The purpose of this study was to characterize a collection of 103 multidrug resistance IncF plasmids recovered from Escherichia coli of food producing and companion animals between 2003 and 2012. A total of 103 incF plasmids were characterized using an established PCR-based IncF replicon sequence typing (RST) system to identify FII, FIA, and FIB (FAB) groups. Plasmids were also analyzed using-restriction fragment length polymorphism (RFLP). Antibiotic Resistance determinants blaCTX-M, plasmid-mediated quinolone resistance (PMQR) genes and rmtB and plasmid addiction systems (PAS) were identified by PCR screening. A total of 20 different RSTs from 103 IncF plasmids were identified. The groups F2 and F33 with the RST formulae A-: B- were the most frequently encountered types (63.1%). The antibiotic resistance genes (ARGs) blaCTX-M, rmtB, and oqxB were carried by 82, 37, and 34 IncF plasmids, respectively. Most of these plasmids carried more than one resistance gene (59.2%, 61/103). The IncF plasmids also had a high frequency of addiction systems (mean 2.54) and two antisense RNA-regulated systems (hok–sok and srnBC) and a protein antitoxin-regulated system (pemKI) were the most prevalent. Not surprisingly, RFLP profiles among the IncF plasmids were diverse even though some shared identical IncF-RSTs. This is the first extensive study of IncF plasmid-positive E. coli isolates from animals in China. Our results demonstrate that IncF is the most prevalent plasmid family in E. coli plasmids and they commonly carry multiple resistance determinants that render them resistant to different antibiotic classes simultaneously. IncF plasmids also harbor addiction systems, promoting their stability and maintenance in the bacterial host, under changing environmental conditions.

BackgroundThe concurrence of mcr and carbapenemase genes among Enterobacteriaceae has been a great clinical concern. In our study, we aimed to investigate the prevalence of mcr-positive carbapenem-resistant Enterobacteriaceae (CRE) in fresh vegetables and shed light on the possibility of transmission of mcr-positive CRE via fresh vegetables.MethodsIn this study, 712 fresh vegetable samples from 10 provinces in China were collected between May 2017 and Dec 2018 and were screened for mcr and carbapenemase genes. Antibiotic susceptibilities for isolates co-harboring carbapenemase genes and mcr were determined by an agar dilution or a broth microdilution method. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) analysis were also performed. Transferability of the carbapenemase/mcr-bearing plasmids was determined by conjugation, replicon typing and S1-PFGE-Southern blotting. The sequences of these plasmids were analyzed by using whole-genome sequencing with Illumina Hiseq platform.ResultsTwo E. coli isolates concomitantly carrying mcr-1 and blaNDM-5/9 from leaf rape and spinach, respectively, were found and both isolates showed multidrug resistance. Notably, mcr-1-positive 690 harboring blaNDM-5 and 701 carrying blaNDM-9 belonged to ST156 and ST2847, respectively, similar to the prevalent MLST types of E. coli co-carrying mcr-1 and blaNDM from avian in our previous study. mcr-1 was on ~33-kb IncX4 plasmid or ~60-kb IncI2 plasmid, while blaNDM-5/9 was on ~46-kb IncX3 plasmid or ~120-kb untypable plasmid. The plasmids were highly similar to those from animals and clinical patients reported in various countries.Conclusion: E. coli isolates concomitantly carrying mcr-1 and blaNDM-5/9 in fresh vegetables may serve as a direct source of pathogens in humans, and such discovery in fresh vegetables emphasizes the importance of prompt surveillance and intervention in limiting the spread of E. coli co-carrying blaNDM and mcr-1. To our knowledge, this is the first report of Enterobacteriaceae co-carrying blaNDM and mcr-1 in fresh vegetables.

dWe sequenced a novel conjugative multidrug resistance IncF plasmid, p42-2, isolated from Escherichia coli strain 42-2, previously identified in China. p42-2 is 106,886 bp long, composed of a typical IncFII-type backbone (ϳ54 kb) and one distinct acquired DNA region spanning ϳ53 kb, harboring 12 antibiotic resistance genes [bla CTX-M-55 , oqxA, oqxB, fosA3, floR, tetA(A), tetA(R), strA, strB, sul2, aph(3=)-II, and ⌬bla TEM-1 ]. The spread of these multidrug resistance determinants on the same plasmid is of great concern and, because of coresistance to antibiotics from different classes, is therapeutically challenging. Widespread antibiotic resistance poses an enormous threat for human and animal health worldwide (1). This problem has been exacerbated in recent years with the emergence of multidrug resistance (MDR) plasmids conferring resistance to most classes of antimicrobials (2). Plasmids of the incompatibility F (IncF) group, representing one of the most frequently encountered plasmid types (3), have frequently been associated with MDR phenotypes, including extended-spectrum ␤-lactamases (ESBLs) and plasmid-mediated quinolone resistance (PMQR) genes (4, 5). In a previous study of ours, IncF plasmids recovered from Escherichia coli strains of food-producing and companion animals were investigated, and most of them carried numerous resistance determinants, such as bla CTX-M , rmtB, oqxAB, and floR (4, 6). The spread of these multiresistance plasmids has prompted worldwide concern because of coresistance to multiple antimicrobial agents that facilitates the survival of bacteria under the selective pressure of antibiotics. Herein, we analyzed a common subtype IncF plasmid, F33:AϪ:BϪ (FII, FIA, FIB [FAB] formula); this plasmid, designated p42-2, contains 12 different resistance genes and was fully sequenced, and the data were compared with those of other IncF plasmids.E. coli 42-2 was recovered from the feces of a healthy duck in Guangzhou, China. Conjugation was performed by mixing E. coli 42-2 and E. coli C600 in a liquid medium and isolating for E. coli C600 (p42-2) by selection on MacConkey agar containing streptomycin (1,000 mg/liter) and cefotaxime (2 mg/liter) as previously described (4, 6). p42-2 was extracted from the E. coli C600 transconjugant using a commercial kit (Qiagen midikit; Qiagen, Germany). Sequencing of p42-2 was performed on an Illumina IIx genome analyzer with a 500-bp paired-end library (approximately 100 million available reads, 935-fold genome coverage) and a 2,000-bp paired-end library (ϳ337 million available reads, 3,150-fold genome coverage). These raw data were assembled by SOAPdenovo (7). Gene prediction and annotation were performed using RAST tools (8). The sequence comparison and map generation were performed using BLAST (http://blast.ncbi.nlm.nih.gov) and Easyfig version 2.1 (9).Plasmid p42-2 confers resistance to ampicillin, chloramphenicol, kanamycin, streptomycin, sulfonamides/trimethoprim, tetracycline, olaquindox, fosfomycin, cephalosporin, and florfenicol. Because of its ...