Epidemic Plasmids Research Articles

Genomic insights into epidemic plasmids carrying bla CTX-M and mcr-1 genes in Escherichia coli from Lebanese broiler production.

In a previous nationwide survey in the Lebanese broiler production, multidrug-resistant CTX-M-producing E. coli were found to carry the mobile colistin resistance gene mcr-1. To investigate the mobile genetic supports responsible for the spread of these resistance genes among E. coli in healthy broilers in Lebanon. Thirty-three bla CTX-M and mcr-1 positive E. coli of various sequence types from 17 broilers farms were subjected to conjugation assays. Long-read sequencing (Oxford Nanopore Technologies) and hybrid assembly were performed to determine complete plasmid sequences and their phylogenetic diversity. Twenty-nine conjugative IncFII plasmids harboured the extended-spectrum β-lactamase genes bla CTX-M-3 (n = 25) or bla CTX-M-55 (n = 4). Highly related IncF2:A-:B-/bla CTX-M-3 plasmids differing only through IS-mediated genetic rearrangements in antibiotic resistance gene clusters were found in genetically diverse E. coli strains isolated from distant farms. The mobile colistin resistance genes mcr-1.1 and mcr-1.26 were carried by IncX4 and IncI2 plasmids. Worryingly, in one isolate, the ISEcp1-bla CTX-M-55 transposable unit was found integrated in a mcr-1.26-carrying IncX4 plasmid. Beside expanded cephalosporins and colistin resistances, all E. coli isolates were multidrug-resistant with different additional resistances against aminoglycosides, (fluoro)quinolones, fosfomycin, phenicols, sulphonamides, tetracycline and trimethoprim. Closely related blaCTX-M-3/55-borne IncF2:A-:B- plasmids harbouring variable MDR regions and mcr-1 carrying IncX4 plasmids are widely disseminated in the E. coli population of healthy broilers in Lebanon. Further surveillance programmes of antimicrobial resistance and interventions to reduce the abusive use of medically important antibiotics are necessary to limit the spread of resistances in food-producing animals in Lebanon.

Carriage Rate of Enterobacterales Resistant to Extended-Spectrum Cephalosporins in the Tunisian Population.

Enterobacterales resistant to extended-spectrum cephalosporins (ESC) are a marker of the antimicrobial resistance (AMR) burden. They are infecting humans, but the intestinal microbiota can also be transiently colonized without developing symptoms. Healthy carriage can promote silent dissemination of resistant bacteria, and data on this colonization are often lacking. Between 2021 and 2023, a sampling of healthy Tunisian people was carried out. Fecal samples (n = 256) were plated on selective agar, and all collected isolates were characterized by phenotypic (antibiograms) and genomic (whole-genome sequencing) methods. A total of 26 (26/256, 10.2%) isolates were collected, including 24 Escherichia coli and 2 Klebsiella pneumoniae. In total, 17 isolates (15 E. coli and 2 K. pneumoniae) presented an ESBL phenotype conferred by the blaCTX-M-15 gene, and 9 E. coli isolates presented an AmpC phenotype conferred by the blaDHA-1 gene. K. pneumoniae belonged to ST1564 and ST313, while E. coli belonged to diverse STs including the pandemic ST131 clone. Clonally related ST349 E. coli isolates carrying the blaDHA-1 gene were found in nine individuals. In parallel, four blaCTX-M-15 -positive E. coli isolates carried this ESC-resistance gene on an epidemic plasmid IncF/F-:A-:B53 previously identified in Tunisian pigeons and fish. These findings highlight the spread of genetically diverse ESC-resistant Enterobacterales as well as an epidemic plasmid in Tunisia, emphasizing the need for antimicrobial stewardship to limit the transmission of these resistances in the Tunisian population.

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).

Genomic investigation of multispecies and multivariant blaNDM outbreak reveals key role of horizontal plasmid transmission.

