Multi-locus variable number of tandem repeat analysis for rapid and accurate typing of virulent multidrug resistant Escherichia coli clones.

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 ß-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.

Characterization of Enterobacteriaceae producing OXA-48-like carbapenemases in the UK.

OBJECTIVES: To characterize UK clinical isolates of Enterobacteriaceae producing OXA-48-like carbapenemases and to compare their resistance plasmids. METHODS: Twenty-six enterobacteria producing OXA-48-like enzymes were studied. These were from 22 diverse hospitals in the UK. Isolates of Escherichia coli and Klebsiella pneumoniae were assigned to clonal lineages by multilocus sequence typing. Carbapenemase genes and their genetic environments were characterized by PCR and sequencing. Resistance plasmids were transferred by transformation or conjugation and compared by restriction analysis and PCR for genes encoding critical plasmid functions. RESULTS: Thirteen isolates of K. pneumoniae, 10 E. coli and 2 Enterobacter cloacae harboured a classical bla(OXA-48) gene; the K. pneumoniae isolates belonged to 11 sequence types (STs) and the E. coli to 7 STs, including ST131 and ST38. The bla(OXA-48) genes were located within either Tn1999 or Tn1999.2 transposons on related ≈ 50 kb or ≈ 62 kb plasmids, which lacked other resistance genes or, in one isolate, on an ≈ 140 kb plasmid that also encoded OXA-9 and CTX-M group-9 ß-lactamases. One India-linked K. pneumoniae isolate had a bla(OXA-181) gene in association with an ISEcp1 insertion sequence on a 7 kb plasmid. CONCLUSIONS: Horizontal transfer of related plasmids has facilitated the spread of OXA-48 carbapenemase into multiple strains of several Enterobacteriaceae species. The clonal diversity of the producers suggests repeated introduction into the UK. Low carbapenem MICs for some producers complicates detection and creates a risk for unrecognized spread.

Dissemination of pCT-like IncK plasmids harboring CTX-M-14 extended-spectrum ß-lactamase among clinical Escherichia coli isolates in the United Kingdom.

IncK plasmids encoding CTX-M-14 extended-spectrum ß-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.

Characterization of plasmids encoding extended-spectrum ß-lactamases and their addiction systems circulating among Escherichia coli clinical isolates in the UK.

OBJECTIVES: To characterize plasmids encoding extended-spectrum ß-lactamases (ESBLs) from a recent UK collection of clinical Escherichia coli isolates. METHODS: The isolates comprised 118 ESBL producers referred from 54 laboratories. Plasmids were transferred by electroporation, and their incompatibility groups, associated addiction systems and resistance genes with the flanking genetic environments were identified by PCR or sequencing. RESULTS: Seventy isolates had plasmids encoding CTX-M-15 (n = 53), CTX-M-14 (n =9), CTX-M-27 (n = 1), CTX-M-3 (n = 2) and SHV-12 (n = 5) ESBLs that were transformable; non-transformable ESBLs were mainly CTX-M enzymes (42/48). Most transformable bla(CTX-M-15) genes (43/53) were harboured on single replicon or multireplicon IncF plasmids, with IncFIA4-FIB1-FII31 (n = 11) and IncFIA1-FII2 (n = 15) being most frequent; the latter included eight pEK499 plasmids, typical of UK epidemic strain A. Plasmids harbouring bla(CTX-M-14) belonged variously to IncF, IncI1 and IncHI2 types, and 16 encoding CTX-M or SHV enzymes were non-typeable. Only IncF plasmid types carried the addiction systems sought and those with bla(CTX-M-15) frequently harboured bla(OXA-1) and aac(6')-Ib-cr, and often transferred trimethoprim and tetracycline resistance; those with bla(CTX-M-14) encoded trimethoprim, sulphonamide, streptomycin and tetracycline resistance. Most ESBL genes were associated with the well-known mobile elements ISEcp1 and IS26, but nearly half (23/55) of the ISEcp1 sequences upstream of bla(CTX-M-15) were interrupted by an IS26 at various positions. CONCLUSIONS: Most ESBLs (70/118) were encoded by transformable plasmids, although a sizable minority could not be transformed. The majority of transformable plasmids (51/70; 72.9%) were diverse multiresistant IncF types possessing multiple addiction systems. The spread of bla(CTX-M-15) can be attributed not just to clonal expansion, but also to the horizontal dissemination of related plasmids.

False extended-spectrum {beta}-lactamase phenotype in clinical isolates of Escherichia coli associated with increased expression of OXA-1 or TEM-1 penicillinases and loss of porins.

