Prevalence, Antimicrobial Resistance, and Genomic Characterization of Salmonella Typhimurium Isolated in Hengyang, China (2020-2023).

Nat'ralists observe, a FleaHath smaller Fleas that on him prey, And these have smaller yet to bite 'em,

The main work of this study is to analyze antimicrobial resistant E. coli based on whole genome sequencing data. Three stages were included for isolates from different sources (ready-to-eat food, retail raw meats and human patients). 
\nAt the first stage, a retrospective study for antimicrobial resistant E. coli in ready-to-eat (RTE) food sold in retail food premises in Singapore was performed in collaboration with Environmental Health Institute under NEA. A total of 99 E. coli isolates from poultry-based dishes (n=77) and fish-based dishes (n=22) were obtained between 2009 and 2014 during the surveillance project. All the isolates were included for disk diffusion testing for antimicrobial susceptibility testing. Of the 99 isolates, 24 (24.2%) were resistant to at least one antimicrobial agent. These isolates were then subjected to broth microdilution testing against 33 antimicrobial agents, including β-lactams, aminoglycosides, tetracycline, fluoroquinolones and polymyxin E (colistin), to determine the minimum inhibitory concentration (MIC) of isolates. Whole genome sequence (WGS) was carried out on the strains in order to correlate resistant phenotypes to putative antimicrobial resistance-related genes. Of the 24 isolates, 15 (62.5%) were found to be resistant to three or more classes of antimicrobials and thus were defined as multi-drug resistant strains. Two isolates (8.3%) were confirmed as Extended-Spectrum β-lactamase (ESBL)- producing E. coli by double disk synergy test. Based on WGS data, online analysis tool ResFinder detected 7 classes of antimicrobial resistance genes and resistance-related chromosomal point mutations in 19 of the 24 E. coli isolates. By analyzing the WGS contigs using BLASTn and KmerFinder, ESBL genes and transferable colistin resistance gene mcr-1 (2/24) and mcr-5 (1/24) were determined to be located on plasmids, which could pose a greater risk of AMR transfer among bacteria. Mutations were detected in four isolates within genes previously shown to confer resistance to quinolones (gyrA and parE) and tetracycline (rrsB). Prediction of AMR using WGS data was evaluated for six antimicrobials including ampicillin, chloramphenicol, colistin, fluoroquinolones, tetracycline and trimethoprim. The evaluation indicates WGS–based genotype and phenotype showed high consistency, however, for some antimicrobial resistance whose mechanism is not totally clear yet (such as colistin), there is challenge for resistance gene detection.
\nTo have a better understanding of genetic environment and mobility of colistin resistance gene mcr-5.1, the isolates carrying mcr-5.1 were sequenced using long-read sequencing technology. The plasmid sequences were assembled into a closed circle using both long-read sequencing data and short-read sequencing data. The blasting result showed the closest plasmid sequence to pSGMCR103 in NCBI is plasmid pYD786-3 (accession number KU254580.1) with 77% query coverage and 99% identity, which was carried by one E. coli isolate from human urine in USA. They share antimicrobial resistance gene aph(3’)-la, aadA1 (aminoglycoside resistance), blaTEM-176 (beta-lactam resistance) and sul3 (sulphonamide resistance). Gene mcr-5.1 was harbored on a Tn3 transposon-like element, which is similar with pSE13-SA01718 (accession number KY807921.1) carried by a Salmonella isolate reported before. Also, other insertion elements such as IS5, IS6, IS91, IS256 family were found on the plasmid, which may indicate the recombination activity of the plasmid. Moreover, the mobility of this plasmid was confirmed by the conjugation experiment. The frequency of conjugation after 24 hours is 10-6.
\nAfter knowing the AMR profile in ready-to-eat food in Singapore, at the second stage, an important resistance type was studied: Extended-Spectrum Beta-Lactamase (ESBL)-caused resistance to most of beta-lactams. We collected 634 meat samples including chicken, pork and beef from 97 supermarkets and 65 wet markets in Singapore during June 2017-October 2018. The samples were enriched before bacteria isolation. Presumptive ESBLs were screened by Brilliance TM ESBL Agar and confirmed by Double Disk Synergy Test (DDST). E. coli isolates were identified by EMB agar and indole test. The genomic DNA of ESBL-producing E. coli were extracted and sent for WGS. Besides the analysis for AMR genes, MLST, annotation and genetic environment, these sequence collection was also compared with sequence data of ESBL E. coli