Abstract

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 blaOXA-48 gene; the K. pneumoniae isolates belonged to 11 sequence types (STs) and the E. coli to 7 STs, including ST131 and ST38. The blaOXA-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 β-lactamases. One India-linked K. pneumoniae isolate had a blaOXA-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.

Introduction

Carbapenems have the broadest spectrum of all β-lactam antibiotics and are increasingly used to treat infections caused by otherwise multidrug-resistant Gram-negative bacteria. Consequently, emerging resistance to carbapenems is a major public health concern, especially when it involves acquired, horizontally transmissible carbapenemases. These enzymes are diverse, belonging to classes A, B or D of the Ambler β-lactamase classification. Acquired class D carbapenemases are encountered most often in isolates of Acinetobacter spp., where OXA-23, -40 and -58 variants are all important; however, the genes encoding the OXA-48-like subgroup, which originally escaped from environmental bacteria of the genus Shewanella,1,2 are increasingly detected in clinical Enterobacteriaceae.

OXA-48 β-lactamase was initially identified in Turkey,3 but there are now multiple reports of its production, mainly by Klebsiella pneumoniae isolates, from countries in the Middle East, North Africa and, increasingly, Europe.4–10 Hospital outbreaks by producers of this carbapenemase have been described in Turkey6 and, more recently, in the UK,11 France,12 Spain,13 Ireland,14 The Netherlands15 and Belgium.16

K. pneumoniae of sequence type (ST) 395 has been implicated in the European spread of OXA-48 carbapenemase,17 although the blaOXA-48 gene has been detected in other clones and, indeed, other genera. The gene is usually located on transposon Tn1999,5 which has been found in OXA-48-positive Enterobacteriaceae of diverse geographic origin on highly related plasmids of ∼62 kb.4–6,12,13,18,19 A recently described variant, OXA-181, from India, differs from OXA-48 by just four amino acids, but its gene is associated with an ISEcp1 element on a distinct transposon, Tn2013, carried on much smaller plasmids of ∼7.6 kb.20 It is inferred to represent a separate genetic escape from Shewanella spp.2

From 2007 to the end of 2011, the Health Protection Agency (HPA) detected OXA-48-like carbapenemases in 94 referred isolates of Enterobacteriaceae from the UK, including 74 Klebsiella spp., 17 Escherichia coli, 2 Enterobacter cloacae and 1 Citrobacter sp.; these were among 993 isolates of Enterobacteriaceae that were confirmed to produce a carbapenemase in this 5year period (N. Woodford, unpublished data).21 We sought to characterize a representative group of these in greater detail.

Materials and methods

Twenty-six of 94 isolates known to produce OXA-48-like carbapenemases (based on PCR results) and representing referrals from diverse UK hospitals (n = 22) in 2007–11 were selected from the collection of carbapenemase producers held by the HPA's Antibiotic Resistance Monitoring and Reference Laboratory. They comprised 14 isolates of K. pneumoniae, 10 E. coli and two E. cloacae from urine (14), blood (5), wounds (4), drain fluid (2) and sputum (1). Twenty-three were from separate inpatients and three—K. pneumoniae KP16, E. coli EC25 and E. cloacae ENT26—were from urine samples collected from a single patient in outpatient clinic and general practitioner (GP) settings over a period of 3–4 months. Only 4/24 patients were known to have recent travel history—two to Turkey, one to India and one to Pakistan—though we cannot exclude unreported travel by others.

MICs were determined by BSAC agar dilution and were interpreted according to BSAC/EUCAST criteria (www.eucast.org). The presence of a blaOXA-48-like carbapenemase gene was confirmed by PCR followed by sequencing.22 PCR was also used to screen for ISEcp1 and IS1999 elements, as previously described.22 The presence of other β-lactamase genes—blaTEM, blaSHV, blaCTX-M group-1, blaCTX-M group-9, blaOXA-1 and blaOXA-9—was also sought, as these genes have been previously reported in Enterobacteriaceae producing OXA-48 enzyme.5–7,11,12 Multilocus sequence typing (MLST) was used to assess the relatedness of K. pneumoniae (www.pasteur.fr/recherche/genopole/PF8/mlst) and E. coli (http://mlst.ucc.ie/) isolates, with STs assigned using online database tools.

Plasmids were extracted by alkaline lysis and transformed into electro-competent E. coli DH5α cells (Bioline, London, UK). Transformants were selected on nutrient agar by picking colonies growing within zones of inhibition of piperacillin/tazobactam 75 + 10 μg discs and screening these for blaOXA-48-like by PCR. Plasmids were re-extracted from transformants and compared after digestion with EcoR1 (Sigma-Aldrich, Poole, UK). PCR-based replicon typing (PBRT) was used to identify plasmid incompatibility groups.23 The repA, traU and parA genes encoding proteins involved in plasmid replication, transfer and partitioning were sought in transformants or, where necessary, in the clinical isolates, using published primers to compare plasmids with the IncL/M plasmid pOXA-48a.18

Associations of blaOXA-48-like genes with insertion sequences were tested by PCR mapping,21 followed by sequencing of selected amplicons. In addition, conjugation experiments were performed to assess the self-transferability of plasmids carrying the blaOXA-48-like genes, using selected clinical isolates and/or transformants as donors and E. coli J53 as the recipient. Transconjugants were selected on nutrient agar supplemented with rifampicin 250 mg/L and ampicillin 100 mg/L.

