High prevalence and genomic characteristics of carbapenem-resistant Enterobacteriaceae and colistin-resistant Enterobacteriaceae from large-scale rivers in China.

The widespread presence of carbapenem-resistant Enterobacteriaceae (CRE) and mcr-positive Escherichia coli (MCREC) poses a huge threat to both animal and human health. River water environments are vital reservoirs of antibiotic resistance genes, however, the prevalence and characteristics of CRE and MCREC from large-scale rivers in China have not been reported. In the current study, we sampled 86 rivers from four cities in Shandong Province, China in 2021 and analyzed the prevalence of CRE and MCREC. The blaNDM/blaKPC-2/mcr-positive isolates were characterized with methods including PCR, antimicrobial susceptibility testing, conjugation, replicon typing, whole-genome sequencing and phylogenetic analysis. We found that the prevalence of CRE and MCREC in 86 rivers was 16.3% (14/86) and 27.9% (24/86), respectively and eight rivers carried both mcr-1 and blaNDM/blaKPC-2. A total of 48 Enterobacteriaceae isolates (10 ST11 Klebsiella pneumoniae with blaKPC-2, 12 blaNDM-positive E. coli and 26 MCREC carrying only mcr-1) were obtained in this study and 47 displayed multidrug resistance (MDR). Notably, 10 of the 12 blaNDM-positive E. coli isolates also harbored the mcr-1 gene. The blaKPC-2 gene was located within mobile element ISKpn27-blaKPC-2-ISKpn6 on novel F33:A-:B- non-conjugative MDR plasmids in ST11 K. pneumoniae. The dissemination of blaNDM was mediated by transferable MDR IncB/O plasmids or IncX3 plasmids while mcr-1 was primarily disseminated by highly similar IncI2 plasmids. Notably, these waterborne IncB/O, IncX3 and IncI2 plasmids were all highly similar to previously identified plasmids from animal and human isolates. A phylogenomic analysis revealed that the CRE and MCREC isolates from water environments might be derived from animals and trigger infections in humans. The high prevalence of CRE and MCREC in large-scale environmental rivers is alarming and needs sustained surveillance due to the potential risk for transmission to humans via the food chain (irrigation) or direct contact.

Whole-Genome Analysis of blaNDM-Bearing Proteus mirabilis Isolates and mcr-1-Positive Escherichia coli Isolates Carrying blaNDM from the Same Fresh Vegetables in China.

The global spread of colistin or carbapenem-resistant Enterobacteriaceae (CRE) has been a pressing threat to public health. Members of Enterobacteriaceae, especially Proteus mirabilis and Escherichia coli, have been prevalent foodborne pathogens and such pathogens from fresh vegetables have triggered foodborne illness in China. However, reports about CRE, especially P. mirabilis from fresh vegetables, are still lacking. In this study, we identified five blaNDM-positive P. mirabilis and five blaNDM-positive generic E. coli concurrently from five fresh vegetables in two markets from China, and four of the five E. coli also carried mcr-1. The 10 isolates were characterized with methods including antimicrobial susceptibility testing, conjugation, whole-genome sequencing and phylogenetic analysis. All 10 isolates were multidrug-resistant (MDR). blaNDM-5 in five E. coli isolates and one P. mirabilis carrying blaNDM-5 was located on similarly transferable IncX3 plasmids, while transferably untypable plasmids were the carriers of blaNDM-1 in four P. mirabilis isolates from different types of vegetables/markets. mcr-1 in the four blaNDM-5-positive E. coli was located on similarly non-conjugative IncHI2 MDR plasmids lacking transfer region. Notably, ISCR1 complex class 1 integron capable of capturing blaNDM-1 was found on all untypable plasmids from P. mirabilis, and five copies of ISCR1 complex class 1 integron containing blaNDM-1 even occurred in one P. mirabilis, which showed high-level carbapenem resistance. Plasmid and phylogenetic analysis revealed that the blaNDM-positive P. mirabilis and E. coli from fresh vegetables might be derived from animals and transmitted to humans via the food chain. The concurrence of blaNDM-positive P. mirabilis and E. coli carrying both mcr-1 and blaNDM in different types of fresh vegetables eaten raw is alarming and threatens food safety. Sustained surveillance of these foodborne pathogens among fresh vegetables is urgent to ensure the health of food consumers. We report for the first time the concurrence of blaNDM-positive P. mirabilis and mcr-1-bearing E. coli carrying blaNDM from the same fresh vegetables.

