CRISPR-AMRtracker: A novel toolkit to monitor the antimicrobial resistance gene transfer in fecal microbiota.

The spread of antibiotic resistance genes (ARGs), particularly those carried on plasmids, poses a major risk to global health. However, the extent and frequency of ARGs transfer in microbial communities among human, animal, and environmental sectors is not well understood due to a lack of effective tracking tools. We have developed a novel fluorescent tracing tool, CRISPR-AMRtracker, to study ARG transfer. It combines CRISPR/Cas9 fluorescence tagging, fluorescence-activated cell sorting, 16S rRNA gene sequencing, and microbial community analysis. CRISPR-AMRtracker integrates a fluorescent tag immediately downstream of ARGs, enabling the tracking of ARG transfer without compromising the host cell's antibiotic susceptibility, fitness, conjugation, and transposition. Notably, our experiments demonstrate that sfGFP-tagged plasmid-borne mcr-1 can transfer across diverse bacterial species within fecal samples. This innovative approach holds the potential to illuminate the dynamics of ARG dissemination and provide valuable insights to shape effective strategies in mitigating the escalating threat of antibiotic resistance.

Genomics analysis of KPC-2 and NDM-5-producing Enterobacteriaceae in migratory birds from Qinghai Lake, China.

The study examined the epidemiological characteristics of carbapenem-resistant Enterobacteriaceae (CRE) isolated from migratory birds and surroundings in Qinghai Lake, China. We identified 69 (15.7%) CRE isolates from a total of 439 samples including 29 (6.6%) blaNDM-5 Escherichia coli and 40 (9.1%) blaKPC-2 Klebsiella pneumoniae. WGS analysis indicated that ST746, ST48, ST1011, and ST167 were the primary sequence types (ST) for blaNDM-5 E. coli, while all blaKPC-2 K. pneumoniae were ST11 and harbored numerous antibiotic resistance gene types including blaCTX-M, qnrS, and rmtB. A phylogenetic tree based on core genomes revealed that blaNDM-5 E. coli was highly heterogeneous while the blaKPC-2 K. pneumoniae was highly genetically similar within the group and to human Chinese isolates. IncX3, IncHI2, and IncFIB-HI2 plasmid replicon types were associated with blaNDM-5 spread, while IncFII-R and IncFII plasmids mediated blaKPC-2 spread. We also identified IncFII-R hybrid plasmids most likely formed by IS26-mediated integration of IncFII into IncR plasmid backbones. This also facilitated the persistence of IncFII-R plasmids and antibiotic resistance genes including blaKPC-2. In addition, all of the blaKPC-2 K. pneumoniae isolates harbored a pLVKP-like virulence plasmid carrying a combination of two or more hypervirulence markers that included peg-344, iroB, iucA, rmpA, and rmpA2. This is the first description of ST11 K. pneumoniae that co-carried blaKPC-2- and pLVKP-like virulence plasmids from migratory birds. The blaKPC-2 K. pneumoniae carried by migratory birds displayed high genetic relatedness to human isolates highlighting a high risk of transmission of these K. pneumoniae. KEY POINTS: • Multidrug resistance plasmids (blaKPC-2, bla436NDM-5, bla CTX-M, qnrS, and rmtB). • Co-occurrence of plasmid-mediated resistance and virulence genes. • High similarity between migratory bird genomes and humans.

Comparison of the prevalence and molecular characteristics of fosA3 and fosA7 among Salmonella isolates from food animals in China.

