Ruichao Li

Genetic characterization of mcr-1-bearing plasmids to depict molecular mechanisms underlying dissemination of the colistin resistance determinant

Objectives To analyse and compare mcr-1-bearing plasmids from animal Escherichia coli isolates, and to investigate potential mechanisms underlying dissemination of mcr-1. Methods Ninety-seven ESBL-producing E. coli strains isolated from pig farms in China were screened for the mcr-1 gene. Fifteen mcr-1-positive strains were subjected to molecular characterization and bioinformatic analysis of the mcr-1-bearing plasmids that they harboured. Results Three major types of mcr-1-bearing plasmids were recovered: IncX4 (∼33 kb), IncI2 (∼60 kb) and IncHI2 (∼216–280 kb), among which the IncX4 and IncI2 plasmids were found to harbour the mcr-1 gene only, whereas multiple resistance elements including blaCTX-M, blaCMY, blaTEM, fosA, qnrS, floR and oqxAB were detected, in various combinations, alongside mcr-1 in the IncHI2 plasmids. The profiles of mcr-1-bearing plasmids in the test strains were highly variable, with coexistence of two mcr-1-bearing plasmids being common. However, the MIC of colistin was not affected by the number of mcr-1-carrying plasmids harboured. Comparative analysis of the plasmids showed that they contained an mcr-1 gene cassette with varied structures (mcr-1-orf, ISApl1-mcr-1-orf and Tn6330), with the IncHI2 type being the most active in acquiring foreign resistance genes. A novel transposon, Tn6330, with the structure ISApl1-mcr-1-orf-ISApl1 was found to be the key element mediating translocation of mcr-1 into various plasmid backbones through formation of a circular intermediate. Conclusions The mcr-1 gene can be disseminated via multiple mobile elements including Tn6330, its circular intermediate and plasmids harbouring such elements. It is often co-transmitted with other resistance determinants through IncHI2 plasmids. The functional mechanism of Tn6330, a typical composite transposon harbouring mcr-1, should be further investigated.

Complete genetic analysis of plasmids carrying mcr-1 and other resistance genes in an Escherichia coli isolate of animal origin.

Objectives: To investigate the genetic features of three plasmids recovered from an MCR-1 and ESBL-producing Escherichia coli strain, HYEC7, and characterize the transmission mechanism of mcr-1. Methods: The genetic profiles of three plasmids were determined by PCR, S1-PFGE, Southern hybridization and WGS analysis. The ability of the mcr-1-bearing plasmid to undergo conjugation was also assessed. The mcr-1-bearing transposon Tn6330 was characterized by PCR and DNA sequencing. Results: Complete sequences of three plasmids were obtained. A non-conjugative phage P7-like plasmid, pHYEC7-mcr1, was found to harbour the mcr-1-bearing transposon Tn6330, which could be excised from the plasmid by generating a circular intermediate harbouring mcr-1 and the ISApl1 element. The insertion of the circular intermediate into another plasmid, pHYEC7-IncHI2, could form pHNSHP45-2, the original IncHI2-type mcr-1-carrying plasmid that was reported. The third plasmid, pHYEC7-110, harboured two replicons, IncX1 and IncFIB, and comprised multiple antimicrobial resistance mobile elements, some of which were shared by pHYEC7-IncHI2. Conclusions: The Tn6330 element located in the phage-like plasmid pHYEC7-mcr1 could be excised from the plasmid and formed a circular intermediate that could be integrated into plasmids containing the ISApl1 element. This phenomenon indicated that Tn6330 is a key element responsible for widespread dissemination of mcr-1 among various types of plasmids and bacterial chromosomes. The dissemination rate of such an element may be further enhanced upon translocation into phage-like vectors, which may also be transmitted via transduction events.

Characterization of an IncA/C Multidrug Resistance Plasmid in Vibrio alginolyticus

ABSTRACT Cephalosporin-resistant Vibrio alginolyticus was first isolated from food products, with β-lactamases encoded by blaPER-1, blaVEB-1, and blaCMY-2 being the major mechanisms mediating their cephalosporin resistance. The complete sequence of a multidrug resistance plasmid, pVAS3-1, harboring the blaCMY-2 and qnrVC4 genes was decoded in this study. Its backbone exhibited genetic homology to known IncA/C plasmids recoverable from members of the family Enterobacteriaceae, suggesting its possible origin in Enterobacteriaceae.