Acquisition of a novel conjugative multidrug-resistant hypervirulent plasmid leads to hypervirulence in clinical carbapenem-resistant Klebsiella pneumoniae strains.

The co-occurrence of plasmid-mediated multidrug resistance and hypervirulence in epidemic carbapenem-resistant Klebsiella pneumoniae has emerged as a global public health issue. In this study, an ST23 carbapenem-resistant hypervirulent K. pneumoniae (CR-HvKP) strain VH1-2 was identified from cucumber in China and harbored a novel hybrid plasmid pVH1-2-VIR. The plasmid pVH1-2-VIR carrying both virulence and multidrug-resistance (MDR) genes was likely generated through the recombination of a virulence plasmid and an IncFIIK conjugative MDR plasmid in clinical ST23 18622 isolated from a sputum sample. The plasmid pVH1-2-VIR exhibited the capacity for transfer to the clinical ST11 carbapenem-resistant K. pneumoniae (CRKP) strain via conjugation assay. Acquisition of pVH1-2-VIR plasmid directly converted a CRKP into CR-HvKP strain characterized by hypermucoviscosity, heightened virulence for Galleria mellonella larvae, and increased colonization ability in the mouse intestine. The emergence of such a hybrid plasmid may expedite the spread of CR-HvKP strains, posing a significant risk to human health.

Prevalence and Antibiotic Resistance Characteristics of Extraintestinal Pathogenic Escherichia coli among Healthy Chickens from Farms and Live Poultry Markets in China.

Chicken products and chickens with colibacillosis are often reported to be a suspected source of extraintestinal pathogenic Escherichia coli (ExPEC) causing several diseases in humans. Such pathogens in healthy chickens can also contaminate chicken carcasses at the slaughter and then are transmitted to humans via food supply; however, reports about the ExPEC in healthy chickens are still rare. In this study, we determined the prevalence and characteristics of ExPEC isolates in healthy chickens in China. A total of 926 E. coli isolates from seven layer farms (371 isolates), one white-feather broiler farm (78 isolates) and 17 live poultry markets (477 isolates from yellow-feather broilers) in 10 cities in China, were isolated and analyzed for antibiotic resistance phenotypes and genotypes. The molecular detection of ExPEC among these healthy chicken E. coli isolates was performed by PCRs, and the serogroups and antibiotic resistance characteristics of ExPEC were also analyzed. Pulsed-field gel electrophoresis (PFGE) and Multilocus sequence typing (MLST) were used to analyze the genetic relatedness of these ExPEC isolates. We found that the resistance rate for each of the 15 antimicrobials tested among E. coli from white-feather broilers was significantly higher than that from brown-egg layers and that from yellow-feather broilers in live poultry markets (p < 0.05). A total of 22 of the 926 E. coli isolates (2.4%) from healthy chickens were qualified as ExPEC, and the detection rate (7.7%, 6/78) of ExPEC among white-feather broilers was significantly higher than that (1.6%, 6/371) from brown-egg layers and that (2.1%, 10/477) from yellow-feather broilers (p < 0.05). PFGE and MLST analysis indicated that clonal dissemination of these ExPEC isolates was unlikely. Serogroup O78 was the most predominant type among the six serogroups identified in this study, and all the six serogroups had been frequently reported in human ExPEC isolates in many countries. All the 22 ExPEC isolates were multidrug-resistant (MDR) and the resistance rates to ampicillin (100%) and sulfamethoxazole-trimethoprim (100%) were the highest, followed by tetracycline (95.5%) and doxycycline (90.9%). blaCTX-M was found in 15 of the 22 ExPEC isolates including 10 harboring additional fosfomycin resistance gene fosA3. Notably, plasmid-borne colistin resistance gene mcr-1 was identified in six ExPEC isolates in this study. Worryingly, two ExPEC isolates were found to carry both mcr-1 and blaNDM, compromising both the efficacies of carbapenems and colistin. The presence of ExPEC isolates in healthy chickens, especially those carrying mcr-1 and/or blaNDM, is alarming and will pose a threat to the health of consumers. To our knowledge, this is the first report of mcr-1-positive ExPEC isolates harboring blaNDM from healthy chickens.