OBJECTIVES: To investigate the prevalence and molecular characteristics of fosA3 and fosA7 among Salmonella isolates. METHODS: Five hundred and fifty-one Salmonella isolates collected from food animals in China during 2016-19 were screened for fos genes. The drug resistance, serovars, clonal relationships and genetic environments of fosA were compared between fosA7- and fosA3-positive Salmonella. RESULTS: A relatively high prevalence of fosA7 (9.26%) and fosA3 (6.53%) was identified. fosA3 was associated with high-level fosfomycin resistance (≥512 mg/L), while fosA7 conferred relatively low-level resistance that was independent of the presence of glucose-6-phosphate. Additionally, fosA7 could facilitate Salmonella survival under oxidative stress. Both fosA3 and fosA7 were found in diverse serovars and STs, but segregated into distinct groups. The fosA3-positive Salmonella Typhimurium/Salmonella Indiana strains showed close genetic relationships, while fosA7-positive Salmonella Meleagridis/Salmonella Agona/Salmonella Derby showed a relatively high degree of whole-genome sequence heterogeneity. fosA3 was located on conjugative IncHI2 plasmids or chromosomes, while fosA7 was strictly chromosomal. Furthermore, two strains carried large chromosomal fosA7 regions within genomic islands. The fosA3 and fosA7 contigs from our isolates and the NCBI could be segregated into four primary and distinct genomic backbones. IS26 and the antibiotic resistance genes (ARGs) blaCTX-M, blaTEM-1B and rmtB were frequently adjacent to fosA3, while fosA7-carrying contigs generally lacked mobile elements and ARGs. CONCLUSIONS: fosA3 and fosA7 were the primary factors contributing to reduced fosfomycin susceptibility, to different degrees, in these Salmonella isolates. The distinct distributions and molecular characteristics of fosA7 and fosA3 indicated that their origin and evolution in Salmonella were most likely distinct.

Transmission and molecular characteristics of blaNDM-producing Escherichia coli between companion animals and their healthcare providers in Guangzhou, China.

OBJECTIVES: To determine the transmission and molecular characteristics of blaNDM-producing Escherichia coli between companion animals and their healthcare providers at veterinary clinics in Guangzhou, China. METHODS: A total of 359 samples from companion animals and their healthcare providers were collected at 14 veterinary clinics in Guangzhou, China. Genomic characteristics and clonal relationships for blaNDM-positive E. coli and complete plasmid sequences were characterized based on WGS data from combined Illumina and MinION platform reads. RESULTS: Forty-five blaNDM-positive bacteria were recovered from companion animals (n = 43) and their healthcare providers (n = 2) at 10 veterinary clinics. Overall, E. coli (73.3%, 33/45) and Klebsiella pneumoniae (13.3%, 6/45) were the most prevalent species among the seven species of blaNDM-positive bacteria. Four blaNDM variants (blaNDM-1, blaNDM-4, blaNDM-5 and blaNDM-7) were identified in 45 blaNDM-positive bacteria and blaNDM-5 was the most prevalent (77.8%, 35/45). WGS indicated that the most prevalent STs were ST405 (8/33), ST453 (6/33), ST457 (6/33) and ST410 (5/33) among the 33 blaNDM-positive E. coli isolates. Phylogenomics and PFGE analysis revealed that clonal spread of blaNDM-positive ST453 E. coli isolates between companion animals and their healthcare providers was evident. In addition, two novel IncFIB plasmids carrying blaNDM-4 (pF765_FIB and pG908_FIB) were found in this study and indicated that IS26 may promote the horizontal transmission of blaNDM between different plasmid types. CONCLUSIONS: In this study we conducted a large-scale investigation on the prevalence of blaNDM-positive E. coli isolates from companion animals and their healthcare providers and revealed the clonal spread of blaNDM-positive E. coli isolates between these two groups.

Phylogenomic analysis of Salmonella Indiana ST17, an emerging MDR clonal group in China.

OBJECTIVES: To reconstruct the genomic epidemiology and evolution of MDR Salmonella Indiana in China. METHODS: A total of 108 Salmonella Indiana strains were collected from humans and livestock in China. All isolates were subjected to WGS and antimicrobial susceptibility testing. Phylogenetic relationships and evolutionary analyses were conducted using WGS data from this study and the NCBI database. RESULTS: Almost all 108 Salmonella Indiana strains displayed the MDR phenotype. Importantly, 84 isolates possessed concurrent resistance to ciprofloxacin and cefotaxime. WGS analysis revealed that class 1 integrons on the chromosome and IncHI2 plasmids were the key vectors responsible for multiple antibiotic resistance gene (ARG) [including ESBL and plasmid-mediated quinolone resistance (PMQR) genes] transmission among Salmonella Indiana. The 108 Salmonella Indiana dataset displayed a relatively large core genome and ST17 was the predominant ST. Moreover, the global ST17 Salmonella Indiana strains could be divided into five distinct lineages, each of which was significantly associated with a geographical distribution. Genomic analysis revealed multiple antimicrobial resistance determinants and QRDR mutations in Chinese lineages, which almost did not occur in other global lineages. Using molecular clock analysis, we hypothesized that ST17 isolates have existed since 1956 and underwent a major population expansion from the 1980s to the 2000s and the genetic diversity started to decrease around 2011, probably due to geographical barriers, antimicrobial selective pressure and MDR, favouring the establishment of this prevalent multiple antibiotic-resistant lineage and local epidemics. CONCLUSIONS: This study revealed that adaptation to antimicrobial pressure was possibly pivotal in the recent evolutionary trajectory for the clonal spread of ST17 Salmonella Indiana in China.

