We developed a multiplex PCR assay capable of identifying two capsular polysaccharide synthesis sequence types (sequence type 258 [ST258] cps-1 and cps-2) in epidemic Klebsiella pneumoniae ST258 strains. The assay performed with excellent sensitivity (100%) and specificity (100%) for identifying cps types in 60 ST258 K. pneumoniae sequenced isolates. The screening of 419 ST258 clonal isolates revealed a significant association between cps type and K. pneumoniae carbapenemase (KPC) variant: cps-1 is largely associated with KPC-2, while cps-2 is primarily associated with KPC-3.
We assessed the activity of tigecycline (TGC) combined with colistin (COL) against carbapenem-resistant enterobacteria. Synergy occurred in vitro against the majority of isolates, with the exception of Serratia marcescens. In a simple animal model (Galleria mellonella), TGC-COL was superior (P < 0.01) in treating Escherichia coli, Klebsiella pneumoniae, and Enterobacter infections, including those with TGC-COL resistance. Clinical studies are needed to determine whether TGC-COL regimens may be a viable option.
Escherichia coli; enteroaggregative; ESBL; extended-spectrum β-lactamase; extraintestinal; ST38; diarrheagenic E. coli; multiple drug resistance; Germany; the Netherlands; United Kingdom; bacteria
Although the presence of Carbapenemase-producing Enterobacteriaceae (CPE) are extensively documented in North and South America. CPE have not been reported from Curacao. However, recently intercontinental spread was suggested of a KPC carbapenemase producing Klebsiella pneumoniae in a patient in the United Kingdom with previous admission to a hospital in Curacao in 2009.
After the introduction of the CLSI 2010 revised breakpoints, seven patients with carbapenemase-producing Enterobacteriaceae were found in a general hospital in Curacao over a period of 16 months. Four patients carried KPC-2 positive Klebsiella pneumoniae, ST11. Two patients carried KPC-3 positive Klebsiella pneumoniae ST258 and one patient carried a KPC-3 positive Citrobacter freundii. Furthermore, our Klebsiella pneumoniae KPC-2 ST11 strain was matched to the Klebsiella pneumoniae KPC-2 ST11 strain in the United Kingdom.
Introduction of new laboratory methods, and adoption of new guidelines and breakpoints led to the first detection of CPE in Curacao. By matching our Klebsiella pneumoniae KPC-2 ST11 strain to a Klebsiella pneumoniae KPC-2 ST11 strain in the United Kingdom, we suggest that carbapenemase-producing Enterobacteriaceae are probably more prevalent in Curacao than previously recognized.
KPC; Carbapenemases; Enterobacteriaceae
The aim of this study was to determine the ability of a disc susceptibility test using faropenem (10 μg) to predict carbapenemase activity in Enterobacteriaceae. A collection of 166 isolates of carbapenemase-producing Enterobacteriaceae (CPE) and 82 isolates of Enterobacteriaceae that produced other β-lactamases was compiled from diverse sources. Disc susceptibility testing was performed using the CLSI/EUCAST methodology with discs of faropenem (10 μg), temocillin (30 μg), and four carbapenems (each 10 μg). A further prospective evaluation of the faropenem disc susceptibility test was performed using 205 consecutive isolates referred to a United Kingdom reference laboratory in parallel with molecular methods for carbapenemase detection. Of 166 isolates of CPE, 99% showed growth up to the edge of a 10-μg faropenem disc compared with only 6% of other β-lactamase producers (sensitivity, 99%; specificity, 94%). A “double zone” around 10-μg faropenem discs was frequently associated with OXA-48 producers. Of the carbapenems, the most useful agent was imipenem, where a zone diameter of ≤23 mm as a predictor of carbapenemase activity had a sensitivity of 99% and a specificity of 85%. The presence of no zone of inhibition around a 30-μg temocillin disc was a consistent feature of strains producing OXA-48 carbapenemase. For 205 isolates of Enterobacteriaceae referred to a United Kingdom reference laboratory, growth up to a 10-μg faropenem disc correctly identified 84 of 86 carbapenemase producers (98% sensitivity), with a specificity of 87%. Disc susceptibility testing using faropenem (10 μg) is a simple, convenient, and highly predictive screening test for carbapenemase-producing Enterobacteriaceae.
We present a duplex, real-time PCR assay for detection of Klebsiella pneumoniae carbapenemase (blaKPC) and New Delhi metallo-β-lactamase (blaNDM) genes. Accuracy was assessed with 158 Gram-negative bacillary isolates, including 134 carbapenemase producers. The assay had 100% sensitivity and specificity compared with reference methods and a turnaround time of 90 min.