Results

PCR and sequencing confirmed that 25 of the 26 isolates harboured a classical blaOXA-48 gene, although one K. pneumoniae isolate (KP19), from a patient with a history of travel to India, harboured blaOXA-181. All were non-susceptible to ertapenem (MICs 1 to >16 mg/L), ampicillin, amoxicillin/clavulanate, piperacillin and piperacillin/tazobactam (MICs >64 mg/L). However, 18 remained susceptible in vitro to meropenem (MICs ≤4 mg/L) and 13 to imipenem (MICs ≤2 mg/L). Neither OXA-48 nor OXA-181 carbapenemase hydrolyse oxyimino-cephalosporins efficiently,19 yet 21/26 isolates were resistant to cefotaxime (MICs 2 to >256 mg/L), 17 to aztreonam (MICs 4 to >64 mg/L) and 15 to ceftazidime (MICs 2–256 mg/L), reflecting the frequent presence of extended-spectrum β-lactamases (ESBLs) (Table 1). Genes encoding group 1 or 9 CTX-M ESBLs were detected in 10 and 6 isolates, respectively, and these 16 isolates were more resistant to cephalosporins than the remaining 10 isolates, all of which remained susceptible or showed only intermediate susceptibility to cefotaxime and/or ceftazidime (Table 1). Susceptibility to non-β-lactam antibiotics varied: 15 were resistant to ciprofloxacin (MICs 4 to >8 mg/L), 11 to gentamicin (MICs 16 to >32 mg/L), 5 to amikacin (MICs 16 to >64 mg/L) and 5 to tigecycline (MICs 4–16 mg/L). All 26 isolates were susceptible to colistin, with MICs ≤1 mg/L.

Table 1.

Characteristics of clinical isolates of Enterobacteriaceae with OXA-48-like carbapenemases

Isolate Hospital + patient Year Isolation site Travel history ST Carbapenemase Other β-lactamase genes detected by PCR MIC (mg/L)a
 
IPM MEM ETP CTX CAZ CIP GEN TGC 
K. pneumoniae 
 KP01 A1 2007 urine Turkey 147 OXA-48 CTX-M group 1; OXA-1; TEM; SHVb 16 >16 >256 256 >8 
 KP02c B2 2008 wound unknown 353 OXA-48 TEM; SHV >16 64 0.5 0.5 
 KP03 C3 2009 urine unknown 432 OXA-48 OXA-1; SHV 0.25 0.5 0.5 
 KP04 D4 2009 urine unknown 101 OXA-48 CTX-M group 1; OXA-1; TEM; SHV 0.5 256 256 >8 >32 0.5 
 KP05 E5 2009 fluid unknown 383 OXA-48 CTX-M group 1; OXA-1; SHV 16 >256 256 >8 >32 
 KP07 F6 2010 urine unknown 14 OXA-48 CTX-M group 1; SHV >256 64 >32 0.5 
 KP08 G7 2010 blood unknown 858 OXA-48 CTX-M group 1; OXA-1; TEM; SHV 0.5 256 64 >8 32 
 KP09 H8 2010 urine none 17 OXA-48 OXA-1; TEM; SHV 0.5 ≤0.12 
 KP16d I9 2010 urine Turkey 152 OXA-48 SHV 16 ≤0.12 0.5 ≤0.25 
 KP17 J10 2011 fluid unknown 101 OXA-48 CTX-M group 1; OXA-1; TEM; SHV >256 256 >8 0.5 
 KP20 K11 2011 blood unknown 101 OXA-48 CTX-M group 1; OXA-1; OXA-9; TEM; SHV 16 >16 >256 128 >8 >32 16 
 KP22 E12 2011 wound Pakistan 37 OXA-48 CTX-M group 1; OXA-1; SHV 256 >256 >8 16 
 KP24 L13 2011 urine unknown 376 OXA-48 CTX-M group 9; OXA-9; TEM; SHV >16 >256 64 >8 >32 
 KP19 B14 2011 blood India 11 OXA-181 CTX-M group 1; OXA-1; SHV 128 32 >16 >256 64 >8 >32 0.5 
E. coli 
 EC06 H15 2010 wound unknown 648 OXA-48 CTX-M group 1; OXA-1 0.5 >256 32 >8 0.25 
 EC10 M16 2010 blood none 131 OXA-48 None 0.25 >8 0.5 
 EC11 B17 2010 wound unknown 38 OXA-48 CTX-M group 9; TEM >16 256 0.25 16 ≤0.25 
 EC12 N18 2010 urine unknown 10 OXA-48 TEM 0.5 0.25 ≤0.12 ≤0.25 
 EC13 J19 2010 blood unknown 38 OXA-48 CTX-M group 9; TEM 64 >32 >16 256 >8 32 ≤0.25 
 EC15 O20 2010 urine unknown 155 OXA-48 TEM 0.5 0.5 0.25 0.5 ≤0.25 
 EC18 P21 2011 urine unknown 38 OXA-48 CTX-M group 9; TEM 0.5 256 0.25 32 ≤0.25 
 EC21 Q22 2011 urine unknown 88 OXA-48 TEM 16 0.5 ≤0.12 ≤0.25 
 EC23 R23 2011 sputum unknown 38 OXA-48 CTX-M group 9; TEM 128 >32 >16 >256 ≤0.12 
 EC25d I9 2011 urine Turkey 167 OXA-48 TEM 0.5 0.5 0.25 >8 0.5 ≤0.25 
Enterobacter 
 ENT14 D24 2010 urine unknown NA OXA-48 CTX-M group 9 16 32 ≤0.12 
 ENT26d I9 2011 urine Turkey NA OXA-48 None ≤0.12 0.5 ≤0.25 
Isolate Hospital + patient Year Isolation site Travel history ST Carbapenemase Other β-lactamase genes detected by PCR MIC (mg/L)a
 