Molecular Characteristics of Antimicrobial Resistance and Virulence in Klebsiella pneumoniae Strains Isolated from Goose Farms in Hainan, China.

We retrospectively investigated 326 samples that were collected from goose farms in Hainan Province, China, in 2017. A total of 33 carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates were identified from 326 samples, and the 33 CRKP isolates were characterized based on whole-genome sequencing (WGS) data from the Illumina and Oxford Nanopore Technologies (ONT) platforms. All of these 33 CRKP isolates possessed blaNDM-5, and a single isolate coharbored mcr-1 and blaNDM-5, while 4 isolates carried multiple virulence and metal tolerance gene clusters. One CRKP strain (CMG-35-2) was selected for long sequence reading. A hybrid plasmid carrying the virulence, resistance, and metal resistance gene in the strain was found. It possessed 2 backbones [IncFIB(K)-IncFII(K)] within a single plasmid that were closely related to K. pneumoniae plasmids from a human-associated habitat in the United States and from a human isolate in Hong Kong. A mouse abdominal infection model indicated that that strain was of the moderate virulence phenotype. This study revealed that K. pneumoniae on goose farms is an important reservoir for blaNDM-5 and these bacteria are represented by a diversity of sequence types. The heterozygous multiple drug resistance genes carried on plasmids highlighted the genetic complexity of CRKP and the urgent need for continued active surveillance. IMPORTANCE CRKP is one of the most important pathogens, which can cause infection not only in humans but also in waterfowl. The discovery of blaNDM-5-producing K. pneumoniae in waterfowl farms in recent years suggests that waterfowl are an important reservoir for blaNDM-5-producing Enterobacteriaceae. However, there are few studies on the spread of blaNDM-5-producing bacteria in waterfowl farms. Our study showed that the IncX3 plasmid carrying blaNDM-5 in goose farms is widely present in K. pneumoniae isolates and a large number of resistance genes are accumulated in it. We found a transferable IncFIB-FII hybrid plasmid that combines virulence, resistance, and metal resistance genes, which allow transfer of these traits between bacteria in different regions. The results of this study contribute to a better understanding of the prevalence and transmission of carbapenem-resistant K. pneumoniae in goose farms.

Molecular epidemiology of carbapenemase-producing Escherichia coli from duck farms in south-east coastal China.

OBJECTIVES: To determine the dissemination and molecular characteristics of NDM-producing Escherichia coli strains from duck farms in south-east coastal China and their threats to human health. METHODS: A total of 232 NDM-producing E. coli were recovered from 1505 samples collected from 25 duck farms and their surrounding environments in five provinces in China. Resistance genes were confirmed using PCR. Genomic characteristics of the carbapenemase-producing isolates were determined by WGS and bioinformatic analysis. RESULTS: The rate of NDM-positive E. coli detected in samples from the five provinces ranged from 3.7% to 28.5%. There was substantial variation in the prevalence of NDM-positive E. coli from different duck farms in each province studied. Three variants (blaNDM-1, blaNDM-4 and blaNDM-5) were found in 232 NDM-positive E. coli; blaNDM-5 (94.8%, 220/232) was the most prevalent. WGS analysis indicated that ST746, ST48, ST1011 and ST167 E. coli isolates were prevalent in the current study and poultry was likely the primary reservoir for NDM-positive ST746 and ST48 E. coli in China. Phylogenomic analysis showed that NDM-positive E. coli isolates from ducks were closely related to those of human origin. In addition, WGS analysis further revealed that blaNDM co-existed with other antibiotic resistance genes, conferring resistance to nine classes of antimicrobials. CONCLUSIONS: This study revealed that ducks farm in China are an important reservoir for NDM-positive E. coli and STs of the isolates showed obvious distinctive diversities in geographical distribution. The distribution and spread of NDM-positive E. coli in duck farms poses a threat to public health.

