Carbapenemase-producing Salmonella enterica isolates in the UK.

Sir, Most non-typhoidal salmonella infections are self-limiting, but antibiotics can be life-saving for vulnerable patients and invasive disease. Salmonella enterica may exhibit resistance to quinolones and cephalosporins, but carbapenem resistance is rarely reported. Non-duplicate isolates (n1⁄4126) of cefotaxime-resistant (MICs .1 mg/L) non-typhoidal S. enterica were screened by disc testing for ertapenem resistance. Briefly, 0.5 McFarland standard cell suspensions in Iso-Sensitest broth were inoculated onto Mueller– Hinton agar plates and 10 mg ertapenem discs were added. Inhibition zone diameters that measured ≤27 mm after 18 h of incubation at 378C were considered resistant compared with positive controls (not salmonellae) producing NDM-1, KPC, VIM, IMP or OXA-48-like carbapenemases. In addition, 2597 S. enterica isolates from patients with a history of travel to North Africa, the Middle East or the Indian subcontinent (all cephalosporin susceptible) were screened retrospectively. A further 266 S. enterica serovar Typhi, 157 S. enterica serovar Paratyphi A and 10 S. enterica serovar Paratyphi B were screened as part of routine susceptibility testing for high-level temocillin resistance (MICs .128 mg/L; a marker for OXA-48-like enzymes) by breakpoint testing using IsoSensitest agar. The MICs were determined by Etest (bioMérieux, Basingstoke, UK) and interpreted using BSAC criteria (http://bsac. org.uk/wp-content/uploads/2012/02/Version-12-Apr-2013_final. pdf). Carbapenemase genes (KPC, OXA-48-like, NDM, IMP, VIM, GIM, SIM and SPM) were sought by in-house PCRs with gene variants identified by amplicon sequencing. Non-susceptible isolates were used as plasmid donors in electro-transformation to E. coli Alpha-Select cells (Bioline, London, UK) with the transformants selected on Iso-Sensitest agar containing 128 mg/L ampicillin onto which a 10 mg ertapenem disc had been placed. One of the 126 cefotaxime-resistant isolates showed reduced ertapenem susceptibility. S. enterica serovar Senftenberg strain H083340277 isolated in 2008 from the faeces of an outpatient with an unknown travel history was resistant to cefotaxime and ceftazidime (MICs .256 mg/L) and to ertapenem (MIC1⁄42 mg/L), but was susceptible to meropenem and imipenem (MICs1⁄42 mg/L) (Table 1). A blaNDM-1 gene was identified in this isolate. NDM carbapenemases are associated with a diverse range of plasmid backbones; therefore, the total genomic DNA of the NDM plasmid transformant was sequenced to .30× coverage on a HiSeq sequencing system (Illumina, Little Chesterford, UK). The reads were assembled into contigs using VelvetOptimiser (https://github.com/VictorianBioinformatics-Consortium/VelvetOptimiser.git), of which three contigs encoding the NDM plasmid were subtracted from the known E. coli host genome and linked by PCR-based gap closure and Sanger DNA sequencing to determine that blaNDM-1 was located on a 53133 bp complete plasmid sequence. SNP-calling using Bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2) and SAMtools (http://samtools.sourceforge.net) showed 99.7% identity to an IncX3-type blaNDM-1 plasmid from a Raoultella planticola isolated in China (GenBank KF877335), with only 13 SNPs to distinguish them. In both plasmids, blaNDM-1 was flanked upstream by ISAba125 (interrupted by IS5), with the bleomycin resistance gene bleMBL located downstream and with blaSHV-12 as the only other co-resident resistance gene. Temocillin MICs .128 mg/L were observed for two isolates from 2013: S. enterica serovar Typhimurium definitive phage type 193 strain H132980529 from an inpatient with diarrhoea and a history of travel to Africa and S. enterica serovar Paratyphi B phage type Dundee strain H134000562 from an inpatient with diarrhoea and vomiting reporting no recent travel. Both isolates showed reduced susceptibility to cefotaxime (MICs 2 mg/L) and ceftazidime susceptibility (MICs 0.5 mg/L), indicating that there were no co-resident ESBL or AmpC genes; they showed intermediate susceptibility to ertapenem (MICs 1 mg/L), and susceptibility to meropenem and imipenem (MICs 0.25–0.5 mg/L) (Table 1). blaOXA-48 was identified in both isolates. Previous studies have shown that the international dissemination of blaOXA-48 is largely due to a single plasmid, pOXA-48a. PCR for repA, traU and parA, Research letters