Epidemiological characteristics of human- and chicken-derived CTX-M-type extended-spectrum ß-lactamase-producing Escherichia coli from China.

Bacterial resistance to ß-lactams is mainly attributed to CTX-M-type extended-spectrum ß-lactamases (ESBLs). However, the predominant sequence type (ST) of blaCTX-M-carrying Escherichia coli (blaCTX-M-Ec) in chickens, an important food animal, in China and its contribution to human ß-lactam resistance are not investigated. In this study, approximately 1808 chicken-derived strains collected from 10 provinces from 2012 to 2020 were screened for blaCTX-M-Ec, and 222 blaCTX-M-Ec were identified. Antimicrobial susceptibility tests, whole genome sequencing and conjugation experiment were performed. All quality-controlled 136 chicken-derived blaCTX-M-Ec and 1193 human-derived blaCTX-M-Ec genomes were downloaded from NCBI and EnteroBase to comprehensively analyze the prevalence of blaCTX-M-Ec in China. blaCTX-M-55 (153/358, 42.7% in chicken isolates; 312/1193, 26.2% in human isolates) and blaCTX-M-14 (92/358, 25.7% in chicken isolates; 450/1193, 37.7% in human isolates) were dominant in blaCTX-M-Ec. The STs of blaCTX-M-Ec were diverse and scattered, with ST155 (n = 21) and ST152 (n = 120) being the most abundant in chicken- and human-derived isolates, respectively. Few examples indicated that chicken- and human-derived blaCTX-M-Ec have 10 or less core genome single nucleotide polymorphisms (cgSNPs). Genetic environment analysis indicated that ISEcp1, IS26 and IS903B were closely associated with blaCTX-M transfer. The almost identical pc61-55 and pM-64-1161 indicated the possibility of plasmid-mediated transmission of blaCTX-M between humans and chickens. Although the genomes of most blaCTX-M-Ec isolated from chickens and humans were quite different, the prevalence and genetic environment of blaCTX-M variants in both hosts were convergent. CTX-M-mediated resistance is more likely to spread through horizontal gene transmission than bacterial clones.

Novel multidrug resistance genomic islands and transposon carrying blaVEB-1 identified in mcr-positive Aeromonas strains from raw meat in China.

OBJECTIVES: To characterize the genetic environments of ESBL gene blaVEB-1 in mcr-positive Aeromonas strains from raw meat in China. METHODS: Whole genomes of Aeromonas strains were sequenced using the Illumina or Nanopore platforms. Genetic environments of blaVEB-1 were analysed using the BLAST program. RESULTS: The blaVEB-1 gene was detected in five Aeromonas strains carrying the mcr-7-like gene. WGS revealed that all blaVEB-1 genes were located on Aeromonas chromosome, and were carried by two novel different genomic islands named Aeromonas veronii genomic islands AveGI1 and AveGI2, as well as one transposon named Tn7690. AveGI1 is a new member of the Salmonella genomic island 1 family, incorporated into the 3'-end of mnmE (trmE). AveGI2 is a novel genomic island that has a size of 23 180 bp and is incorporated into the 3'-end of syd. The MDR regions of AveGI1 and AveGI2 are two different class 1 integrons containing 10 and five resistance genes, respectively. Tn7690 is a Tn1722 derivative containing In4-type integron and Tn5393, which harbours 10 resistance genes and integrates into different positions on the chromosomes of three strains with the capacity for mobility. CONCLUSIONS: We report chromosomally located novel MDR genomic islands and transposon that carry blaVEB-1 in mcr-positive Aeromonas strains. These genetic elements may mediate the spread of blaVEB-1 in Aeromonas, and may also evolve by capturing new antimicrobial resistance genes or other mobile genetic elements.

Whole-genome sequencing of Escherichia coli from retail meat in China reveals the dissemination of clinically important antimicrobial resistance genes.

