Multiple Antimicrobial Resistance Genes Research Articles

Chromosomal and plasmid localization of ileS2 in high-level mupirocin-resistant Staphylococcus pseudintermedius and Staphylococcus aureus isolated from canine and feline origins.

To characterize the mobile genetic elements and genetic localization of ileS2 in high-level mupirocin-resistant (Hi-MupR) methicillin-resistant Staphylococcus pseudintermedius (MRSP) and MRSA isolates recovered from canine and feline clinical samples. The identification of bacterial species and presence of mecA and ileS2 genes in MRSP and MRSA isolates were performed using MALDI-TOF MS and PCR, respectively. Antimicrobial resistance (AMR) phenotypes were determined by broth microdilution assays. The genome characteristics, ileS2-containing elements and staphylococcal cassette chromosome mec (SCCmec) were illustrated using complete circular genomes obtained from hybrid assembly of Illumina short-reads and Oxford Nanopore Technologies long-reads. These were analysed through phylogenetic and bioinformatics approaches. A total of 18 MRSP clinical isolates and four MRSA clinical isolates exhibited the Hi-MupR phenotype and carried multiple AMR genes, including mecA and ileS2 genes. MRSP ST182-SCCmec V (n = 6) and ST282-ΨSCCmec57395-t10 (n = 4) contained the ileS2 transposable unit associated with IS257 on the chromosome. Three MRSA ST398-SCCmec V-t034/t4652 isolates carried ∼42 kb pSK41-like ileS2 plasmids, whereas similar ileS2 plasmids lacking tra genes were found in MRSP ST282-ΨSCCmec57395-t72/t21 isolates. Furthermore, a new group of ileS2 plasmids, carried by MRSP ST45-ΨSCCmec57395, ST433-ΨSCCmecKW21-t05 and ST2165-SCCmec IV-t06, and by one MRSA ST398-SCCmec V-t034 strain, shared the plasmid backbone with the cfr/fexA-carrying plasmid pM084526_1 in MRSA ST398. This study provides the first evidence of ileS2 integration into the S. pseudintermedius chromosome, which is a rare occurrence in staphylococcal species, and plasmids played a pivotal role in dissemination of ileS2 in both staphylococcal species.

Genomic Insights into Edwardsiella ictaluri: Molecular Epidemiology and Antimicrobial Resistance in Striped Catfish (Pangasianodon hypophthalmus) Aquaculture in Vietnam

Edwardsiella ictaluri is responsible for causing bacillary necrosis (BNP) in striped catfish (Pangasianodon hypophthalmus) in Vietnam. This study offers a comprehensive genomic characterization of E. ictaluri to enhance understanding of the molecular epidemiology, virulence, and antimicrobial resistance. E. ictaluri isolates were collected from diseased striped catfish in the Mekong Delta. The species was confirmed through PCR. Antimicrobial susceptibility testing was conducted using minimum inhibitory concentrations for commonly used antimicrobials. Thirty representative isolates were selected for whole genome sequencing to delineate their genomic profiles and phylogeny. All strains belonged to ST-26 and exhibited genetic relatedness, differing by a maximum of 90 single nucleotide polymorphisms. Most isolates carried multiple antimicrobial resistance genes, with the tet(A) gene present in 63% and floR in 77% of the genomes. The ESBL gene, blaCTX-M-15, was identified in 30% of the genomes. Three plasmid replicon types were identified: IncA, p0111, and IncQ1. The genomes clustered into two clades based on their virulence gene profile, one group with the T3SS genes and one without. The genetic similarity among Vietnamese isolates suggests that disease spread occurs within the Mekong region, underscoring the importance of source tracking, reservoir identification, and implementation of necessary biosecurity measures to mitigate spread of BNP.

Draft genome sequences of clinical mastitis-associated Enterococcus faecalis and Enterococcus faecium carrying multiple antimicrobial resistance genes isolated from dairy cows

