National survey of colistin resistance among carbapenemase-producing Enterobacteriaceae and outbreak caused by colistin-resistant OXA-48-producing Klebsiella pneumoniae, France, 2014

From January 2014 to December 2014, 972 consecutive non-replicate carbapenemase-producing Enterobacteriaceae isolates from colonised or infected patients were collected at the Associated French National Reference Centre as part of the French national survey on antimicrobial resistance. It included 577 Klebsiella spp. (59%), 236 Escherichia coli (24%), 108 Enterobacter spp. (11%), 50 Citrobacter spp. (5%), and a single Salmonella spp. isolate (0.1%). Of 561 K. pneumoniae isolates, 35 were found to be resistant to colistin (6.2%). PFGE analysis revealed a clonal outbreak involving 15 K. pneumoniae isolates belonging to sequence type ST11, recovered in a single hospital in the Picardie region in northern France. Those clonally related isolates showed variable levels of resistance to colistin, ranging from 4 to 64 mg/L. They harboured the blaOXA-48 carbapenemase gene and the blaCTX-M-15 extended-spectrum beta-lactamase gene. Among the 91 Enterobacter cloacae isolates, seven were resistant to colistin and produced different types of carbapenemases. Surprisingly, none of the E. coli and Citrobacter spp. isolates showed resistance to colistin. This national survey including carbapenemase-producing isolates recovered in 2014 reported a high rate of colistin resistance in K. pneumoniae and E. cloacae (6.2% and 7.7%, respectively) in France.


Introduction
Carbapenemase-producing Enterobacteriaceae (CPE) resistant to colistin are increasingly reported. They represent an additional link in the development of pan-drug resistance. However, the epidemiology of colistin resistance among enterobacterial isolates is currently almost unknown in most parts of the world. In Italy, an increase in carbapenemase-producing Enterobacteriaceae has been noted in the past years, but the situation remains unknown in France [1]. The lack of information about the prevalence of colistin resistance among multidrug-resistant enterobacterial isolates derives from several reasons: (i) so far, there has been limited interest in that field, (ii) methods used for determination of colistin susceptibility are not adequate, and (iii) the lack of well-defined breakpoints does not allow precise determination of prevalence. However, the recent identification of a plasmid-borne polymyxin resistance determinant (MCR-1) raised a very serious concern in that resistance to colistin might widely disseminate [2].
The aim of this study was to evaluate retrospectively the prevalence of colistin resistance among a collection of CPE strains recovered in France during a period of one year and to analyse the phenotypic, genotypic features and clonality of the colistin-resistant isolates.

Carbapenemase-producing Enterobacteriaceae isolates
From January to December 2014, 972 consecutive non-duplicate isolates of carbapenemase-producing Enterobacteriaceae were isolated in private laboratories and hospitals in France either by screening for colonisation or by analysing clinical samples in the context of infections. They were recovered from rectal swabs or stools (n = 625), urine samples (n = 250), respiratory tract samples (n = 35), blood samples (n = 22), wounds (n = 24), catheter (n = 7), vaginal swabs (n = 3) and other specimens (n = 6). Those isolates were sent to the Associated French National Reference Centre for characterisation of resistance mechanisms to carbapenems as part of the French antibiotic resistance survey. The 972 carbapenemase-producing Enterobacteriaceae isolates included 577 isolates of Klebsiella spp. (59%), 236 isolates of Escherichia coli (24%), 108 isolates of Enterobacter spp. (11%), 50 isolates of Citrobacter spp. (5%), and a single isolate of Salmonella spp. (0.1%). Species that are naturally resistant to colistin (Proteus spp., Morganella morganii, Providencia spp., and Serratia spp.) had been excluded before the initiation of this study. Only a single isolate per patient was included in the study. All isolates were identified using the Microflex bench-top MALDI-TOF mass spectrometer (Bruker, Champs-sur-Marne, France).

Antimicrobial susceptibility testing
Minimum inhibitory concentrations (MIC) of colistin (CS) were determined using broth microdilution method according to the guidelines of the Clinical Laboratory Standards Institute (CLSI) [3]. As recommended, E. coli ATCC 25922 was used as quality control strain.
The MIC results for colistin and the disk diffusion diameters were interpreted according to susceptibility breakpoints of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [4].

Molecular characterisation
The mgrB genes of K. pneumoniae and Enterobacter spp. isolates were amplified using specific primers ( Table 1), knowing that the MgrB protein is a negative regulator of the PhoPQ two-component system and that alterations in the mgrB gene are commonly involved in acquisition of colistin resistance in K. pneumoniae [5][6][7]. The plasmid-mediated mcr-1 gene encoding colistin resistance was sought as described previously [2]. Detection of extended-spectrum betalactamases (ESBL) and carbapenemases genes was performed with specific primers as described previously [8]. Both strands of the amplification products obtained were sequenced with an ABI 3100 sequencer (Applied Biosystems, Foster City, US). The nucleotide and deduced protein sequences were analysed at the National Centre for Biotechnology Information website (www.ncbi.nlm.nih.gov) by the Basic Local Alignment Search Tool (BLAST) programme.

Genotyping
Genotyping was performed to evaluate the clonal relationship of the colistin-resistant K. pneumoniae and E. cloacae isolates by pulsed-field gel elctrophoresis (PFGE) with XbaI-digested genomic DNA and interpreted according to Tenover criteria [9]. Multilocus sequence typing (MLST) for K. pneumoniae was performed using the simplified protocol at the Institut Pasteur website (http://bigsdb.pasteur.fr/klebsiella/klebsiella.html) [10].