IPM MEM ETP CTX CAZ CIP GEN TGC 
K. pneumoniae 
 KP01 A1 2007 urine Turkey 147 OXA-48 CTX-M group 1; OXA-1; TEM; SHVb 16 >16 >256 256 >8 
 KP02c B2 2008 wound unknown 353 OXA-48 TEM; SHV >16 64 0.5 0.5 
 KP03 C3 2009 urine unknown 432 OXA-48 OXA-1; SHV 0.25 0.5 0.5 
 KP04 D4 2009 urine unknown 101 OXA-48 CTX-M group 1; OXA-1; TEM; SHV 0.5 256 256 >8 >32 0.5 
 KP05 E5 2009 fluid unknown 383 OXA-48 CTX-M group 1; OXA-1; SHV 16 >256 256 >8 >32 
 KP07 F6 2010 urine unknown 14 OXA-48 CTX-M group 1; SHV >256 64 >32 0.5 
 KP08 G7 2010 blood unknown 858 OXA-48 CTX-M group 1; OXA-1; TEM; SHV 0.5 256 64 >8 32 
 KP09 H8 2010 urine none 17 OXA-48 OXA-1; TEM; SHV 0.5 ≤0.12 
 KP16d I9 2010 urine Turkey 152 OXA-48 SHV 16 ≤0.12 0.5 ≤0.25 
 KP17 J10 2011 fluid unknown 101 OXA-48 CTX-M group 1; OXA-1; TEM; SHV >256 256 >8 0.5 
 KP20 K11 2011 blood unknown 101 OXA-48 CTX-M group 1; OXA-1; OXA-9; TEM; SHV 16 >16 >256 128 >8 >32 16 
 KP22 E12 2011 wound Pakistan 37 OXA-48 CTX-M group 1; OXA-1; SHV 256 >256 >8 16 
 KP24 L13 2011 urine unknown 376 OXA-48 CTX-M group 9; OXA-9; TEM; SHV >16 >256 64 >8 >32 
 KP19 B14 2011 blood India 11 OXA-181 CTX-M group 1; OXA-1; SHV 128 32 >16 >256 64 >8 >32 0.5 
E. coli 
 EC06 H15 2010 wound unknown 648 OXA-48 CTX-M group 1; OXA-1 0.5 >256 32 >8 0.25 
 EC10 M16 2010 blood none 131 OXA-48 None 0.25 >8 0.5 
 EC11 B17 2010 wound unknown 38 OXA-48 CTX-M group 9; TEM >16 256 0.25 16 ≤0.25 
 EC12 N18 2010 urine unknown 10 OXA-48 TEM 0.5 0.25 ≤0.12 ≤0.25 
 EC13 J19 2010 blood unknown 38 OXA-48 CTX-M group 9; TEM 64 >32 >16 256 >8 32 ≤0.25 
 EC15 O20 2010 urine unknown 155 OXA-48 TEM 0.5 0.5 0.25 0.5 ≤0.25 
 EC18 P21 2011 urine unknown 38 OXA-48 CTX-M group 9; TEM 0.5 256 0.25 32 ≤0.25 
 EC21 Q22 2011 urine unknown 88 OXA-48 TEM 16 0.5 ≤0.12 ≤0.25 
 EC23 R23 2011 sputum unknown 38 OXA-48 CTX-M group 9; TEM 128 >32 >16 >256 ≤0.12 
 EC25d I9 2011 urine Turkey 167 OXA-48 TEM 0.5 0.5 0.25 >8 0.5 ≤0.25 
Enterobacter 
 ENT14 D24 2010 urine unknown NA OXA-48 CTX-M group 9 16 32 ≤0.12 
 ENT26d I9 2011 urine Turkey NA OXA-48 None ≤0.12 0.5 ≤0.25 