OBJECTIVES: Two clinical isolates of Escherichia coli, EC18 and EC21, were non-susceptible (MICs 4-16 mg/L) to cefpirome and cefepime, with marked synergy with clavulanate, yet were susceptible to cefotaxime and ceftazidime (MICs ≤ 1 mg/L). EC19, from the same patient as EC21, was susceptible to all four cephalosporins. We sought to characterize the molecular basis of resistance in isolates EC18 and EC21. METHODS: PFGE was used to study the genetic relationships of the isolates, and MICs were determined. ß-Lactamases were characterized by PCR, isoelectric focusing (IEF), construction of genomic libraries and sequencing. A double mutant of E. coli J53 was constructed, lacking OmpC and OmpF porins. Plasmids from clinical isolates were transformed into E. coli J53 and J53ΔompCF. Outer membrane proteins (OMPs) were analysed by SDS-PAGE and OmpA by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry. Expression of omp and bla genes was analysed by RT-PCR. RESULTS: Isolates EC19 and EC21 had identical PFGE profiles, whereas EC18 was distinct. PCR and IEF confirmed ß-lactamases with pIs of 5.4 (TEM-1) in EC18 and 7.4 (OXA-1) in both EC19 and EC21. EC18 had bla(TEM-1b) with the strong promoter P5 and lacked OmpC and OmpF. RT-PCR showed stronger expression of bla(OXA-1) in EC21 versus EC19, along with diminished expression of OmpC, though with increased OmpF. Plasmids extracted from EC18 and EC21 conferred increased MICs of cefpirome and cefepime, although susceptibility to cefotaxime and ceftazidime was retained. CONCLUSIONS: The 'cefpiromase' or 'cefepimase' ESBL phenotype of the clinical isolates non-susceptible to cefpirome and cefepime resulted from high expression of TEM-1 or OXA-1 ß-lactamases combined with loss of porins.

ISEcp1-mediated transposition of linked blaCTX-M-3 and blaTEM-1b from the IncI1 plasmid pEK204 found in clinical isolates of Escherichia coli from Belfast, UK.

OBJECTIVES: Linked bla(CTX-M-3)-bla(TEM-1b) genes, as found on the IncI1 plasmid pEK204 prevalent in Belfast, also occur on plasmids belonging to other rep types in Escherichia coli isolated from nursing-home residents in Belfast. We investigated the mechanisms for their joint dissemination among diverse plasmids. METHODS: Plasmid pEK204 was transferred by electroporation into E. coli DH5α harbouring derivative pBAD Myc-His vectors. Transposition experiments were then performed at 37°C. Transposition of bla genes onto the derivative pBAD Myc-His vector was confirmed by sequencing. RESULTS: ISEcp1 mediated transposition of bla(CTX-M-3) alone from pEK204, as well as both bla(CTX-M-3) and bla(TEM-1b) jointly. The 5' and 3' termini of the transposed fragments were identical to or resembled the ISEcp1 IR(L) and IR(R), respectively, thereby replicating the environments previously found in various Belfast clinical plasmids harbouring bla(CTX-M-3). CONCLUSIONS: Simultaneous dissemination of bla(CTX-M-3) and bla(TEM-1b) among plasmids in Belfast's nursing homes is facilitated by ISEcp1-mediated transposition of these bla genes from pEK204-like and other plasmids. Such transposition events are of public health concern, as they potentially allow wider dissemination of CTX-M-3 enzyme than would be possible through the spread of extended-spectrum ß-lactamase-encoding plasmids and strains alone.

Phosphoethanolamine modification of lipid A in colistin-resistant variants of Acinetobacter baumannii mediated by the pmrAB two-component regulatory system.

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-Mg(2+) 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.

Variation in the genetic environments of bla(CTX-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 ß-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.

Isolation of fluoroquinolone-resistant O25b:H4-ST131 Escherichia coli with CTX-M-14 extended-spectrum ß-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 ß-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.

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

OBJECTIVES: Between January 2004 and May 2006 Escherichia coli producing extended-spectrum ß-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.

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 ß-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.

Real-time PCR for detection of the O25b-ST131 clone of Escherichia coli and its CTX-M-15-like extended-spectrum beta-lactamases.

CTX-M-15 has become the most prevalent extended-spectrum beta-lactamase amongst Escherichia coli in many countries during the past decade. Its dominance partly reflects the dissemination of an E. coli O25b:H4 ST131 clone that commonly produces this enzyme. We describe rapid real-time polymerase chain reaction (PCR) assays able to detect E. coli belonging to the ST131 clone and to identify bla(CTX-M-15)-like.

Rapid detection of the O25b-ST131 clone of Escherichia coli encompassing the CTX-M-15-producing strains.

OBJECTIVES: Recently, a CTX-M-15 extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli O25b-ST131 clone, belonging to the B2 phylogenetic group and with a high virulence potential, has been reported all over the world, representing a major public health problem. The present study was carried out to develop a rapid and simple detection assay that identifies members of this clone. METHODS: A total of 627 E. coli isolates of which 373 produced an ESBL, collected across four continents, were screened using a O25b-ST131 clone allele-specific PCR for the pabB gene. RESULTS: One hundred and forty-three ESBL isolates were found positive with the assay. These isolates were all of O25b type and, when studied by multilocus sequence typing (25 cases), were all of ST131. The O25b-ST131 clone was found to produce ESBLs other than CTX-M-15, specifically CTX-M-2, -3, -14, -27, -32 and -61 as well as TEM-24. This clone represents 3% of non-ESBL B2 isolates originating from urinary tract infections in Paris. CONCLUSIONS: We have developed a PCR-based assay that easily identifies a clone with high likelihood of producing ESBLs, including CTX-M-15.