Molecular characteristic of mcr-1 producing Escherichia coli in a Chinese university hospital

Colistin has been considered as a last-line treatment option in severe infections caused by multidrug-resistant (MDR) gram-negative pathogens. However, the emergence of the mobile colistin resistance gene (mcr-1) has challenged this viewpoint. The aim of this study is to explore the prevalence of mcr-1 in Escherichia coli (E. coli) in a Chinese teaching hospital, and investigate their molecular characteristics. A total of 700 E. coli isolates were used to screen mcr-1 by PCR and sequencing in a Chinese university hospital from August 2014 to August 2015. Susceptibility test of mcr-1-producing isolates was determined by Vitek -2 Compact system. 26 virulence factors (VFs), phylogenetic groups, Multi-locus sequence typing (MLST), and DNA Fingerprinting (ERIC-PCR) of strains were investigated by PCR. Four (0.6%) mcr-1 producing E. coli isolates were found in this study. The results of antibiotic susceptibility test showed that all four isolates were resistant to colistin, ciprofloxacin, levofloxacin, cefazolin, and trimethoprim/sulfamethoxazole, and were susceptible to amikacin, ertapenem and imipenem. In addition, all 4 isolates exhibited high-level resistance to aztreonam, cefotaxime and gentamicin. The numbers of VFs contained in mcr-1 positive isolates were no more than 4 in our study. MLST result demonstrated that these isolates were assigned to two sequence types: ST156 and ST167. The result of phylogenetic analysis showed that four mcr-1-positive isolates belong to two phylogenetic groups: A and B1 group. ERIC-PCR showed that four mcr-1 positive strains were categorized into three different genotypes. Our study demonstrated a low prevalence of mcr-1 in E. coli clinical isolates in a Chinese teaching hospital, and we have gained insights into the molecular characteristics of these mcr-1-positive strains. Increasing the surveillance of these infections, as well as taking effective infection control measures are urgently needed to take to control the transmission of mcr-1 gene.


Background
In recent years, colistin has been considered as an effective therapeutic option for the rapid increasing of multidrug-resistant (MDR) gram-negative pathogens [1,2]. However, the prevalence of the mobile colistin resistance gene (mcr-1) in animals and human beings worldwide has challenged this viewpoint [3,4]. Resistance to polymyxins is mainly caused by the modification to bacterial outer membrane, which was usually considered as chromosomally mediated resistance [5,6].
Since it was initially found, plasmid-mediated mcr-1 has been detected widely [3,7]. Nowadays, mcr-1-producing bacteria have been reported in many regions in China [4,8]. Mcr-1 was firstly found in Escherichia coli (E. coli), and now it has been spreading to other Enterobacteriaceae [9]. Several reports showed that the mcr-1 gene could coexist with other resistance genes (such as

Open Access
Annals of Clinical Microbiology and Antimicrobials  [8,10]. Therefore, the emergence and spread of mcr-1 gene among human beings should be given close attention. The aim of this study was to evaluate the prevalence of mcr-1 in E. coli clinical isolates in a Chinese teaching hospital, and to investigate the molecular characteristics of these strains.

DNA extraction
Several colonies were suspended in 50 µl of sterile distilled water for preparing genomic DNA of the isolates, and then the bacterial suspension was heated at 100 °C for 10 min as described previously [13].

MCR-1 detection
mcr-1 gene was screened in E. coli clinical isolates by PCR using primers as previously described [4]. All of the PCR products were sequenced and then compared with known sequences listed in the GenBank (http://www. ncbi.nlm.nih.gov/blast/).

Phylogenetic analysis
The phylogenetic groups (A, B1, B2, and D) of mcr-1 producing E. coli isolates were identified by a triplex PCR as previously described [16].

DNA fingerprinting
Enterobacterial Repetitive Intergenic Consensus Sequences PCR (ERIC-PCR) was applied to typing mcr-1 producing E. coli isolates with the primers ERIC-1 and ERIC-2 [18]. DNA fingerprints were compared by visual inspection, ERIC profiles were regarded as different if there were different bands on visual inspection [19].