New Delhi metallo-β-lactamases (NDMs) are major contributors to the spread of carbapenem resistance globally. In Australia, NDMs were previously associated with international travel, but from 2019 we noted increasing incidence of NDM-positive clinical isolates. We investigated the clinical and genomic epidemiology of NDM carriage at a tertiary-care Australian hospital from 2016 to 2021. We identified 49 patients with 84 NDM-carrying isolates in an institutional database, and we collected clinical data from electronic medical record. Short- and long-read whole genome sequencing was performed on all isolates. Completed genome assemblies were used to assess the genetic setting of blaNDM genes and to compare NDM plasmids. Of 49 patients, 38 (78%) were identified in 2019-2021 and only 11 (29%) of 38 reported prior travel, compared with 9 (82%) of 11 in 2016-2018 (P = .037). In patients with NDM infection, the crude 7-day mortality rate was 0% and the 30-day mortality rate was 14% (2 of 14 patients). NDMs were noted in 41 bacterial strains (ie, species and sequence type combinations). Across 13 plasmid groups, 4 NDM variants were detected: blaNDM-1, blaNDM-4, blaNDM-5, and blaNDM-7. We noted a change from a diverse NDM plasmid repertoire in 2016-2018 to the emergence of conserved blaNDM-1 IncN and blaNDM-7 IncX3 epidemic plasmids, with interstrain spread in 2019-2021. These plasmids were noted in 19 (50%) of 38 patients and 35 (51%) of 68 genomes in 2019-2021. Increased NDM case numbers were due to local circulation of 2 epidemic plasmids with extensive interstrain transfer. Our findings underscore the challenges of outbreak detection when horizontal transmission of plasmids is the primary mode of spread.

First report of blaOXA-181-carrying IncX3 plasmids in multidrug-resistant Enterobacter hormaechei and Serratia nevei recovered from canine and feline opportunistic infections.

blaOXA-181 is a significant carbapenemase-encoding gene, usually associated with an epidemic IncX3 plasmid found in Enterobacterales worldwide. In this article, we revealed six carbapenemase-producing (CP) Enterobacter hormaechei and two CP Serratia nevei strains harboring blaOXA-181-carrying IncX3 and multidrug resistance plasmids recovered from dogs and cats in Thailand. The carriage of these plasmids can promote extensively drug-resistant properties, limiting antimicrobial treatment options in veterinary medicine. Since E. hormaechei and S. nevei harboring blaOXA-181-carrying IncX3 plasmids have not been previously reported in dogs and cats, our findings provide the first evidence of dissemination of the epidemic plasmids in these bacterial species isolated from animal origins. Pets in communities can serve as reservoirs of significant antimicrobial resistance determinants. This situation places a burden on antimicrobial treatment in small animal practice and poses a public health threat.

Clonal and plasmidic dissemination of critical antimicrobial resistance genes through clinically relevant ExPEC and APEC-like lineages (ST) in the dairy cattle population of Québec, Canada.

Antimicrobial resistance can be effectively limited by improving the judicious use of antimicrobials in food production. However, its effect on the spread of AMR genes in animal populations is not well described. In the province of Québec, Canada, a new legislation implemented in 2019 has led to an unprecedented reduction in the use of critical antimicrobials in dairy production. We aimed to investigate the potential link between ESBL/AmpC E. coli isolated before and after legislation and to determine the presence of plasmids carrying genes responsible for critical AMR. We collected fecal samples from calves, cows, and manure pit from 87 Québec dairy farms approximately 2 years before and 2 years after the legislation came into effect. The whole genomes of 183 presumptive ESBL/AmpC E. coli isolated after cefotaxime enrichment were sequenced. Their phylogenetic characteristics (MLST, serogroup, cgMLST) and the presence of virulence and resistance genes and replicons were examined. A maximum likelihood phylogenetic tree was constructed based on single nucleotide polymorphism (SNPs). We identified 10 clonal lineages (same cgMLST) and 7 clones (SNPs ≤ 52). Isolates belonging to these clones could be found on different farms before and after the legislation, strongly suggesting a clonal spread of AMR genes in the population during this 4-year period. All isolates were multidrug resistant (MDR), with clone 2 being notable for the presence of macrolide, fluoroquinolone, and third-generation cephalosporin resistance genes. We also identified clinically relevant ExPEC (ST10) and APEC-like lineages (ST117, ST58, ST88) associated with the presence of ExPEC and APEC virulence genes, respectively. Our data also suggests the presence of one epidemic plasmid belonging to the IncY incompatibility group and carrying qnrs1 and blaCTX-M-15. We demonstrated that AMR genes spread through farms and can persist over a 4-year period in the dairy cattle population through both plasmids and E. coli clones, despite the restriction of critical antimicrobial use. MDR ExPEC and APEC-like STs are present in the normal microbiota of cattle (more frequently in calves). These data increase our knowledge on gene dissemination dynamics and highlight the fact that biosecurity measures should be enhanced in this industry to limit such dissemination.