Escherichia coli is one of the important reservoirs of antimicrobial resistance genes (ARG), which often causes food-borne diseases and clinical infections. Contamination with E. coli carrying clinically important antimicrobial resistance genes in retail meat products can be transmitted to humans through the food chain, posing a serious threat to public health. In this study, a total of 330 E. coli strains were isolated from 464 fresh meat samples from 17 food markets in China, two of which were identified as enterotoxigenic and enteropathogenic E. coli. Whole genome sequencing revealed the presence of 146 different sequence types (STs) including 20 new STs, and 315 different clones based on the phylogenetic analysis, indicating the high genetic diversity of E. coli from retail meat products. Antimicrobial resistance profiles showed that 82.42 % E. coli were multidrug-resistant strains. A total of 89 antimicrobial resistance genes were detected and 12 E. coli strains carried clinically important antimicrobial resistance genes blaNDM-1, blaNDM-5, mcr-1, mcr-10 and tet(X4), respectively. Nanopore sequencing revealed that these resistance genes are located on different plasmids with the ability of horizontal transfer, and their genetic structure and environment are closely related to plasmids isolated from humans. Importantly, we reported for the first time the presence of plasmid-mediated mcr-10 in E. coli from retail meat. This study revealed the high genetic diversity of food-borne E. coli in retail meat and emphasized their risk of spreading clinically important antimicrobial resistance genes.

Differential analysis of IBV-infected primary chicken embryonic fibroblasts and immortalized DF-1.

Infectious bronchitis virus (IBV), the causative agent of infectious bronchitis, is responsible for major economic losses in the poultry industry worldwide. While IBVs can usually be passaged in primary chicken embryonic fibroblasts (CEFs), most of the wild ones cannot adapt to passaged cell lines. In this study, the wild strain CK/CH/MY/2020 was used to infect primary CEF and immortalize DF-1 CEF cells. Results indicated that IBV was able to cause lesions and pass onto CEF, but not DF-1. Indeed, the virus could enter DF-1 cells and synthesize the associated structural gene but could not assemble into complete viral particles for release. Furthermore, transcriptome sequencing analysis showed significant differences in gene expression between CEF and DF-1 cells after viral infection, although the corresponding antiviral responses could be activated in both cell types. The biggest difference was in terms of the amino acid biosynthesis pathway and the cytokine receptor interaction pathway, which were significantly and specifically activated in CEF. This could actually explain why intact viruses can be assembled but not in DF-1. In addition, SLBP and P2RX7 affect the replication of IBV's structural genes to some extent. Overall, IBV can enter CEF and DF-1 cells, but the complex intracellular cytokine interactions affect the assembly and release of viral particles. The insight will be useful for the study of IBV through in vitro transmission and pathogenesis. IMPORTANCE: Infectious bronchitis virus (IBV) is responsible for high morbidity and mortality as well as substantial economic losses worldwide. Transcriptome sequencing of IBV-infected chicken embryonic fibroblast and DF-1 cells revealed that the virus elicits antiviral immunity in cells after viral infection, but IBV cannot activate DF-1 cells to produce sufficient amounts of viral structures to assemble into complete virions, which may be caused by the interactions between cytokines. The study of IBV cellular adaptations is important for vaccine development and investigation of the pathogenesis of IBV.

Characterization and risk assessment of novel SXT/R391 integrative and conjugative elements with multidrug resistance in Proteus mirabilis isolated from China, 2018-2020.