First Detection of AmpC β-Lactamase blaCMY-2 on a Conjugative IncA/C Plasmid in a Vibrio parahaemolyticus Isolate of Food Origin

ABSTRACT Vibrio parahaemolyticus is an important causative agent of gastroenteritis, with the consumption of contaminated seafood being the major transmission route. Resistance to penicillin is common among V. parahaemolyticus strains, whereas cephalosporin resistance remains rare. In an attempt to assess the current prevalence and characteristics of antibiotic resistance of this pathogen in common food samples, a total of 54 (17% of the total samples) V. parahaemolyticus strains were isolated from 318 meat and seafood samples purchased from supermarkets and wet markets in Shenzhen, China, in 2013. These isolates exhibited high-level resistance to ampicillin, yet they were mostly susceptible to other antimicrobials, except for two that were resistant to extended-spectrum cephalosporins. The β-lactamase gene blaPER-1 was detectable in one strain, V. parahaemolyticus V43, which was resistant to both third- and fourth-generation cephalosporins. Compared to other blaPER-1-positive V. parahaemolyticus strains reported in our previous studies, strain V43 was found to harbor an ∼200-kb conjugative plasmid carrying genes that were different from the antimicrobial resistance genes reported from the previous studies. The β-lactamase gene blaCMY-2 was detectable for the first time in another V. parahaemolyticus isolate, V4, which was resistant to third-generation cephalosporins. This blaCMY-2 gene was shown to be located in an ∼150-kb IncA/C-type conjugative plasmid with a genetic structure consisting of traB-traV-traA-ISEcp1-blaCMY-2-blc-sugE-encR-orf1-orf2-orf3-orf4-dsbC-traC, which is identical to that of other IncA/C conjugative plasmids in Enterobacteriaceae, albeit with a different size. These findings indicate that the transmission of extended-spectrum-β-lactamase (ESBL) and AmpC β-lactamase genes via conjugative plasmids can mediate the development of extended-spectrum cephalosporin resistance in V. parahaemolyticus, thereby posing a potential threat to public health.

A Novel PCR-Based Approach for Accurate Identification of Vibrio parahaemolyticus.

A PCR-based assay was developed for more accurate identification of Vibrio parahaemolyticus through targeting the blaCARB-17 like element, an intrinsic β-lactamase gene that may also be regarded as a novel species-specific genetic marker of this organism. Homologous analysis showed that blaCARB-17 like genes were more conservative than the tlh, toxR and atpA genes, the genetic markers commonly used as detection targets in identification of V. parahaemolyticus. Our data showed that this blaCARB-17-specific PCR-based detection approach consistently achieved 100% specificity, whereas PCR targeting the tlh and atpA genes occasionally produced false positive results. Furthermore, a positive result of this test is consistently associated with an intrinsic ampicillin resistance phenotype of the test organism, presumably conferred by the products of blaCARB-17 like genes. We envision that combined analysis of the unique genetic and phenotypic characteristics conferred by blaCARB-17 shall further enhance the detection specificity of this novel yet easy-to-use detection approach to a level superior to the conventional methods used in V. parahaemolyticus detection and identification.

CARB-17 Family of β-Lactamases Mediates Intrinsic Resistance to Penicillins in Vibrio parahaemolyticus

ABSTRACT Vibrio parahaemolyticus is commonly resistant to ampicillin, yet the mechanisms underlying this phenomenon are not clear. In this study, a novel class A carbenicillin-hydrolyzing β-lactamase (CARB) family of β-lactamases, blaCARB-17, was identified and found to be responsible for the intrinsic penicillin resistance in V. parahaemolyticus. Importantly, blaCARB-17-like genes were present in all 293 V. parahaemolyticus genome sequences available in GenBank and detectable in all 91 V. parahaemolyticus food isolates, further confirming the intrinsic nature of this gene.

IncHI2 Plasmids Are the Key Vectors Responsible for oqxAB Transmission among Salmonella Species.