Emergence of plasmid-mediated high-level tigecycline resistance gene tet(X4) in Enterobacterales from retail aquatic products

The spread of antimicrobial-resistant microbes and genes in various foods poses a significant threat to public health. Of particular global concern is the plasmid-mediated tigecycline resistance gene tet(X4), which, while identified in various sources, has not hitherto been reported in aquatic products. This study aimed to investigate the occurrence and characterization of tigecycline-resistant strains from aquatic products. A total of 73 nonrepetitive seafood samples were purchased from 26 farmers’ markets to detect tigecycline-resistant strains. Of these, nine Escherichia coli strains (comprising two ST58, one ST195, ST10, ST48, ST88, ST877, ST1244, ST14462) and one Citrobacter meridianamericanus, recovered from nine (12.33 %, 9/73) seafood samples (fish, n = 7; shrimp, clam and crab, n = 1 respectively), were positive for the tet(X4). Notably, phylogenetic analysis showed that E. coli ST195, a common ST carrying tet(X4), has a close phylogenetic relationship (23∼48 SNPs) with 32 tet(X4)-harboring E. coli ST195 isolates (isolated from pigs, animal foods, vegetable, and humans) deposited in NCBI database. Additionally, E. coli ST58 was closely (2 SNPs) related to one tet(X4)-positive E. coli strain from retail vegetables documented in the NCBI database. Whole genome sequencing and bioinformatic analysis revealed that tet(X4) genes were located on IncX1 (7 E. coli) or hybrid plasmid IncFIA(HI1)/IncHI1B(R27)/IncHI1A (2 E.coli and one C. meridianamericanus). These plasmids displayed high homology with those of plasmids from other sources deposited in GenBank database. These findings underscore the role of epidemic clones and plasmids in driving the dissemination of tet(X4) gene within Enterobacterales of aquatic products origin. To the best of our knowledge, this is the first report of tet(X4)-positive Enterobacterales from aquatic products. The pervasive propagation of tet(X4) gene facilitated by epidemic plasmids and clones across food animals, food products, humans, and the environment presents a serious threat to public health.

Multidrug-Resistant Escherichia coli Isolate of Chinese Bovine Origin Carrying the blaCTX-M-55 Gene Located in IS26-Mediated Composite Translocatable Units.

Elevated detection rates of the blaCTX-M-55 gene in animals have been reported as a result of antibiotic misuse in clinics. To investigate the horizontal transfer mechanism of blaCTX-M-55 and its associated mobile genetic elements (MGEs), we isolated 318 nonrepetitive strains of Escherichia coli (E. coli) from bovine samples in Xinjiang and Gansu provinces, China. All E. coli strains were screened for the CTX-M-55 gene using PCR. The complete genomic data were sequenced using the PacBio triplet sequencing platform and corrected using the Illumina data platform. The genetic environment of the plasmids carrying the resistance blaCTX-M-55 gene was mapped using the software Easyfig2.2.3 for comparison. The results showed that all blaCTX-M-55-positive strains were resistant to multiple antibiotics. Five strains of Escherichia coli carry the blaCTX-M-55 gene, which is adjacent to other resistance genes and is located on the IncHI2-type plasmid. Four of the five blaCTX-M-55-harbor strains carried translocatable units (TUs). All the donor bacteria carrying the blaCTX-M-55 genes could transfer horizontally to the recipient (E. coli J53 Azr). This study demonstrates that the transmission of blaCTX-M-55 is localized on IS26-flanked composite transposons. The cotransmission and prevalence of blaCTX-M-55 with other MDR resistance genes on epidemic plasmids require enhanced monitoring and control.

Nosocomial dissemination of blaIMP-4 among Klebsiella pneumoniae by horizontal gene transfer and clonal spread: the epidemic IncN plasmids and the emerging high-risk IMP-4-producing ST101 clone.