Occurrence and Transmission of blaNDM-Carrying Enterobacteriaceae from Geese and the Surrounding Environment on a Commercial Goose Farm.

We investigated the prevalence and transmission of NDM-producing Enterobacteriaceae in fecal samples of geese and environmental samples from a goose farm in southern China. The samples were cultivated on MacConkey agar plates supplemented with meropenem. Individual colonies were examined for blaNDM, and blaNDM-positive bacteria were characterized based on whole-genome sequencing (WGS) data from the Illumina and Oxford Nanopore Technologies (ONT) platforms. Of 117 samples analyzed, the carriage rates for New Delhi metallo-ß-lactamase (NDM)-positive Enterobacteriaceae were 47.1, 18, and 50% in geese, inanimate environments (sewage, soil, fodder, and dust), and mouse samples, respectively. Two variants (blaNDM-1 and blaNDM-5, in 4 and 40 isolates, respectively) were found among 44 blaNDM-positive Enterobacteriaceae; these variants belonged to eight species, and Escherichia coli was the most prevalent (50%). WGS analysis revealed that blaNDM coexisted with diverse antibiotic resistance genes (ARGs). Population structure analysis showed that most E. coli and Enterobacter sp. isolates were highly heterogeneous, while most Citrobacter sp. and P. stuartii isolates possessed extremely high genetic similarities. In addition, blaNDM-5-positive ST4358/ST48 E. coli isolates were found to be clonally spread between geese and the environment and were highly genetically similar to those reported from ducks, farm environments, and humans in China. Plasmid analysis indicated that IncX3 pHNYX644-1-like (n = 40) and untypeable pM2-1-like plasmids (n = 4) mediated blaNDM spread. pM2-1-like plasmids possessed diverse ARGs, including blaNDM-1, the arsenical and mercury resistance operons, and the maltose operon. Our findings revealed that the goose farm is a reservoir for NDM-positive Enterobacteriaceae The blaNDM contamination of wild mice and the novel pM2-1-like plasmid described here likely adds to the risk for dissemination of blaNDM and associated resistance genes.IMPORTANCE Carbapenem-resistant bacteria, in particular NDM-producing Enterobacteriaceae, have become a great threat to global public. These bacteria have been found not only in hospital and community environments but also among food animal production chains, which are recognized as reservoirs for NDM-producing Enterobacteriaceae However, the dissemination of NDM-producing bacteria in waterfowl farms has been less well explored. Our study demonstrates that the horizontal spread of blaNDM-carrying plasmids and the partial clonal spread of blaNDM-positive Enterobacteriaceae contribute to the widespread contamination of blaNDM in the goose farm ecosystem, including mice. Furthermore, we found a novel and transferable blaNDM-1-carrying multidrug resistance (MDR) plasmid that possessed multiple environmental adaptation-related genes. The outcomes of this study contribute to a better understanding of the prevalence and transmission of blaNDM-carrying Enterobacteriaceae among diverse niches in the farm ecosystem.

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.

Diverse and Flexible Transmission of fosA3 Associated with Heterogeneous Multidrug Resistance Regions in Salmonella enterica Serovar Typhimurium and Indiana Isolates.

We identified fosA3 at a rate of 2.6% in 310 Salmonella isolates from food animals in Guangdong province, China. The fosA3 gene was genetically linked to diverse antibiotic resistance genes (ARGs), including mcr-1, blaCTX-M-14/55, oqxAB, and rmtB These gene combinations were embedded in heterogeneous fosA3-containing multidrug resistance regions on the transferable ST3-IncHI2 and F33:A-:B- plasmids and the chromosome. This indicated a great flexibility of fosA3 cotransmission with multiple important ARGs among Salmonella species.

Global clonal spread of mcr-3-carrying MDR ST34 Salmonella enterica serotype Typhimurium and monophasic 1,4,[5],12:i:- variants from clinical isolates.