ObjectivesThe emergence of antimicrobial-resistant and mastitis-associated Enterococcus faecalis and Enterococcus faecium is of great concern due to the huge economic losses associated with enterococcal infections. Here we report the draft genome sequences of Enterococcus faecalis and Enterococcus faecium strains which were isolated from raw milk samples obtained from mastitis-infected cows in Bangladesh. MethodsStrains were isolated, identified and Genomic DNA was sequenced using Illumina NextSeq 550 platform. The assembled contigs were analyzed for virulence, antimicrobial resistance genes, and multi-locus sequence type. The genomes were compared to previously reported Enterococcus faecalis and Enterococcus faecium genomes to generate core genome phylogenetic trees. ResultsEnterococcus faecalis strain BR-MHR218Efa and Enterococcus faecium strain BR-MHR268Efe belonged to multilocus sequence type ST-190 and ST-22, respectively. Both sequence types seem to represent relatively rare sequence types. BR-MHR268Efe harbored only one antibiotic resistance gene encoding resistance towards macrolides (lsa(A)), while BR-MHR218Efa harbored ten different antibiotic resistance genes encoding resistance to aminoglycosides (ant[6]-Ia, aph(3′)-III), sulphonamides (aac(6′)-II), lincosamides (lnu(B)), macrolides (erm(B)), MLSB antibiotics (msr(C)), tetracyclines (tet(M), tet(L)), trimethoprim (dfrG) and pleuromutilin-lincosamide-streptogramin A (lsa(E)).The virulence gene composition was different in the two isolates. BR-MHR218Efa harbored only two virulence genes involved in adherence (acm, scm). BR-MHR268Efe harbored eight complete virulence operons including three operons involved in adherence (Ace, Ebp pili, EfaA), two operons involved in biofilm formation (BopD, Fsr) and three exoenzymes (gelatinase, hyaluronidase, SprE). ConclusionsThe genome sequences of strains BR-MHR268Efe and BR-MHR218Efa will serve as a reference point for molecular epidemiological studies of mastitis-associated Enterococcus faecalis and Enterococcus faecium. Additionally, the findings will help the understand the complex antimicrobial resistant livestock Enterococci.

Sporadic clone Escherichia coli ST615 as a vector and reservoir for dissemination of crucial antimicrobial resistance genes.

There is scarce information concerning the role of sporadic clones in the dissemination of antimicrobial resistance genes (ARGs) within the nosocomial niche. We confirmed that the clinical Escherichia coli M19736 ST615 strain, one of the first isolates of Latin America that harbors a plasmid with an mcr-1 gene, could receive crucial ARG by transformation and conjugation using as donors critical plasmids that harbor bla CTX-M-15, bla KPC-2, bla NDM-5, bla NDM-1, or aadB genes. Escherichia coli M19736 acquired bla CTX-M-15, bla KPC-2, bla NDM-5, bla NDM-1, and aadB genes, being only blaNDM-1 maintained at 100% on the 10th day of subculture. In addition, when the evolved MDR-E. coli M19736 acquired sequentially bla CTX-M-15 and bla NDM-1 genes, the maintenance pattern of the plasmids changed. In addition, when the evolved XDR-E. coli M19736 acquired in an ulterior step the paadB plasmid, a different pattern of the plasmid's maintenance was found. Interestingly, the evolved E. coli M19736 strains disseminated simultaneously the acquired conjugative plasmids in different combinations though selection was ceftazidime in all cases. Finally, we isolated and characterized the extracellular vesicles (EVs) from the native and evolved XDR-E. coli M19736 strains. Interestingly, EVs from the evolved XDR-E. coli M19736 harbored bla CTX-M-15 though the pDCAG1-CTX-M-15 was previously lost as shown by WGS and experiments, suggesting that EV could be a relevant reservoir of ARG for susceptible bacteria. These results evidenced the genetic plasticity of a sporadic clone of E. coli such as ST615 that could play a relevant transitional link in the clinical dynamics and evolution to multidrug/extensively/pandrug-resistant phenotypes of superbugs within the nosocomial niche by acting simultaneously as a vector and reservoir of multiple ARGs which later could be disseminated.

Global transmission of extended-spectrum cephalosporin resistance in Escherichia coli driven by epidemic plasmids