Proteus mirabilis can transfer transposons, insertion sequences, and gene cassettes to the chromosomes of other hosts through SXT/R391 integrative and conjugative elements (ICEs), significantly increasing the possibility of antibiotic resistance gene (ARG) evolution and expanding the risk of ARGs transmission among bacteria. A total of 103 strains of P. mirabilis were isolated from 25 farms in China from 2018 to 2020. The positive detection rate of SXT/R391 ICEs was 25.2% (26/103). All SXT/R391 ICEs positive P. mirabilis exhibited a high level of overall drug resistance. Conjugation experiments showed that all 26 SXT/R391 ICEs could efficiently transfer to Escherichia coli EC600 with a frequency of 2.0 × 10-7 to 6.0 × 10-5. The acquired ARGs, genetic structures, homology relationships, and conservation sequences of 26 (19 different subtypes) SXT/R391 ICEs were investigated by high-throughput sequencing, whole-genome typing, and phylogenetic tree construction. ICEPmiChnHBRJC2 carries erm (42), which have never been found within an SXT/R391 ICE in P. mirabilis, and ICEPmiChnSC1111 carries 19 ARGs, including clinically important cfr, blaCTX-M-65, and aac(6')-Ib-cr, making it the ICE with the most ARGs reported to date. Through genetic stability, growth curve, and competition experiments, it was found that the transconjugant of ICEPmiChnSCNNC12 did not have a significant fitness cost on the recipient bacterium EC600 and may have a higher risk of transmission and dissemination. Although the transconjugant of ICEPmiChnSCSZC20 had a relatively obvious fitness cost on EC600, long-term resistance selection pressure may improve bacterial fitness through compensatory adaptation, providing scientific evidence for risk assessment of horizontal transfer and dissemination of SXT/R391 ICEs in P. mirabilis.IMPORTANCEThe spread of antibiotic resistance genes (ARGs) is a major public health concern. The study investigated the prevalence and genetic diversity of integrative and conjugative elements (ICEs) in Proteus mirabilis, which can transfer ARGs to other hosts. The study found that all of the P. mirabilis strains carrying ICEs exhibited a high level of drug resistance and a higher risk of transmission and dissemination of ARGs. The analysis of novel multidrug-resistant ICEs highlighted the potential for the evolution and spread of novel resistance mechanisms. These findings emphasize the importance of monitoring the spread of ICEs carrying ARGs and the urgent need for effective strategies to combat antibiotic resistance. Understanding the genetic diversity and potential for transmission of ARGs among bacteria is crucial for developing targeted interventions to mitigate the threat of antibiotic resistance.

Prevalence and genetic diversity of multidrug-resistant Salmonella Typhimurium monophasic variant in a swine farm from China.

Salmonella 4,[5],12:i:-, a monophasic variant of S. Typhimurium, has become a global serovar causing animal and human infections since its first emergence in the late 1980's. Several previous studies showed the increasing prevalence of S. 4,[5],12:i:- in China, most of which were from swine with multidrug resistance (MDR) profiles. However, the molecular characteristic and evolution of S. 4,[5],12:i:- in the same swine farm are still unknown. In this study, a total of 54 S. enterica strains were isolated from different fattening pigs aged 1, 3, and 6 months, most of which belonged to S. 4,[5],12:i:-. Whole-genome sequencing revealed that all 45 S. 4,[5],12:i:- strains belonged to ST34 and were further divided into two different ribosomal STs and nine different core-genome STs. Phylogenetic analysis of 286 S. 4,[5],12:i:- strains in China, including 241 from the EnteroBase Salmonella database, revealed the genetic diversity of S. 4,[5],12:i:- and indicated that S. 4,[5],12:i:- in this swine farm might have multiple origins. Three different IncHI2 plasmids carrying various resistance genes were characterized by nanopore sequencing and could be conjugated to Escherichia coli. The colistin resistance gene mcr-1 and ESBLs gene blaCTX - M-14 were co-located on the chromosome of one strain. The dynamic changes in antimicrobial resistance regions and transferability of IncHI2 plasmids, as well as the chromosomal location of resistance genes, facilitated the diversity of the antimicrobial resistance characteristics in S. 4,[5],12:i:-. Since the swine farm is regarded as the important reservoir of MDR S. 4,[5],12:i:-, the prevalence and evolution of S. 4,[5],12:i:- from swine farms to pig products and humans should be continually monitored.

Florfenicol and oxazolidone resistance status in livestock farms revealed by short- and long-read metagenomic sequencing.