IPM, imipenem; MEM, meropenem; ETP, ertapenem; CTX, cefotaxime; CAZ, ceftazidime; CIP, ciprofloxacin; GEN, gentamicin; TGC, tigecycline; NA, not applicable.

aAll isolates were resistant to piperacillin/tazobactam (MICs >64 mg/L) and were susceptible to colistin (MICs ≤1 mg/L).

bblaSHV may be chromosomal and inherent in Klebsiella spp.

cAvailable from the National Collection of Type Cultures (accession NCTC 13442).

dIsolates of K. pneumoniae, E. coli and Enterobacter sp. from a single patient in an outpatient or GP setting.

MLST showed that the 13 OXA-48-positive K. pneumoniae isolates belonged to diverse STs: three to ST101; one to each of STs 14, 17, 37, 147, 152, 353, 376, 383 and 432; and one to a new ST, 858, which is a single locus variant of ST17. Isolate KP19, with OXA-181 carbapenemase, belonged to ST11. Similarly the 10 OXA-48-positive E. coli isolates were clonally diverse: four belonged to ST38, and one to each of STs 10, 88, 131, 155, 167 and 648.

Carbapenemase-encoding plasmids were transformed into E. coli DH5α using DNA extracted from 21/25 OXA-48-positive isolates and from the single OXA-181 producer, with the presence of blaOXA-48-like confirmed in the transformants by PCR. Despite repeated attempts, transformants were not obtained using DNA extractions from any of the four E. coli isolates belonging to ST38. Most (20/21) of the OXA-48 transformants and the single OXA-181 transformant had reduced susceptibility to carbapenems, resistance to ampicillin, amoxicillin/clavulanate, piperacillin and piperacillin/tazobactam (MICs 32 to >64 mg/L), and susceptibility to the other agents tested (Table 2). Eighteen of the 21 OXA-48-positive transformants harboured a single plasmid of ∼62 kb, and 2 harboured a smaller plasmid of ∼50 kb. The remaining OXA-48-positive transformant (T24) additionally gained resistance to expanded-spectrum cephalosporins, aztreonam and aminoglycosides. It contained an ∼140 kb plasmid with blaOXA-9 and a group 9 blaCTX-M gene along with blaOXA-48, consistent with its broader resistance phenotype. The OXA-181-positive transformant gained a plasmid of ∼7 kb and acquired resistance only to carbapenems (MICs >16–128 mg/L), penicillins and penicillin/β-lactamase inhibitor combinations.

Table 2.

Characteristics of transformants of E. coli DH5α with OXA-48-like carbapenemases

Donor ST of donor Transformed plasmid (kb) PCR for ‘pOXA-48a genes’
 
Carbapenemase contexta Other bla in transformant MICs (mg/L)b
 
parA repA traU IPM MEM ETP CTX CAZ 
DH5α (recipient) — — — — — — 0.125 ≤0.06 ≤0.06 ≤0.125 ≤0.125 
KP01 147 ∼62 + + + Tn1999 with blaOXA-48 none 0.25 0.25 ≤0.125 0.25 
KP05 383 ∼62 + + + Tn1999 with blaOXA-48 none 0.125 0.5 ≤0.125 ≤0.125 
KP02 353 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.25 0.25 0.25 0.25 
KP03 432 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.25 0.25 0.5 0.25 
KP04 101 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.25 0.5 0.25 ≤0.125 
KP07 14 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.125 0.5 0.25 ≤0.125 
KP08 858 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.125 0.5 0.25 ≤0.125 
KP09 17 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.125 0.25 0.25 ≤0.125 
KP16d 152 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.25 0.25 ≤0.125 
KP17 101 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.25 0.5 ≤0.125 ≤0.125 
KP20 101 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.125 0.5 0.25 ≤0.125 
KP22 37 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.25 0.5 ≤0.125 ≤0.125 
KP24 376 ∼140 + + + Tn1999.2 with blaOXA-48 blaCTX-M-9, blaOXA-9 0.25 0.5 256 
KP19 11 ∼7 − − − Tn2013 with blaOXA-181 none 0.25 0.25 ≤0.125 
EC10 131 ∼50 − − Tn1999 with blaOXA-48 none 0.5 
EC11 38 none obtained +c c c Tn1999 with blaOXA-48 NA — — — — — 
EC13 38 none obtained +c c c Tn1999 with blaOXA-48 NA — — — — — 
EC18 38 none obtained +c c c Tn1999 with blaOXA-48 NA — — — — — 
EC23 38 none obtained +c c c Tn1999 with blaOXA-48 NA — — — — — 
EC06 648 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.125 0.5 0.25 0.25 
EC12 10 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.125 0.5 0.25 0.25 
EC15 155 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.125 0.5 0.25 ≤0.125 
EC21 88 ∼50 + + + Tn1999.2 with blaOXA-48 none 0.5 0.5 0.25 
EC25d 167 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.5 0.5 0.25 
ENT14 NA ∼62 + + + Tn1999.2 with blaOXA-48 none 0.125 0.25 0.25 ≤0.125 
ENT26d NA ∼62 + + + Tn1999.2 with blaOXA-48 none 0.25 0.5 0.25 ≤0.125 
Donor ST of donor Transformed plasmid (kb) PCR for ‘pOXA-48a genes’
 