The putative virulence and antimicrobial resistance gene contents of extended spectrum β-lactamase (ESBL)-positive E. coli (n=629) isolated between 2005 and 2009 from humans, animals and animal food products in Germany, The Netherlands and the UK were compared using a microarray approach to test the suitability of this approach with regard to determining their similarities. A selection of isolates (n=313) were also analysed by multilocus sequence typing (MLST). Isolates harbouring blaCTX-M-group-1 dominated (66%, n=418) and originated from both animals and cases of human infections in all three countries; 23% (n=144) of all isolates contained both blaCTX-M-group-1 and blaOXA-1-like genes, predominantly from humans (n=127) and UK cattle (n=15). The antimicrobial resistance and virulence gene profiles of this collection of isolates were highly diverse. A substantial number of human isolates (32%, n=87) did not share more than 40% similarity (based on the Jaccard coefficient) with animal isolates. A further 43% of human isolates from the three countries (n=117) were at least 40% similar to each other and to five isolates from UK cattle and one each from Dutch chicken meat and a German dog; the members of this group usually harboured genes such as mph(A), mrx, aac(6’)-Ib, catB3, blaOXA-1-like and blaCTX-M-group-1. forty-four per cent of the MLST-typed isolates in this group belonged to ST131 (n=18) and 22% to ST405 (n=9), all from humans. Among animal isolates subjected to MLST (n=258), only 1.2% (n=3) were more than 70% similar to human isolates in gene profiles and shared the same MLST clonal complex with the corresponding human isolates. The results suggest that minimising human-to-human transmission is essential to control the spread of ESBL-positive E. coli in humans.
To assess the level of research funding awarded to UK institutions specifically for antimicrobial resistance-related research and how closely the topics funded relate to the clinical and public health burden of resistance.
Databases and web sites were systematically searched for information on how infectious disease research studies were funded for the period 1997–2010. Studies specifically related to antimicrobial resistance, including bacteriology, virology, mycology and parasitology research, were identified and categorized in terms of funding by pathogen and disease and by a research and development value chain describing the type of science.
The overall dataset included 6165 studies receiving a total investment of £2.6 billion, of which £102 million was directed towards antimicrobial resistance research (5.5% of total studies, 3.9% of total spend). Of 337 resistance-related projects, 175 studies focused on bacteriology (40.2% of total resistance-related spending), 42 focused on antiviral resistance (17.2% of funding) and 51 focused on parasitology (27.4% of funding). Mean annual funding ranged from £1.9 million in 1997 to £22.1 million in 2009.
Despite the fact that the emergence of antimicrobial resistance threatens our future ability to treat many infections, the proportion of the UK infection-research spend targeting this important area is small. There are encouraging signs of increased investment in this area, but it is important that this is sustained and targeted at areas of projected greatest burden. Two areas of particular concern requiring more investment are tuberculosis and multidrug-resistant Gram-negative bacteria.
antibiotics; antifungal; antiviral; antiparasitic
A microarray capable of detecting genes for resistance to 75 clinically relevant antibiotics encompassing 19 different antimicrobial classes was tested on 132 Gram-negative bacteria. Microarray-positive results correlated >91% with antimicrobial resistance phenotypes, assessed using British Society for Antimicrobial Chemotherapy clinical breakpoints; the overall test specificity was >83%. Microarray-positive results without a corresponding resistance phenotype matched 94% with PCR results, indicating accurate detection of genes present in the respective bacteria by microarray when expression was low or absent and, hence, undetectable by susceptibility testing. The low sensitivity and negative predictive values of the microarray results for identifying resistance to some antimicrobial resistance classes are likely due to the limited number of resistance genes present on the current microarray for those antimicrobial agents or to mutation-based resistance mechanisms. With regular updates, this microarray can be used for clinical diagnostics to help accurate therapeutic options to be taken following infection with multiple-antibiotic-resistant Gram-negative bacteria and prevent treatment failure.
IncK plasmids encoding CTX-M-14 extended-spectrum β-lactamase (ESBL) and highly related to plasmid pCT were detected in 13 of 67 (19%) human clinical isolates of Escherichia coli with a group 9 CTX-M-type ESBL from the United Kingdom and in 2 quality assurance isolates. None of these E. coli strains was related to the cattle strain from which pCT was originally characterized.
Clinical isolates of Klebsiella pneumoniae producing NDM-1 carbapenemase from India (n = 22), the United Kingdom (n = 13), and Sweden (n = 4) were subjected to multilocus sequence typing (MLST), automated repetitive sequence-based PCR (rep-PCR), serotyping, virulence gene screening, and plasmid replicon typing. The most frequently detected MLST sequence types (STs) were ST14 (n = 13; all serotype K2), ST11, ST149, ST231, and ST147. The correlation between MLST and automated rep-PCR was excellent. IncA/C was the most frequently detected plasmid replicon type (n = 14). ST14, ST11, and other successful clones may be important for the dissemination of blaNDM-1.