Antibiotic resistance genes (ARGs) as a novel type of environmental pollutant pose a health risk to humans. Oxazolidinones are one of the most important antibiotics for the treatment of Gram-positive bacterial infections in humans. Although oxazolidinones are not utilized in the livestock industry, florfenicol is commonly used on farms to treat bacterial infections, which may contribute to the spread of the cfr, optrA, and poxtA genes on farms. Using metagenomics sequencing, we looked into the antibiotic resistome context of florfenicol and oxazolidinone in 10 large-scale commercial farms in China. We identified 490 different resistance genes and 1,515 bacterial genera in the fecal samples obtained from 10 farms. Florfenicol-resistant Kurthia, Escherichia, and Proteus were widely present in these samples. The situation of florfenicol and oxazolidone resistance in pig farms is even more severe. The total number of genes and the abundance of drug resistance genes were higher in pigs than in chickens, including optrA and poxtA. All the samples we collected had a high abundance of fexA and floR. Through nanopore metagenomic analysis of the genetic environment, we found that plasmids, integrative and conjugative element (ICE), and transposons (Tn7-like and Tn558) may play an important role in the spread of floR, cfr, and optrA. Our findings suggest that florfenicol and oxazolidinone resistance genes have diverse genetic environments and are at risk of co-transmission with, for example, tetracycline and aminoglycoside resistance genes. The spread of florfenicol- and oxazolidinone-resistant bacteria on animal farms should be continuously monitored.

Whole genome sequence of Proteus mirabilis ChSC1905 strain harbouring a new SXT/R391-family ICE.

OBJECTIVES: The aim of this study was to characterise the whole genome sequence of a multidrug-resistant (MDR) Proteus mirabilis strain ChSC1905 isolated from a swine farm in China. METHODS: The genome was sequenced by Illumina NovaSeq and Oxford Nanopore platforms, and it was assembled via Canu v.1.5. The acquired antimicrobial resistance genes (ARGs) were identified by ResFinder. A conjugation experiment was carried out to determine the mobilisation of integrative and conjugative element. RESULTS: Strain ChSC1905 exhibited a MDR phenotype. The genome of strain ChSC1905 was 4 038 038 bp in length with a GC content of 39.1%, which contained 3645 coding sequences and 110 RNA genes. A total of 23 acquired ARGs were identified, among which 21 ARGs including the clinically important resistance genes blaCTX-M-65, cfr, fosA3, and aac(6')-Ib-cr were located on a SXT/R391 integrative and conjugative element (ICE). BLAST analysis showed that this new SXT/R391-family ICE (ICEPmiChnChSC1905 of 143 689 bp) was involved in sequence inversion mediated by ISVsa3 and genetic rearrangement mediated by IS26, and it could be transferred to E. coli EC600. CONCLUSION: In this study, we report the genome sequence of MDR P. mirabilis strain ChSC1905 that harboured a novel SXT/R391-family ICE (ICEPmiChnChSC1905) involved in genetic rearrangement in China, which promotes the diversity of ICE and should receive more attention.

Whole genome sequence of Salmonella Rissen SCSW714, a porcine strain harbouring a novel multidrug-resistant Tn7-like pco- and sil-containing transposon.

OBJECTIVES: The aim of this study was to characterise the whole genome sequence of multidrug resistance (MDR) Salmonella Rissen strain SCSW714 of swine origin. METHODS: The whole genome of SCSW714 was sequenced using the Illumina Hiseq platform combined with the Nanopore PromethION platform and assembled by software Unicycler. NCBI Prokaryotic Genome Annotation Pipeline was used to annotate the genome of SCSW714. The sequence type (ST) as well as antimicrobial resistance genes were determined by MLST 2.0 and ResFinder 4.1, respectively. RESULTS: The chromosome of SCSW714 was 4 928 262 bp in size with a GC content of 52.1%. Strain SCSW714 contained a total of 4759 genes, including 4531 protein-coding sequences, 108 pseudogenes and 120 RNAs. It belonged to ST469 and carried six resistance genes including tet(A), dfrA12, sul3, aadA2, aadA1 and blaTEM-1b. All of the six resistance genes were carried by a novel MDR Tn7-pco-sil transposon designated as Tn6777. Tn6777 was stable in S. Rissen and could be excised from S. Rissen chromosome. CONCLUSION: We report a complete genome sequence of S. Rissen and characterised a novel MDR Tn7-like pco- and sil-containing transposon for the first time. The excision of Tn6777 suggests that Tn6777 has functional activity and may promote the co-spreading of metal and antimicrobial resistance genes. The complete genome sequence of S. Rissen strain SCSW714 provides valuable information for tracing the potential spread from swine to humans.

Characterisation of novel Tn7-derivatives and Tn7-like transposon found in Proteus mirabilis of food-producing animal origin in China.

