Complete Genetic Analysis of Plasmids Carried by Two Nonclonal blaNDM-5- and mcr-1-Bearing Escherichia coli Strains: Insight into Plasmid Transmission among Foodborne Bacteria

ABSTRACT Our objective was to characterize the genetic features of plasmids harbored by two genetically related, MCR-1 and NDM-5-producing Escherichia coli strains recovered from a chicken meat sample. The genetic profiles of all plasmids harbored by the two test strains, namely, 1106 and 1107, were determined by whole-genome sequencing, S1-pulsed-field gel electrophoresis (PFGE), Southern hybridization, and bioinformatics analysis. The transferability of plasmids harbored by the two strains was assessed by filter mating assay. Strains 1106 and 1107 were resistant to almost all the antibiotics, including colistin and fosfomycin, but remained susceptible to amikacin and tigecycline. The plasmids of p1107-NDM-5 and p1106-NDM-5 both contain a class I integron which lacks the ISAba125 element. The backbone of p1106-IncFII exhibited a high degree of similarity with that of p1106-NDM-5 and p1107-NDM-5, implying that events of plasmid fusion and resolution were involved in the formation of the two plasmids. The plasmids p1106-IncHI2MCR and p1107-IncHI2MCR belong to an IncHI2 replicon type, with three copies of ISApl1 being observed in p1106-IncHI2MCR, implying that the mcr-1 gene was transferable among bacteria that reside in the same food matrix. In this study, p1106-IncFIB, p1107-99K, p1107-111K, and p1107-118K were all found to be phage-like plasmids, with p1106-IncFIB and p1107-118K containing several virulence genes, including iroBCDEN, iucABCD, sitABCD, hlyF, and iss. Surprisingly, resistance genes such as aph(3′)-Ia, sul3, and aac(3′)-IId could also be found in p1107-118K, but resistance genes were not detected in other phage-like plasmids. In conclusion, enhanced surveillance is required to monitor and control the dissemination of various resistance determinants among foodborne pathogens. IMPORTANCE Carbapenem and colistin are last-resort antibiotics used to treat serious clinical infections caused by multidrug-resistant (MDR) bacterial pathogens. Plasmids encoding resistance to carbapenems and colistin have been reported in clinical pathogens in recent years, and yet few studies reported cocarriage of mcr and blaNDM genes in Escherichia coli strains of food origin. How plasmids encoding these two important resistance determinants are being evolved and transmitted in bacterial pathogens is not well understood. In this study, we investigated the genetic features of plasmids harbored by two nonclonal, mcr-1- and blaNDM-5-bearing E. coli strains (1106 and 1107) recovered from a fresh chicken meat sample to understand and provide evidence of the level and dynamics of MDR plasmid transmission. Our data confirmed that active plasmid fusion and resolution events were involved in the formation of plasmids that harbor multiple resistance genes, which provide insights into the further control of plasmid evolution in bacterial pathogens.


Bacterial isolation
E. coli isolates 1106 and 1107 were recovered from a chicken meat sample purchased from a supermarket in Shenzhen, Guangdong Province, China in March 27 th , 2017. However, they were obtained in two different ways for the reason described as follows. In our previous studies on isolation of Salmonella from meat samples, we found that some of the colonies picked up from XLT4 plates were E. coli. These E. coli isolates were phenotypically very different from E. coli isolates that we isolated from the same meat products using MacConkey agar plates.
Although we did not understand the underlying reason for discrepancy of colony morphology in E. coli isolated by the two methods, we intended to simultaneously use these two methods for isolation of E. coli during our surveillance. In brief, 25 g of chicken meat sample were placed in a sterile homogeneous bag containing 50 ml of sterilized saline. Strain 1106 was obtained after a loopful of food suspension was spread onto MacConkey agar plates supplemented with 0.5μg/ml meropenem, followed by incubation at 37℃ overnight. Strain 1107 was obtained by the following procedure. Briefly, 1ml of homogenate was transferred to Lactose broth which was then incubated at 42℃ for 12-16 h. One milliliter of this pre-enriched broth was transferred to Rappaport-Vassiliadis broth (RV broth) and incubated overnight at 37℃. A loopful of the culture was inoculated onto the XLT4 agar plate supplemented with 0.5μg/ml meropenem. After incubation at 37 ℃ overnight, a colony with typical E. coli morphology was selected. Both 1106 and 1107 were identified to be E. coli by MALDI-TOF MS using a Bruker MicroFlex LT mass spectrometer (Bruker Daltonics) and API20E test strip (BioMerieux, Inc).

Antimicrobial susceptibility tests
Antimicrobial susceptibility tests for strain 1106 and 1107 were performed using the agar dilution method according to the Clinical and Laboratory Standards Institute (CLSI)(1).
Antibiotics used were listed in Table 1. Resistance breakpoints were interpreted according to CLSI recommendations. E. coli strain ATCC 25922 was used as the quality control strain.

