Prevalence of mcr-type genes among colistin-resistant Enterobacteriaceae collected in 2014-2016 as part of the INFORM global surveillance program

A set of 908 clinically derived colistin-resistant Enterobacteriaeae isolates collected worldwide in 2014–2016 were screened for the presence of the plasmid-borne mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 genes. In total 3.2% (29/908) of the collection were positive for mcr, including 27 Escherichia coli, 1 Klebsiella pneumoniae and 1 Enterobacter cloacae. Twenty-four isolates possessed genes from the mcr-1 family, including the original mcr-1 (n = 22), as well as mcr-1.2 (n = 1) and mcr-1.5 (n = 1), which each differ from mcr-1 by encoding single amino acid variations. Genes from the mcr-3 family were found in isolates from Thailand, including mcr-3.1 (n = 3) and mcr-3.2 (n = 1). An E. coli isolated from a patient with a urinary tract infection in Colombia contained the recently discovered mcr-5. The full colistin-resistant collection was tested against a panel of antimicrobial agents with ceftazidime-avibactam and tigecycline exhibiting the highest activity.


Introduction
Use of colistin, which became clinically available in 1959, has historically played a minor role as an anti-infective therapy due to its nephrotoxicity, as well as the availability of alternative antimicrobial agents [1]. However, the recent proliferation of multi-drug resistant (MDR) Gram-negative pathogens in the clinical setting threatens the efficacy of antibiotics across all classes. To bolster the number of so called "last resort" antimicrobial agents, polymyxins such as colistin are once again being administered clinically due to their potential effectiveness against MDR infections [2]. Until 2015, all characterized colistin resistance mechanisms were chromosomally encoded and thus only limited vertical transmission of resistance was envisioned [3]. However, the discovery by Liu, et al. [4] of the plasmid-borne phosphoethanolamine transferase resistance determinant mcr-1 revealed a mechanism for horizontal spread. MCR-1 and MCR-2, a protein with 80.7% identity to MCR-1 [5], have now been reported in Enterobacteriaceae worldwide [6][7][8]. In 2017, three additional MCR protein variants have been a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 described, MCR-3 [9], MCR-4 [10] and MCR-5 [11], all isolated from hosts with agricultural origins. To gain further insight into the global prevalence of mcr in enteric bacteria isolated from human clinical samples, colistin-resistant isolates from a large international surveillance study were examined for the presence of these genes.

Material and methods
The INFORM (International Network for Optimal Resistance Monitoring) global surveillance program monitors antimicrobial resistance to a variety of pathogens isolated from intraabdominal, urinary tract, skin/soft tissue, lower respiratory tract and, as of 2014, blood infections [12]. During 2014-2016, the program received a total of 44,407 isolates of Enterobacteriaceae including those collected by 87 medical center laboratories located in 18 countries in Europe (n = 21,461), 36 medical center laboratories in 9 countries in the Asia/Pacific region (n = 7,215), 24 medical center laboratories in 6 countries in Latin America (n = 7,180), 17 medical center laboratories in 5 countries in the Middle East/Africa region (n = 3,707) and 26 medical center laboratories in the United States (n = 4,844). All isolate species identifications were confirmed in the central laboratory by MALDI-TOF MS (Bruker Daltonics, Waltham, Massachusetts). Not including Serratia spp. and members of the tribe Proteeae (genera Proteus, Providencia and Morganella), which are intrinsically colistin non-susceptible, 934 isolates were found to be resistant to colistin by broth microdilution [13] at an MIC ! 4 μg/mL, which is the EUCAST resistance breakpoint for the Enterobacteriaceae [14]. Of these, 908 isolates were available to screen, as no isolates could be obtained from China in 2014-2016 or Hong Kong in 2015-2016 due to export restrictions. The species composition of the complete set included Citrobacter freundii (n = 6), Citrobacter koseri (n = 3), Enterobacter aerogenes (n = 18), Enterobacter asburiae (n = 143), Enterobacter cancerogenus (n = 1), Enterobacter cloacae (n = 165), Enterobacter kobei (n = 11), Escherichia coli (n = 64), Hafnia alvei (n = 1), Klebsiella oxytoca (n = 13), Klebsiella pneumoniae (n = 481) and Klebsiella variicola (n = 2).

