Prospective study on human fecal carriage of Enterobacteriaceae possessing mcr-1 and mcr-2 genes in a regional hospital in Hong Kong

Background Human fecal carriage of Enterobacteriaceae possessing mobilized colistin resistance genes (mcr-1 and mcr-2) remains obscure in Hong Kong. As part of routine surveillance on emerging antibiotic resistance, we conducted a prospective study on this topic in a regional hospital in Hong Kong. Methods From October 31 to November 25, 2016, all fecal specimens submitted for routine analysis were included in this surveillance study. These comprised 672 consecutive routine fecal specimens collected from 616 individuals. Fecal specimens were screened for colistin-resistant Enterobacteriaceae by culture-based method, and the presence of mcr-1 and mcr-2 genes in resistant isolates was identified by polymerase chain reaction and Sanger sequencing. Whole genome sequencing (WGS) of mcr-1-possessing Escherichia coli strains was facilitated using Illumina® MiSeq® followed by sequence analysis with appropriate bioinformatics tools. Results Fourteen mcr-1-positive E. coli strains were isolated from 14 separate individuals (2.08% of total fecal specimens), with 9 of them being asymptomatic, healthy clients coming for health assessment. No mcr-2-possessing Enterobacteriaceae was identified. Colistin minimum inhibitory concentrations of these mcr-1-positive isolates ranged from 2 to 4 μg/mL. All these isolates were susceptible to carbapenems with 2 being extended spectrum β-lactamase producers. WGS data revealed that these isolates belonged to at least 12 different sequence types (STs) and possessed diversified plasmid replicons, virulence and acquired antibiotic resistance genes. Further study on an E. coli ST201 strain (Pasteur scheme) revealed coexistence of 47,818-bp IncP-1 and 33,309-bp IncX4 types of mcr-1 plasmids, which was a combination of stability and high transmissibility. Conclusions To the best of our knowledge, this is the first study on human fecal carriage of Enterobacteriaceae possessing mcr-1 and mcr-2 genes in Hong Kong. Our data further revealed asymptomatic carriage of mcr-1-possessing Enterobacteriaceae by both patients and healthy individuals. This is alarming considering wide diversity and high transmissibility of mcr-1 plasmids, which potentially facilitate emergence of pan-drug-resistant bacteria in future infection. This also highlights the importance of surveillance on emerging antibiotic resistance, especially for patients under intensive care.


Background
Recent reports on plasmid-mediated colistin resistance of Enterobacteriaceae from research groups worldwide have heralded concerns about emergence of 'superbugs' which are resistant to this last resort of treatment [1][2][3]. With broad range of compatible plasmids and bacterial hosts [4], mobilized colistin resistance gene (mcr-1) has successfully disseminated globally. The presence of mcr-1-carrying Enterobacteriaceae in food animals worldwide [1,5,6] further facilitates transmission of mcr-1 to human. Escherichia coli has been the major bacterial host of mcr-1 gene, and recent study has unraveled significant geographical clustering with regional spread of IncHI2 and IncI2 types of mcr-1 plasmids in Europe and Asia, respectively [7].
In Hong Kong, Wong and coworkers reported a mcr-1 positive rate of 0.4% among 1,324 Enterobacteriaceae isolates from patients, which included 2 asymptomatic carriers [8]. At present, no prevalence data on human fecal carriage of mcr-1-and mcr-2-possessing Enterobacteriaceae has been documented in our region. As a routine surveillance on emerging antibiotic resistance, we conducted a prospective study on this topic in a regional hospital in Hong Kong.

Collection of human fecal specimens
The goal of this surveillance study was to have a gross picture on (1) fecal carriage of mcr-1and mcr-2-harboring Enterobacteriaceae among patients/ clients using our service, (2) characteristics of the carriers and (3) phenotypic and molecular traits of the positive strains. This information is important for infection control and risk assessment. From October 31 to November 25, 2016, all fecal specimens submitted for routine analysis were included in this surveillance study. These comprised 672 consecutive routine fecal specimens collected from 616 individuals, including 79 fecal specimens from 67 outpatients, 171 fecal specimens from 144 inpatients and 422 fecal specimens from 418 asymptomatic clients coming for health assessment. Forty out of 144 inpatients had been screened for vancomycin-resistant enterococci (VRE), which is routinely performed for inpatients who have been hospitalized in/ outside Hong Kong, underwent VRE screening in other hospitals, undergone surgical operation overseas or staying in a nursing home within past 6 months. After routine testing, leftover fecal specimens were used for surveillance screening.