Results and discussion
In this study, four isolates (0.6%) were confirmed to carry mcr-1 gene, which is lower than previous study [4]. The age of the patients ranged between 38 and 80 years. These mcr-1 producing strains were isolated from two different wards (Table 1). Two strains were isolated from the same patient. The clinical data of patients with mcr-1 positive E. coli infection were shown in Table 2.
Mcr-1 was usually found to be co-localized with other resistance genes on plasmids, such as ESBL genes and carbapenemase genes [20], which might increase the emergence of pan-drug resistance. In our study, the results of antimicrobial susceptibility test showed a high drug resistance in the mcr-1-producing isolates. All of the mcr-1 positive isolates were resistance to at least 3 different kinds of antibiotics (Table 1).
All four mcr-1 positive strains detected in our study were resistant to colistin and the MICs ranged from 4 to 16 μg/ml. It will be worrisome once mcr-1 coexists with other resistant genes, especially carbapenemase genes because of limited therapeutic options [20]. Previous studies revealed that mcr-1 co-produced with carbapenem-resistant genes in E. coli [8,21]. Fortunately, all of them were susceptible to carbapenems (IPM and ETP), which probably indicated that no carbapenem-resistant genes coexisted with mcr-1. Result of ERIC-PCR (Fig. 1) showed that four mcr-1 positive strains were categorized into three different genotypes, one of which contained 2 strains (from the same patient). These isolates which have different patterns suggest that they were non-clonal transmission. In a previous study, two mcr-1 positive E. coli isolates from a single fowl were belonging to phylogenetic B1 and D group [22]. The mcr-1-producing isolates in this study were belonged to phylogenetic groups A and B1, which were mainly distributed among human commensal E. coli isolates [23]. The mcr-1 producing isolates were assigned by MLST to two different sequence types: ST156 and ST167 (Table 1), which was similar to previous reports in other studies from China [8,22]. E. coli ST156 has been found that it has connection with different ESBL genes [24,25]. ST167 was belonged to ST10 complex and regarded as prevalent ST among ESBL-producing E. coli from human and animal sources [26]. In addition, E. coli ST167 was reported to be closely related to bla NDM , which needed closely concern of spreading [27]. The similar molecular characterizations illustrated that mcr-1 positive isolates detected from the same department in our study were clonally related.
VFs in E. coli were associated with colonization, bacterial fitness and virulence [28]. VFs include five main groups: (1) adhesions; (2) toxins; (3) siderophores; (4) capsule production and (5) protections and invasions.  Clinical E. coli strains often carry multiple VFs, and isolates belonging to groups A and B1 often have less VFs than those belonging to phylogroups B2 and D [28]. To the best of our knowledge, there is no study concerning about VFs in mcr-1 producing E. coli. In our study, mcr-1 producing isolates contained less than 4 different VFs (Table 1). Only five different kinds of VFs had been detected in our mcr-1 positive isolates, which included fimH, fyuA, traT, iutA and cvaC. fimH is one of the most commonly VFs present in E. coli, which encodes the adhesion subunit of type 1 fimbriae and related to colonization [15]. Lee et al. reported that fyuA, traT, and iutA were found to be independent predictors for pathogenicity. Meanwhile, traT and iutA were thought to be closely related to ESBL genes [29]. Pitout et al. found that cvaC was only present in non-CTX-M-producing isolates [30]. Previous reports suggested that antibiotic resistance has negative association with virulence factors [31], which could be interpreted by the loss of VFs associated with mutation to resistance [32]. It is noteworthy that two mcr-1 positive E. coli strains were isolated from the same patient but at different time ( Table 1). Results of MLST and ERIC-PCR revealed that these isolates had identical genetic background. Result of antimicrobial susceptibility test showed that they had similar antibiograms. We speculate that the two isolates probably originated from a same source.
In conclusion, we have revealed a low prevalence of mcr-1 in E. coli clinical isolates in a Chinese teaching hospital, and presented detailed molecular characteristics of these isolates. The presence of mcr-1 in E. coli clinical isolates suggests that it will pose a threat to public healthcare. Effective infection control measures are urgently needed to take to control the transmission of mcr-1 gene.