Conjugation, S1-PFGE and Southern hybridization
The transferability of resistance phenotypes was confirmed by filter mating assays performed as previously described, with slight modification (2). Briefly, cultures of donor strains (E. coli 1106 and 1107) and the recipient strain (sodium-azide-resistant E. coli strain J53) were mixed at a ratio of 4:1, inoculated onto a filter membrane which was then placed on LB agar medium without antibiotics, and incubated at 37℃ for 16 hours. Transconjugants were selected on MacConkey agar plates supplemented with meropenem (1μg/mL) and sodium azide (200μg/mL) or on Eosin Methylene Blue Agar containing sodium azide (100μg/mL) and colistin (2μg/mL). The genetic relatedness of strain 1106 and 1107 were determined by PFGE using the Chef-Mapper pulsed-field electrophoresis system (Bio-Rad, USA) upon XbaI digestion. The BioNumerics (Applied Maths) system was used to perform cluster analysis of PFGE patterns. S1-nuclease PFGE was performed to characterize the plasmids that conferred were assigned using online databases ( http://bigsdb.pasteur.fr/) for E. coli.

Plasmid sequencing and bioinformatics analyses
To determine the complete nucleotide sequences of the plasmids harbored by E. coli 1106 and 1107, plasmids were extracted from E. coli strain 1106 and 1107 by using the QIAGEN Plasmid Midi Kit (Qiagen, Valencia, CA). The quality of plasmid DNA was measured by Nanodrop and subjected to short-read and long-read sequencing of Illumina Nextseq 500 and ONT MinION sequencers to obtain accurate complete plasmid sequences. Library preparation and data analysis were performed according to published methods with minor modification(3). The sequence accuracy of raw Nanopore read is below 97%, and consensus accuracy of assembly with high sequencing coverage could reach up to 99%, but still did not meet the high-quality genome sequences requirements. We therefore used hybrid assembly strategy combining shortread Illumina data and long-read Nanopore data with Unicycler (v0.4.4) to generate highquality sequences. In Unicycler, the Pilon tool was used to correct assemblies with accurate short-read Illumina data. This quality control steps could be retrieved from the software manual (4). Alignment of MDR plasmids and nanopore long reads were visualized by the Easyfig and BRIG tools(5, 6). All complete sequences were submitted to NCBI GenBank database with accession numbers as listed in Table 2.

Supplementary results
Apart from the plasmids described above in these two E. coli strains, strain 1106 also harbored another plasmid and 1107 harbored three more plasmids. One plasmid from strain 1106, designated as p1106-IncFIB, and three plasmids from strain 1107 designated as p1107-99K, p1107-111K and p1107-118K respectively, were all bacteriophage like plasmids.
Bacteriophage, one of mobile genetic elements involved in horizontal gene transfer in microorganisms, could be integrated into the chromosome or transmitted to the new host through self-replication (7). Plasmid p1106-IncFIB was 190,401bp in size, with a GC content of 49.8%. It exhibits 99% nucleotide similarity with two ColV plasmids including the plasmid p1ColV5155(CP005931)(8) carried by E. coli strain IMT5155 at 90% coverage and pAPEC-O1-ColBM (DQ381420)(9) carried by an avian pathogenic E. coli (APEC) strain O1 at 63% coverage (Fig. S6). The plasmid comprises a virulence region and a large transfer region; the virulence region contains several virulence genes associated with APEC strains, including the iroBCDEN genes encoding the siderophore receptor, the iucABCD and iutA genes of the aerobactin iron transport system, the sitABCD genes of the manganese ABC transport system, and the hlyF and iss genes of the salmochelin operon. Such structure was similar to those of p1ColV5155 and pAPEC-O1-ColBM (8,9) (Fig. S6). However, p1106-IncFIB lacked the cvaA and cvaB genes of ColV operon, indicating that p1106-IncFIB might not belong to ColV plasmids. On the other hand, the 32kb transfer region of p1106-IncFIB was also structurally similar to a phage-harboring MDR plasmid, pKP12226 (KP453775), which was carried by a K. pneumoniae strain isolated in South Korea(10) (Fig. S6).
BLASTN analysis revealed that p1107-99K exhibited the highest similarity with the plasmid RCS47 harbored by an E. coli strain isolated in Paris, and that both plasmids belonged to an unknown incompatibility group. RCS47 was found to harbor a blaSHV-2 gene(11), however, no resistance genes were detected in p1107-99K, of which 81% of the sequence was identical to P1 bacteriophage (AF234172)(12) (Fig. S7). The sequence of plasmid p1107-111K was similar to that of the E. coli plasmid LF82 (CU638872) (13), which was associated with Crohn's disease and at a high query coverage of 89% and a bacteriophage-like plasmid pECOH89(HG530657) (14) from an E. coli strain containing the ISEcp1-blaCTX-M-15 transposition unit at 84% coverage (Fig. S8). Nevertheless, p1107-118K exhibited high sequence similarity to an IncHI2 plasmid pP2-3T (MG014722) (15) and an IncFII plasmid pSMS35_130 (CP000971) (16). A large region of ~35kb that contained representative virulence genes including sitABCD, iroBCDEN, ompT, hlyF, colicinM and iss genes was observed, in which the corresponding gene clusters are also located in pP2-3T. Worryingly, some resistance genes such as aph(3')-Ia, sul3 and aac(3')-IId were also found in p1107-118K (Fig. S9). It was noteworthy that phage-like sequences are more commonly reported in E. coli and Acinetobacter baumannii strains (10,17), thus dissemination of resistance genes-bearing phage-like plasmids among various bacterial pathogens is a new concern.