OBJECTIVES: To investigate the prevalence and transmission of mcr-3 among Salmonella enterica serotype Typhimurium and 1,4,[5],12:i:-. METHODS: A total of 4724 clinical Salmonella isolates were screened for the presence of mcr-3 in China during 2014-19. The clonal relationship of the mcr-3-positive isolates and their plasmid contents and complete sequence were also characterized based on WGS data from the Illumina and MinION platforms. RESULTS: We identified 10 mcr-3-positive isolates, and all were MDR, mostly resistant to colistin, cefotaxime, ciprofloxacin, doxycycline and florfenicol. mcr-3 was co-present with blaCTX-M-55-qnrS1 on hybrid ST3-IncC-FII conjugatable plasmids (n = 6) and an ST3-IncC non-conjugatable plasmid (n = 1) and embedded into a pCHL5009T-like IncFII plasmid on the Salmonella chromosome (n = 3). Four distinctive genetic contexts surrounded mcr-3 and all but one were closely related to each other and to the corresponding region of IncFII plasmid pCHL5009T. IS15DI was most likely the vehicle for integration of mcr-3-carrying IncFII plasmids into ST3-IncC plasmids and the chromosome and for shaping the MDR regions. In addition, a phylogenetic tree based on the core genome revealed a unique Salmonella lineage (≤665 SNPs) that contained these 10 mcr-3-positive isolates and another 38 (33 from patients) mcr-3-positive Salmonella from five countries. In particular, most of the 51 mcr-3-positive isolates belonged to ST34 and harboured diverse antibiotic resistance genes (ARGs), including mcr-3-blaCTX-M-55-qnrS1, and possessed similar ARG profiles. CONCLUSIONS: Our findings revealed global clonal spread of MDR ST34 Salmonella from clinical isolates co-harbouring mcr-3 with blaCTX-M-55 and qnrS1 and a flexibility of mcr-3 co-transmittance with other ARGs mediated by mobile genetic elements.

Complete Nucleotide Sequence of a Novel Plasmid Bearing the High-Level Tigecycline Resistance Gene tet(X4).

We reported the complete nucleotide sequence of a tet(X4)-carrying plasmid, pSTB20-1T, from a tigecycline-resistant Escherichia coli isolate in China. Sequence analysis indicated that pSTB20-1T contains a hybrid plasmid backbone and a tet(X4)-containing multidrug resistance region, likely originated through recombination of multiple plasmids. tet(X4) was flanked by two ISCR2, which may be responsible for tet(X4) mobilization. The occurrence and transmission of this novel hybrid plasmid may exacerbate the spread of the clinically significant tet(X4) gene.

Identification of novel Tet(X6)-Tet(X2) recombinant variant in Elizabethkingia meningoseptica from a bullfrog farm and downstream river in China.

Introduction: The dissemination of strains producing tetracyclines monooxygenase Tet(X) from breeding farms to the natural environment poses a potential threat to public health. Methods: Antimicrobial susceptibility testing and WGS were performed to identify resistance phenotypes and genotypes. Cloning experiments, sequence alignment, and homology modeling were used to characterize the function and formation mechanisms of the recombinant variant. The mobilization potential of Tet(X) was assessed by collinearity analysis, conjugation experiments, and phylogenetic analysis. Results: Three tet(X)-producing Elizabethkingia meningoseptica strains were isolated from bullfrog breeding ponds, the sewage outlet, and downstream river in Zhejiang Province, China. These strains carry a novel Tet(X) variant, differing from Tet(X6) by seven residues, and possess the ability to degrade tetracyclines. Interestingly, the novel Tet(X) is a recombinant variant formed by homologous recombination of Tet(X6) and the C-terminal of Tet(X2). Further analysis revealed that Tet(X6) formed several Tet(X) variants, including Tet(X5), through homologous recombination. The novel tet(X) gene is located on a circularizable integrative and conjugative element (ICEEmeChn3), with ISwz1 participating in the recombination of its multi-drug resistance region, potentially facilitating the mobilization and recombination of tet(X) in early hosts. These three strains were clonally transmitted and shared a close genetic relationship (SNP < 62) with a clinically-sourced strain isolated from the same province. Discussion: To our knowledge, this is the first report of homologous recombination between Tet(X) variants with differing activities. These clonal strains provide evidence of the transmission of tet(X)-positive strains from aquaculture sewage to the natural environment, highlighting the need to strengthen the monitoring and management of this emerging farming model.

Epidemiological and genetic characterization of multidrug-resistant non-O1 and non-O139 Vibrio cholerae from food in southern China.