Extended-spectrum cephalosporins (ESCs) are third and fourth generation cephalosporin antimicrobials used in humans and animals to treat infections due to multidrug-resistant (MDR) bacteria. Resistance to ESCs (ESC-R) in Enterobacterales is predominantly due to the production of extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamases (AmpCs). The dynamics of ESBLs and AmpCs are changing across countries and host species, the result of global transmission of ESC-R genes. Plasmids are known to play a key role in this dissemination, but the relative importance of different types of plasmids is not fully understood. In this study, Escherichia coli with the major ESC-R genes blaCTX-M-1, blaCTX-M-15, blaCTX-M-14 (ESBLs) and blaCMY-2 (AmpC), were selected from diverse host species and other sources across Canada, France and Germany, collected between 2003 and 2017. To examine in detail the vehicles of transmission of the ESC-R genes, long- and short-read sequences were generated to obtain complete contiguous chromosome and plasmid sequences (n=192 ESC-R E.coli). The types, gene composition and genetic relatedness of these plasmids were investigated, along with association with isolate year, source and geographical origin, and put in context with publicly available plasmid sequences. We identified five epidemic resistance plasmid subtypes with distinct genetic properties that are associated with the global dissemination of ESC-R genes across multiple E.coli lineages and host species. The IncI1 pST3 blaCTX-M-1 plasmid subtype was found in more diverse sources than the other main plasmid subtypes, whereas IncI1 pST12 blaCMY-2 was more frequent in Canadian and German human and chicken isolates. Clonal expansion also contributed to the dissemination of the IncI1 pST12 blaCMY-2 plasmid in ST131 and ST117 E.coli harbouring this plasmid. The IncI1 pST2 blaCMY-2 subtype was predominant in isolates from humans in France, while the IncF F31:A4:B1 blaCTX-M-15 and F2:A-:B- blaCTX-M-14 plasmid subtypes were frequent in human and cattle isolates across multiple countries. Beyond their epidemic nature with respect to ESC-R genes, in our collection almost all IncI1 pST3 blaCTX-M-1 and IncF F31:A4:B1 blaCTX-M-15 epidemic plasmids also carried multiple antimicrobial resistance (AMR) genes conferring resistance to other antimicrobial classes. Finally, we found genetic signatures in the regions surrounding specific ESC-R genes, identifying the predominant mechanisms of ESC-R gene movement, and using publicly available databases, we identified these epidemic plasmids from widespread bacterial species, host species, countries and continents. We provide evidence that epidemic resistance plasmid subtypes contribute to the global dissemination of ESC-R genes, and in addition, some of these epidemic plasmids confer resistance to multiple other antimicrobial classes. The success of these plasmids suggests that they may have a fitness advantage over other plasmid types and subtypes. Identification and understanding of the vehicles of AMR transmission are crucial to develop and target strategies and interventions to reduce the spread of AMR. This project was supported by the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR), through the Medical Research Council (MRC, MR/R000948/1), the Canadian Institutes of Health Research (CFC-150770), and the Genomics Research and Development Initiative (Government of Canada), the German Federal Ministry of Education and Research (BMBF) grant no. 01KI1709, the French Agency for food environmental and occupational health & safety (Anses), and the French National Reference Center (CNR) for antimicrobial resistance. Support was also provided by the Biotechnology and Biological Sciences Research Council (BBSRC) through the BBSRC Institute Strategic Programme Microbes in the Food ChainBB/R012504/1 and its constituent project BBS/E/F/000PR10348 (Theme 1, Epidemiology and Evolution of Pathogens in the Food Chain).

Phenotypic and Genotypic Characterization of Pan-Drug-Resistant Klebsiella pneumoniae Isolated in Qatar.

In secondary healthcare, carbapenem-resistant Enterobacterales (CREs), such as those observed in Klebsiella pneumoniae, are a global public health priority with significant clinical outcomes. In this study, we described the clinical, phenotypic, and genotypic characteristics of three pan-drug-resistant (PDR) isolates that demonstrated extended resistance to conventional and novel antimicrobials. All patients had risk factors for the acquisition of multidrug-resistant organisms, while microbiological susceptibility testing showed resistance to all conventional antimicrobials. Advanced susceptibility testing demonstrated resistance to broad agents, such as ceftazidime-avibactam, ceftolozane-tazobactam, and meropenem-vaborbactam. Nevertheless, all isolates were susceptible to cefiderocol, suggested as one of the novel antimicrobials that demonstrated potent in vitro activity against resistant Gram-negative bacteria, including CREs, pointing toward its potential therapeutic role for PDR pathogens. Expanded genomic studies revealed multiple antimicrobial-resistant genes (ARGs), including blaNMD-5 and blaOXA derivative types, as well as a mutated outer membrane porin protein (OmpK37).

Unraveling the diversity and dissemination dynamics of antimicrobial resistance genes in Enterobacteriaceae plasmids across diverse ecosystems.

The objective of this study was to investigate the antimicrobial resistance genes (ARGs) in plasmids of Enterobacteriaceae from soil, sewage, and feces of food-producing animals and humans. The plasmid sequences were obtained from the NCBI database. For the identification of ARG, comprehensive antibiotic resistance database (CARD), and ResFinder were used. Gene conservation and evolution were investigated using DnaSP v.6. The transfer potential of the plasmids was evaluated using oriTfinder and a MOB-based phylogenetic tree was reconstructed using Fastree. We identified a total of 1064 ARGs in all plasmids analyzed, conferring resistance to 15 groups of antibiotics, mostly aminoglycosides, beta-lactams, and sulfonamides. The greatest number of ARGs per plasmid was found in enterobacteria from chicken feces. Plasmids from Escherichia coli carrying multiple ARGs were found in all ecosystems. Some of the most abundant genes were shared among all ecosystems, including aph(6)-Id, aph(3'')-Ib, tet(A), and sul2. A high level of sequence conservation was found among these genes, and tet(A) and sul2 are under positive selective pressure. Approximately 62% of the plasmids carrying at least one ARG were potentially transferable. Phylogenetic analysis indicated a potential co-evolution of Enterobacteriaceae plasmids in nature. The high abundance of Enterobacteriaceae plasmids from diverse ecosystems carrying ARGs reveals their widespread distribution and importance.