OBJECTIVES: This study aimed to clarify the characteristics of Tn7-derivatives transposons in MDR Proteus mirabilis strains isolated from anal swabs of chicken and swine in China from 2015-2020. METHODS: The Tn7 tnsA gene was screened in 207 P. mirabilis isolates by polymerase chain reaction (PCR). These strains were subjected to antimicrobial susceptibility testing. Illumina Hiseq (200 × coverage) was used for genome sequencing. Transposon maps were completed by PCR and Sanger sequencing and analysed by BLAST. RESULTS: The Tn7 tnsA gene was detected in 21 strains by PCR. Eight novel Tn7-derivatives, named Tn6667, Tn6668, Tn6669, Tn6670, Tn7095, Tn7096, Tn7097 and Tn7098, were characterised. Three types of hybrid class 2/1 integrons were found at the right end of Tn7 derivatives. A novel Tn7-like transposon Tn6666 with an active integrase gene intI2, whose transposition module shows 93% nucleotide identity to the corresponding region of Tn7, was characterised in three strains. Tn6666 is also found next to Tn7097 or Tn7098 in the chromosomes of two clonally related P. mirabilis strains. The number of resistance genes carried by the novel transposons varied from 1 to 18. A novel variant of class A extended-spectrum beta-lactamase gene, blaPER-16, with eight base substitutions compared with blaPER-12, was harboured by Tn7098. CONCLUSION: Our study characterised diverse novel Tn7-derivatives and a new Tn7-like transposon in P. mirabilis. An active integrase gene intI2 might promote the diversification of Tn7-like transposons. More attention should be paid to the prevalence and evolution of Tn7-derivatives and Tn7-like transposons and antimicrobial resistance genes they carry.

Contamination of Proteus mirabilis harbouring various clinically important antimicrobial resistance genes in retail meat and aquatic products from food markets in China.

Proteus mirabilis is an opportunistic pathogen frequently associated with nosocomial infection and food poisoning cases. Contamination of P. mirabilis in retail meat products may be important transmission routes for human infection with P. mirabilis. In this study a total of 89 P. mirabilis strains were isolated from 347 samples in 14 food markets in China and subjected to whole-genome sequencing. Phylogenetic analysis showed that all 89 strains were divided into 81 different clones (SNPs >5), indicating high genetic diversity of P. mirabilis in food markets. Antimicrobial susceptibility testing showed that 81 (91.01%) strains displayed multidrug resistance profiles. Seventy-three different resistance genes (or variants) were found, including various clinically important antimicrobial resistance genes aac(6')-Ib-cr (77.53%), bla CTX-M (39.33%), fosA3 (30.34%), as well as multiresistance gene cfr (4.50%), tigecycline resistance gene cluster tmexCD3-toprJ1 (4.50%) and carbapenemase gene bla NDM-1 (1.12%). Diverse genetic elements including Tn7 transposon, plasmid, SXT/R391 integrative conjugative element were associated with the horizontal transfer of cfr. tmexCD3-toprJ1 and bla NDM-1 were located on ICEPmiChnJZ26 and Salmonella genomic island 1, respectively. Our study emphasized high contamination of P. mirabilis harbouring various clinically important antimicrobial resistance genes in retail meat and aquatic products, which might be an important issue in terms of food safety and human health.

Clonal spread and horizontal transfer mediate dissemination of phenicol-oxazolidinone-tetracycline resistance gene poxtA in enterococci isolates from a swine farm in China.