Occurrence and characterization of KPC-2-producing ST11 Klebsiella pneumoniae isolate and NDM-5-producing Escherichia coli isolate from the same horse of equestrian clubs in China.

Carbapenem-resistant Enterobacteriaceae (CRE) have been rapidly increasing among animals in many countries and have been a great threat to public health. Horse riding is becoming increasingly popular worldwide; however, reports of CRE producing NDM or KPC-2, two prevalent types of carbapenemases, from horses of equestrian club are extremely scarce and KPC-2-producing Klebsiella pneumoniae in animals is still rarely characterized. In this study, we identified four NDM-5-producing Escherichia coli isolates from horses in equestrian club in Qingdao, China, and one horse possessing NDM-5-producing E. coli also carried ST11 KPC-2-producing K. pneumoniae. Transferability of the plasmids producing carbapenemases was determined by conjugation, and the sequences of all CRE isolates and their transconjugants were then analysed by using whole-genome sequencing. blaNDM-5 was located on a highly similar ~ 46 kb self-transmissible IncX3 plasmid in all isolates, and these plasmids were nearly identical to IncX3 plasmids from different bacterial species of clinical patients in several countries, even including plasmid from clinical E. coli in Qingdao, China. The chromosome of the ST11 K. pneumoniae in this study was highly similar to ST11 clinical K. pneumoniae reported worldwide including the ST11 KPC-2-producing WCHKP020098 from Chengdu, China, and the blaKPC-2 -bearing plasmid in our study was a novel F33:A-:B- non-conjugative multidrug resistance plasmid. The presence of CRE from horses in equestrian club is alarming due to the potential for transmitting these isolates to humans during horse riding, and the prevalence of CRE among equestrian clubs in the whole country requires further monitoring.

Co-occurrence of Plasmid-Mediated Tigecycline and Carbapenem Resistance in Acinetobacter spp. from Waterfowls and Their Neighboring Environment.

Tigecycline serves as one of the antibiotics of last resort to treat multidrug-resistant (including carbapenem-resistant) pathogens. However, the recently emerged plasmid-mediated tigecycline resistance mechanism, Tet(X), challenges the clinical efficacy of this class of antibiotics. In this study, we detected 180 tet(X)-harboring Acinetobacter isolates (8.9%, n = 180) from 2,018 samples collected from avian farms and adjacent environments in China. Eighteen tet(X)-harboring isolates (10.0%) were found to cocarry the carbapenemase gene blaNDM-1, mostly from waterfowl samples (94.4%, 17/18). Interestingly, among six Acinetobacter strains, tet(X) and blaNDM-1 were found to colocalize on the same plasmids. Moreover, whole-genome sequencing (WGS) revealed a novel orthologue of tet(X) in the six isolates coharboring tet(X) and blaNDM-1 Inverse PCR suggested that the two tet(X) genes form a single transposable unit and may be cotransferred. Sequence comparison between six tet(X)- and blaNDM-1-coharboring plasmids showed that they shared a highly homologous plasmid backbone even though they were isolated from different Acinetobacter species (three from Acinetobacter indicus, two from Acinetobacter schindleri, and one from Acinetobacter lwoffii) from various sources and from different geological regions, suggesting the horizontal genetic transfer of a common tet(X)- and blaNDM-1-coharboring plasmid among Acinetobacter species in China. Emergence and spread of such plasmids and strains are of great clinical concern, and measures must be implemented to avoid their dissemination.