isolated from community in Singapore for phylogenetic study based on SNPs. A total of 225 ESBL-producing E. coli were isolated from 184 samples. The prevalence of ESBL in chicken, pork and beef was 51.2% (109/213), 26.9% (58/216), 7.3% (15/205), respectively. The most common AMR genes in all 225 ESBL isolates were beta-lactam-resistance genes (100%), aminoglycoside resistance gens (92.4%), sulphonamide resistance genes (86.2%). In terms of beta-lactam resistance genes, 172 of isolates (76.4%) carry blaCTX-M genes, 102 (45.3%) of isolates carry blaTEM genes and 52 of isolates (23.1%) carry blaSHV genes. Besides these most common three beta-lactamase genes, blaCMY-2, blaOXA and blaDHA were also found. Gene blaCTX-M-55 (57/225, 25.3%) and blaCTX-M-65 (40/225, 17.8%) were the most frequent ESBL genes. Among all these classes of antimicrobials, beta-lactam-resistance genes and aminoglycoside resistance genes exhibit great variety. The last-resort antimicrobial colistin resistance mcr genes exist in 15.6% of all isolates (33 isolates carry mcr-1, one carries mcr-3.1 and one carries mcr-5). Phylogeny tree based on SNPs of our isolates and previous ESBL isolates from human community in Singapore shows obvious separate human clusters and food clusters, however, two E. coli isolates from human fell into food clusters and showed high similarity with our isolates from meats, which indicates the possible transmission of resistant E. coli from meats to human may exist. Occurrence of AMR genes especially for last resort drug resistance genes was observed, raising concerns on food safety and public health.
\nAt the last stage, we applied WGS to the analysis of clinical isolates. We collaborated with the university in Thailand to get 28 ESBL-producing E. coli isolated from diarrhea patients hospitalized at the Phayao Ram Hospital in Thailand. Result shows all E. coli carried CTX-Ms beta-lactamase (including CTX-M-14, CTX-M-15, CTX-M-27, CTX-M-55), and half of these isolates (14/28) belong to the important pathogenic cluster ST131. CTX-M-55s were detected only in non-ST131s. Two serotypes O16:H5 (6/14) and O25:H4 (8/14) were observed in ST131 isolates. Generally, ST131 isolates showed different virulence factor patterns with non-ST131 isolates. BLAST results indicate that for half of ST131 isolates, blaCTX-M genes are located on chromosome adjacent to insert sequences (IS). For the other half ST131s, blaCTX-M genes are located on plasmids. Besides CTX-Ms, other beta-lactamases such as TEM-1B, OXA-1 and CMY-2 were also observed in our study. Phylogenetic analysis for a global collection and our clinical isolates in Thailand based on SNPs showed the closest isolates with our isolates are from Thailand, Singapore, Australia, Laos and New Zealand. A special strain cluster O16:H5-ST131 was found from 2015-2017 as well as other previous Thailand studies. These isolates showed high similarity in term of serotype, MLST, virulence factor / AMR patterns, and phylogeny, which indicates the persistence and spread of this cluster. This study provides an insight on characteristics of clinical ESBL-producing E. coli with special focus on ST131 in Thailand.
\nIt is noteworthy that four isolates in our study showed same serotype, ST (O16:H5-ST131), same virulence factor pattern (cnf1, iha, sat, senB) and even resistance gene pattern. In terms of SNP analysis, these isolates were located the same cluster 3 and the SNP difference range 17-20, which indicate the high similarity of these strains. But actually they are isolated in different years from 2015-2017, respectively. In cluster 3, other four isolates from Thailand (ERR1218557, ERR1218609, ERR1218624, ERR1218628) in a previous study also showed less than 30 SNPs difference with our four isolates. This also strengthens the hypothesis that these isolates diverged from one ancestor and this cluster of isolates are persistent in Thailand in recent years. This strain should raise our attention of further spread. The phylogenetic study indicates that the closest isolates with Thailand ST131 isolates are from Oceania and Southeast Asia. Our data based on whole genome add more evidence on ubiquity of ESBL-ST131 E. coli in Thailand. The co-existence of multi-resistance and multi-virulence factors adds challenges to clinical treatment. Although resistances to carbapenem and/or colistin are rare in this study, more epidemiology studies are needed to verify their actual prevalence. 
\nOverall speaking, WGS is a useful tool for prevalence and epidemiological analysis and source tracking. With the development of sequencing technology and the decrease of cost, whole-genome-based analysis is becoming more and more necessary for AMR study.