This study reports a comprehensive epidemiological and genetic analysis of V. cholerae strains, specifically non-O1/non-O139 serogroups, isolated from animal-derived food samples in Guangdong province from 2015 to 2019. A total of 21 V. cholerae strains were obtained, which exhibited high resistance rates for nalidixic acid (57.14 %, 12/21), ampicillin (33.33 %, 7/21), and ciprofloxacin (19.05 %, 4/21). The quinolone resistance-related gene, qnrVC, was prevalent in 80.95 % (17/21) of the isolates. Additionally, chromosomally mediated quinolone-resistance mutations, including mutations in GyrA at position 83 (S83I) and ParC at position 85 (S85L), were detected in 47.62 % of the isolates. The combination of target mutation and qnrVC genes was shown to mediate resistance or intermediate resistance to ciprofloxacin in V. cholerae. Furthermore, an IncC-type conjugative plasmid carrying thirteen antibiotic resistance genes, including genes conferring resistance to two clinically important antibiotics, cephalosporins and fluoroquinolones, was identified in the shrimp-derived strain Vc516. While none of our food isolates harbored the toxigenic CTX- and TCP-encoding genes, they did possess genes encoding toxins such as HlyA and Autoinducer-2. Notably, some V. cholerae strains from this study exhibited a close genetic relationship with clinical strains, suggesting their potential to cause human infections. Taken together, this study provides a comprehensive view of the epidemiological features and genetic basis of antimicrobial resistance and virulence potential of V. cholerae strains isolated from food in southern China, thereby advancing our understanding of this important pathogen.

Prevalence and genomic characterization of the Bacillus cereus group strains contamination in food products in Southern China.

The Bacillus cereus group, as one of the important opportunistic foodborne pathogens, is considered a risk to public health due to foodborne diseases and an important cause of economic losses to food industries. This study aimed to gain essential information on the prevalence, phenotype, and genotype of B. cereus group strains isolated from various food products in China. A total of 890 strains of B. cereus group bacteria from 1181 food samples from 2020 to 2023 were identified using the standardized detection method. These strains were found to be prevalent in various food types, with the highest contamination rates observed in cereal flour (55.8 %) and wheat/rice noodles (45.7 %). The tested strains exhibited high resistance rates against penicillin (98.5 %) and ampicillin (98.9 %). Strains isolated from cereal flour had the highest rate of meropenem resistance (7.8 %), while strains from sausages were most resistant to vancomycin (16.8 %). A total of 234 out of the 891 B. cereus group strains were randomly selected for WGS analysis, 18.4 % of which displayed multidrug resistance. The species identification by WGS analysis revealed the presence of 10 distinct species within the B. cereus group, with B. cereus species being the most prevalent. The highest level of species diversity was observed in sausages. Notably, B. anthracis strains lacking the anthrax toxin genes were detected in flour-based food products and sausages. A total of 20 antibiotic resistance genes have been identified, with ß-lactam resistance genes (bla1, bla2, BcI, BcII, and blaTEM-116) being the most common. The B. tropicus strains exhibit the highest average number of virulence genes (23.4). The diarrheal virulence genes nheABC, hblACD, and cytK were found in numerous strains. Only 4 of the 234 (1.7 %) sequenced strains contain the ces gene cluster linked to emetic symptoms. These data offer valuable insights for public health policymakers on addressing foodborne B. cereus group infections and ensuring food safety.

Early detection of OXA-232-producing Klebsiella pneumoniae in China predating its global emergence.

Antibiotic resistance is a global health issue, with Klebsiella pneumoniae (KP) posing a particular threat due to its ability to acquire resistance to multiple drug classes rapidly. OXA-232 is a carbapenemase that confers resistance to carbapenems, a class of antibiotics often used as a last resort for treating severe bacterial infections. The study reports the earliest known identification of six OXA-232-producing KP strains that were isolated in Zhejiang, China, in 2008 and 2009 within a hospital, two years prior to the first reported identification of OXA-232 in France. The four KP strains carry the OXA-232 gene and exhibit hypervirulent loci, suggesting a broader temporal and geographical spread and integration of this resistance and virulence than previously recognized with implications for public health. Global analysis of all OXA-232-bearing KP strains revealed that OXA-232-encoding plasmids are conservative, while the strains were very diverse suggesting the plasmid mediated transmission of this carbapenemase genes. Importantly, a large proportion of the OXA-232-bearing KP strains also carried virulence plasmids, in particular the recent emergence of ST15 type of KP that carried both OXA-232-encoding plasmids and hypervirulent (hv) plasmids in China since 2019, highlighting the importance of the emergence of this type of KP strains in clinical setting. The early detection and investigations of OXA-232 in these strains warrants the retrospective studies to uncover the true timeline of antibiotic resistance spread, which could provide valuable insights for shaping future strategies to tackle the global health crisis.