The emergence of the phenicol-oxazolidinone-tetracycline resistance gene poxtA becomes a significant challenge for public health, since it confers a decreased susceptibility not only to the last resort drug linezolid, but also to florfenicol and doxycycline widely used in veterinary medicine. To determine the dissemination mechanism of poxtA in enterococci isolates from different healthy pigs in the swine farm, a total of 178 florfenicol-resistant enterococci isolates were collected from 400 fresh faecal swabs in a swine farm in China. The poxtA gene was detected in 11 (6.18 %) enterococci isolates, including 8 E. faecium, 2 E. hirae and 1 E. casseliflavus isolates. Whole genome sequencing indicated that the eight poxtA-harbouring E. faecium strains belonged to four different sequence types, including ST156 and three new STs, ST1818, ST1819 and ST1820. Five out of the 11 poxtA-positive enterococci isolates also harboured optrA gene. Moreover, E. casseliflavus strain DY31 co-harboured poxtA, optrA and cfr. Seven different poxtA-harbouring plasmids were obtained through Nanopore combined with Illumina sequencing. The poxtA-harbouring plasmids exhibited high genetic variation, six out of which belonged to rep2 plasmid of Inc18 family. The poxtA gene was flanked by IS1216E in the left and/or right ends.The optrA and cfr genes were located on different plasmids, respectively, but those genes could be co-transferred with poxtA gene into the recipient E. faecalis strain by electrotransformation. Our study highlights that both clonal spread and horizontal transfer mediated by Inc18 plasmid and IS1216E promote the dissemination of poxtA in enterococci isolates from different healthy pigs in the swine farm.

Tracking Salmonella enterica by whole genome sequencing of isolates recovered from broiler chickens in a poultry production system.

Salmonella enterica is a major cause of foodborne diseases, and is also an important pathogenic bacterium in poultry industry. Whole genome sequencing (WGS) has become a crucial molecular typing technology used for the surveillance of the pathogenic bacteria. In the present study, we adopted WGS for tracking transmission of S. enterica in the production chain of broiler chickens. A total of 74 S. enterica strains were isolated from the different steps of breeding and slaughtering in a large production enterprise in Sichuan Province, China. The isolation rate of Salmonella was the highest in procedure of defeathering (50.0%) and evisceration (36.7%). Serotype identification showed that 74 Salmonella isolates included 7 serotypes, among which Mbandaka accounted for the highest proportions (35.1%). WGS revealed that 74 strains belonged to 7 different sequence types (STs), as well as 7 different ribosomal STs and 35 core genome STs. cgMLST-based Minimum Spanning Trees and phylogenetic tree based on the SNPs indicated that three serotypes, Mbandaka, Indiana and Kentucky, could be clonally transmitted between broiler farm and slaughterhouse. Heterogeneous resistant phenotypes and genotypes were found in two serotypes, Indiana and Kentucky. Our study indicated WGS in an accurate tool for molecular typing of S. enterica. Routine surveillance of S. enterica in the production chain of broiler chickens is needed.

Whole genome sequence of a New Delhi metallo-ß-lactamase 1-producing Proteus mirabilis isolate SNYG35 from broiler chicken in China.

OBJECTIVES: The global spread of the New Delhi metallo-ß-lactamase 1 (NDM-1) gene poses a significant challenge to worldwide public health. Here, we characterize the whole genome of NDM-1-producing Proteus mirabilis isolate SNYG35 of broiler chicken origin in China. METHODS: The genome of SNYG35 was sequenced using a PacBio RS II sequencing instrument and Illumina HiSeq platform. SMRT cell data were assembled independently using HGAP4 and Canu v1.6, and were further polished with Illumina data using Pilon v1.22. The presence of antimicrobial resistance genes was identified using CGE ResFinder 3.2. A conjugation experiment was performed using the sodium azide-resistant Escherichia coli J53AziR strain as the recipient. RESULTS: The chromosome of SNYG35 is 4 014 504 bp in size and consists of one chromosome and one plasmid named pSNYG35. It contains 3646 coding sequences, 82 tRNA genes, 22 rRNAs, and four non-coding RNAs. Besides blaNDM-1, SNYG35 harbours 26 different antimicrobial resistance genes including ESBL gene blaCTX-M-65 as well as fluoroquinolone and aminoglycoside resistance gene aac(6')-Ib-cr. The blaNDM-1-harbouring pSNYG35 is a pPrY2001-like plasmid and shares highest nucleotide identity to pHFK418-NDM. It carries a Tn1696-like multidrug-resistant region harbouring 12 different antimicrobial resistance genes, and could be transferred to E. coli J53. CONCLUSIONS: Here, we report for the first time the whole genome sequence of a NDM-1-producing P. mirabilis isolate from broiler chicken in China, which provides valuable information for tracing the potential transmission of NDM-1-producing P. mirabilis from broiler chicken to humans, as well as revealing the spread and evolution of blaNDM-1-harbouring pPrY2001-like plasmids.