One hundred E. coli isolates from Norway
(n = 37), Sweden (n = 24), UK
(n = 20) and Spain (n = 19), producing
CTX-M-type - (n = 84), or SHV-12
(n = 4) extended spectrum β-lactamases, or the plasmid
mediated AmpC, CMY-2 (n = 12), were typed using multi-locus
sequence typing (MLST) and multi-locus variable number of tandem repeat analysis
(MLVA). Isolates clustered into 33 Sequence Types (STs) and 14 Sequence Type
Complexes (STCs), and 58 MLVA-Types (MTs) and 25 different MLVA-Type Complexes
(MTCs). A strong agreement between the MLST profile and MLVA typing results was
observed, in which all ST131-isolates (n = 39) and most of
the STC-648 (n = 10), STC-38 (n = 9),
STC-10 (n = 9), STC-405 (n = 8) and
STC-23 (n = 6) isolates were clustered distinctly into
MTC-29, -36, -20, -14, -10 and -39, respectively. MLVA is a rapid and accurate
tool for genotyping isolates of globally disseminated virulent multidrug
resistant E. coli lineages, including ST131.
A total of 2,841 clinical isolates of Klebsiella pneumoniae from intra-abdominal infections worldwide were collected in the Study for Monitoring Antimicrobial Resistance Trends (SMART) during 2008 and 2009. Overall, 22.4% of isolates had extended-spectrum β-lactamases (ESBLs). The most active antibiotics among the 11 tested were imipenem, amikacin, and ertapenem, though even these, like all other comparators, were less consistently active against ESBL-positive isolates than against ESBL-negative isolates. Globally, 6.5% of isolates were ertapenem resistant based on the June 2010 clinical breakpoints published by the Clinical and Laboratory Standards Institute, with MICs of ≥1 μg/ml. Molecular characterization of 43 isolates with ertapenem MICs of ≥4 μg/ml showed that they variously produced CTX-M or SHV ESBLs combined with altered impermeability and/or had KPC (n = 28), OXA-48 (n = 3), or VIM (n = 1) carbapenemases. Further monitoring of ertapenem susceptibility and molecular characterization of ertapenem-resistant isolates are needed.
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.
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.
We assessed the ability of three commercial systems to infer carbapenem resistance mechanisms in 39 carbapenemase-producing and 16 other carbapenem-resistant Enterobacteriaceae. The sensitivity/specificity values for “flagging” a likely carbapenemase were 100%/0% (BD Phoenix), 82 to 85%/6 to 19% (MicroScan), and 74%/38% (Vitek 2), respectively. OXA-48 producers were poorly detected, but all systems reliably detected isolates with KPC and most with metallo-carbapenemases.
An Escherichia coli isolate with New Delhi metallo-β-lactamase was isolated from a patient with pyelonephritis and prostatitis who returned to Canada after recent hospitalization in India. The patient was successfully treated with ertapenem and fosfomycin. This patient highlights the role of international travel in the spread of antimicrobial drug resistance and blaNDM-1.
Urinary tract infection; New Delhi metallo-β-lactamase; Escherichia coli; bacteria; antimicrobial drug resistance; carbapenems; nosocomial; Canada; expedited; dispatch
Expression of ampC, oprD, mexA, mexC, mexE, and mexX was studied in 25 Pseudomonas aeruginosa isolates from cystic fibrosis patients, including 14 isolates of the Liverpool epidemic strain. Overexpressed mexA or ampC and reduced oprD were associated with β-lactam resistance. A specific combination of mexR, nalC, and nalD mutations occurred in 11 Liverpool strain isolates, including 7 with upregulated mexA.
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.
Enterobacteriaceae isolates were studied from two major centres in India—Chennai (south India), Haryana (north India)—and those referred to the UK's 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.
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.
The potential of NDM-1 to be a worldwide public health problem is great, and co-ordinated international surveillance is needed.
European Union, Wellcome Trust, and Wyeth.