Emergence of two Escherichia coli strains co-harboring mcr-1 and bla NDM in fresh vegetables from China.

BACKGROUND: The 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. METHODS: In 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. RESULTS: Two E. coli isolates concomitantly carrying mcr-1 and bla NDM-5/9 from leaf rape and spinach, respectively, were found and both isolates showed multidrug resistance. Notably, mcr-1-positive 690 harboring bla NDM-5 and 701 carrying bla NDM-9 belonged to ST156 and ST2847, respectively, similar to the prevalent MLST types of E. coli co-carrying mcr-1 and bla NDM from avian in our previous study. mcr-1 was on ~33-kb IncX4 plasmid or ~60-kb IncI2 plasmid, while bla NDM-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 bla NDM-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 bla NDM and mcr-1. To our knowledge, this is the first report of Enterobacteriaceae co-carrying bla NDM and mcr-1 in fresh vegetables.

Whole genome sequencing of NDM-1-producing serotype K1 ST23 hypervirulent Klebsiella pneumoniae in China.

PURPOSE: The emergence and spread of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) is causing worldwide concern, whereas NDM-producing hvKP is still rare. Here we report the complete genome sequence characteristics of an NDM-1-producing ST23 type clinical hvKP in PR China. METHODOLOGY: Capsular polysaccharide serotyping was performed by PCR. The complete genome sequence of isolate 3214 was obtained using both the Illumina Hiseq platform and Pacbio RS platform. Multilocus sequence type was identified by submitting the genome sequence to mlst 2.0 and the antimicrobial resistance genes and plasmid replicons were identified using ResFinder and PlasmidFinder, respectively. Transferability of the blaNDM-1-bearing plasmid was determined by conjugation experiment, S1 pulsed-field gel electrophoresis and Southern hybridization. RESULTS: Isolate 3214 was classified to ST23 and belonged to the K1 capsular serotype. The isolate's total genome size was 6 171 644 bp with a G+C content of 56.39 %, consisting of a 5 448 209 bp chromosome and seven plasmids. The resistome included 18 types of antibiotic resistance genes. Fourteen resistance genes including blaNDM-1 and blaCTX-M-14 were located on plasmids and five also including blaCTX-M-14 were in the chromosome. Plasmid pNDM_3214 carrying blaNDM-1 harboured six types of resistance genes surrounded by insertion sequences and was conjugative. The worldwide pLVPK-like virulence plasmid harbouring rmpA2 and rmpA was also found in this isolate. CONCLUSION: This study provides basic information of phenotypic and genomic features of ST23 CR-hvKP isolate 3214. Our data highlights the potential risk of spread of NDM-1-producing ST23 hvKP.

Colistin-Resistant mcr-Positive Enterobacteriaceae in Fresh Vegetables, an Increasing Infectious Threat in China.