Genomic characterization, antimicrobial resistance and virulence profiles of Klebsiella pneumoniae isolated from mink in Northern China.

ABSTRACTPurposeUrinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are a major public health concern due to their recurrent nature and antibiotic resistance. Biofilm formation plays a crucial role in UPEC persistence, yet the genetic mechanisms underlying this process remain poorly understood. This study employs next-generation sequencing (NGS) to investigate the genomic characteristics of biofilm-forming, multidrug-resistant (MDR) UPEC isolates, with a focus on antimicrobial resistance (AMR), virulence factors, and mobile genetic elements.MethodsFive biofilm-forming MDR UPEC isolates were selected for whole-genome sequencing (WGS) using the Illumina NovaSeq 6000 platform. Genome assembly and annotation were performed using SPAdes and Prokka. Multilocus sequence typing (MLST) and serotyping were conducted to determine genetic diversity, while AMR genes were identified using ResFinder. Virulence factors and biofilm-related genes were analyzed through PathogenFinder, and mobile genetic elements, including plasmids and insertion sequences, were characterized.ResultsGenomic analysis revealed significant diversity among isolates, with MLST identifying high-risk sequence types such as ST131, known for its strong association with MDR and virulence. AMR profiling indicated resistance to multiple antibiotics, including beta-lactams, aminoglycosides, and fluoroquinolones. All isolates harbored virulence genes associated with adhesion, immune evasion, and biofilm formation. Mobile genetic elements, particularly IncF-type plasmids and insertion sequences, were detected across isolates, suggesting a role in horizontal gene transfer of resistance traits. Biofilm-associated genes correlated with biofilm production capabilities, reinforcing their role in UPEC persistence.ConclusionThis study provides critical insights into the genetic landscape of biofilm-forming UPEC, highlighting the role of mobile elements in antibiotic resistance dissemination. The findings underscore the importance of genomic surveillance and the need for novel therapeutic strategies targeting biofilm-mediated resistance to combat recurrent UTIs.

Salmonella enterica is an important foodborne pathogen worldwide. Ciprofloxacin and extended-spectrum cephalosporins are the common first-line antimicrobial drugs for the treatment of salmonellosis, antimicrobial resistance genes for which are mostly transferred via plasmids. The goal of this work was to perform genomic analysis of plasmids from foodborne S. enterica isolates obtained in Russia based on whole-genome sequencing. In the current study, 11 multidrug-resistant samples isolated in 2021 from 8 regions of Russia were selected based on their resistance to ciprofloxacin and third-generation cephalosporins (CIP-3rd). Whole-genome short-read sequencing (WGS) was performed for all isolates; the samples belonged to five different sequence types (ST32, ST469, ST11, ST142, and ST548) which had different profiles of antimicrobial resistance (AMR) and virulence genes. We have performed additional long-read sequencing of four representative S. enterica isolates, which showed that they carried pESI-like megaplasmids of 202-280 kb length harboring extended-spectrum β-lactamase genes, fluoroquinolone, tetracycline, and aminoglycosides resistance genes, as well as several virulence determinants. We believe that the WGS data obtained will greatly facilitate further studies of foodborne S. enterica isolates epidemiology in terms of their self-transmissible plasmid composition that mediated antimicrobial resistance and virulence determinants conferring selective advantages of this important bacterial pathogen.