We determined the complete nucleotide sequences of three plasmids that encode CTX-M extended-spectrum β-lactamases (ESBLs) in pulsed-field gel electrophoresis-defined United Kingdom variants (strains A, C, and D) of the internationally prevalent Escherichia coli O25:H4-ST131 clone. Plasmid pEK499 (strain A; 117,536 bp) was a fusion of type FII and FIA replicons and harbored the following 10 antibiotic resistance genes conferring resistance to eight antibiotic classes: blaCTX-M-15, blaOXA-1, blaTEM-1, aac6′-Ib-cr, mph(A), catB4, tet(A), and the integron-borne dfrA7, aadA5, and sulI genes. pEK516 (strain D; 64,471 bp) belonged to incompatibility group IncFII and carried seven antibiotic resistance genes: blaCTX-M-15, blaOXA-1, blaTEM-1, aac6′-Ib-cr, catB4, and tet(A), all as in pEK499. It also carried aac3-IIa, conferring gentamicin resistance, and was highly related to pC15-1a, a plasmid encoding the CTX-M-15 enzyme in Canada. By contrast, pEK204 (strain C; 93,732 bp) belonged to incompatibility group IncI1 and carried only two resistance genes, blaCTX-M-3 and blaTEM-1. It probably arose by the transposition of Tn3 and ISEcp1-blaCTX-M-3 elements into a pCOLIb-P9-like plasmid. We conclude that (i) United Kingdom variants of the successful E. coli ST131 clone have acquired different plasmids encoding CTX-M ESBLs on separate occasions, (ii) the blaCTX-M-3 and blaCTX-M-15 genes on pEK204 and pEK499/pEK516 represent separate escape events, and (iii) IncFII plasmids harboring blaCTX-M-15 have played a crucial role in the global spread of CTX-M-15 ESBLs in E. coli.
The current β-lactamase classifications have reached a high level of complexity, making them less accessible to clinicians, infection control professionals, hospital management and politicians. From the clinical perspective, a revised comprehensible nomenclature scheme is therefore needed. The term extended-spectrum β-lactamases (ESBLs) has reached a broader audience over time, but is currently restricted to functional class 2be/molecular class A, clavulanic acid inhibited enzymes with activity against extended-spectrum cephalosporins. The proposed new classification expands the definition of ESBL to other clinically important acquired β-lactamases with activity against extended-spectrum cephalosporins and/or carbapenems. The classical class A ESBLs have been designated ESBLA in this classification, whereas plasmid-mediated AmpC and OXA-ESBLs are classed as miscellaneous ESBLs (ESBLM). Lastly, the carbapenemases have been designated ESBLCARBA, ESBLs with hydrolytic activity against carbapenems. We believe that this simplified classification may encourage new groups of healthcare professionals to engage in the effort to prevent the spread of acquired β-lactamases.
classification; ESBLs; metallo-β-lactamases; OXA; KPC
A novel Klebsiella pneumoniae carbapenemase (KPC) variant, designated blaKPC-5, was discovered in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate from Puerto Rico. Characterization of the upstream region of blaKPC-5 showed significant differences from the flanking regions of other blaKPC variants. Comparison of amino acid sequences with those of other KPC enzymes revealed that KPC-5 was an intermediate between KPC-2 and KPC-4, differing from KPC-2 by a single amino acid substitution (Pro103→Arg), while KPC-4 contained Pro103→Arg plus an additional amino acid change (Val239→Gly). Transformation studies with an Escherichia coli recipient strain showed differences in the properties of the KPC variants. KPC-4 and KPC-5 both had pIs of 7.65, in contrast with the pI of 6.7 for KPC-2. KPC-2 transformants were less susceptible to the carbapenems than KPC-4 and KPC-5 transformants. These data correlated with higher rates of imipenem hydrolysis for KPC-2 than for KPC-4 and KPC-5. However, KPC-4 and KPC-5 transformants had higher ceftazidime MICs, and the enzymes from these transformants had slightly better hydrolysis of this drug than KPC-2. KPC-4 and KPC-5 were more sensitive than KPC-2 to inhibition by clavulanic acid in both susceptibility testing and hydrolysis assays. Thus, KPC enzymes may be evolving through stepwise mutations to alter their spectra of activity.
Mycotic aneurysms account for a small proportion of all aneurysms. Escherichia coli a gram-negative organism, is recognised as a rare cause of aortic aneurysm. We report two cases of mycotic aneurysm caused by the same strain of multi-resistant Escherichia coli. The purpose of this case report is to highlight the possibility that this strain may be associated with an increased risk of endovascular infection especially in extra-aortic sites. These aneurysms can be difficult to detect and can have serious consequences.
In case one, the patient presented with symptoms and signs of septicaemia secondary to a urinary tract infection. Despite adequate treatment the patient continued with pyrexia and raised inflammatory markers, therefore a series of CT scans of the abdomen and thorax were performed, which revealed two intra-thoracic pseudo-aneurysms with associated haematomas. In case two, the patient also developed Escherichia coli septicaemia. On day 44 he developed a swelling on the right side of his neck. An ultrasound scan showed a pseudoaneurysm of the right common carotid artery.
Whilst a case report cannot prove that a heightened risk exists, we suggest that it is an area worthy of further surveillance. We recommend when older patients with atheromatosis develop prolonged Escherichia coli septicaemia, the possibility of an infected aneurysm should be borne in mind.