Salmonella Virchow) from 10 patients; 3 isolates of E. coli from 2 patients; and 2 isolates of Salmonella Paratyphi B var Java from poultry meat imported from the EU. The mcr-1 gene was located on diverse plasmids belonging to the IncHI2, IncI2 and IncX4 replicon types and its association with ISApl1 varied. Six mcr-1-positive S. enterica isolates were from patients who had recently travelled to Asia. CONCLUSIONS Analysis of WGS data allowed rapid confirmation of the presence of the plasmid-mediated colistin resistance gene mcr-1 in diverse genetic environments and plasmids. It has been present in E. coli and Salmonella spp. harboured by humans in England and Wales since at least 2012.

Diversity of carbapenem resistance mechanisms in Acinetobacter baumannii from a Taiwan hospital: spread of plasmid-borne OXA-72 carbapenemase.

OBJECTIVES We investigated the molecular epidemiology of carbapenem-resistant Acinetobacter baumannii from a Taiwanese hospital and determined the mechanisms responsible for resistance. METHODS Ninety-two consecutive meropenem-resistant A. baumannii isolates collected between January 2005 and June 2007 were screened for genes encoding OXA carbapenemases, metallo-beta-lactamases and for the carO gene encoding an outer membrane protein. PFGE was used to define clonal relatedness. PCR mapping was used to examine the linkage of insertion sequences and bla(OXA) genes. Southern hybridization of plasmid extracts and I-CeuI-restricted chromosomal DNA was used to locate bla(OXA-24-like) genes. Sequences of selected bla(OXA-24-like) and carO genes were determined and loss of CarO expression was confirmed by SDS-PAGE. RESULTS Most (70/92, 76%) isolates belonged to one of three PFGE pulsotypes, indicating clonal spread. Fifty-nine isolates, including the majority of those of pulsotypes I and III, produced OXA-72 carbapenemase. The bla(OXA-72) gene was located on a 54 kb plasmid in selected isolates. Thirty-three (36%) isolates, including all 16 of pulsotype II, had ISAba1 preceding the bla(OXA-51-like) gene, promoting its expression. In addition to OXA-72 carbapenemase, two pulsotype I and three pulsotype III isolates did not produce CarO protein as the carO gene was disrupted by insertion of an ISAba1 element. Two isolates of a minor pulsotype had a bla(OXA-58-like) gene and a single PFGE-unique isolate had a bla(OXA-23-like) gene. CONCLUSIONS Although diverse mechanisms were identified, production of OXA-72 carbapenemase was the most common mechanism of carbapenem resistance in A. baumannii from this Taiwanese hospital. The plasmidic location of the gene had facilitated its spread to multiple strains.

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.

AdeABC-mediated efflux and tigecycline MICs for epidemic clones of Acinetobacter baumannii

OBJECTIVES Tigecycline non-susceptibility in individual Acinetobacter baumannii isolates has been associated with up-regulation of the resistance-nodulation-division (RND)-type efflux system, AdeABC. We sought to relate variation in the expression of this system to differences in modal tigecycline MIC among prevalent A. baumannii clones. The role of AdeABC in the emergence of tigecycline resistance during therapy was also investigated for two representatives of the prevalent UK lineage, OXA-23 clone 1. METHODS Clonal type was defined by PFGE and expression of adeABC by real-time RT-PCR. Laboratory mutants were selected in vitro by exposing a susceptible clinical isolate to increasing tigecycline concentrations. The adeB gene was inactivated by the directed integration of a suicide plasmid containing an internal fragment of the target gene. RESULTS Higher modal tigecycline MICs for particular clones correlated with elevated expression of adeABC. Expression of this operon was also increased in the two post-therapy, tigecycline-resistant clinical isolates and in a laboratory mutant as compared with their pre-exposure, tigecycline-susceptible counterparts. Interruption of adeB in a tigecycline-resistant clinical isolate restored full susceptibility to tigecycline. CONCLUSIONS Differences in expression of adeABC contribute to both inter- and intra-clone variation in tigecycline MICs. Tigecycline resistance can arise during therapy, mediated by up-regulation of AdeABC.