Prevalence and molecular characteristics of intestinal pathogenic Escherichia coli isolated from diarrheal pigs in Southern China.

Intestinal pathogenic Escherichia coli (InPEC) is one of the most common causes of bacterial diarrhea in farm animals, including profuse neonatal diarrhea and post weaning diarrhea (PWD) in piglets. In this study, we investigated the prevalence of InPEC and associated primary virulence factors among 543 non-duplicate E. coli isolates from diarrheal pigs from 15 swine farms in southern China. Six major virulence genes associated with InPEC were identified among 69 (12.71 %) E. coli isolates and included est (6.62 %), K88 (4.79 %), elt (3.68 %), eae (1.47 %), stx2 (0.92 %) and F18 (0.55 %). Three pathotypes of InPEC were identified including ETEC (8.10 %), EPEC (1.29 %) and STEC/ETEC (0.92 %). In particular, K88 was only found in ETEC from breeding farms, whereas F18 was only present in STEC/ETEC hybrid from finishing farms. Whole genome sequence analysis of 37 E. coli isolates revealed that InPEC strains frequently co-carried multiple antibiotic resistance gene (ARG). est, elt and F18 were also found to co-locate with ARGs on a single IncFIB/IncFII plasmid. InPEC isolates from different pathotypes also possessed different profiles of virulence genes and antimicrobial resistance genes. Population structure analysis demonstrated that InPEC isolates from different pathotypes were highly heterogeneous whereas those of the same pathotype were extremely similar. Plasmid analysis revealed that K88 and/or est/elt were found on pGX18-2-like/pGX203-2-like and pGX203-1-like IncFII plasmids, while F18 and elt/est, as well as diverse ARGs were found to co-locate on IncFII/IncFIB plasmids with a non-typical backbone. Moreover, these key virulence genes were flanked by or adjacent to IS elements. Our findings indicated that both clonal expansion and horizontal spread of epidemic IncFII plasmids contributed to the prevalence of InPEC and the specific virulence genes (F4, F18, elt and est) in the tested swine farms.

Characterization of Acinetobacter baumannii at a tertiary hospital in Guangzhou: a genomic and clinical study.

Acinetobacter baumannii (AB) infections have become a global public health concern due to the continued increase in the incidence of infection and the rate of resistance to carbapenems. This study aimed to investigate the genomic features of AB strains recovered from a tertiary hospital and assess the clinical implications of the findings. A total of 217 AB strains were collected between 2016 and 2018 at a tertiary hospital in Guangzhou, with 183 (84.33%) being carbapenem-resistant AB (CRAB), with the main mechanism being the carriage of the blaOXA-23 gene. The overall mortality rate of patients caused by such strains was 15.21% (n = 33). Artificial lung ventilation and the use of meropenem were mortality risk factors in AB-infected patients, while KL2 AB infection was negatively associated. Core genome multilocus sequence typing and clustering analysis were performed on the integrated AB genome collection from the NCBI database and this study to illustrate the population structure among China. The results revealed diverse core genome profiles (n = 17) among AB strains from China, and strains from this single hospital exhibited most of the core genome profiles (n = 13), suggesting genetic variability within the hospital and transmission across the country. These findings show that the high transmission potential of the CRAB strains and meropenem usage that confers a selective advantage of CRAB clinically are two major factors that pose significant challenges to the effective clinical management of AB infections. Understanding the genetic features and transmission patterns of clinical AB strains is crucial for the effective control of infections caused by this pathogen.

Antimicrobial resistance and population genomics of emerging multidrug-resistant Salmonella 4,[5],12:i:- in Guangdong, China.