Complete genome sequences of a Canadian strain of enteroaggregative Escherichia coli (EAEC) with multiple metals and antimicrobial resistance genes isolated from municipal waste-activated sludge.

Enteroaggregative Escherichia coli (EAEC) is an emerging food-borne pathogen causing acute or persistent diarrhea in humans. Here, we report the complete genome sequence of a strain of EAEC with multiple metals and antimicrobial resistance genes isolated from a waste-activated sludge collected from a Canadian municipal wastewater treatment plant.

Genome-centric analyses of 165 metagenomes show that mobile genetic elements are crucial for the transmission of antimicrobial resistance genes to pathogens in activated sludge and wastewater.

Antimicrobial resistance (AMR) threatens the health of humans, animals, and natural ecosystems. In our study, an analysis of 165 metagenomes from wastewater revealed antibiotic-targeted alteration, efflux, and inactivation as the most prevalent AMR mechanisms. We identified several genera correlated with multiple ARGs, including Klebsiella, Escherichia, Acinetobacter, Nitrospira, Ottowia, Pseudomonas, and Thauera, which could have significant implications for AMR transmission. The abundance of bacA, mexL, and aph(3")-I in the genomes calls for their urgent management in wastewater. Our approach could be applied to different ecosystems to assess the risk of potential pathogens containing ARGs. Our findings highlight the importance of managing AMR in wastewater and can help design measures to reduce the transmission and evolution of AMR in these systems.

An investigation of the presence and antimicrobial susceptibility of Enterobacteriaceae in raw and cooked kibble diets for dogs in the United Kingdom.

Raw meat diets (RMD) for dogs are an increasingly popular alternative pet food choice, however studies worldwide have demonstrated them to be contaminated with zoonotic and antimicrobial resistant (AMR) bacteria, including bacteria resistant to critically important antibiotics. Despite this, few data exist surrounding the presence of these bacteria in RMD in the United Kingdom. The present study aimed to identify the most commonly selected RMD and non-raw diets (NRMD) by United Kingdom dog owners. Additionally, it investigated the presence of AMR-Enterobacteriaceae in samples of pre-prepared RMD and cooked commercial kibble dog foods. An online survey investigating diet preferences of United Kingdom dog owners was open for 6 weeks between February-March 2020. From this, the top 10 brands of pre-prepared raw and cooked kibble diets were ascertained and 134 samples purchased (110 RMD, 24 kibble) and subjected to microbiological testing. Bacterial enumeration of E. coli and other Enterobacteriaceae was undertaken, and the presence of Salmonella spp. and AMR-E. coli within samples determined. Whole genome sequencing was undertaken on Salmonella spp. and third-generation cephalosporin-resistant 3GCR-E. coli isolates. Pre-prepared RMD was most commonly selected by dog owners who fed RMD, and cooked commercial complete dry food was most frequently fed by owners who fed NRMD. Damaged and leaking packaging was observed in samples of RMD, alongside variability in information provided surrounding product traceability. Counts of E. coli and other Enterobacteriaceae exceeding >5,000 CFU/g were identified in samples of RMD. AMR-, extended-spectrum beta-lactamase (ESBL)-producing and 3GCR-E. coli was isolated from 39, 14 and 16% of RMD samples, respectively. Multiple antimicrobial resistance genes were identified in 3GCR-E. coli isolates. Of the ESBL encoding genes, blaCTX-M-15 was most commonly identified. S. enterica was isolated from 5% of RMD samples. No Enterobacteriaceae were isolated from any of the cooked kibble samples. The present study suggests that pre-prepared RMD available for dogs in the United Kingdom can be contaminated with zoonotic and AMR-Enterobacteriaceae. RMDs, therefore, are potentially an important One Health concern. Veterinary and medical professionals, pet food retailers and pet owners should be aware of these risks; and stringent hygiene measures should be practiced if owners choose to feed RMD.

Emergence and genomic characteristics of multi-drug-resistant Salmonella in pet turtles and children with diarrhoea.