The presence of mobilized colistin resistance (mcr) genes is a global concern. However, data concerning mcr in fresh vegetables, a reservoir for antibiotic resistance genes, are still rare. In this study, mcr genes were analysed in 528 vegetable samples from 53 supermarkets or farmer's markets in 23 cities of 9 provinces in China, and the mcr-positive Enterobacteriaceae were characterized. Nineteen (3.6%) samples carried one or more mcr-positive isolates, and the highest three detection rates were found in carrot, pak choi and green pepper. Twenty-four mcr-1-positive isolates (23 Escherichia coli and one Enterobacter cloacae) were obtained, and E. coli isolates showed high genetic diversity. Different multilocus sequence type (MLST) isolates were also observed within the same sample. All 24 isolates showed multidrug resistance, and 14 carried blaCTX-M genes. Most isolates harbored similarly conjugative IncX4-type (∼33 kb) or IncI2-type (∼60 kb) mcr-1-bearing plasmids. The sequenced prevalent IncX4 plasmid and IncI2 plasmid from tomato were similar to the relevant plasmids from animals and clinical isolates in various countries. mcr-1-bearing IncHI2/ST3 plasmid highly similar to that carrying 14 resistance genes from E. coli of chicken was also observed. In conclusion, a high prevalence of mcr-1 in fresh vegetables was found in China, and the dissemination of mcr-1 was mediated by similar IncX4 or IncI2 plasmids. The plasmids from vegetables showed high similarity to plasmids from clinical isolates, indicating MCR-1-producers in ready-to-eat vegetables may pose a huge threat to public health and measures need to be taken to ensure food safety.

Characterization of Highly Prevalent Plasmids Coharboring mcr-1, oqxAB, and blaCTX-M and Plasmids Harboring oqxAB and blaCTX-M in Escherichia coli Isolates from Food-Producing Animals in China.

The emergence and spread of multidrug resistance (MDR) plasmids carrying the colistin resistance gene mcr-1 has become a major public health concern. However, there is a paucity of data regarding the prevalence of mcr-1 plasmids concomitantly carrying blaCTX-M and oqxAB, an efflux pump that confers resistance to multiple agents. In this study, we determined the prevalence and characteristics of plasmids coharboring mcr-1, oqxAB, and blaCTX-M as well as those harboring oqxAB and blaCTX-M in Escherichia coli from food-producing animals. We isolated 493 E. coli strains, and mcr-1, blaNDM, and blaCTX-M were present in 140 (28.4%), 51 (10.3%), and 195 (39.6%) of the isolates, respectively. The two most prevalent plasmid-mediated quinolone resistance genes were oqxAB (34.5%) and qnrS (29.4%). Nine IncHI2/ST3 plasmids co-carrying mcr-1, oqxAB, and blaCTX-M were found, and similar IncHI2/ST3 plasmids mediated dissemination of these resistance genes. Two sequenced MDR IncHI2/ST3 plasmids coharboring mcr-1, oqxAB, and blaCTX-M showed high similarity to reference plasmid pHNSHP45-2, although they were from different regions in China. Colocalization of oqxAB and blaCTX-M on the same plasmid was found in 28 isolates, including the nine plasmids harboring mcr-1. The co-dissemination of oqxAB and blaCTX-M was mediated by diverse F33:A-:B- plasmids and similar IncHI2/ST3 plasmids. Pulsed-field gel electrophoresis and multilocus sequence typing analysis of donor isolates revealed heterogeneous patterns indicating that clonal dissemination was unlikely. The high incidence of similar IncHI2/ST3 plasmids simultaneously possessing mcr-1, oqxAB, and blaCTX-M poses a great threat to public health.

A novel experimental intraperitoneal infection model for Haemophilus parasuis in neutropenic guinea pigs.