To determine the genomic features of IMP-4-producing Klebsiella pneumoniae isolates recovered from paediatric patients and the transmission dynamics of blaIMP-4. IMP-producing K. pneumoniae isolates were collected from paediatric patients in Shanghai Children's Medical Center from 2013 to 2020. WGS was performed for all isolates, and the complete genomes of three IMP-4-producing isolates were generated. The distribution of blaIMP-4-harbouring plasmids was determined, and a conjugation assay was employed to investigate the horizontal transfer of blaIMP-4-harbouring plasmids. We collected 21 blaIMP-carrying K. pneumoniae isolates, with IMP-4 (16/21, 76.2%) as the predominant subtype, followed by IMP-8 (n = 3) and IMP-26 (n = 2). IMP-4-producing isolates displayed a diverse population structure and all blaIMP-4 genes were located on plasmids, including IncN (n = 9), IncHI5 (n = 5), IncFII(K) (n = 1) and IncFII(pKP91) (n = 1), although only IncN plasmids were conjugative. Clonal transmission of ST101 strains carrying IncHI5 blaIMP-4-harbouring plasmids was observed, and the acquisition of blaIMP-4 by the international high-risk ST101 clone constituted a novel combination of ST101 clone and carbapenemase genes. Plasmid analysis demonstrated that the conjugal transfer of the IncHI5 blaIMP-4-harbouring plasmid might be blocked by the ST101 bacterial host. The horizontal transfer of IncN plasmids and clonal spread of the international high-risk ST101 clone facilitated the nosocomial dissemination of blaIMP-4 among K. pneumoniae. The emerging IMP-4-producing ST101 clone displays diverse combinations of carbapenemase genes, and this clone could be a continually evolving threat and warrants prospective monitoring.

Dissemination of IncI plasmid encoding bla CTX-M-1 is not hampered by its fitness cost in the pig's gut.

Multiresistance plasmids belonging to the IncI incompatibility group have become one of the most pervasive plasmid types in extended-spectrum beta-lactamase-producing Escherichia coli of animal origin. The extent of the burden imposed on the bacterial cell by these plasmids seems to modulate the emergence of "epidemic" plasmids. However, in vivo data in the natural environment of the strains are scarce. Here, we investigated the cost of a bla CTX-M-1-IncI1 epidemic plasmid in a commensal E. coli animal strain, UB12-RC, before and after oral inoculation of 15 6- to 8-week- old specific-pathogen-free pigs. Growth rate in rich medium was determined on (i) UB12-RC and derivatives, with or without plasmid, in vivo and/or in vitro evolved, and (ii) strains that acquired the plasmid in the gut during the experiment. Although bla CTX-M-1-IncI1 plasmid imposed no measurable burden on the recipient strain after conjugation and during the longitudinal carriage in the pig's gut, we observed a significant difference in the bacterial growth rate between IncI1 plasmid-carrying and plasmid-free isolates collected during in vivo carriage. Only a few mutations on the chromosome of the UB12-RC derivatives were detected by whole-genome sequencing. RNA-Seq analysis of a selected set of these strains showed that transcriptional responses to the bla CTX-M-1-IncI1 acquisition were limited, affecting metabolism, stress response, and motility functions. Our data suggest that the effect of IncI plasmid on host cells is limited, fitness cost being insufficient to act as a barrier to IncI plasmid spread among natural population of E. coli in the gut niche.

An IncN-ST7 epidemic plasmid mediates the dissemination of carbapenem-resistant Klebsiella pneumoniae in a neonatal intensive care unit in China over 10 years

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has widely disseminated globally, but its epidemiological characterization and clinical significance in paediatric patients are not well understood. In this study, we aimed to trace the dissemination dynamics of CRKP in the neonatal intensive care unit (NICU) of a tertiary hospital over a 10-y period. We collected 67 non-duplicate K. pneumoniae species complex isolates from the NICU with patient metadata during 2009-2018. Antimicrobial susceptibility was determined by the agar or broth microdilution method. Risk factors for CRKP-positive patients were identified by univariate and multivariate analysis. Genetic characterization was dissected by whole-genome sequencing. Plasmid transmissibility, stability, and fitness were assessed. Thirty-four of 67 isolates (50.75%) were identified as CRKP. Premature rupture of membranes, gestational age, and invasive procedures are independent risk factors for CRKP-positive patients. The annual isolation rate of CRKP varied between 0% and 88.9%, and multiple clonal replacements were observed during the study period, which could be largely due to the division of the NICU. All but one CRKP produced IMP-4 carbapenemase, which was encoded by an IncN-ST7 epidemic plasmid, suggesting that the IncN-ST7 plasmid mediated the CRKP dissemination in the NICU over 10 y. The same plasmid was found in several CRKP isolates from adult patients, of which two ST17 isolates from the neurosurgery department shared a high homology with the ST17 isolates from the NICU, indicating possible cross-departmental transmission. Our study highlights the urgent need for infection control measures targeting high-risk plasmids like IncN-ST7.