Pet turtles are a well-recognized source of human salmonellosis, posing a threat to human health, particularly children who commonly keep pet turtles. To date, the genomic characteristics of Salmonella among pet turtles and children has not been well described. We investigated the prevalence, antimicrobial resistance (AMR) and genomic characteristics of Salmonella from pet turtles in Beijing, China. In total, 9.6 % (46/480) of pet turtles were positive for Salmonella with S. Thompson being the dominant serovar (19/46) in 2019. Moreover, 80.4 % of Salmonella were multi-drug resistant (MDR) and 60.7 % were resistant to ampicillin, streptomycin, sulfonamides and tetracycline (ASSuT). We further compared the genomes of S. Thompson isolates from pet turtles (n=19) with those from children with diarrhoea (n=28) in the same region and year, most of which were sequence type (ST)26, with one novel ST7937 identified from a child-associated isolate. S. Thompson isolates from children with diarrhoea exhibited less resistance than isolates from pet turtles. Most MDR isolates possessed multiple AMR genes, including the AmpC β-lactamase-encoding genes bla DHA-15 and bla DHA-1 which co-occurred with the IncA/C and IncHI plasmid replicon types. To the best of our knowledge, this is the first time that the bla DHA-15 gene has been detected from Salmonella. Several pet turtle-associated S. Thompson isolates comprised phylogenetically close clusters with those from children with diarrhoea (<20 SNP differences). Bayesian analysis demonstrated that the Chinese ST26 S. Thompson strains had a recent evolutionary history and evolved into two major clades, with one clade acquiring various resistant plasmids. Our findings revealed the emergence of MDR Salmonella among pet turtles in China and provided evidence for the interspecies transmission of S. Thompson.

Rapid Detection of Antimicrobial Resistance Genes in Critically Ill Children Using a Custom TaqMan Array Card.

Bacteria are identified in only 22% of critically ill children with respiratory infections treated with antimicrobial therapy. Once an organism is isolated, antimicrobial susceptibility results (phenotypic testing) can take another day. A rapid diagnostic test identifying antimicrobial resistance (AMR) genes could help clinicians make earlier, informed antimicrobial decisions. Here we aimed to validate a custom AMR gene TaqMan Array Card (AMR-TAC) for the first time and assess its feasibility as a screening tool in critically ill children. An AMR-TAC was developed using a combination of commercial and bespoke targets capable of detecting 23 AMR genes. This was validated using isolates with known phenotypic resistance. The card was then tested on lower respiratory tract and faecal samples obtained from mechanically ventilated children in a single-centre observational study of respiratory infection. There were 82 children with samples available, with a median age of 1.2 years. Major comorbidity was present in 29 (35%) children. A bacterial respiratory pathogen was identified in 13/82 (16%) of children, of which 4/13 (31%) had phenotypic AMR. One AMR gene was detected in 49/82 (60%), and multiple AMR genes were detected in 14/82 (17%) children. Most AMR gene detections were not associated with the identification of phenotypic AMR. AMR genes are commonly detected in samples collected from mechanically ventilated children with suspected respiratory infections. AMR-TAC may have a role as an adjunct test in selected children in whom there is a high suspicion of antimicrobial treatment failure.

Analysis of resistance genes of carbapenem-resistant Providencia rettgeri using whole genome sequencing

ObjectiveThis study aimed to investigate the clinical infection characteristics and analyze the resistance gene carrying status of carbapenem-resistant Providencia rettgeri via whole genome sequencing (WGS).MethodsCarbapenem-resistant P. rettgeri were collected from clinical patients between January 2020 and December 2021, and their susceptibility to 19 antimicrobial drugs was determined using the VITEK 2 Compact system and Kirby–Bauer (KB) disk diffusion method. The Illumina platform was used to perform WGS of the P. rettgeri isolates, and the resistance genes carried by the Carbapenem-resistant P. rettgeri strains were detected via ABRicate software. The phylogenetic tree was constructed by thirty-four strains including twenty-eight strains downloaded from NCBI database and the carbapenem-resistant six P. rettgeri strains in this study. Which based on genomic single nucleotide polymorphism (SNP) to understand the affinities of the carbapenem-resistant P. rettgeri strains.ResultsSix carbapenem-resistant P. rettgeri strains were isolated from five different clinical departments using the blood, urine, sputum, and secretion specimens. These infected patients are middle-aged and elderly people with a history of severe trauma, tumors, hypertension, and various other underlying diseases, and invasive procedures. Antimicrobial sensitivity testing showed that all strains presented resistance to ampicillin-sulbactam, ceftazidime, ciprofloxacin, levofloxacin, and ertapenem, whereas they exhibited full susceptibility to cefepime and amikacin. Most strains demonstrated high resistance to β-lactams, aminoglycosides, and sulfonamides. Thirty-five resistance genes were identified by ABRicate. All carbapenem-resistant P. rettgeri strains carried aminoglycoside, fluoroquinolone, chloramphenicol, rifampicin, sulfonamide, and β-lactam resistance genes, and most importantly, all strains possessed the carbapenem resistance gene blaNDM−1. The six P. rettgeri strains in this study and the 28 carbapenem-resistant P. rettgeri strains from the NCBI database were divided into four evolutionary groups. The WF3643, WF3849, WF3822, and WF3821 strains in this study were in the same evolutionary group (clade A), while the closely related WF3099 and WF3279 strains were in different evolutionary groups (clade B and clade D), respectively. The WF3099 strain was distantly related to the other five strains.ConclusionCarbapenem-resistant P. rettgeri strains were mostly isolated from middle-aged and older patients with a history of surgery or serious underlying diseases, and they were found to cause multisystem infections. All Carbapenem-resistant P. rettgeri strains in this study carried blaNDM−1 and multiple antimicrobial drug resistance genes. Furthermore, the P. rettgeri strains in this study were closely related, suggesting the possibility of nosocomial infections. Therefore, our study highlights the need for research on P. rettgeri to control the spread of these nosocomial infections.