Carbapenemase contexta Other bla in transformant MICs (mg/L)b
 
parA repA traU IPM MEM ETP CTX CAZ 
DH5α (recipient) — — — — — — 0.125 ≤0.06 ≤0.06 ≤0.125 ≤0.125 
KP01 147 ∼62 + + + Tn1999 with blaOXA-48 none 0.25 0.25 ≤0.125 0.25 
KP05 383 ∼62 + + + Tn1999 with blaOXA-48 none 0.125 0.5 ≤0.125 ≤0.125 
KP02 353 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.25 0.25 0.25 0.25 
KP03 432 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.25 0.25 0.5 0.25 
KP04 101 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.25 0.5 0.25 ≤0.125 
KP07 14 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.125 0.5 0.25 ≤0.125 
KP08 858 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.125 0.5 0.25 ≤0.125 
KP09 17 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.125 0.25 0.25 ≤0.125 
KP16d 152 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.25 0.25 ≤0.125 
KP17 101 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.25 0.5 ≤0.125 ≤0.125 
KP20 101 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.125 0.5 0.25 ≤0.125 
KP22 37 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.25 0.5 ≤0.125 ≤0.125 
KP24 376 ∼140 + + + Tn1999.2 with blaOXA-48 blaCTX-M-9, blaOXA-9 0.25 0.5 256 
KP19 11 ∼7 − − − Tn2013 with blaOXA-181 none 0.25 0.25 ≤0.125 
EC10 131 ∼50 − − Tn1999 with blaOXA-48 none 0.5 
EC11 38 none obtained +c c c Tn1999 with blaOXA-48 NA — — — — — 
EC13 38 none obtained +c c c Tn1999 with blaOXA-48 NA — — — — — 
EC18 38 none obtained +c c c Tn1999 with blaOXA-48 NA — — — — — 
EC23 38 none obtained +c c c Tn1999 with blaOXA-48 NA — — — — — 
EC06 648 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.125 0.5 0.25 0.25 
EC12 10 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.125 0.5 0.25 0.25 
EC15 155 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.125 0.5 0.25 ≤0.125 
EC21 88 ∼50 + + + Tn1999.2 with blaOXA-48 none 0.5 0.5 0.25 
EC25d 167 ∼62 + + + Tn1999.2 with blaOXA-48 none 0.5 0.5 0.25 
ENT14 NA ∼62 + + + Tn1999.2 with blaOXA-48 none 0.125 0.25 0.25 ≤0.125 
ENT26d NA ∼62 + + + Tn1999.2 with blaOXA-48 none 0.25 0.5 0.25 ≤0.125 

IPM, imipenem; MEM, meropenem; ETP, ertapenem; CTX, cefotaxime; CAZ, ceftazidime; NA, not applicable.

aDeduced from donor isolates for EC11, 13, 18 and 23.

bAll transformants were resistant to piperacillin/tazobactam (MICs >64 mg/L), used for selection.

cPCR for pOXA-48a genes was performed on clinical isolates since no transformants were available.

dDonor isolates of K. pneumoniae, E. coli and Enterobacter sp. from a single patient.

Plasmid restriction analysis (data not shown) indicated that the ∼62 kb OXA-48-encoding plasmids were all highly related, whereas the ∼50 kb and ∼140 kb plasmids appeared dissimilar (Table 2). All of these plasmids were transferable by conjugation to E. coli J53. However, no OXA-48-positive transconjugants were obtained when E. coli of ST38 were used as donors, which is consistent with the lack of transformants from these isolates. The 7 kb OXA-181-encoding plasmid had a distinct restriction profile (KP19) and also could not be transferred by conjugation.

None of the plasmids could be assigned to an incompatibility group by the PBRT method. Plasmid pOXA-48a, which is an IncL/M-type plasmid, albeit not typeable by the PBRT method,18 shares three critical functional genes (repA, traU and parA) with other sequenced IncL/M plasmids such as pCTX-M-3 and pHK-NDM.18 We detected the repA, traU and parA genes of plasmid pOXA-48a19 in 20/21 transformants with OXA-48 enzyme, suggesting that these 20 plasmids had an IncL/M-pOXA-48a-like backbone. The remaining OXA-48 transformant, with a 50 kb plasmid from isolate EC10, and the four E. coli clinical isolates of ST38 gave products only with parA primers (Table 2). The transformant from KP19 with OXA-181 enzyme gave no PCR products with any of the primers.