Characterization of IncC Plasmids in Enterobacterales of Food-Producing Animals Originating From China.

Incompatibility group C (IncC) plasmids have received attention due to their broad host range and because they harbor key antibiotic resistance genes. Because these resistance genes can spread from food-producing animals to human, the proliferation of these plasmids represents a public health risk. In this study, a total of 20 IncC plasmids were collected from food-producing animals in China, and characterized by Oxford Nanopore Technologies long-read sequencing. Based on four key differences of the IncC backbone, 4 IncC plasmids were classified as type 1, 15 were classified as type 1/2 hybrid, and one was classified as type 2. The 15 type 1/2 hybrids were further divided into 13 type 1/2a and 2 type 1/2b, based on sequence differences arising from different homologous recombination events between type 1 and type 2 IncC backbones. Genome comparison of accessory resistance modules showed that different IncC plasmids exhibited various phenotypes via loss and gain of diverse modules, mainly within the bla CMY -carrying region, and two antibiotic resistance islands designated ARI-A and ARI-B. Interestingly, in addition to insertion and deletion events, IS26 or IS1294-mediated large sequence inversions were found in the IncC genome of the 4 type1/2a plasmids, suggesting that insertion sequence-mediated rearrangements also promote the diversity of the IncC genome. This study provides insight into the structural diversification and multidrug resistance of IncC plasmids identified from food-producing animals in China.

Whole genome sequence of Enterococcus gallinarum EG81, a porcine strain harbouring the oxazolidinone-phenicol resistance gene optrA with chromosomal and plasmid location.

OBJECTIVE: The aim of this study was to characterise the whole genome sequence of linezolid-intermediate Enterococcus gallinarum strain EG81 of swine origin in China. METHODS: Whole genome of EG81 was sequenced using Illumina MiSeq platform combined with the Nanopore PromethION platform, and assembled de novo using Canu v1.5. NCBI Prokaryotic Genome Annotation Pipeline (PGAP) was used to annotate the genome of EG81. Antimicrobial resistance genes were identified using CGE ResFinder 3.2. RESULTS: The genome of EG81 consists of one 3,433,237-bp chromosome and two plasmids, pEG81-1 (51,632 bp) and pEG81-2 (3425 bp). A total of 3285 coding sequences and 80 RNA genes were predicted by PGAP. The oxazolidinone-phenicol resistance gene optrA is located on both the chromosome and plasmid pEG81-1 associated with Tn554 and Tn558, respectively. In addition, EG81 harbours vanC1XY (vancomycin resistance), fexA (phenicol), dfrG (trimethoprim), aadD, ant(6)-Ia and ant(9)-Ia (aminoglycoside), erm(A) and erm(B) (macrolide), and tet(L) and tet(M) (tetracycline). CONCLUSION: Here, we first report the oxazolidinone-phenicol gene optrA in E. gallinarum that is intrinsically resistant to vancomycin, which poses a great threat to public health. The genome sequence of E. gallinarum EG81 provides valuable information for the dissemination of optrA among vancomycin-resistant enterococci.

Prevalence and transmission of antimicrobial-resistant Staphylococci and Enterococci from shared bicycles in Chengdu, China.