INTRODUCTION: Haemophilus parasuis, one of the major swine pathogens, has at least fifteen different types, all of which have significant economic effects on the global swine industry. The aim of this study was to establish an experimental intraperitoneal infection model for H. parasuis in neutropenic guinea pigs. METHODS: Intraperitoneal administration of cyclophosphamide and Haemophilus parasuis was conducted in guinea pigs. Clinical signs, gross pathology, and histopathology were observed in neutropenic guinea pigs infected with H. parasuis. RESULTS: Intraperitoneal administration of 100 mg/kg cyclophosphamide led to immunosuppression with white blood cells, lymphocytes, and neutrophils all <1000 mm3, while no histological tissue damage was observed. Intraperitoneal administration of 109 colony-forming units (CFU) of H. parasuis led to typical respiratory symptoms, 90% morbidity, and 20% mortality in a 72 h-period. Bacteriological screening revealed that multiple organs, including the heart, liver, spleen, lungs, kidneys, and blood, were infected with H. parasuis. The threshold loads of bacteria in blood and the lungs were (7.04 ±â€¯0.53)log10 CFU/mL and (6.24 ±â€¯0.62)log10 CFU/g, respectively, at 3 d after infection. Gross pathology examination showed celiac effusion, intestinal mucosal hemorrhage, and liver, spleen, or lung swelling, necrosis, and hemorrhage. Congestion, mild interstitial pneumonia, inflammatory exudation, and endothelial cell proliferation were observed in the histological examination. DISCUSSION: All the results suggest that we have established an experimental intraperitoneal infection model for H. parasuis in neutropenic guinea pigs. It is especially useful as a tool for pharmacokinetics, pharmacodynamics, or a pharmacokinetics/pharmacodynamics (PK/PD) model of antimicrobial agents against respiratory disease.

Characteristics of Carbapenem-Resistant Enterobacteriaceae in Ready-to-Eat Vegetables in China.

Vegetables harboring bacteria resistant to antibiotics are a growing food safety issue. However, data concerning carbapenem-resistant Enterobacteriaceae (CRE) in ready-to-eat fresh vegetables is still rare. In this study, 411 vegetable samples from 36 supermarkets or farmer's markets in 18 cities in China, were analyzed for CRE. Carbapenemase-encoding genes and other resistance genes were analyzed among the CRE isolates. Plasmids carrying carbapenemase genes were studied by conjugation, replicon typing, S1-PFGE southern blot, restriction fragment length polymorphism (RFLP), and sequencing. CRE isolates were also analyzed by pulsed-field gel electrophoresis (PFGE). Ten vegetable samples yielded one or more CRE isolates. The highest detection rate of CRE (14.3%, 4/28) was found in curly endive. Twelve CRE isolates were obtained and all showed multidrug resistance: Escherichia coli, 5; Citrobacter freundii, 5; and Klebsiella pneumoniae, 2. All E. coli and C. freundii carried blaNDM, while K. pneumoniae harbored blaKPC-2. Notably, E. coli with blaNDM and ST23 hypervirulent Klebsiella pneumoniae (hvKP) carrying blaKPC-2 were found in the same cucumber sample and clonal spread of E. coli, C. freundii, and K. pneumoniae isolates were all observed between vegetable types and/or cities. IncX3 plasmids carrying blaNDM from E. coli and C. freundii showed identical or highly similar RFLP patterns, and the sequenced IncX3 plasmid from cucumber was also identical or highly similar (99%) to the IncX3 plasmids from clinical patients reported in other countries, while blaKPC-2 in K. pneumoniae was mediated by similar F35:A-:B1 plasmids. Our results suggest that both clonal expansion and horizontal transmission of IncX3- or F35:A-:B1-type plasmids may mediate the spread of CRE in ready-to-eat vegetables in China. The presence of CRE in ready-to-eat vegetables is alarming and constitutes a food safety issue. To our knowledge, this is the first report of either the C. freundii carrying blaNDM, or K. pneumoniae harboring blaKPC-2 in vegetables. This is also the first report of ST23 carbapenem-resistant hvKP strain in vegetables.

High Incidence of Escherichia coli Strains Coharboring mcr-1 and blaNDM from Chickens.

This study investigated the characteristics of Escherichia coli isolates carrying mcr-1-blaNDM from a chicken farm in China. Of the 78 E. coli isolates, 21 clonally unrelated isolates carried mcr-1-blaNDM Diverse IncI2 plasmids disseminated mcr-1, while the dissemination of blaNDM was mediated by diverse IncB/O plasmids. More striking was the colocalization of resistance genes mcr-1 and blaNDM-4 in an IncHI2/ST3 plasmid, which might pose a great challenge for public health.