The genetic environments of the blaOXA-48-like genes were determined using combinations of primers specific for an insertion sequence and the carbapenemase gene. Sequencing indicated that the blaOXA-48 genes were flanked by upstream and downstream copies of IS1999; in six isolates the upstream copy was intact, corresponding to transposon Tn1999,22 whereas 19 had a Tn1999.2 transposon in which the IS1999 sequence is disrupted by an IS1R element.6 The single OXA-181-positive isolate lacked IS1999, and had an ISEcp1 element immediately upstream of the blaOXA-181 gene, as reported previously.20

K. pneumoniae KP16, E. coli EC25 and E. cloacae ENT26 were recovered from the urinary tract of the same patient, but exhibited different susceptibility profiles to cephalosporins, reflecting the presence of different additional β-lactamases (Table 1). All carried indistinguishable 70 kb plasmids, with blaOXA-48 as part of a Tn1999.2 element. Notably EC25 and ENT26 were isolated 3–4 months after KP16.

Discussion

We have shown that OXA-48 carbapenemase is becoming scattered in the UK, not only in K. pneumoniae, but also in E. coli and E. cloacae. Producers from multiple UK hospitals represented diverse STs. We did not detect K. pneumoniae ST395, which has been implicated in clonal outbreaks elsewhere in Europe,17 but did find E. coli ST38, reported previously with OXA-48 carbapenemase in France.18,19 The data support multiple origins or repeated introduction to the UK of bacteria with the blaOXA-48 gene.

Despite this clonal diversity, we demonstrated that emergence of OXA-48 carbapenemase in the UK has been facilitated by horizontal transfer between strains and genera of successful and related plasmids, most of which were similar to pOXA-48a18 and others identified previously in many countries, especially in Europe and the Mediterranean region.4–6,8,12,13 Consistent with the molecular epidemiological evidence, the ∼50 kb, ∼62 kb and ∼140 kb plasmids were all transferable in vitro by conjugation. Isolation of three OXA-48-positive isolates of K. pneumoniae, E. coli and E. cloacae from a single patient further suggests that this plasmid can transfer successfully in vivo. The location of blaOXA-48 and its associated Tn1999.2 element in E. coli of ST38 requires further study since plasmids could not be transferred from any of four isolates by transformation or conjugation; this contrasts with a previous report in which ST38 E. coli transferred blaOXA-48 in association with a 62 kb plasmid.18,19

Plasmids encoding OXA-48 carbapenemase often do not carry other resistance determinants,19 and so confer resistance only to penicillin/inhibitor combinations and carbapenems. Therefore, some OXA-48 producers remain susceptible to oxyimino-cephalosporins, which offer potential therapeutic options in these cases. The resulting carbapenem-resistant, cephalosporin-susceptible phenotype is not attributed reliably to carbapenemase production by automated systems,24 and is perhaps not believed by some microbiologists who encounter it in classical susceptibility testing; consequently there is concern that such strains may pass unrecognized in diagnostic laboratories. Accurate detection is further complicated, since many OXA-48 producers remain susceptible or intermediate in vitro to imipenem and meropenem. Recently Glupczynski et al.16 have suggested that temocillin could be a sensitive screening tool for difficult-to-detect OXA-48-producing Enterobacteriaceae in Belgium, which is noteworthy, as temocillin is also used in the UK. A combination of an OXA-48-encoding plasmid with other resistance mechanisms, principally ESBLs, leads to broader resistance and more reliable detection, but leaves fewer therapeutic options. Of concern, we identified a larger OXA-48-encoding plasmid that also carried a gene for a group 9 CTX-M ESBL in one isolate of K. pneumoniae. A further concern is our detection of blaOXA-48 in an E. coli isolate of the uropathogenic clone ST131, which has achieved notoriety for its role in the rapid global dissemination of ESBLs, especially CTX-M-15, including in community settings.25 The present finding adds to a growing number of reports that this clone can host carbapenemases, also including NDM and KPC types.26,27 Its potential to serve as a vehicle for hospital-community spread of carbapenemases is profoundly alarming.

This is also the first known report of a bacterial isolate producing OXA-181 carbapenemase in the UK. The isolate came from a patient with recent travel to India, supporting the link, demonstrated by others,2,28 between this resistance determinant and the Indian subcontinent; it has also been identified in New Zealand, in a patient previously hospitalized in Nepal and Thailand.29 Perhaps significantly, this isolate displayed higher levels of carbapenem resistance than most producing the OXA-48 enzyme.

