Rapid communications Genesis of a KPC-producing Klebsiella pneumoniae after in vivo transfer from an imported Greek strain

We document here the in vivo transfer of bla(KPC-2) between intensive care unit-acquired and a commensal strain of K. pneumoniae in a French patient after his repatriation from Greece. This first report of an emerging KPC-producing strain in France raises further concerns about the spread of carbapenem resistance among Enterobacteriaceae.


Introduction
Carbapenems are the cornerstone of therapy against multidrug-resistant (MDR) enterobacteria, notably those expressing extended-spectrum β-lactamases (ESBL).To date, enterobacterial strains producing Ambler class A Klebsiella pneumoniae carbapenemases (KPC) remain very scarce in western European countries and correspond almost exclusively to imported clones from endemic areas, namely, the United States, Israel and Greece [1].The blaKPC genes are located in a set of plasmid-borne Tn4401-type transposons [2], with recent evidence of interspecies conjugative transfer [3,4].Here, we provide the first evidence of in vivo transfer of bla KPC-2 between two K. pneumoniae strains from a single patient, one imported from Greece and the other from the commensal flora, leading to the emergence of a new KPC-2producing strain in France.

Case report and study
A French man in his 70s who was travelling in Greece was admitted to the intensive care unit (ICU) of a hospital in Athens on 30 April 2009 (day 0) for intestinal bleeding complicated by haemorrhagic shock and multiple organ failure.Several nosocomial infections occurred during his five-week long ICU stay in Athens, including a catheter-related bloodstream infection (BSI, day 25) due to a carbapenem-resistant K. pneumoniae strain that was also resistant to fluoroquinolones, co-trimoxazole, and aminoglycosides except gentamicin.This episode resolved after catheter removal and a one-week course of intravenous colistin.Subsequent clinical improvement allowed medical repatriation in France, and the patient was transferred to the ICU of a hospital in Paris (day 42).Intestinal carriage of MDR enterobacteria was routinely screened at admission by plating a rectal swab on ChromID ESBL medium (BioMérieux).One carbapenem-resistant K. pneumoniae strain (CHA-1) was isolated, and expressed the same co-resistances as the one involved in the BSI episode (Table ).As the patient had never been hospitalised previously, we assume that he acquired the CHA-1 in the ICU in Athens.
The patient was discharged to a general medical ward on day 62.He did not receive carbapenems or other β-lactams after his transfer from Greece.On day 92, a second rectal swab was cultured on ChromID ESBL medium.Overnight growth yielded K. pneumoniae for which subsequent antibiotic susceptibility testing showed two distinct phenotypes.Subculturing recovered CHA-1 and another K. pneumoniae strain designated as CHA-2 (Table ).According to the latest breakpoints published by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [5], CHA-2 was resistant to ertapenem, intermediate susceptible to doripenem, and susceptible to imipenem and meropenem (Table ).
Strains CHA-1 and CHA-2 were PCR-tested for all major β-lactamase-encoding genes, with subsequent sequencing of the PCR products.Both CHA-1 and CHA-2 carried bla KPC-2 and bla TEM-1 .In addition, the Ambler class B carbapenemase-encoding gene bla VIM-1 was detected in strain CHA-1 (Table ).

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We suspected that the strain CHA-2 had emerged by an in vivo co-transfer of bla KPC-2 /bla TEM-1 from the ICUacquired strain CHA-1 to a recipient wild-type commensal strain of K. pneumoniae.This hypothesis was supported by several facts: Firstly, conjugation assays in mixed broth cultures using the rifampicin-resistant Escherichia coli J53 strain as recipient and either CHA-1 or CHA-2 as donors resulted in bla KPC-2/ bla TEM-1 -positive J53 transconjugants (conjugation frequency: 10 -7 to 10 -8 ), suggesting co-transfer of a plasmid carrying both genes.After extraction using the CompactPrep Plasmid Midi Kit (Qiagen), plasmids from both transconjugants yielded identical EcoRI-digestion patterns, arguing that CHA-1 and CHA-2 strains harboured the same bla KPC-2 / bla TEM-1 -carrying plasmid.bla VIM-1 could not be transferred from CHA-1, as already experienced elsewhere [6].Secondly, the swab from day 92 was re-plated on Drigalski agar.Twenty-five suspected K. pneumoniae were isolated, and those that did not grow on subcultures on ChromID ESBL medium were identified and tested for β-lactam susceptibility.Sixteen wildtype isolates of K. pneumoniae were thus collected and all yielded identical patterns in an enterobacterial repetitive intergenic consensus (ERIC)-PCR, suggesting that they were duplicates of a single wild-type K. pneumoniae strain, designated as BW1 (Table ).Pulsed-field gel electrophoresis (PFGE) patterns of strains CHA-1, CHA-2 and BW1 were then compared to those of all KPC-2-producing pulsotypes of K. pneumoniae isolated to date in Greece (Figure ).
The result indicated that (i) strain CHA-1 belonged to a KPC-2/VIM-1-coproducing pulsotype that is currently spreading in Greek hospitals (pulsotype C) in parallel with the pulsotype A that is the predominant KPC-2-producing pulsotype in Greece [6,7], (ii) CHA-2 did not match with any of the described Greek pulsotypes and (iii) KPC-2-producing strain CHA-2 and a Obtained by conjugation assays using a rifampin-resistant mutant of BW1 selected on Szybalski gradients (BW1m, MIC of rifampin > 250 mg/L) as recipient and CHA-1 as donor, and subsequent isolation on Drigaslki agar supplemented with cefotaxime (1mg/L) plus rifampin (250mg/L).b The co-expression of VIM-1 and KPC-2 contributes to explain the higher MICs of β-lactams in strain CHA-1 when compared to the blaVIM-1egative/blaKPC-2-positive strain CHA-2; c MIC: minimal inhibitory concentrations, as defined by E-test d CLA: clavulanic acid (2 mg/L) e TZP: tazobactam (4 mg/L) wild-type strain BW1 displayed strictly identical XbaIfingerprints, except for one band of approximately 100 kb also observed in CHA-1 that may correspond to the bla KPC-2 /bla TEM-1 -carrying plasmid.These data supported the role of BW1, the dominant wild-type K. pneumoniae strain within the digestive flora, as the bla KPC-2 -negative precursor of CHA-2.Lastly, we confirmed that the bla KPC-2 /bla TEM-1 -carrying plasmid was transferable from CHA-1 to a rifampicin-resistant BW1 strain obtained on a Szybalski gradient (Table ).Some limitations are yet to be considered since we cannot strictly exclude that CHA-2 could have been acquired in Greece and could have been missed in the swab taken on day 42 at admission in France.Likewise, we cannot exclude that acquisition of CHA-2 could have occurred in France although reports on KPC-producing strains remain scarce to date.This report raises further concerns about the diffusion of carbapenem resistance among enterobacteria.
Indeed, that imported strains from endemic areas are able to spread blaKPC genes -even in the absence of β-lactam selective pressure, as in this patient -is worrisome, most notably for western European countries where the incidence of KPC-producing pathogens is still low.Greek pulsotype F * CHA-2 and BW1 pulsotypes only differ by a ~100-kb band deemed to match the bla KPC-2 -carrying plasmid (also harboured by strains CHA-1 and 1780) [6].PFGE: pulsed-field gel electrophoresis.

Table
Antibiotic resistance phenotypes and acquired bla gene contents of enterobacterial strains described in this study bla: beta lactamase; ICU: intensive care unit; NA: not applicable.