Screening of colistin-resistant Enterobacteriaceae in fecal specimens
The concept of Blackburn and coworkers' method for screening carbapenem-resistant bacteria was adopted [9], with some modifications. Briefly, fecal specimens with approximate sizes of 10 μL inoculation loop were homogenized in 500 μL sterile, double-deionized water and centrifuged at 1,500 rpm for 30 s. An aliquot of 10 μL supernatant from each sample was inoculated on chromID® CPS® Elite agar (bioMérieux, Marcy, I'Etoile, France) and a piece of polymyxin B disc (PB300, Oxoid, Basingstoke, Hampshire, England) was applied on the area of inoculation, followed by overnight incubation at 35 o C in ambient air. Bacterial growth around polymyxin B disc, namely inside the inhibition zone of approximately 11 mm in diameter (taking the Clinical and Laboratory Standards Institute (CLSI) guidelines' interpretative criteria of polymyxin B for Pseudomonas aeruginosa as reference), was subjected to polymerase chain reaction (PCR) targeting mcr-1 and mcr-2 genes.
Bacterial identification of PCR-positive isolates PCR-positive colony suspensions were subcultured on CPS agar and incubated at 35 o C overnight in ambient air. Recovered bacterial colonies were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS, Microflex LT, Bruker Daltonik, Bremen, Germany). Briefly, minute amount of bacterial colony was smeared on target plate and covered with 1 μL of 10 mg/mL matrix solution (αcyano-4-hydroxycinnamic acid, Bruker Daltonik, Bremen, Germany). The dried samples were subjected to MS measurement after successful calibration with Bacterial Test Standard (Bruker Daltonik, Bremen, Germany). Spectra were obtained with an accelerating voltage of 20 kV in linear mode with m/z range of 2,000 to 20,000 Da, followed by analysis with MALDI Biotyper version 3.0 and reference library version 3.1.2.0 (Bruker Daltonik, Bremen, Germany).

Bioinformatics analysis of WGS data
WGS short reads were assembled into contigs using SPAdes version 3.10.1 [11]. E. coli sequence types (STs) were determined using MLST databases of The University of Warwick [12] and PubMLST [13]. Acquired resistance genes were identified using ResFinder 2.1 [14]. Virulence genes were detected using VirulenceFinder 1.5 [15]. Alignment of contigs to appropriate reference sequences was performed using Mauve version 2.4.0 [16] followed by post-assembly gap filling by PCR and Sanger sequencing. Prokka version 1.11 [17] was used for plasmid annotation with manual editing. Plasmid replicons and insertion sequences were identified using PlasmidFinder 1.3 [18] and IS finder [19]. Plasmid genetic maps were generated using SnapGene® Viewer 4.1. Sequence comparisons between plasmids were generated using BLAST Ring Image Generator (BRIG) [20].