In summary, the scatter and diversity of Enterobacteriaceae producing OXA-48 carbapenemase in the UK demonstrates that this easily missed resistance is probably distributed more widely than is currently recognized. The ‘Mediterranean region’, including North African countries and the Middle East are the ‘cradle’ of blaOXA-48, but international studies will be needed to confront its continued diffusion, especially in view of the population movements triggered by unrest and revolution in North Africa. There is already a published report of its import, via a war casualty, from Libya to Slovenia,30 with similar cases seen recently in the UK (Antibiotic Resistance Monitoring & Reference Laboratory [ARMRL], data on file).

Funding

This work was supported by funding from the European Community (TROCAR contract HEALTH-F3-2008-223031).

Transparency declarations

D. M. L. has shareholdings in AstraZeneca, Dechra, Eco Animal Health, GlaxoSmithKline, Pfizer and Merck, within diversified portfolios; has accepted grants, speaking invitations and conference invitations from GlaxoSmithKline, Merck, Pfizer, Novartis and AstraZeneca; has recent or ongoing consultancy with Achaogen, Astellas, AstraZeneca, Basilea, Bayer, Cubist, Kalidex and Tetraphase; and has ongoing contract work for Achaogen, Basilea, Cubist, Meiji and Merck. N. W. has received research grants and/or conference support from numerous pharmaceutical companies, including those that manufacture carbapenems. Other authors: none to declare.

References

1
Poirel
L
Heritier
C
Nordmann
P
Chromosome-encoded Ambler class D β-lactamase of Shewanella oneidensis as a progenitor of carbapenem-hydrolyzing oxacillinase
Antimicrob Agents Chemother
 , 
2004
, vol. 
48
 (pg. 
348
-
51
)
2
Potron
A
Poirel
L
Nordmann
P
Origin of OXA-181, an emerging carbapenem-hydrolyzing oxacillinase, as a chromosomal gene in Shewanella xiamenensis
Antimicrob Agents Chemother
 , 
2011
, vol. 
55
 (pg. 
4405
-
7
)
3
Poirel
L
Heritier
C
Tolun
V
, et al.  . 
Emergence of oxacillinase-mediated resistance to imipenem in Klebsiella pneumoniae
Antimicrob Agents Chemother
 , 
2004
, vol. 
48
 (pg. 
15
-
22
)
4
Goren
MG
Chmelnitsky
I
Carmeli
Y
, et al.  . 
Plasmid-encoded OXA-48 carbapenemase in Escherichia coli from Israel
J Antimicrob Chemother
 , 
2011
, vol. 
66
 (pg. 
672
-
3
)
5
Carrer
A
Poirel
L
Yilmaz
M
, et al.  . 
Spread of OXA-48-encoding plasmid in Turkey and beyond
Antimicrob Agents Chemother
 , 
2010
, vol. 
54
 (pg. 
1369
-
73
)
6
Carrer
A
Poirel
L
Eraksoy
H
, et al.  . 
Spread of OXA-48-positive carbapenem-resistant Klebsiella pneumoniae isolates in Istanbul, Turkey
Antimicrob Agents Chemother
 , 
2008
, vol. 
52
 (pg. 
2950
-
4
)
7
Gulmez
D
Woodford
N
Palepou
MF
, et al.  . 
Carbapenem-resistant Escherichia coli and Klebsiella pneumoniae isolates from Turkey with OXA-48-like carbapenemases and outer membrane protein loss
Int J Antimicrob Agents
 , 
2008
, vol. 
31
 (pg. 
523
-
6
)
8
Moquet
O
Bouchiat
C
Kinana
A
, et al.  . 
Class D OXA-48 carbapenemase in multidrug-resistant enterobacteria, Senegal
Emerg Infect Dis
 , 
2011
, vol. 
17
 (pg. 
143
-
4
)
9
Huang
TD
Bogaerts
P
Berhin
C
, et al.  . 
Rapid emergence of carbapenemase-producing Enterobacteriaceae isolates in Belgium
Euro Surveill
 , 
2011
, vol. 
16
  
pii=19900
10
Kalpoe
JS
al Naiemi
N
Poirel
L
, et al.  . 
Detection of an Ambler class D OXA-48-type β-lactamase in a Klebsiella pneumoniae strain in The Netherlands
J Med Microbiol
 , 
2011
, vol. 
60
 (pg. 
677
-
8
)
11
Thomas
CP
Elamin
N
Doumith
M
, et al.  . 
Hospital outbreak of Klebsiella pneumoniae producing OXA-48 carbapenemase in the United Kingdom
Abstracts of the Forty-ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, USA, 2009
Washington, DC, USA
American Society for Microbiology
 