Phenotypic and genotypic antimicrobial resistance correlation and plasmid characterization in Salmonella spp. isolates from Italy reveal high heterogeneity among serovars.

The spread of antimicrobial resistance among zoonotic pathogens such as Salmonella is a serious health threat, and mobile genetic elements (MGEs) carrying antimicrobial resistance genes favor this phenomenon. In this work, phenotypic antimicrobial resistance to commonly used antimicrobials was studied, and the antimicrobial resistance genes (ARGs) and plasmid replicons associated with the resistances were determined. Eighty-eight Italian Salmonella enterica strains (n = 88), from human, animal and food sources, isolated between 2009 and 2019, were selected to represent serovars with different frequency of isolation in human cases of salmonellosis. The presence of plasmid replicons was also investigated. Resistances to sulphonamides (23.9%), ciprofloxacin (27.3%), ampicillin (29.5%), and tetracycline (32.9%) were the most found phenotypes. ARGs identified in the genomes correlated with the phenotypical results, with blaTEM-1B, sul1, sul2, tetA and tetB genes being frequently identified. Point mutations in gyrA and parC genes were also detected, in addition to many different aminoglycoside-modifying genes, which, however, did not cause phenotypic resistance to aminoglycosides. Many genomes presented plasmid replicons, however, only a limited number of ARGs were predicted to be located on the contigs carrying these replicons. As an expectation of this, multiple ARGs were identified on contigs with IncQ1 plasmid replicon in strains belonging to the monophasic variant of Salmonella Typhimurium. In general, high variability in ARGs and plasmid replicons content was observed among isolates, highlighting a high level of heterogeneity in Salmonella enterica. Irrespective of the serovar., many of the ARGs, especially those associated with critically and highly important antimicrobials for human medicine were located together with plasmid replicons, thus favoring their successful dissemination.

Genomic traits of multidrug resistant enterotoxigenic Escherichia coli isolates from diarrheic pigs.

Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) infections poses a significant challenge in global pig farming. To address this issue, the study was conducted to identify and characterize 19 ETEC isolates from fecal samples of diarrheic pigs sourced from large-scale farms in Sichuan Province, China. Whole-genome sequencing and bioinformatic analysis were utilized for identification and characterization. The isolates exhibited substantial resistance to cefotaxime, ceftriaxone, chloramphenicol, ciprofloxacin, gentamicin, ampicillin, tetracycline, florfenicol, and sulfadiazine, but were highly susceptible to amikacin, imipenem, and cefoxitin. Genetic diversity among the isolates was observed, with serotypes O22:H10, O163orOX21:H4, and O105:H8 being dominant. Further analysis revealed 53 resistance genes and 13 categories of 195 virulence factors. Of concern was the presence of tet(X4) in some isolates, indicating potential public health risks. The ETEC isolates demonstrated the ability to produce either heat-stable enterotoxin (ST) alone or both heat-labile enterotoxin (LT) and ST simultaneously, involving various virulence genes. Notably, STa were linked to human disease. Additionally, the presence of 4 hybrid ETEC/STEC isolates harboring Shiga-like toxin-related virulence factors, namely stx2a, stx2b, and stx2e-ONT-2771, was identified. IncF plasmids carrying multiple antimicrobial resistance genes were prevalent, and a hybrid ETEC/STEC plasmid was detected, highlighting the role of plasmids in hybrid pathotype emergence. These findings emphasized the multidrug resistance and pathogenicity of porcine-origin ETEC strains and the potential risk of epidemics through horizontal transmission of drug resistance, which is crucial for effective control strategies and interventions to mitigate the impact on animal and human health.

Whole genome sequence analysis of Aeromonas spp. isolated from ready-to-eat seafood: antimicrobial resistance and virulence factors.