Klebsiella pneumoniae
Of 561 K. pneumoniae isolates, 35 were found to be resistant to colistin (6%). Fifteen of the 35 colistinresistant K. pneumoniae isolates were recovered from a single hospital in the Picardie region, northern France ( Figure 1). We could not obtain the exact dates of their isolations due to the retrospective nature of the study. These isolates had mostly been recovered from rectal swab specimens, but also from a catheter, a urinary sample, a wound exudate and a respiratory specimen (isolates 1 to 15, Table 2). PFGE analysis revealed that the 15 isolates were clonally related ( Figure 2, Table 2). The clone was of the ST11 type, and was susceptible only to cefoxitin, amikacin and fosfomycin (Table 2). A single isolate among these 15 was susceptible to tigecycline. The 15 isolates harboured both the blaOXA-48 carbapenemase gene, and the blaCTX-M-15 extendedspectrum beta-lactamase (ESBL) gene, and the MICs for colistin ranged from 4 to 64 mg/L ( Table 2).
The single carbapenem-resistant E. asburiae strain was resistant to colistin. It had an MIC of colistin above 128 mg/L and produced the VIM-1 carbapenemase (isolate 43, Table 2). All Enterobacter spp. isolates had a wild-type mgrB gene, leaving unexplained the colistin resistance mechanism (E. cloacae and E. asburiae) ( Table 2).
Of the eight colistin-resistant Enterobacter spp. isolates, four were non-susceptible to cefepime and tigecycline, and three were non-susceptible to ciprofloxacin, trimethoprim-sulfamethoxazole and gentamicin ( Figure 3B). Amikacin and fosfomycin were the most active agents against colistin-resistant E. cloacae (all seven isolates were susceptible) ( Figure 3B).

Other species
None of the E. coli (n = 236) and Citrobacter spp. (n = 50) isolates were resistant to colistin.

Discussion
We describe here a clonal outbreak involving 15 K. pneumoniae isolates recovered from a single hospital in the Picardie region in northern France. This outbreak was caused by a colistin-resistant OXA-48 and CTX-M-15-producing K. pneumoniae of ST11 type that was susceptible only to cefoxitin, amikacin and fosfomycin. Surprisingly, those clonally related isolates had variable MIC values for colistin ranging from 4 to 64 mg/L. An ST11 clone co-producing OXA-48 and CTX-M-15 was responsible for a large outbreak involving 44 patients in a hospital in Madrid, Spain, from 2009 to 2014 but only 3.4% of the isolates were resistant to colistin [11].
Sequencing of the mgrB gene revealed mgrB alterations which are likely to be responsible for colistin resistance as described previously [5][6][7]. Interestingly, the three strains belonging to the single clone M recovered in the Provence-Alpes Côtes-d'Azur region had different mechanisms of mgrB inactivation ( Table 2).
The occurrence of such different mechanisms of colistin resistance among clonally related isolates indicates that it is not the product of clonal dissemination of a single colistin-resistant K. pneumoniae strain, but rather clonal dissemination of a carbapenemase-producing isolate, which has acquired colistin resistance thereafter.
The rates of colistin resistance among the carbapenemase-producing isolates were 7.7% for Enterobacter spp. and 3.6% for K. pneumoniae isolates (excluding the isolates responsible for the outbreak in the Picardie region). The resistance rate observed among the carbapenemase-producing K. pneumoniae isolates was much lower than the high rates reported in the neighbouring countries of southern Europe such as Spain (20%) [19] and Italy (43%) [1].
None of the 236 carbapenemase-producing E. coli isolates were colistin-resistant or carried the mcr-1 gene. This is surprising considering that a recent report of the French antimicrobial resistance Resapath surveillance network identified the plasmid-borne mcr-1 gene in 21% of ESBL-producing E. coli isolates recovered from faeces of veal calves in France between 2005 and mid-2014 [22]. The plasmid-borne mcr-1 colistin resistance gene has also been found in many neighbouring countries of France, for example among ESBL-producing Enterobacteriaceae isolates recovered from river water and imported vegetable samples in Switzerland [23], in E. coli isolates recovered from calves and piglets in Belgium [24], in swine and human wound infections in Germany [25], and in food and human bloodstream infections in Denmark [26]. The mcr-1 gene was also detected in Salmonella enterica from food samples in Portugal [27] and France [28]. An E. coli isolate coharbouring the blaVIM-1 carbapenemase gene and the mcr-1 gene was described in Switzerland [29] and an isolate co-producing NDM-9 and MCR-1 was reported from China [30]. We believe that the plasmid carrying the mcr-1 gene might be currently more prevalent among ESBL-producing isolates than among carbapenemase-producing isolates in human samples, which would explain why we did not identify this gene in our collection of carbapenemase-producing isolates.
Amikacin and fosfomycin were most effective against the colistin and carbapenem-resistant K. pneumoniae (susceptibility rates of 80% and 75%, respectively) and

Conclusion
This national survey on carbapenemase-producing isolates recovered in 2014 discovered a high rate of colistin resistance in K. pneumoniae and E. cloacae (6.2% and 7.7%, respectively) in France. These resistance rates remain much lower than those observed in other European countries such as Greece, Italy and Spain.
No plasmid-encoded mcr-1 gene was identified here. Therefore it seems that it is still possible to control the spread of those multidrug-resistant isolates based on accurate identification of colistin resistance and isolation of plasmid-encoded MCR-1 producers. Amikacin and fosfomycin remained the antibiotic agents most effective against those isolates which were resistant to polymyxins and produced a carbapenemase.