Abstract C2-648
12
Cuzon
G
Ouanich
J
Gondret
R
, et al.  . 
Outbreak of OXA-48-positive carbapenem-resistant Klebsiella pneumoniae isolates in France
Antimicrob Agents Chemother
 , 
2011
, vol. 
55
 (pg. 
2420
-
3
)
13
Pitart
C
Sole
M
Roca
I
, et al.  . 
First outbreak of a plasmid-mediated carbapenem-hydrolyzing OXA-48 β-lactamase in Klebsiella pneumoniae in Spain
Antimicrob Agents Chemother
 , 
2011
, vol. 
55
 (pg. 
4398
-
401
)
14
O'Brien
DJ
Wrenn
C
Roche
C
, et al.  . 
First isolation and outbreak of OXA-48-producing Klebsiella pneumoniae in an Irish hospital, March to June 2011
Euro Surveill
 , 
2011
, vol. 
16
  
pii=19921
15
RIVM—National Institute for Public Health and the Environment
Combatting the Superbug Klebsiella OXA-48 Outbreak in a Dutch Hospital
   
)
16
Glupczynski
Y
Huang
TD
Bouchahrouf
W
, et al.  . 
Rapid emergence and spread of OXA-48-producing carbapenem-resistant Enterobacteriaceae isolates in Belgian hospitals
Int J Antimicrob Agents
 , 
2012
, vol. 
39
 (pg. 
168
-
72
)
17
Potron
A
Kalpoe
J
Poirel
L
, et al.  . 
European dissemination of a single OXA-48-producing Klebsiella pneumoniae clone
Clin Microbiol Infect
 , 
2011
, vol. 
17
 (pg. 
E24
-
6
)
18
Poirel
L
Bernabeu
S
Fortineau
N
, et al.  . 
Emergence of OXA-48-producing Escherichia coli clone ST38 in France
Antimicrob Agents Chemother
 , 
2011
, vol. 
55
 (pg. 
4937
-
8
)
19
Poirel
L
Bonnin
RA
Nordmann
P
Genetic features of the widespread plasmid coding for the carbapenemase OXA-48
Antimicrob Agents Chemother
 , 
2012
, vol. 
56
 (pg. 
559
-
62
)
20
Potron
A
Nordmann
P
Lafeuille
E
, et al.  . 
Characterization of OXA-181, a carbapenem-hydrolyzing class D β-lactamase from Klebsiella pneumoniae
Antimicrob Agents Chemother
 , 
2011
, vol. 
55
 (pg. 
4896
-
9
)
21
Anonymous
Carbapenemase-producing Enterobacteriaceae in the UK, 2003–2011
Health Protect Rep
 , 
2011
, vol. 
5
  
issue 24 (17 June)
22
Aubert
D
Naas
T
Heritier
C
, et al.  . 
Functional characterization of IS1999, an IS4 family element involved in mobilization and expression of β-lactam resistance genes
J Bacteriol
 , 
2006
, vol. 
188
 (pg. 
6506
-
14
)
23
Carattoli
A
Bertini
A
Villa
L
, et al.  . 
Identification of plasmids by PCR-based replicon typing
J Microbiol Methods
 , 
2005
, vol. 
63
 (pg. 
219
-
28
)
24
Woodford
N
Eastaway
AT
Ford
M
, et al.  . 
Comparison of BD Phoenix, Vitek 2, and MicroScan automated systems for detection and inference of mechanisms responsible for carbapenem resistance in Enterobacteriaceae
J Clin Microbiol
 , 
2010
, vol. 
48
 (pg. 
2999
-
3002
)
25
Rogers
BA
Sidjabat
HE
Paterson
DL
Escherichia coli O25b-ST131: a pandemic, multiresistant, community-associated strain
J Antimicrob Chemother
 , 
2011
, vol. 
66
 (pg. 
1
-
14
)
26
Poirel
L
Hombrouck-Alet
C
Freneaux
C
, et al.  . 
Global spread of New Delhi metallo-β-lactamase 1
Lancet Infect Dis
 , 
2010
, vol. 
10
 pg. 
832
 
27
Morris
D
Boyle
F
Ludden
C
, et al.  . 
Production of KPC-2 carbapenemase by an Escherichia coli clinical isolate belonging to the international ST131 clone
Antimicrob Agents Chemother
 , 
2011
, vol. 
55
 (pg. 
4935
-
6
)
28
Castanheira
M
Deshpande
LM
Mathai
D
, et al.  . 
Early dissemination of NDM-1- and OXA-181-producing Enterobacteriaceae in Indian hospitals: report from the SENTRY antimicrobial surveillance program, 2006–2007
Antimicrob Agents Chemother
 , 
2011
, vol. 
55
 (pg. 
1274
-
8
)
29
Williamson
DA
Heffernan
H
Sidjabat
H
, et al.  . 
Intercontinental transfer of OXA-181-producing Klebsiella pneumoniae into New Zealand
J Antimicrob Chemother
 , 
2011
, vol. 
66
 (pg. 
2888
-
90
)
30
Pirs
M
Andlovic
A
Cerar
T
, et al.  . 
A case of OXA-48 carbapenemase-producing Klebsiella pneumoniae in a patient transferred to Slovenia from Libya, November 2011
Euro Surveill
 , 
2011
, vol. 
16
  
pii = 20042