Molecular ecology of highest priority critically important antibiotic resistant Escherichia coli from mammals housed at an urban zoo.

Zoos are environments where species of highly valued animals are kept largely separated from others and the wider world. We report the molecular ecology of critically important antibiotic resistant (ABR) Escherichia coli carried by 28 mammalian species housed in a zoo located in an urban residential district. Over 3 months we collected 167 faecal samples from captive mammals and processed for E. coli resistant to third-generation cephalosporins (3GC-R) and fluoroquinolones (FQ-R). Isolates were sequenced using Illumina. We identified high rates of faecal sample-level positivity, with 50%, 57% and 36% of mammalian species excreting 3GC-R, FQ-R or dual 3GC-R/FQ-R E. coli, respectively. Isolates represented multiple ST and ABR mechanisms; CTX-M-15 and CMY-2 dominated for 3GC-R, and target-site mutation caused 75% of FQ-R. We identified multiple examples of ABR E. coli transmission between mammalian species in separate enclosures, and a variant of the epidemic plasmid pCT within the zoo. There was no evidence for ABR E. coli leaving the zoo, based on comparative analysis with E. coli from humans, cattle and dogs isolated from the 50 × 50 km region in which the zoo is located. Amoxicillin/clavulanate was the most widely used antibiotic in the zoo, and we identified four widely disseminated amoxicillin/clavulanate resistance mechanisms, including a previously unreported inhibitor-resistant TEM, and the carbapenemase OXA-181. We conclude that the zoo studied here is a 'melting pot' for the selection and circulation of 3GC-R and FQ-R E. coli, but these circulating E. coli appear captive within the zoo.

Exploring the epidemiology of mcr genes, genetic context and plasmids in Enterobacteriaceae originating from pigs and humans on farms in Thailand.

In veterinary medicine, colistin has been widely used as therapeutic and prophylactic agent, and for growth promotion. However, colistin has been re-introduced into treatment of human MDR bacterial infections. We assessed the characteristics and spread of plasmid-borne colistin resistance among healthy pigs, workers with animal-contact and their household members in Thailand. WGS and MIC data of 146 mcr-positive isolates from a cross-sectional One Health study were analysed. Long-read sequencing and conjugation were performed for selected isolates. mcr-carrying isolates were detected in 38% of pooled-pig samples and 16% of human faecal samples. Of 143 Escherichia coli and three Escherichia fergusonii, mcr-1, mcr-3, and mcr-9 variants were identified in 96 (65.8%), 61 (41.8%) and one (0.7%) isolate, respectively. Twelve E. coli co-harboured two mcr variants (mcr-1 and mcr-3). Clonal transmission was detected in five out of 164 farms. mcr-1 was mostly harboured by epidemic IncX4 and IncHI1 plasmids (89.9%). Conversely, mcr-3 was harboured by a range of different plasmids. Comparative plasmid studies suggested IncP and IncFII plasmids as possible endemic mcr-3 plasmids in Asian countries. Moreover, mcr-3 was associated with different mobile genetic elements including TnAs2, ISKpn40 and IS26/15DI. Detected genetic signatures (DRs) indicated recent mcr-3 transpositions, underlining the mobilizable nature of the mcr-3 cassette. The epidemiology of mcr and the possible evolution of successful plasmids and transposition modules should be carefully monitored. Of special concern is the growing number of different horizontal gene transferring pathways encompassing various transposable modules the mcr genes can be shared between bacteria.