Shared bicycles are prevailing in China but the extent to which they contribute to maintaining and transmitting pathogens and antibiotic-resistant bacteria remain largely unknown. To fill the knowledge gap, herein, swab samples (n = 963) were collected from handlebars of shared bicycles in areas of hospital, school, metro station (n = 887) and riders (n = 76) in Chengdu, China. Staphylococci (n = 241) and Enterococci (n = 69) were widely distributed across sampling locations at a frequency of 2.3%-12.9%, and 0.08%-5.5%, respectively. Bicycle or rider-borne Gram-positive bacteria were frequently resistant to clinically important antibiotics including linezolid, fosfomycin, and vancomycin, and a significant portion of these isolates (3.4%-16.6% for Staphylococci and 0.1%-13.8% for Enterococci) indicated multidrug resistance. Nineteen Staphylococcus aureus isolates were identified in this collection and 52.6% of which were considered as methicillin-resistant S. aureus. Whole genome sequencing further characterized 26 antimicrobial resistance genes (ARGs) including fosB, fusB, and lnu(G) in S. aureus and 21 ARGs including optrA in Enterococci. Leveraging a complementary approach with conventional MLST, whole genome SNP and MLST analyses, we present that genetically closely-related bacteria were found in bicycles and riders across geographical-distinct locations suggesting bacterial transmission. Further, five new ST types 5697-5701 were firstly characterized in S. aureus. ST 942 and ST 1640 are new ST types observed in E. faecalis, and E. faecium, respectively. Our results highlighted the risk of shared bicycle system in disseminating pathogens and antibiotic resistance which warrants effective disinfections.

Identification of Proteus genomic island 2 variants in two clonal Proteus mirabilis isolates with coexistence of a novel genomic resistance island PmGRI1.

OBJECTIVES: To characterize the MDR genomic islands (GIs) in Proteus mirabilis isolates. METHODS: Two P. mirabilis strains (C55 and C74) of chicken origin were subjected to WGS (HiSeq and PacBio) and the MDR GIs were determined. RESULTS: P. mirabilis strains C55 and C74 are clonal strains and harbour different Proteus genomic island 2 (PGI2) variants (PGI2-C55 and PGI2-C74). The MDR region of PGI2-C55 is composed of two class 1 integrons, separated by a region containing seven copies of IS26 and eight resistance genes, including blaCTX-M-3 and fosA3. The region in PGI2-C74 is a complete In4-type class 1 integron, harbouring five gene cassettes (dfrA16, blaCARB-2, aadA2, cmlA1 and aadA1). In addition, C55 and C74 carry an SXT/R391 integrative and conjugative element (ICEPmiJpn1), harbouring blaCMY-2, and a novel 50.46 kb genomic resistance island named PmGRI1-C55. PmGRI1-C55 harbours a tyrosine-type recombinase/integrase that might be responsible for the integration of PmGRI1-C55 at the 3' end of tRNA-Sec. It carries an MDR region derived from Tn2670 that harbours a Tn21 region and carries six resistance genes (catA1, blaTEM-1b, aphA1a, sul2, strA and strB). Blast analysis showed diverse PmGRI1 variants in P. mirabilis and Escherichia coli strains. CONCLUSIONS: The finding of the two new PGI2 variants highlights that the homologous recombination between shared components of class 1 integrons and transposition by IS26 promote the diversity of MDR regions in PGI2. PmGRI1 is a new GI that carries various resistance genes identified in P. mirabilis and E. coli.

Integration of the blaNDM-1 carbapenemase gene into a novel SXT/R391 integrative and conjugative element in Proteus vulgaris.

OBJECTIVES: To characterize the genetic environment of the carbapenem resistance determinant in Proteus vulgaris of swine origin. METHODS: The carbapenem-resistant P. vulgaris strain BC22 was isolated from a faecal swab from a diseased pig with diarrhoea in Sichuan Province of China in 2018. The presence of carbapenemase genes was screened by PCR. WGS and bioinformatics analysis were performed to analyse the genetic environment of the carbapenem resistance determinant. RESULTS: P. vulgaris strain BC22 was found to harbour the carbapenemase gene blaNDM-1. WGS data revealed that blaNDM-1 was located in a truncated ISAba125 composite transposon. The carbapenem resistance gene blaNDM-1 and 20 other resistance genes, including the multiresistance gene cfr and the bifunctional aminoglycoside/quinolone resistance gene aac(6')-lb-cr, were located in a novel SXT/R391 integrative and conjugative element (ICE). This new SXT/R391 ICE of 148.7 kb was chromosomally located, and could be transferred to Escherichia coli. CONCLUSIONS: Here, we report a carbapenemase gene, blaNDM-1, integrated into an SXT/R391 ICE. Our study highlights that this SXT/R391 ICE may facilitate the dissemination of clinically important resistance genes such as blaNDM-1, cfr and aac(6')-lb-cr.