Salmonella 4,[5],12:i:-, a monophasic variant of Salmonella Typhimurium, has emerged as a global cause of multidrug-resistant salmonellosis and has become endemic in many developing and developed countries, especially in China. Here, we have sequenced 352 clinical isolates in Guangdong, China, during 2009-2019 and performed a large-scale collection of Salmonella 4,[5],12:i:- with whole genome sequencing (WGS) data across the globe, to better understand the population structure, antimicrobial resistance (AMR) genomic characterization, and transmission routes of Salmonella 4,[5],12:i:- across Guangdong. Salmonella 4,[5],12:i:- strains showed broad genetic diversity; Guangdong isolates were found to be widely distributed among the global lineages. Of note, we identified the formation of a novel Guangdong clade (Bayesian analysis of population structure lineage 1 [BAPS1]) genetically diversified from the global isolates and likely emerged around 1990s. BAPS1 exhibits unique genomic features, including large pan-genome, decreased ciprofloxacin susceptibility due to mutation in gyrA and carriage of plasmid-mediated quinolone resistance (PMQR) genes, and the multidrug-resistant IncHI2 plasmid. Furthermore, high genetic similarity was found between strains collected from Guangdong, Europe, and North America, indicating the association with multiple introductions from overseas. These results suggested that global dissemination and local clonal expansion simultaneously occurred in Guangdong, China, and horizontally acquired resistance to first-line and last-line antimicrobials at local level, underlying emergences of extensive drug and pan-drug resistance. Our findings have increased the knowledge of global and local epidemics of Salmonella 4,[5],12:i:- in Guangdong, China, and provided a comprehensive baseline data set essential for future molecular surveillance.IMPORTANCESalmonella 4,[5],12:i:- has been regarded as the predominant pandemic serotype causing diarrheal diseases globally, while multidrug resistance (MDR) constitutes great public health concerns. This study provided a detailed and comprehensive genome-scale analysis of this important Salmonella serovar in the past decade in Guangdong, China. Our results revealed the complexity of two distinct transmission modes, namely global transmission and local expansion, circulating in Guangdong over a decade. Using phylogeography models, the origin of Salmonella 4,[5],12:i:- was predicted from two aspects, year and country, that is, Salmonella 4,[5],12:i:- emerged in 1983, and was introduced from the UK, and subsequently differentiated into the local endemic lineage circa 1991. Additionally, based on the pan-genome analysis, it was found that the gene accumulation rate in local endemic BAPS 1 lineage was higher than in other lineages, and the horizontal transmission of MDR IncHI2 plasmid associated with high resistance played a major role, which showed the potential threat to public health.

IncHI1 plasmids are epidemic vectors that mediate transmission of tet(X4) in Escherichia coli isolated from China.

Introduction: This study aimed to investigate the genetic factors promoting widespread Q6 dissemination of tet(X4) between Escherichia coli and to characterize the genetic contexts of tet(X4). Methods: We isolated E. coli from feces, water, soil and flies collected across a large-scale chicken farm in China in 2020. Antimicrobial susceptibility testing and PFGE typing were used to identify tigecycline resistance and assess clonal relationships among isolates. Plasmids present and genome sequences were analyzed by conjugation, S1 pulsed-field gel electrophoresis (PFGE), plasmid stability testing and whole-genome sequencing. Results: A total of 204 tigecycline-resistant E. coli were isolated from 662 samples. Of these, we identified 165 tet(X4)-carrying E. coli and these strains exhibited a high degree of multidrug resistance. Based on the geographical location distribution of the sampled areas, number of samples in each area and isolation rate of tigecycline-resistant strains and tet(X4)-carrying isolates, 72 tet(X4)-positive isolates were selected for further investigation. Tigecycline resistance was shown to be mobile in 72 isolates and three types of tet(X4)-carrying plasmids were identified, they were IncHI1 (n = 67), IncX1 (n = 3) and pO111-like/IncFIA(HI1) (n = 2). The pO111-like/IncFIA(HI1) is a novel plasmid capable of transferring tet(X4). The transfer efficiency of IncHI1 plasmids was extremely high in most cases and IncHI1 plasmids were stable when transferred into common recipient strains. The genetic structures flanked by IS1, IS26 and ISCR2 containing tet(X4) were complex and varied in different plasmids. Discussion: The widespread dissemination of tigecycline-resistant E. coli is a major threat to public health. This data suggests careful use of tetracycline on farms is important to limit spread of resistance to tigecycline. Multiple mobile elements carrying tet(X4) are in circulation with IncHI1 plasmids the dominant vector in this setting.