Aeromonas are widespread in aquatic environments and are considered emerging pathogens in humans and animals. Multidrug resistant (MDR) Aeromonas circulating in the aquatic environment and food production chain can potentially disseminate antimicrobial resistance (AMR) to humans via the foodborne route. In this study, we aimed to investigate AMR and virulence factors of 22 Aeromonas strains isolated from ready-to-eat (RTE) seafood. A multilocus phylogenetic analysis (MLPA) using the concatenated sequences of six housekeeping genes (gyrB, rpoD, gyrA, recA, dnaJ, and dnaX) in the 22 Aeromonas genomes and average nucleotide identity (ANI) analysis revealed eight different species; A. caviae, A. dhakensis, A. hydrophila, A. media, A. rivipollensis, A. salmonicida, A. bestiarum, and A. piscicola. The presence of virulence genes, AMR genes and mobile genetic elements (MGEs) in the Aeromonas genomes was predicted using different databases. Our data showed that the genes responsible for adherence and motility (Msh type IV pili, tap type IV pili, polar flagella), type II secretion system (T2SS) and hemolysins were present in all strains, while the genes encoding enterotoxins and type VI secretion system (T6SS) including major effectors were highly prevalent. Multiple AMR genes encoding β-lactamases such as cphA and blaOXA were detected, and the distribution of those genes was species-specific. In addition, the quinolone resistance gene, qnrS2 was found in a IncQ type plasmid of the A. rivopollensis strain A539. Furthermore, we observed the co-localization of a class I integron (intl1) with two AMR genes (sul1 and aadA1), and a Tn521 transposon carrying a mercury operon in A. caviae strain SU4-2. Various MGEs including other transposons and insertion sequence (IS) elements were identified without strongly associating with detected AMR genes or virulence genes. In conclusion, Aeromonas strains in RTE seafood were potentially pathogenic, carrying several virulence-related genes. Aeromonas carrying multiple AMR genes and MGEs could potentially be involved in the dissemination and spread of AMR genes to other bacterial species residing in the same environment and possibly to humans. Considering a One-Health approach, we highlight the significance of monitoring AMR caused by Aeromonas circulating in the food chain.

Characterization of microbial community and antibiotic resistome in intra urban water, Wenzhou China.

The present study investigated the water quality index, microbial composition and antimicrobial resistance genes in urban water habitats. Combined chemicals testing, metagenomic analyses and qualitative PCR (qPCR) were conducted on 20 locations, including rivers from hospital surrounds (n = 7), community surrounds (n = 7), and natural wetlands (n = 6). Results showed that the indexes of total nitrogen, phosphorus, and ammonia nitrogen of hospital waters were 2-3 folds high than that of water from wetlands. Bioinformatics analysis revealed a total of 1,594 bacterial species from 479 genera from the three groups of water samples. The hospital-related samples had the greatest number of unique genera, followed by those from wetlands and communities. The hospital-related samples contained a large number of bacteria associated with the gut microbiome, including Alistipes, Prevotella, Klebsiella, Escherichia, Bacteroides, and Faecalibacterium, which were all significantly enriched compared to samples from the wetlands. Nevertheless, the wetland waters enriched bacteria from Nanopelagicus, Mycolicibacterium and Gemmatimonas, which are typically associated with aquatic environments. The presence of antimicrobial resistance genes (ARGs) that were associated with different species origins in each water sample was observed. The majority of ARGs from hospital-related samples were carried by bacteria from Acinetobacter, Aeromonas and various genera from Enterobacteriaceae, which each was associated with multiple ARGs. In contrast, the ARGs that were exclusively in samples from communities and wetlands were carried by species that encoded only 1 to 2 ARGs each and were not normally associated with human infections. The qPCR showed that water samples of hospital surrounds had higher concentrations of intI1 and antimicrobial resistance genes such as tetA, ermA, ermB, qnrB, sul1, sul2 and other beta-lactam genes. Further genes of functional metabolism reported that the enrichment of genes associated with the degradation/utilization of nitrate and organic phosphodiester were detected in water samples around hospitals and communities compared to those from wetlands. Finally, correlations between the water quality indicators and the number of ARGs were evaluated. The presence of total nitrogen, phosphorus, and ammonia nitrogen were significantly correlated with the presence of ermA and sul1. Furthermore, intI1 exhibited a significant correlation with ermB, sul1, and blaSHV, indicating a prevalence of ARGs in urban water environments might be due to the integron intI1's diffusion-promoting effect. However, the high abundance of ARGs was limited to the waters around the hospital, and we did not observe the geographical transfer of ARGs along with the river flow. This may be related to water purifying capacity of natural riverine wetlands. Taken together, continued surveillance is required to assess the risk of bacterial horizontal transmission and its potential impact on public health in the current region.

Antimicrobial Resistance Profiles and Co-Existence of Multiple Antimicrobial Resistance Genes in mcr-Harbouring Colistin-Resistant Enterobacteriaceae Isolates Recovered from Poultry and Poultry Meats in Malaysia.