Results and discussion
In total, mcr was detected in 29 isolates ( [9] and one possessing the gene coding for the single amino acid variant, MCR-3.2 (T488I). An E. coli strain from Colombia was shown to carry the recently discovered mcr-5 gene [11]. No mcr-2 or mcr-4 genes were identified. As part of the INFORM surveillance program, organisms non-susceptible to meropenem, resistant to ceftazidime, and/or positive for ESBL activity qualify for β-lactamase gene screening. Thirteen of the 29 mcr positive isolates qualified and were screened for genes encoding acquired ESBLs, AmpC β-lactamases, serine carbapenemases (bla KPC , bla OXA-48 , bla GES ), and metallo-βlactamases by PCR and DNA sequencing, as previously described [16]. Nine mcr-positive isolates were found to carry CTX-M-type ESBLs either alone or in combination with AmpC-type β-lactamases and/or original-spectrum β-lactamases (OSBL) of the TEM or SHV type. Four possessed a CMY-2 AmpC-type enzyme either alone or with a TEM-OSBL, and in one case with a CTX-M-161 enzyme. None of the mcr-positive isolates carried carbapenemases. Of note, each of the four mcr-3 gene family-harboring isolates also carried the CTX-M-55 ESBL variant, known to be common in Asia especially in E. coli isolated from veterinary sources [17].
The mcr-positive E. coli were distributed among several lineages, with the ST10 clonal complex (including ST167, ST744 and ST48) the most abundant (n = 6). mcr-harboring E. coli from this group has been reported on numerous occasions, for example ST10 from human clinical samples in China [21], ST744 from human and cattle-associated samples in Europe [22,23], ST167 from human infections in Spain and China [24,25], as well as ST48 from hospital sewage and human clinical samples, in China and Switzerland, respectively [26,27]. Additional worldwide clones previously shown to harbor mcr were also confirmed here, and include ST641 [28], ST410 [29,30], and ST156 [31,32]. Our screening identified two mcr-harboring ST117 E. coli (and a ST117 single-locus variant with a novel fumC), one of which carried the MCR-3.2 gene. ST117 is a clonal group associated with poultry disease [33] and mcrtype genes have only rarely been observed in this clone [27,34]. Of particular interest, one isolate from Brazil typed as a single locus variant (novel purA) of the pathogenic E. coli ST131 [35]. ST131 often exhibits an extended spectrum β-lactamase (ESBL) phenotype and frequently possess CTX-M-15; however, this Brazilian isolate was susceptible to third-generation cephalosporins. In general, the fact that mcr-type genes have been found in E. coli of such diverse STs from food, human and animal specimens suggests the spread of these genes is linked more to successful plasmids and mobile elements rather than single specific E. coli clones [27].
Overall, the prevalence of mcr observed here is in accordance with previous reports from large global surveillance studies. For example, Castanheira, et al. noted that 4.9% (19/390) of a  worldwide colistin-resistant collection of E. coli and K. pneumoniae from the SENTRY program contained mcr-1, and 32.3% (19/59) of the resistant E. coli contained this gene [36]. mcr was also enriched in the colistin-resistant E. coli population examined here, as 42.2% (27/64) of the resistant isolates from this species harbored mcr with the remainder presumably possessing a chromosomally-encoded resistance determinant. It should be noted that mcr has been discovered in isolates susceptible to colistin [37], so the actual frequency of occurrence could be higher. In this study, mcr-1 was observed exclusively in E. coli except for an E. cloacae isolate originating from Portugal. Until recently, mcr-1 positive E. cloacae were only reported from Asia [38,39]; however, the geographic range was expanded with the discovery of a clinical E. cloacae isolate with mcr-1 in France [40]. The mcr-3 harboring E. coli and K. pneumoniae from Thailand confirm the previous report of the presence of this gene in clinical isolates from this country [9]. Finally, finding mcr-5 in a Colombian E. coli clinical isolate expands both its geographic and host range, as at the time of this writing mcr-5 has only been confirmed in Salmonella enterica Paratyphi B isolated from food animals and food products in Germany, and in E. coli from porcine clinical specimens in Japan [41]. This gene was found in silico to be present the genome of a Cupriavidus gilardii from the U.S., and mcr-5 has been reported to be located on a unique Tn3-type transposon in both S. enterica Paratyphi B and C. gilardii [11]. Although we did not sequence this complete region, the forward mcr-5 flanking primer utilized to amplify the full coding region overlaps the 3' end of the chromate reductase gene, chrB, directly upstream of mcr-5 in the Tn3-type transposon, and the reverse flanking primer anneals to the 5' portion of the MFS-type transporter gene, immediately downstream of mcr-5 in the transposon arrangement [11], suggesting a similar genetic orientation in this Colombian strain.
In summary, this report confirms the global spread of mcr. Notably we did not find the coexistence of mcr with any carbapenemase genes, although co-carriage is being increasingly reported, including mcr-1 with bla NDM in Enterobacteriaceae from the U.S. and China [32,[42][43][44][45][46], as well as mcr-1 and bla KPC in isolates from Singapore [47]. Continual surveillance of this recently recognized threat to public health is warranted as MDR bacteria that acquire mcr will leave few treatment options.