Results and Discussion
Fecal carriage of mcr-1-/ mcr-2-possessing Enterobacteriaceae Seventy nine out of 672 fecal specimens revealed bacterial growth inside the inhibition zone of polymyxin B disc and subjected to colony PCR, with 14 specimens being mcr-1-positive (2.08%) and none being mcr-2-positive. Results of MALDI-TOF MS showed that all 14 isolates were E. coli (top identification scores ranged from 2.271 to 2.526) which were consistent with the appearance of bacterial colonies on CPS agars (red-colored). These E. coli strains were isolated from 14 separate individuals including 3 inpatients, 2 outpatients and 9 asymptomatic clients coming for health assessment, with age from 1 to 58 years (Table 1). Regarding available clinical/ epidemiological information of these mcr-1 carriers, Subject 1 and 10 had received antibiotic treatment other than colistin prior to fecal specimen collection. For others, none had been prescribed with antibiotics in our hospital. We could not access any information on antibiotic usage outside our hospital. Subject 3 had been hospitalized in another hospital within 6 months prior to admission. None of these mcr-1-possessing E. coli strains caused diseases in their human hosts. In general, no apparent correlation was observed between mcr-1 fecal carriage and the available clinical/ epidemiological information.
The fecal carriage data further revealed asymptomatic carriage of mcr-1-possessing Enterobacteriaceae by both patients and healthy individuals. The mcr-1 positive rate estimated in this study (2.08%) was much higher than that reported by another local research group (0.4%) [8], which implies the prevalence was higher than expected in Hong Kong. This difference in mcr-1 positive rate might be explained by different screening targets adopted, with the study by Wong and coworkers focusing on clinical isolates whereas our study screening fecal specimens. As fecal specimens normally contain numerous strains of Enterobacteriaceae per sample, the chance of isolating mcr-1-harboring strains might be increased. Compared with human fecal carriage data of other regions, the mcr-1 positive rate estimated in this study was lower than that of mainland China (4.9 -6.2%) [21,22] and higher than that of several European countries (Switzerland, 0% [23]; Western France, 0% [24]; The Netherlands, 0.35% [25]). This is not surprising considering high prevalence of mcr-1-harboring Enterobacteriaceae among livestock animals in mainland China [26], which is a major supplier of food animals in Hong Kong.
On the other hand, no mcr-2-carrying Enterobacteriaceae was isolated. In another large-scale study in Japan, mcr-2 gene was not detected among 9,306 E. coli strains isolated from healthy animals [27]. Considering these prevalence data together with reports of zero human fecal carriage from various European countries [23][24][25], it appears that mcr-2-carrying Enterobacteriaceae has not been disseminated globally at present.
The range of colistin MICs in this study was 2 to 4 μg/mL by BMD, which appeared to be narrower than that of mcr-1-positive E. coli isolates causing human diseases (4 to 16 μg/mL by BMD) reported by various research groups [28][29][30][31]. Colistin MICs determined by VITEK® 2 AST were consistent with that of BMD for 35.7% of the isolates, whereas MICs were 2-to > 4-fold higher than that of BMD for the remainder. Albeit underestimation of colistin MIC was not observed in this study, the suitability of using VITEK® 2 AST for colistin MIC determination awaits further evaluation due to its high very major error rate (36%) revealed by a recent study [32]. In addition, as 21.4% of the isolates displayed colistin MICs lower than the resistant breakpoint (> 2 μg/mL) published by The European Committee on Antimicrobial Susceptibility Testing (EUCAST), we echo the suggestion of using a lower susceptible breakpoint of < 1 μg/mL by Chew and coworkers [32] for improving detection of mcr-1-possessing Enterobacteriaceae. On the other hand, it was noticeable that more than half of the isolates (57.1%) displayed a 'baseline' insusceptibility to 5 or more antibiotics, and 2 of which were ESBL producers. The choice of usable antibiotics may be further limited for these isolates upon acquisition of antibiotic resistance genetic elements, for instance, carbapenemase genes.

Molecular characteristics of mcr-1-positive isolates
The results are summarized in Table 2.

mcr-1 gene sequences
The mcr-1 gene sequences of 12 isolates were 100% identical to that of E. coli strain SHP45 (NG_050417), with exception of the isolate from Subject 4 harboring C>T substitution at nucleotide position 27 and the isolate from Subject 2 with mixed nucleotides (R) at position 1263, indicating possible coexistence of 'wild type' and 1263G>A variant in the same bacterium. Both of these nucleotide substitutions were silent mutations.

Plasmid incompatibility groups
Eight plasmid replicon types were identified in 10 isolates, with IncI1 (21.4%), IncX4 (21.4%) and IncN (21.4%) being the predominant incompatibility groups, followed by IncQ1 (14.3%), IncP1 (7.1%), IncR (7.1%), IncI2 (7.1%) and IncHI2 (7.1%). Previous study has shown that IncX4, IncI2 and IncHI2 incompatibility groups comprised 90.4% of identified mcr-1 plasmids [7]. Significant geographical clustering was observed with regional spread of IncI2 and IncHI2 types of mcr-1 plasmids in Asia and Europe, respectively, while there was no significant difference between distribution of these major replicon types from different sources of isolation [7]. In this study, albeit further work is needed to confirm the genomic locations of mcr-1 genes, plasmid replicon types were diversified and not confined to IncI2, which was detected in a single isolate from Subject 11. Further study with larger cohort of isolates from different sources is needed to determine local characteristics of mcr-1 plasmid incompatibility groups.
The two IncP-1 GenBank sequences originated from mainland China. The 57,278-bp plasmid pMCR_1511 (KX377410) was isolated from Klebsiella pneumoniae was identified in Salmonella enterica subsp. enterica. pMCR_1511 was used as reference for comparative sequence analysis (Fig. 3). The IncP-1 plasmid of this study was very similar to pMCR16_P053 (99% sequence homology), while both of them were different from reference by the absence of 9 genes and insertion sequences, including bla TEM (non-ESBL), ble (bleomycin resistance), dgkA (diacylglycerol kinase), eptA (phosphoethanolamine transferase), higA (antitoxin), higB-1 (probable mRNA interferase), ybaQ (transcriptional regulator), IS26 and ISApl1.  Fig. 3 Comparative sequence analysis of IncP-1 type of mcr-1 plasmids. The circular image was generated with BRIG using pMCR_1511 (KX377410) as reference. The 2 inner rings reveal GC content and GC skew and the outer ring represents the coding sequences (CDS) of reference. Unlabeled CDS represents genes encoding hypothetical proteins Fig. 4 Comparative sequence analysis of IncX4 type of mcr-1 plasmids. The circular image was generated with BRIG using pFS170G (KX711707) as reference. The 2 inner rings reveal GC content and GC skew and the outer ring represents the CDS of reference. Unlabeled CDS represents genes encoding hypothetical proteins The 30 IncX4 GenBank sequences were diversified in geographical origins (from 10 regions, with 18 entries from Asia, 8 from America and 4 from Europe). There were 2 ranges of plasmid sizes, with 27 sequences between 31,229 and 34,997 bp (< 40,000 bp) and 3 sequences from 42,941 to 48,350 bp (> 40,000 bp). The hosts of these plasmids included E. coli (86.7%), K. pneumoniae (6.7%) and S. enterica subsp. enterica (6.7%). Plasmid pFS170G (KX711707) was used as reference for comparative sequence analysis (Fig. 4). Majority of these sequences were very similar, and the IncX4 plasmid of this study shared highest homology (99%) with pICBE-C3AM (KY770024). IS26 was the predominant type of insertion sequence (78.6%). Carbapenemase/ ESBL genes were absent in most of these plasmids (96.8%), while pCQ02-121 (KU647721) was the only plasmid harboring carbapenemase gene (bla NDM-5 ). In fact, it was an IncX3-IncX4 hybrid plasmid identified in an E. coli isolate [39], which might account for its difference from majority.