The co-existence of the colistin resistance (mcr) gene with multiple drug-resistance genes has raised concerns about the possibility of the development of pan-drug-resistant bacteria that will complicate treatment. This study aimed to investigate the antibiotic resistance profiles and co-existence of antibiotic resistance genes among the colistin-resistant Enterobacteriaceae isolates recovered from poultry and poultry meats. The antibiotic susceptibility to various classes of antibiotics was performed using the Kirby-Bauer disk diffusion method and selected antimicrobial resistance genes were detected using PCR in a total of 54 colistin-resistant Enterobacteriaceae isolates including Escherichia coli (E. coli) (n = 32), Salmonella spp. (n = 16) and Klebsiella pneumoniae (K. pneumoniae) (n = 6) isolates. Most of the isolates had multi-drug resistance (MDR), with antibiotic resistance against up to seven classes of antibiotics. All mcr-harbouring, colistin-resistant Enterobacteriaceae isolates showed this MDR (100%) phenotype. The mcr-1 harbouring E. coli isolates were co-harbouring multiple antibiotic resistance genes. The seven most commonly identified resistance genes (blaTEM, tetA, floR, aac-3-IV, aadA1, fosA, aac(6_)-lb) were detected in an mcr-1-harbouring E. coli isolate recovered from a cloacal swab. The mcr-5 harbouring Salmonella spp. isolate recovered from poultry meats was positive for blaTEM, tetA, floR, aac-3-IV, fosA and aac(6_)-lb genes. In conclusion, the colistin-resistant Enterobacteriaceae with mcr genes co-existing multiple clinically important antimicrobial resistance genes in poultry and poultry meats may cause potential future threats to infection treatment choices in humans and animals.

Inter-species diversity and functional genomic analyses of closed genome assemblies of clinically isolated, megaplasmid-containing Enterococcus raffinosus Er676 and ATCC49464.

Enterococcus raffinosus is an understudied member of its genus possessing a characteristic megaplasmid contributing to a large genome size. Although less commonly associated with human infection compared to other enterococci, this species can cause disease and persist in diverse niches such as the gut, urinary tract, blood and environment. Few complete genome assemblies have been published to date for E. raffinosus . In this study, we report the complete assembly of the first clinical urinary E. raffinosus strain, Er676, isolated from a postmenopausal woman with history of recurrent urinary tract infection. We additionally completed the assembly of clinical type strain ATCC49464. Comparative genomic analyses reveal inter-species diversity driven by large accessory genomes. The presence of a conserved megaplasmid indicates it is a ubiquitous and vital genetic feature of E. raffinosus . We find that the E. raffinosus chromosome is enriched for DNA replication and protein biosynthesis genes while the megaplasmid is enriched for transcription and carbohydrate metabolism genes. Prophage analysis suggests that diversity in the chromosome and megaplasmid sequences arises, in part, from horizontal gene transfer. Er676 demonstrated the largest genome size reported to date for E. raffinosus and the highest probability of human pathogenicity. Er676 also possesses multiple antimicrobial resistance genes, of which all but one are encoded on the chromosome, and has the most complete prophage sequences. Complete assembly and comparative analyses of the Er676 and ATCC49464 genomes provide important insight into the inter-species diversity of E. raffinosus that gives it its ability to colonize and persist in the human body. Investigating genetic factors that contribute to the pathogenicity of this species will provide valuable tools to combat diseases caused by this opportunistic pathogen.

In-vitro activity of oral third-generation cephalosporins plus clavulanate against ESBL-producing Enterobacterales isolates from the MERINO trial

Extended-spectrum-beta-lactamase (ESBL)-producing Enterobacterales as a cause of community-acquired uncomplicated urinary tract infection (UTI) is on the rise. Currently, there are minimal oral treatment options. New combinations of existing oral third-generation cephalosporins paired with clavulanate may overcome resistance mechanisms seen in these emerging uropathogens. Ceftriaxone-resistant Escherichia coli and Klebsiella pneumoniae containing CTX-M-type ESBLs or AmpC, in addition to narrow-spectrum OXA and SHV enzymes, were selected from blood culture isolates obtained from the MERINO trial. Minimum inhibitory concentration (MIC) values of third-generation cephalosporins (cefpodoxime, ceftibuten, cefixime, cefdinir), both with and without clavulanate, were determined. One hundred and one isolates were used with ESBL, AmpC and narrow-spectrum OXA genes (e.g. OXA-1, OXA-10) present in 84, 15 and 35 isolates, respectively. Susceptibility to oral third-generation cephalosporins alone was very poor. Addition of 2 mg/L clavulanate reduced the MIC50 values (cefpodoxime MIC50 2 mg/L, ceftibuten MIC50 2 mg/L, cefixime MIC50 2 mg/L, cefdinir MIC50 4 mg/L) and restored susceptibility (33%, 49%, 40% and 21% susceptible, respectively) in a substantial number of isolates. This finding was less pronounced in isolates co-harbouring AmpC. In-vitro activity of these new combinations may be limited in real-world Enterobacterales isolates co-harbouring multiple antimicrobial resistance genes. Pharmacokinetic/pharmacodynamic data would be useful for further evaluation of their activity.