ABSTRACT This study reported and analyzed the complete sequences of two oqxAB-bearing IncHI2 plasmids harbored by a clinical Salmonella enterica serovar Typhimurium strain and an S. Indiana strain of animal origin, respectively. In particular, pA3T recovered from S. Indiana comprised the resistance determinants oqxAB, aac(6′)Ib-cr, fosA3, and blaCTX-M-14. Further genetic screening of 63 oqxAB-positive Salmonella isolates revealed that the majority carried IncHI2 plasmids, confirming that such plasmids play a pivotal role in dissemination of oqxAB in Salmonella spp.

Complete Nucleotide Sequence of a Conjugative Plasmid Carrying blaPER-1

ABSTRACT The nucleotide sequence of a self-transmissible plasmid pVPH1 harboring blaPER-1 from Vibrio parahaemolyticus was determined. pVPH1 was 183,730 bp in size and shared a backbone similar to pAQU1 and pAQU2, differing mainly in an ∼40-kb multidrug resistance (MDR) region. A complex class 1 integron was identified together with ISCR1 and blaPER-1 (ISCR1-blaPER-1-gst-abct-qacEΔ1-sul1), which was shown to form a circular intermediate playing an important role in the dissemination of blaPER-1.

Selection of target mutation in rat gastrointestinal tract E. coli by minute dosage of enrofloxacin.

It has been suggested that bacterial resistance is selected within a mutation selection window of antibiotics. More recent studies showed that even extremely low concentration of antibiotic could select resistant bacteria in vitro. Yet little is known about the exact antibiotic concentration range that can effectively select for resistant organisms in animal gastrointestinal (GI) tract. In this study, the effect of different dosages of enrofloxacin on resistance and mutation development in rat GI tract E. coli was investigated by determining the number of resistant E. coli recoverable from rat fecal samples. Our data showed that high dose antibiotic treatment could effectively eliminate E. coli with single gyrA mutation in the early course of treatment, yet the eradication effects diminished upon prolonged treatment. Therapeutic and sub-therapeutic dose (1/10 and 1/100 of therapeutic doses) of enrofloxacin could effectively select for mutation in GI tract E. coli at the later course of enrofloxacin treatment and during the cessation periods. Surprisingly, very low dose of enrofloxacin (1/1000 therapeutic dose) could also select for mutation in GI tract E. coli at the later course of enrofloxacin treatment, only with slightly lower efficiency. No enrofloxacin-resistant E. coli could be selected at all test levels of enrofloxacin during long term treatment and the strength of antibiotic treatment does not alter the overall level of E. coli in rat GI tract. This study demonstrated that long term antibiotic treatment seems to be the major trigger for the development of target mutations in GI tract E. coli, which provided insight into the rational use of antibiotics in animal husbandry.

Genome analysis of clinical multilocus sequence Type 11 Klebsiella pneumoniae from China

The increasing prevalence of KPC-producing Klebsiella pneumoniae strains in clinical settings has been largely attributed to dissemination of organisms of specific multilocus sequence types, such as ST258 and ST11. Compared with the ST258 clone, which is prevalent in North America and Europe, ST11 is common in China but information regarding its genetic features remains scarce. In this study, we performed detailed genetic characterization of ST11 K. pneumoniae strains by analyzing whole-genome sequences of 58 clinical strains collected from diverse geographic locations in China. The ST11 genomes were found to be highly heterogeneous and clustered into at least three major lineages based on the patterns of single-nucleotide polymorphisms. Exhibiting five different capsular types, these ST11 strains were found to harbor multiple resistance and virulence determinants such as the blaKPC-2 gene, which encodes carbapenemase, and the yersiniabactin-associated virulence genes irp, ybt and fyu. Moreover, genes encoding the virulence factor aerobactin and the regulator of the mucoid phenotype (rmpA) were detectable in six genomes, whereas genes encoding salmochelin were found in three genomes. In conclusion, our data indicated that carriage of a wide range of resistance and virulence genes constitutes the underlying basis of the high level of prevalence of ST11 in clinical settings. Such findings provide insight into the development of novel strategies for prevention, diagnosis and treatment of K. pneumoniae infections.