Emergence of Colistin Resistance Gene mcr-1 in Cronobacter sakazakii Producing NDM-9 and in Escherichia coli from the Same Animal.

We report the presence of mcr-1 in Escherichia coli and carbapenem-resistant Cronobacter sakazakii from the same diseased chicken. The mcr-1 gene linked with ISApl1 was located on two different IncI2 plasmids, including one multidrug plasmid in E. coli, whereas fosA3-blaNDM-9 was on an IncB/O plasmid in C. sakazakii The development of the fosA3-blaNDM-9 resistance region was mediated by IS26 The colocation of mcr-1 or blaNDM-9 with other resistance genes will accelerate the dissemination of the two genes.

Complete Sequence of the FII Plasmid p42-2, Carrying blaCTX-M-55, oqxAB, fosA3, and floR from Escherichia coli.

We 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 (∼54 kb) and one distinct acquired DNA region spanning ∼53 kb, harboring 12 antibiotic resistance genes [blaCTX-M-55, oqxA, oqxB, fosA3, floR, tetA(A), tetA(R), strA, strB, sul2, aph(3')-II, and ΔblaTEM-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.

IncF plasmid diversity in multi-drug resistant Escherichia coli strains from animals in China.

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 bla CTX-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) bla CTX-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.

Two genes conferring resistance to Pythium stalk rot in maize inbred line Qi319.

Stalk rots are destructive diseases in maize around the world, and are most often caused by the pathogen Pythium, Fusarium and other fungi. The most efficient management for controlling stalk rots is to breed resistant cultivars. Pythium stalk rot can cause serious yield loss on maize, and to find the resistance genes from the existing germplasm is the basis to develop Pythium-resistance hybrid lines. In this study, we investigated the genetic resistance to Pythium stalk rot in inbred line Qi319 using F2 and F2:3 population, and found that the resistance to Pythium inflatum in Qi319 was conferred by two independently inherited dominant genes, RpiQI319-1 and RpiQI319-2. Linkage analysis uncovered that the RpiQI319-1 co-segregated with markers bnlg1203, and bnlg2057 on chromosome 1, and that the RpiQI319-2 locus co-segregated with markers umc2069 and bnlg1716 on chromosome 10. The RpiQI319-1 locus was further mapped into a ~500-kb interval flanked by markers SSRZ33 and SSRZ47. These results will facilitate marker-assisted selection of Pythium stalk rot-resistant cultivars in maize breeding. To our knowledge, this is the first report on the resistance to P. inflatum in the inbred line Qi319, and is also the first description of two independently inherited dominant genes conferring the resistance of Pythium stalk rot in maize.

Co-spread of oqxAB and blaCTX-M-9G in non-Typhi Salmonella enterica isolates mediated by ST2-IncHI2 plasmids.

In this study, 130 non-Typhi Salmonella enterica isolates from chickens were analysed for the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants as well as the co-existence of oqxAB and extended-spectrum ß-lactamase (ESBL) genes. The genes oqxAB, aac(6')-Ib-cr, blaCTX-M-9G and blaTEM were present alone or in combination in 40 (30.8%), 40 (30.8%), 55 (42.3%) and 2 (1.5%) isolates, respectively. Most of the oqxAB-blaCTX-M-9G-positive isolates (17/28) carried transferable ST2-IncHI2 plasmids containing an oqxAB cassette and blaCTX-M-14 flanked by insertion sequences IS10 or ISEcp1 upstream and IS903 downstream. The oqxAB-blaCTX-M-9G-positive isolates from a local area showed similar pulsed-field gel electrophoresis (PFGE) patterns, whilst the isolates from different areas were genetically divergent, suggesting that both clonal expansion in local areas and horizontal transmission contributed to the spread of ST2-IncHI2 plasmids containing oqxAB and blaCTX-M-14. This is the first report on the prevalence of ST2-IncHI2 plasmids concomitantly carrying oqxAB and blaCTX-M-14 in Salmonella and also the first description of the genetic environment of oqxAB-blaCTX-M. The genetic linkage of oqxAB-blaCTX-M-9G in non-Typhi Salmonella likely facilitates the spread of antibiotic-resistant Salmonella and poses a threat for clinical treatment of salmonellosis.