Simple SummaryWorldwide, antimicrobial resistance (AMR) is of major concern for human and animal health since infections with multidrug-resistant bacteria are often more challenging and costly. In the family Staphyloccocaceae, the species Staphylococcusaureus in particular was reported to cause severe infections. Although most of the other Staphylococcaceae members were not shown to cause severe illnesses, the transmission of AMR genes to harmful species might take place. Therefore, the monitoring of AMR potential in different environments is of high relevance. Mammaliicocci on dairy farms might represent such an AMR gene reservoir. Thus, in this study, the AMR potential of mammaliicocci isolates from German dairy farms was investigated. Whole-genome sequencing (WGS) of the isolates was conducted to evaluate the phylogenetic relationship of the isolates and analyze AMR genes. In addition, antimicrobial susceptibility testing was performed to compare the AMR genotype with the phenotype. It turned out that mammaliicocci may harbor large numbers of different AMR genes and exhibit phenotypic resistance to various antibiotics. Since some AMR genes are likely located on mobile genetic elements, such as plasmids, AMR gene transmission between members of the Staphylococcaceae family might occur.Mammaliicocci might play a major role in antimicrobial resistance (AMR) gene transmission between organisms of the family Staphylococcaceae, such as the potentially pathogenic species Staphylococcus aureus. The interest of this study was to analyze AMR profiles of mammaliicocci from German dairy farms to evaluate the AMR transmission potential. In total, 65 mammaliicocci isolates from 17 dairy farms with a history of MRSA detection were analyzed for AMR genotypes and phenotypes using whole genome sequencing and antimicrobial susceptibility testing against 19 antibiotics. The various genotypic and phenotypic AMR profiles of mammaliicocci from German dairy farms indicated the simultaneous occurrence of several different strains on the farms. The isolates exhibited a non-wildtype phenotype to penicillin (58/64), cefoxitin (25/64), chloramphenicol (26/64), ciprofloxacin (25/64), clindamycin (49/64), erythromycin (17/64), fusidic acid (61/64), gentamicin (8/64), kanamycin (9/64), linezolid (1/64), mupirocin (4/64), rifampicin (1/64), sulfamethoxazol (1/64), streptomycin (20/64), quinupristin/dalfopristin (26/64), tetracycline (37/64), tiamulin (59/64), and trimethoprim (30/64). Corresponding AMR genes against several antimicrobial classes were detected. Linezolid resistance was associated with the cfr gene in the respective isolate. However, discrepancies between genotypic prediction and phenotypic resistance profiles, such as for fusidic acid and tiamulin, were also observed. In conclusion, mammaliicocci from dairy farms may carry a broad variety of antimicrobial resistance genes and exhibit non-wildtype phenotypes to several antimicrobial classes; therefore, they may represent an important source for horizontal gene transfer of AMR genes to pathogenic Staphylococcaceae.

IntroductionThe antimicrobial resistance (AMR) mobilome plays a key role in the dissemination of resistance genes encoded by mobile genetics elements (MGEs) including plasmids, transposons (Tns), and insertion sequences (ISs). These MGEs contribute to the dissemination of multidrug resistance (MDR) in enteric bacterial pathogens which have been considered as a global public health risk.MethodsTo further understand the diversity and distribution of AMR genes and MGEs across different plasmid types, we utilized multiple sequence-based computational approaches to evaluate AMR-associated plasmid genetics. A collection of 1,309 complete plasmid sequences from Gammaproteobacterial species, including 100 plasmids from each of the following 14 incompatibility (Inc) types: A/C, BO, FIA, FIB, FIC, FIIA, HI1, HI2, I1, K, M, N, P except W, where only 9 sequences were available, was extracted from the National Center for Biotechnology Information (NCBI) GenBank database using BLAST tools. The extracted FASTA files were analyzed using the AMRFinderPlus web-based tools to detect antimicrobial, disinfectant, biocide, and heavy metal resistance genes and ISFinder to identify IS/Tn MGEs within the plasmid sequences.Results and DiscussionIn silico prediction based on plasmid replicon types showed that the resistance genes were diverse among plasmids, yet multiple genes were widely distributed across the plasmids from enteric bacterial species. These findings provide insights into the diversity of resistance genes and that MGEs mediate potential transmission of these genes across multiple plasmid replicon types. This notion was supported by the observation that many IS/Tn MGEs and resistance genes known to be associated with them were common across multiple different plasmid types. Our results provide critical insights about how the diverse population of resistance genes that are carried by the different plasmid types can allow for the dissemination of AMR across enteric bacteria. The results also highlight the value of computational-based approaches and in silico analyses for the assessment of AMR and MGEs, which are important elements of molecular epidemiology and public health outcomes.