Emergence of blaSHV-12 and qnrS1 encoded on IncX3 plasmids: Changing epidemiology of extended-spectrum ß-lactamases among Enterobacterales isolated from broilers

The occurrence of extended-spectrum ß-lactamase (ESBL) producing Enterobacterales in broilers represents a risk to public health because of the possibility of transmission of ESBL-producers and/or blaESBL genes via the food chain or within settings where human-animal interfaces exist. This study assessed the occurrence of ESBL-producers among faecal samples of broilers at slaughter. Isolates were characterised by multilocus sequence typing (MLST), antimicrobial susceptibility testing (AST), and whole genome sequencing (WGS). The flock prevalence, determined by sampling crates of 100 poultry flocks, was 21%. The predominant blaESBL gene was blaSHV-12 identified in 92% of the isolates. A variety of E. coli and K. pneumoniae sequence types (STs) were identified, including extraintestinal pathogenic (ExPEC) E. coli ST38, avian pathogenic E. coli (APEC) ST10, ST93, ST117 and ST155, and nosocomial outbreak clone K. pneumoniae ST20. WGS characterized a subset of 15 isolates, including six Escherichia coli, four Klebsiella pneumoniae, one K. grimontii, one K. michiganensis, one K. variicola, and one Atlantibacter subterranea. Fourteen isolates carried identical or closely related 46338-54929 bp IncX3 plasmids encoding blaSHV-12 and qnrS1. One E. coli isolate carried the 46338 bp IncX3 plasmid integrated chromosomally into ydbD. The blaSHV-12 gene has replaced the previously predominant blaCTX-M-1 in ESBL-producing Enterobacterales from broilers in Switzerland. Broilers may play a role in the dissemination of blaSHV-12 and qnrS1 associated with epidemic IncX3 plasmids, representing a risk to human and animal health.

The Significance of Epidemic Plasmids in the Success of Multidrug-Resistant Drug Pandemic Extraintestinal Pathogenic Escherichia coli.

Epidemic IncF plasmids have been pivotal in the selective advantage of multidrug-resistant (MDR) extraintestinal pathogenic Escherichia coli (ExPEC). These plasmids have offered several advantages to their hosts that allowed them to coevolve with the bacterial host genomes and played an integral role in the success of ExPEC. IncF plasmids are large, mosaic, and often contain various types of antimicrobial resistance (AMR) and virulence associated factor (VAF) genes. The presence of AMR, VAF genes, several addition/restriction systems combined with truncated transfer regions, led to the fixation of IncF plasmids in certain ExPEC MDR clones, such as ST131 and ST410. IncF plasmids entered the ST131 ancestral lineage in the mid 1900s and different ST131 clade/CTX-M plasmid combinations coevolved over time. The IncF_CTX-M-15/ST131-C2 subclade combination emerged during the early 2000s, spread rapidly across the globe, and is one of the greatest clone/plasmid successes of the millennium. The ST410-B3 subclade containing blaCTX-M-15 incorporated the NDM-5 carbapenemase gene into existing IncF platforms, providing an additional positive selective advantage that included the carbapenems. A "plasmid-replacement" clade scenario occurred in the histories of ST131 and ST410 as different subclades gained different AMR genes on different IncF platforms. The use of antimicrobial agents will generate selection pressures that enhance the risks for the continuous emergence of MDR ExPEC clone/IncF plasmid combinations. The reasons for clade/IncF replacements and associations between certain clades and specific IncF plasmid types are unknown. Such information will aid in designing management and prevention strategies to combat AMR.

First detection of tet(X4)-positive Enterobacterales in retail vegetables and clonal spread of Escherichia coli ST195 producing Tet(X4) in animals, foods, and humans across China and Thailand

The mobile tigecycline-resistant gene tet(X4), which confers resistance to all tetracyclines, has been identified in bacterial isolates from various sources. However, there are no reports on the occurrence of tet(X4) in bacterial isolates of ready-to-eat fresh vegetables. In this study, 113 vegetable samples from farmers' markets were screened for tigecycline-resistant strains. Ten Escherichia coli (two ST195, two ST48, and one ST10, ST58, ST88, ST394, ST641, and ST101) and one Klebsiella pneumoniae (ST327) recovered from nine vegetable samples (7.96 %) were identified as carrying tet(X4). The core genome sequences of the two E. coli ST195 isolates showed a close relationship (14-41 single-nucleotide polymorphisms) with 31 tet(X4)-bearing E. coli ST195 isolates from humans, pigs, pork, and bird in China and Thailand, and the 33 E. coli ST195 isolates producing Tet(X4) shared similar resistance genes and plasmid replicons. Nanopore sequencing and conjugation experiments confirmed that the tet(X4) genes were located on the hybrid plasmids IncFIA-HI1A-HI1B (n = 6), IncX1 (n = 3), and IncFII2 (n = 1) in E. coli, and IncFII plasmid in K. pneumoniae. IncFIA-HI1A-HI1B and IncX1 plasmids shared highly similar structures with plasmids from various sources in the GenBank database. This is the first study to report the observation of tet(X4)-positive bacteria in retail vegetables. The epidemic clones and plasmids contribute to tet(X4) dissemination in vegetables. The clonal spread of Tet(X4)-producing E. coli ST195 across multiple niches and countries could pose a potential threat to public health.