Characterization of plasmids carrying oqxAB in bla(CTX-M)-negative Escherichia coli isolates from food-producing animals.

To study the characteristics of plasmids harboring oqxAB among bla(CTX-M)-negative Escherichia coli isolates and search for oqxAB-harboring plasmids similar to plasmids carrying oqxAB-bla(CTX-M) reported previously, conjugation experiment was performed for 115 randomly selected oqxAB-positive but bla(CTX-M)-negative E. coli isolates from diseased animals in Guangdong, China. S1 nuclease pulsed-field gel electrophoresis (PFGE) and southern blotting experiments were performed to investigate the location of oqxAB and other resistance genes. The EcoRI digestion profiles of the plasmids with oqxAB were also analyzed. The clonal relatedness of donor isolates was investigated by PFGE. In this study, 32 oqxAB transconjugants were successfully obtained and most transconjugants showed multidrug resistances. Eleven replicon combination types were found in these transconjugants. floR and oqxAB were found on the same plasmids in all nine transconjugants resistant to florfenicol. The sequences between floR and oqxAB were identical in most transconjugants and the two genes were both linked with tnp in insertion sequences. Nine F18:A-:B1 plasmids with only oqxAB shared identical EcoRI digestion profiles and the profiles were also identical with that of a plasmid carrying oqxAB-bla(CTX-M) found previously. Co-transfer of plasmids carrying oqxAB and fosA3, respectively, was also observed in one isolate. This study demonstrates the dissemination of oqxAB among bla(CTX-M)-negative E. coli isolates was mainly mediated by identical F18:A-:B1 plasmids. A novel arrangement of regions between floR and oqxAB might play an important role in the dissemination of floR-oqxAB. This is the first description of the genetic environment of the relationship between oqxAB and floR in E. coli.

Comparison of plasmids coharboring 16s rrna methylase and extended-spectrum ß-lactamase genes among Escherichia coli isolates from pets and poultry.

A total of 247 Escherichia coli isolates (148 from diseased or dead poultry and 99 from diseased pets in the People's Republic of China) were screened for extended-spectrum ß-lactamase (ESBL) determinants by PCR and sequencing. Then, 16S rRNA methylase genes were detected among ESBL-producing isolates. Clonal relatedness of the E. coli isolates was examined by pulsed-field gel electrophoresis. Conjugation experiments were performed to investigate the association of 16S rRNA methylases and ESBLs, and plasmid contents were also characterized. Among 247 E. coli isolates, 74 (29.96%) isolates were positive for blaCTX-M genes, 42 from pets (12 from cats and 30 from dogs) and 32 from poultry (12 from chickens and 20 from ducks). The most common CTX-M type in isolates from pets was blaCTX-M-14, whereas blaCTX-M-27 was the most common for poultry. rmtB was dectected in 39 of the 74 blaCTX-M-positive isolates, 18 from pets and 21 from poultry. One strain from a pet was found to harbor blaCTX-M-14, blaCTX-M-15, and rmtB. blaCTX-M and rmtB were found to be colocated on the same transferable plasmid in 16 isolates. These genes were on the same or similar plasmids (eight F2:A-:B- and two IncN) in isolates from ducks, whereas they were colocated on the similar F2:A-:B- or similar F33:A-:B- plasmids in isolates of pets origin. In conclusion, similar F2:A-:B- plasmids and similar F33:A-:B- plasmids are responsible for the dissemination of both rmtB and blaCTX-Mgenes in E. coli isolates from poultry and pets, respectively.