The presence of mobile genetic elements in Salmonella isolated from a chicken farm constitutes a potential risk for the appearance of emerging bacteria present in the food industry. These elements contribute to increased pathogenicity and antimicrobial resistance through genes that are related to the formation of biofilms and resistance genes contained in plasmids, integrons, and transposons. One hundred and thirty-three Salmonella isolates from different stages of the production line, such as feed manufacturing, hatchery, broiler farm, poultry farm, and slaughterhouse, were identified, serotyped and sequenced. The most predominant serotype was Salmonella Infantis. Phylogenetic analyses demonstrated that the diversity and spread of strains in the pipeline are serotype-independent, and that isolates belonging to the same serotype are very closely related genetically. On the other hand, Salmonella Infantis isolates carried the pESI IncFIB plasmid harboring a wide variety of resistance genes, all linked to mobile genetic elements, and among carriers of these plasmids, the antibiograms showed differences in resistance profiles and this linked to a variety in plasmid structure, similarly observed in the diversity of Salmonella Heidelberg isolates carrying the IncI1-Iα plasmid. Mobile genetic elements encoding resistance and virulence genes also contributed to the differences in gene content. Antibiotic resistance genotypes were matched closely by the resistance phenotypes, with high frequency of tetracycline, aminoglycosides, and cephalosporins resistance. In conclusion, the contamination in the poultry industry is described throughout the entire production line, with mobile genetic elements leading to multi-drug resistant bacteria, thus promoting survival when challenged with various antimicrobial compounds.

Enterotoxigenic E. coli (ETEC) bacteria frequently cause watery diarrhoea in newborn and weaned pigs. Plasmids carrying genes of different enterotoxins and fimbrial adhesins, as well as plasmids conferring antimicrobial resistance are of prime importance in the epidemiology and pathogenesis of ETEC. Recent studies have revealed the significance of the porcine ETEC plasmid pTC, carrying tetracycline resistance gene tet(B) with enterotoxin genes. In contrast, the role of tet(A) plasmids in transferring resistance of porcine ETEC is less understood. The objective of the present study was to provide a comparative analysis of antimicrobial resistance and virulence gene profiles of porcine post-weaning ETEC strains representing pork-producing areas in Central Europe and in the USA, with special attention to plasmids carrying the tet(A) gene. Antimicrobial resistance phenotypes and genotypes of 87 porcine ETEC strains isolated from cases of post-weaning diarrhoea in Austria, the Czech Republic, Hungary and the Midwest USA was determined by disk diffusion and by PCR. Central European strains carrying tet(A) or tet(B) were further subjected to molecular characterisation of their tet plasmids. Results indicated that > 90% of the ETEC strains shared a common multidrug resistant (MDR) pattern of sulphamethoxazole (91%), tetracycline (84%) and streptomycin (80%) resistance. Tetracycline resistance was most frequently determined by the tet(B) gene (38%), while tet(A) was identified in 26% of all isolates with wide ranges for both tet gene types between some countries and with class 1 integrons and resistance genes co-transferred by conjugation. The virulence gene profiles included enterotoxin genes (lt, sta and/or stb), as well as adhesin genes (k88/f4, f18). Characterisation of two representative tet(A) plasmids of porcine F18(+) ETEC from Central Europe revealed that the IncF plasmid (pES11732) of the Czech strain (~120 kb) carried tet(A) in association with catA1 for chloramphenicol resistance. The IncI1 plasmid (pES2172) of the Hungarian strain (~138 kb) carried tet(A) gene and a class 1 integron with an unusual variable region of 2,735 bp composed by two gene cassettes: estX-aadA1 encoding for streptothricin-spectinomycin/streptomycin resistance exemplifying simultaneous recruitment, assembly and transfer of multidrug resistance genes by the tet(A) plasmid of porcine ETEC. By this we provide the first description of IncF and IncI1 type plasmids of F18(+) porcine enterotoxigenic E. coli responsible for cotransfer of the tet(A) gene with multidrug resistance. Additionally, the unusual determinant estX, encoding for streptothricin resistance, is first reported here in porcine enterotoxigenic E. coli.