Whole genome sequence analysis of the first reported isolate of Salmonella Agona carrying blaCTX-M-55 gene in Brazil

This study analyzes the genomic findings of the first report of Salmonella isolate carrying the blaCTX-M-55 gene, recovered from a bacteremic patient from Brazil. A bacterial isolate positive for the blaCTX-M-55 gene was submitted to antimicrobial susceptibility testing by disk diffusion and epsilometric test. Whole genome sequencing was performed using Illumina technology. Conjugation assay was performed; plasmid sizes determined by S1-PFGE and plasmid content were investigated by hybrid assembly after MinION long reads sequencing. Isolate 288_18 was identified as sequence type ST13, resistant to ampicillin, cefotaxime, ceftazidime, cefepime, ceftriaxone, and aztreonam. A transferable IncFII plasmid sized approximately 67 kb was found to carry the blaTEM-1 and blaCTX-M-55 in a module consisting of IS26-blaTEM-1B-WbuC-blaCTX-M-55-IS26. In addition, an 117 kb IncI1plasmid was also identified in the 288_18 isolate, but without additional resistance genes. To the best of our knowledge, this is the first report of blaCTX-M-55 in Salmonella isolated from human infection in Brazil. The occurrence of blaCTX-M-55 in the IncFII epidemic plasmid in a relevant clinical human isolate of Salmonella Agona underscores the urgent need for enhanced and effective continuous surveillance for controlling its dissemination.

Characterization of Escherichia coli and Other Enterobacterales Resistant to Extended-Spectrum Cephalosporins Isolated from Dairy Manure in Ontario, Canada.

Extended-spectrum cephalosporins (ESCs) resistance genes, such as blaCTX-M, blaCMY, and blaSHV, have been found regularly in bacteria from livestock. However, information on their distribution in dairy cattle in Canada and on the associated genome sequences of ESC-resistant Enterobacterales is sparse. In this study, the diversity and distribution of ESC-resistant Escherichia coli throughout manure treatments in six farms in Southern Ontario were assessed over a one-year period, and their ESC-resistance plasmids were characterized. The manure samples were enriched using selective media. The resulting isolates were screened via polymerase chain reaction for blaCTX-M, blaCMY, and blaSHV. No E. coli carrying blaSHV were detected. Escherichia coli (n = 248) carrying blaCTX-M or blaCMY underwent whole-genome sequencing using an Illumina MiSeq/NextSeq. These isolates were typed using multilocus sequence typing (MLST) and their resistance gene profiles. A subset of E. coli (n = 28) were sequenced using Oxford Nanopore Technologies. Plasmids were assembled using Unicycler and characterized via the resistance genes pattern, replicon type, plasmid MLST, phylogenetic analysis, and Mauve alignments. The recovery of ESC-resistant Enterobacterales (18 species, 8 genera) was drastically reduced in manure outputs. However, multiple treatment stages were needed to attain a significant reduction. 62 sequence types were identified, with ST10, ST46, ST58, ST155, ST190, ST398, ST685, and ST8761 being detected throughout the treatment pipeline. These STs overlapped with those found on multiple farms. The ESC-resistance determinants included CTX-M-1, -14, -15, -17, -24, -32, -55, and CMY-2. The plasmids carrying blaCTX-M were more diverse than were the plasmids carrying blaCMY. Known "epidemic plasmids" were detected for both blaCTX-M and blaCMY. IMPORTANCE The increase in antimicrobial resistance is of concern for human and animal health, especially when resistance is conferred to extended-spectrum cephalosporins, which are used to treat serious infections in both human and veterinary medicine. Bacteria carrying extended-spectrum cephalosporin resistance genes, including blaCTX-M and blaCMY, are frequently found in dairy manure. Manure treatment influences the loads and diversity of bacteria, including those carrying antimicrobial resistance genes, such as Enterobacterales and Escherichia coli. Any bacteria that survive the treatment process are subsequently applied to the environment. Enterobacterales carrying blaCTX-M or blaCMY can contaminate soil and crops consumed by humans and animals, thereby increasing the potential for antimicrobial resistance genes to integrate into the human gut microflora through horizontal gene transfer. This furthers the dissemination of resistance. Therefore, it is imperative to understand the effects manure treatments have on ESC-resistance in environmentally applied manure.