Implications from coexistence of IncP-1 and IncX4 plasmids
Coexistence of IncP-1 and IncX4 types of mcr-1 plasmids in the same E. coli isolate was indeed a combination of stability and high transmissibility -the broad-host-range IncP-1 plasmids can transfer to, replicate in and stably maintained in virtually all species of Gram-negative bacteria [40,41], while IncX4 plasmids are self-transferable at high frequency (~10 -1 to~10 -4 ) which have contributed to intercontinental spread of mcr-1 genes [42]. These 2 incompatibility groups of mcr-1 plasmids could be harbored by at least E. coli, K. pneumoniae and S. enterica subsp. enterica, and the list is expected to be expanding. Another aspect of concern is the potential of IncP-1 and IncX4 plasmids to acquire a wide spectrum of antibiotic resistance genes, for instance, ESBL and carbapenemase genes [39,[43][44][45], which may contribute to pan-drug resistance under selective pressure [46,47]. Nevertheless, our study was not the sole report on Enterobacteriaceae harboring more than 1 copy of mcr-1 gene [42,48]. Interestingly, colistin MICs of these isolates appeared to be unaffected by gain of extra copies of mcr-1 gene, which was coherent with the finding in our study. While the rationale behind this phenomenon remains obscure, it may reflect the active nature of transposons carrying mcr-1 gene, for instance, ISApl1 transposons [49]. The mechanism behind mobilization of mcr-1 genes and its integration into a variety of plasmids warrant further investigation.

Limitations of this study
Our study had several limitations. First, we presented the surveillance data of a regional hospital. Further study with larger cohort of experimental subjects from representative geographical locations is warranted to have a more complete picture on prevalence and characteristics of mcr-possessing Enterobacteriaceae in Hong Kong. Second, we could not assess any information regarding antibiotic usage by experimental subjects outside our hospital. In addition, albeit not within the scope of this study, further curation and analysis of WGS data can provide insights into actual genomic locations of mcr-1 in remainder E. coli isolates.

Conclusions
In this study, we have further revealed asymptomatic carriage of mcr-1-harboring Enterobacteriaceae by both patients and healthy individuals, with fecal carriage of 2.08% which was higher than expected in our region. This asymptomatic carriage is alarming because it potentially facilitates acquisition of mcr-1 by multidrug-resistant bacteria in future infection, which severely limits the choice of usable antibiotics for treatment. Prevalence of mcr-2-harboring Enterobacteriaceae, on the contrary, is believed to be very low at present. This surveillance data on emerging antibiotic resistance is important for infection control, especially for patients under intensive care. In addition, we have unraveled the phenotypic and molecular traits of 14 mcr-1-possessing E. coli strains. Further studies on mobilization and resistance mechanisms of this recently-discovered resistance gene family may shed light on strategies limiting emergence and spread of pan-drug-resistant bacteria.