Simple SummaryPhysical injury or microbial infection can cause mastitis, an inflammatory reaction of the udder tissue in the mammary gland of cows. Globally, mastitis is a leading source of significant financial losses for dairy farms. Despite several attempts over the past several years to manage mastitis, efficient and long-lasting control methods or instruments have not yet been created. The current investigation used MALDI-TOF MS and 16S rRNA PCR for the identification of Staphylococcus isolates from the milk of cows with subclinical mastitis (SCM), and further screened them for determination of their antimicrobial resistance (AMR) and virulence gene profiles. Our results uncovered that 33.13% of the cows had subclinical mastitis, while the quarter-level prevalence was 54%. Furthermore, MALDI-TOF MS and 16S rRNA PCR assay and sequencing identified Staphylococcus aureus as the dominant bacteria. An antimicrobial resistance susceptibility test showed that 86% of the isolates were resistant to penicillin, while antimicrobial resistance and virulence genes showed that the isolates carried mostly the mecA- and Lg G-binding region genes. The results of this study demonstrate the need for earlier diagnosis and surveillance of SCM and Staphylococcus species in the studied area.Staphylococcus species are amongst the bacteria that cause bovine mastitis worldwide, whereby they produce a wide range of protein toxins, virulence factors, and antimicrobial-resistant properties which are enhancing the pathogenicity of these organisms. This study aimed to detect Staphylococcus spp. from the milk of cattle with subclinical mastitis using MALDI-TOF MS and 16S rRNA PCR as well as screening for antimicrobial resistance (AMR) and virulence genes. Our results uncovered that from 166 sampled cows, only 33.13% had subclinical mastitis after initial screening, while the quarter-level prevalence was 54%. Of the 50 cultured bacterial isolates, MALDI-TOF MS and 16S rRNA PCR assay and sequencing identified S. aureus as the dominant bacteria by 76%. Furthermore, an AMR susceptibility test showed that 86% of the isolates were resistant to penicillin, followed by ciprofloxacin (80%) and cefoxitin (52%). Antimicrobial resistance and virulence genes showed that 16% of the isolates carried the mecA gene, while 52% of the isolates carried the Lg G-binding region gene, followed by coa (42%), spa (40%), hla (38%), and hlb (38%), whereas sea and bap genes were detected in 10% and 2% of the isolates, respectively. The occurrence of virulence factors and antimicrobial resistance profiles highlights the need for appropriate strategies to control the spread of these pathogens.

Antimicrobial resistance and genomic characterization of Escherichia coli isolated from mink in northern China.

Acinetobacter baumannii is a major cause of nosocomial infection, and the incidence of extensively drug-resistant A. baumannii (XDRAB) infections has dramatically increased worldwide. In this study, we aimed to explore the complete genome sequence of XDRAB 329, ST1166/98 (Oxford/Pasteur), which is an outbreak clone from a hospital in Thailand. Whole-genome sequencing (WGS) was performed using short-read Illumina and long-read PacBio sequencing, and a conjugation assay of its plasmid was performed. The complete genome sequence of A. baumannii AB329 revealed a circular chromosome 3,948,038 bp in length with 39% GC content. Antibiotic resistance genes (ARGs), including beta-lactam resistance (blaOXA-51, blaADC-25, blaOXA-23, blaTEM-1D), aminoglycoside resistance (aph(3′)-Ia, aph(3″)-Ib, aph(6)-Id, armA), tetracycline resistance (tet(B), tet (R)), macrolide resistance (mph(E), msr(E)), and efflux pumps, were found. Mobile genetic elements (MGEs) analysis of A. baumannii AB329 revealed two plasmids (pAB329a and pAB329b), three prophages, 19 genomic islands (GIs), and 33 insertion sequences (ISs). pAB329a is a small circular plasmid of 8,731 bp, and pAB329b is a megaplasmid of 82,120 bp. aph(3′)-VIa was detected in pAB329b, and a major facilitator superfamily (MFS) transporter was detected in the prophage. Acinetobacter baumannii resistance island 4 (AbaR4) harboring tetracycline and aminoglycoside resistance was detected in the genome of A. baumannii AB329. pAB329b, which belongs to Rep-type GR6 (plasmid lineage LN_1), is a conjugative plasmid with the ability to transfer an aminoglycoside resistance gene to sodium azide-resistant A. baumannii. This study provides insights into the features of the MGEs of XDRAB, which are the main reservoir and source of dissemination of ARGs.