Carbapenem resistance in Acinetobacter pittii isolates mediated by metallo-β-lactamases.

To characterize the genetic environment of metallo-β-lactamases (MBL) in carbapenem-resistant clinical Acinetobacter pittii isolates. Seventeen carbapenem-resistant A. pittii isolates harbouring an MBL were collected between 2010 and 2015 in Germany. Antimicrobial susceptibility testing was performed using agar dilution. Presence of MBLs was confirmed by PCR and their genetic location determined by S1-pulsed-field gel electrophoresis followed by Southern blot hybridization. Whole-genome sequencing was performed using the Miseq and MinION platforms. Isolates were typed using an ad hoc core genome MLST scheme. Conjugation into A. baumannii was tested by broth mating. In 10 isolates the MBL was plasmid-encoded and in seven isolates chromosomally encoded. blaGIM-1 and blaVIM-2 were plasmid-encoded, blaVIM-4 was chromosomally encoded, while blaNDM-1 was chromosomally encoded in four and plasmid-encoded in three isolates. Seven of ten plasmids were conjugative into A. baumannii. Although most isolates were unrelated, the backbones of the MBL-encoding plasmid showed >99% similarity and only differed in the MBL-encoding area. blaNDM-1-harbouring plasmids were highly similar to other plasmids from Acinetobacter isolates worldwide while the blaVIM-2- and blaGIM-1-encoding plasmids have not been described. These data show the existence of a promiscuous plasmid circulating in A. pittii isolates in Germany that differs only in the MBL-encoding region. Its plasmid backbone has been found globally among multiple Acinetobacter spp. These data should raise awareness of an epidemic conjugative plasmid that has independently acquired MBLs. We should also consider that future comparative plasmid analysis will look beyond solely the resistome and include the mobile elements carrying the resistance genes.

Whole Genome Sequencing Reveals Presence of High-Risk Global Clones of Klebsiella pneumoniae Harboring Multiple Antibiotic Resistance Genes in Multiple Plasmids in Mwanza, Tanzania.

Klebsiella pneumoniae is an important multidrug-resistant (MDR) pathogen, causing both community- and healthcare-associated infections. The resistance is due to the continuous accumulation of multiple antibiotic-resistance-genes (ARGs) through spontaneous genomic mutations and the acquisition of conjugative plasmids. This study presents antibiotics resistance genes, plasmids replicons, and virulence genes of K. pneumoniae isolates from clinical specimens in a tertiary hospital, Mwanza, Tanzania. Whole genome sequencing (WGS) of 34 K. pneumoniae was performed, using an Illumina NextSeq 500, followed by in silco analysis. A total of 34 extended-spectrum beta-lactamase-producing K. pneumoniae, isolated from blood samples from neonatal units were whole-genome sequenced. Of these, 28 (82.4%) had an identified sequence type (ST), with ST14 (39.3%, n = 11) being frequently identified. Moreover, 18 (52.9%) of the bacteria harbored at least one plasmid, from which a total of 25 plasmid replicons were identified with a predominance of IncFIB(K) 48.0% (n = 12). Out of 34 sequenced K. pneumoniae, 32 (94.1%) were harboring acquired antibiotic/biocides-resistance-genes (ARGs) with a predominance of blaCTX-M-15 (90.6%), followed by oqxB (87.5%), oqxA (84.4%), blaTEM-1B (84.4%) and sul2 (84.4%). Interestingly, we observed the ColRNAI plasmid-replicon (n = 1) and qacE gene (n = 4) for the first time in this setting. Global high-risk clones of K. pneumoniae isolates carry multiple ARGs in multiple plasmid-replicons. Findings from this study warrant genomic-based surveillance to monitor high-risk global clones, epidemic plasmids and ARGs in low- and middle-income countries.