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.

Mouse adaptation of the H9N2 avian influenza virus causes the downregulation of genes related to innate immune responses and ubiquitin-mediated proteolysis in mice.

H9N2 avian influenza viruses sporadically infect humans worldwide. These viruses have also contributed internal genes to H5N1, H5N6, H7N9, and H10N8 viruses, which have been isolated from humans with infections and are a substantial public health threat. To investigate the potential pathogenic mechanism of the H9N2 virus, we performed serial lung-to-lung passage of an avirulent H9N2 avian influenza virus (A/Chicken/Shandong/416/2016 [SD/416]) in mice to increase the pathogenicity of this virus. We generated a mouse-adapted (MA) virus that exhibited increased viral titers in the lungs, caused severe lung damage in mice, and induced body weight loss in mice; however, the avirulent parental virus did not cause any clinical symptoms in infected mice. Global gene expression analysis was performed and indicated that the transcriptional responses of these viruses were distinct. The lungs of mice infected with the MA virus exhibited the downregulation of genes related to innate immunity and ubiquitin-mediated proteolysis, which was not seen in infections with the avirulent parental virus. These data indicated that the MA virus might evade immune surveillance and changed its replication capacity to increase the viral replication level and pathogenicity. Our study demonstrates that host factors play an important role in the adaptive evolution of influenza virus in new hosts.

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.

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.

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.

High prevalence and transmission of blaNDM-positive Escherichia coli between farmed ducks and slaughtered meats: An increasing threat to food safety.

The emergence of carbapenem-resistant bacteria especially carbapenem-resistant Escherichia coli (CREC) in food animals poses a serious threat to food safety and public health. Reports about the dissemination of carbapenem-resistant bacteria along the food animal production chain are scattered and mainly focus on swine and chicken. Abuse of antibiotics in duck farms is common especially in China which has the largest duck production industry, however, the CREC transmission between farmed ducks and slaughtered meats remains unclear and the role of slaughterhouse in disseminating CREC among duck meats remains largely unknown. Herein, we collected 251 fecal samples from five typical duck farms along with 125 slaughtered meat samples (25 from each farm) in the corresponding slaughterhouse in Anhui Province, China, in December 2018. All samples were screened for CREC isolates which were analyzed for the presence of carbapenemase genes and colistin resistance gene mcr. The resistance profiles, transferability, pulsed-field gel electrophoresis (PFGE), whole-genome sequencing and phylogenetic analysis of the CREC isolates from both ducks and meats were further characterized. This is the first report presenting the high prevalence of blaNDM-positive CREC isolates in ducks from duck farms (57.8 %) and slaughtered meats (33.6 %) in the corresponding slaughterhouse. Among the 203 blaNDM-positive CREC isolates obtained in this study, 19.2 % harbored mcr-1 and all CREC isolates showed resistance to nearly all currently available antibiotics (except tigecycline). Of note, mcr-1 was found in 17.8 % of the meat-derived CREC carrying blaNDM. Based on the PFGE analysis, clonal spread of blaNDM-positive CREC including some also carrying mcr-1 was found between farmed ducks and slaughtered duck meats even from different farms. Special attention should be paid to the clonal dissemination of meat-derived CREC within the slaughterhouse, which contributed to the high prevalence of blaNDM in slaughtered meats. Additionally, horizontal transmission mainly mediated by transferable blaNDM-5-bearing IncX3 plasmids, untypable blaNDM-1-bearing plasmids and mcr-1-bearing IncHI2 plasmids further facilitated the rapid spread of such multidrug-resistant strains. Notably, the blaNDM-bearing plasmids and mcr-1-bearing plasmids in CREC from meats were highly similar to those from animals and humans. More worryingly, the phylogenomic analysis showed that CREC isolates from both ducks and corresponding meats clustered with previously reported human CREC isolates carrying mcr-1 in different geographical areas including China. These findings further prove that the CREC and resistance plasmids in farmed ducks could transmit to meats even from different farms via the slaughterhouse and then trigger infections in humans. The high prevalence and clonal transmission of CREC isolates including those also carrying mcr-1 between ducks and meats are alarming, and urgent control measures are required to reduce the dissemination of such organisms.

Spread of oqxAB in Salmonella enterica serotype Typhimurium predominantly by IncHI2 plasmids.

OBJECTIVES: To investigate the prevalence and genetic environment of the multiresistance gene oqxAB in Salmonella enterica serotype Typhimurium isolated from food-producing animals. METHODS: In this study, 63 Salmonella enterica serotype Typhimurium isolates were analysed for the presence of plasmid-mediated quinolone resistance determinants and mutations in the quinolone resistance-determining region by molecular methods (PCR/sequencing). The oqxAB-positive isolates were typed by pulsed-field gel electrophoresis (PFGE). Plasmids carrying oqxAB were studied by conjugation/transformation, replicon typing, Southern hybridization, long-range PCR and restriction fragment length polymorphism (RFLP). RESULTS: The oqxAB, aac(6')-Ib-cr and qnrS1 genes were present alone or in combination in 20 (31.7%), 23 (36.5%) and 1 (1.6%) isolate, respectively. The oqxAB-positive isolates were clonally related, as determined by PFGE. All of the oqxAB-aac(6')-Ib-cr-positive isolates carried transferable IncHI2-type plasmids containing an oqxAB cassette and an incomplete class 1 integron harbouring aac(6')-Ib-cr, blaOXA-1, catB3, arr3, qacEΔ1 and sul1. Meanwhile, 6 of 15 plasmids carrying both oqxAB and aac(6')-Ib-cr showed identical RFLP patterns. CONCLUSIONS: The results suggest that both clonal expansion and horizontal transmission of IncHI2-type plasmids containing oqxAB and aac(6')-Ib-cr may be involved in the spread of oqxAB in Salmonella Typhimurium isolates in food-producing animals in China. There is a great need to monitor the potential dissemination of this multiresistance gene.

Development of ceftriaxone resistance affects the virulence properties of Salmonella enterica serotype Typhimurium strains.

Development of antibiotic resistance may alter the virulence properties of bacterial organisms. In this study, nine clinical ceftriaxone-susceptible Salmonella enterica serotype Typhimurium strains were subjected to stepwise selection with increasing concentrations of ceftriaxone in culture media. Mutations in virulence-associated genes and antibiotic efflux genes were analyzed by polymerase chain reaction (PCR) and DNA sequencing. The expression levels of virulence genes invA and stn as well as efflux pump genes tolC, arcA, and arcB before and after the selection were measured by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The stepwise selection resulted in the development of Salmonella strains that were highly resistant to ceftriaxone. Sequence analysis did not reveal any mutations or deletions in the examined virulence genes and regulatory gene, but a silent mutation (T423C) in acrR (encoding a repressor for the efflux pump) was detected in most of the ceftriaxone-resistant strains. The qRT-PCR revealed increased expression of the AcrAB-TolC efflux pump and decreased expression of invA and stn in the ceftriaxone-resistant strains. Moreover, decreased invasion into cultured epithelial cells and reduced growth rates were observed with the resistant strains. These results suggest that acquisition of ceftriaxone resistance is associated with the overexpression of the AcrAB-TolC efflux pump and leads to reduced virulence in Salmonella Typhimurium.

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.

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.