Background: Chryseobacterium indologenes, an environmental bacterium, is increasingly recognized as an emerging nosocomial pathogen, particularly in Asia, and is often characterized by multidrug resistance. Objectives: This study aimed to investigate the genomic features of clinical C. indologenes isolates from Maharaj Nakorn Chiang Mai Hospital, Thailand, to understand their mechanisms of multidrug resistance, virulence factors, and mobile genetic elements (MGEs). Methods: Twelve C. indologenes isolates were identified, and their antibiotic susceptibility profiles were determined. Whole genome sequencing (WGS) was performed using a hybrid approach combining Illumina short-reads and Oxford Nanopore long-reads to generate complete bacterial genomes. The hybrid assembled genomes were subsequently analyzed to detect antimicrobial resistance (AMR) genes, virulence factors, and MGEs. Results: C. indologenes isolates were primarily recovered from urine samples of hospitalized elderly male patients with underlying conditions. These isolates generally exhibited extensive drug resistance, which was subsequently explored and correlated with genomic determinants. With one exception, CMCI13 showed a lower resistance profile (Multidrug resistance, MDR). Genomic analysis revealed isolates with genome sizes of 4.83–5.00 Mb and GC content of 37.15–37.35%. Genomic characterization identified conserved resistance genes (blaIND-2, blaCIA-4, adeF, vanT, and qacG) and various virulence factors. Phylogenetic and pangenome analysis showed 11 isolates clustering closely with Chinese strain 3125, while one isolate (CMCI13) formed a distinct branch. Importantly, each isolate, except CMCI13, harbored a large genomic island (approximately 94–100 kb) carrying significant resistance genes (blaOXA-347, tetX, aadS, and ermF). The absence of this genomic island in CMCI13 correlated with its less resistant phenotype. No plasmids, integrons, or CRISPR-Cas systems were detected in any isolate. Conclusions: This study highlights the alarming emergence of multidrug-resistant C. indologenes in a hospital setting in Thailand. The genomic insights into specific resistance mechanisms, virulence factors, and potential horizontal gene transfer (HGT) events, particularly the association of a large genomic island with the XDR phenotype, underscore the critical need for continuous genomic surveillance to monitor transmission patterns and develop effective treatment strategies for this emerging pathogen.

BackgroundThe prophylactic use of antimicrobials and zinc oxide (ZnO) in pig production was prohibited by the European Union in 2022 due to potential associations between antimicrobial and heavy metal usage with antimicrobial resistance (AMR) and concerns regarding environmental pollution. However, the effects of their usage on the bacterial AMR profiles on commercial pig farms are still not fully understood and previous studies examining the effect of ZnO have reported contrasting findings. The objective of this study was to examine the effects of antimicrobial and ZnO usage on AMR on commercial pig farms. Faecal and environmental samples were taken on 10 Irish commercial farms, of which 5 farms regularly used ZnO and antimicrobials (amoxicillin or sulphadiazine-trimethoprim) for the prevention of disease. The other 5 farms did not use ZnO or any other form of prophylaxis. Escherichia coli numbers were quantified from all samples using non-supplemented and supplemented Tryptone Bile X-glucuronide agar.ResultsIn total 351 isolates were phenotypically analysed, and the genomes of 44 AmpC/ESBL-producing E. coli isolates from 4 farms were characterised using whole-genome sequencing. Phenotypic analysis suggested higher numbers of multi-drug resistant (MDR) E. coli isolates on farms using prophylaxis. Furthermore, farms using prophylaxis were associated with higher numbers of isolates resistant to apramycin, trimethoprim, tetracycline, streptomycin, and chloramphenicol, while resistance to ciprofloxacin was more associated with farms not using any prophylaxis. Thirty-four of the 44 AmpC/ESBL-producing E. coli strains harboured the blaCTX-M-1 resistance gene and were multi drug resistant (MDR). Moreover, network analysis of plasmids and analysis of integrons showed that antimicrobial and biocide resistance genes were frequently co-located on mobile genetic elements, indicating the possibility for co-selection during antimicrobial or biocide usage as a contributor to AMR occurrence and persistence on farms.ConclusionsThe results of this study showed evidence that antimicrobial and ZnO treatment of pigs post-weaning can favour the selection and development of AMR and MDR E. coli. Co-location of resistance genes on mobile genetic elements was observed. This study demonstrated the usefulness of phenotypic and genotypic detection of antimicrobial resistance by combining sequencing and microbiological methods.

Characterization of mobile resistance elements in extended-spectrum β-lactamase producing gram-negative bacteria from aquatic environment.