Genomic characterization of ST38 NDM-5-producing Escherichia coli isolates from an outbreak in the Czech Republic.

A 2-year national genomic screening in the Czech Republic identified a notable prevalence of the New Delhi metallo-ß-lactamase 5 (NDM-5)-producing Escherichia coli sequence type 38 (ST38) in the city of Brno. Forty-two ST38 E. coli isolates harbored the blaNDM-5 gene on the chromosome. Virulence factors confirmed the persistence of these isolates through biofilm formation. Single Nucleotide Polymorphisms (SNPs)-based phylogeny and CRISPR assay typing showed minimal genomic variations, implying a clonally driven outbreak. Results suggest that this high-risk clone may impose a nationwide problem.

A novel F type plasmid encoding mcr-10 in a clinical Enterobacter ludwigii strain from a tertiary hospital in the Czech Republic.

OBJECTIVE: Here we describe a novel IncFIA plasmid harbouring mcr-10 gene in a clinical Enterobacter ludwigii strain isolated at the University Hospital in Pilsen in the Czech Republic. METHODS: The strain was subjected to antibiotic susceptibility testing. Whole genome sequencing was performed using Illumina for short-read sequencing and Oxford Nanopore Technologies for long-read sequencing followed by hybrid assembly. The resulting genome was used to detect species using average nucleotide identity, resistance genes, plasmid replicon and MLST (using centre for genomic epidemiology databases; ResFinder, PlasmidFinder and MLST, respectively) and virulence genes using VFDB. RESULTS: Τhe strain showed susceptibility against tetracycline, cefuroxime and chloramphenicol, and it was susceptible to the second and third generation of cephalosporins, carbapenems and colistin. Genome analysis identified the strain as E. ludwigii sequence type ST20 and located the mcr-10 gene on an IncFIA (HI1)/IncFII (Yp) plasmid (pI9455333_MCR10; 129 863 bp). Upon blasting the nucleotide sequence of pI9455333_MCR10 against the NCBI database, no similar plasmid sequence was detected, implying a novel plasmid structure. Nevertheless, it showed a partial similarity with pRHBSTW-00123_3 and FDAARGOS 1432, which were detected in Enterobacter cloacae complex (ECC) strains in wastewater samples in 2017 in UK and in 2021 in the United States, respectively, and pEC81-mcr, which was detected in a clinical Escherichia coli strain in 2020 in China. Moreover, I9455333cz genome carried virulence genes coding for curli fibers, fimbrial adherence determinants, siderophore aerobactin, iron uptake proteins and regulators of sigma factor. CONCLUSION: In conclusion, we identified a novel IncF plasmid harbouring mcr-10 gene in a clinical Enterobacter ludwigii strain. To our knowledge, this is the first clinical report of mcr-10 in the Czech Republic.

Changing dynamics of antibiotic resistant Escherichia in Caspian gulls shows the importance of longitudinal environmental studies.

This study is focused on Escherichia spp. isolates resistant to critically important antibiotics (cefotaxime, ciprofloxacin and colistin) among Caspian gull's (Larus cachinnans) chicks nesting in the Nove Mlyny Water Reservoir, Czech Republic. The prevalence of antimicrobial resistance (AMR) in bacteria within wild birds is commonly evaluated using a single sampling event, capturing only a brief and momentary snapshot at a particular location. Therefore, the Caspian gulls in our study were sampled in May 2018 (n = 72) and May 2019 (n = 45), and a water sample was taken from the reservoir (2019). We obtained 197 isolates identified as E. coli by MALDI-TOF MS. A total of 158 representative isolates were whole-genome sequenced, 17 isolates were then reclassified to Escherichia albertii. We observed a higher (86 %; 62/72) occurrence of ESBL/AmpC-producing Escherichia spp. among gulls in 2018 compared to 38 % (17/45) in 2019 (p < 0.00001). The decrease in prevalence was linked to clonal lineage of E. coli ST11893 predominating in 2018 which carried blaCMY-2 and which was not recovered from the gulls in 2019. Oppositely, several Escherichia STs were found in gulls from both years as well as in the water sample including STs commonly recognized as internationally high-risk lineages such as ST10, ST58, ST88, ST117, ST648 or ST744. Phylogenetic analysis of E. coli from EnteroBase from countries where these particular gulls wander revealed that some STs are commonly found in various sources including humans and a portion of them is even closely related (up to 100 SNPs) to our isolates. We demonstrated that the occurrence of AMR in Escherichia can vary greatly in time in synanthropic birds and we detected both, a temporary prevalent lineage and several persistent STs. The close relatedness of isolates from gulls and isolates from EnteroBase highlights the need to further evaluate the risk connected to wandering birds.

The faecal microbiome of the Australian silver gull contains phylogenetically diverse ExPEC, aEPEC and Escherichia coli carrying the transmissible locus of stress tolerance.

Wildlife are implicated in the dissemination of antimicrobial resistance, but their roles as hosts for Escherichia coli that pose a threat to human and animal health is limited. Gulls (family Laridae) in particular, are known to carry diverse lineages of multiple-antibiotic resistant E. coli, including extra-intestinal pathogenic E. coli (ExPEC). Whole genome sequencing of 431 E. coli isolates from 69 healthy Australian silver gulls (Chroicocephalus novaehollandiae) sampled during the 2019 breeding season, and without antibiotic selection, was undertaken to assess carriage in an urban wildlife population. Phylogenetic analysis and genotyping resolved 123 sequence types (STs) representing most phylogroups, and identified diverse ExPEC, including an expansive phylogroup B2 cluster comprising 103 isolates (24 %; 31 STs). Analysis of the mobilome identified: i) widespread carriage of the Yersinia High Pathogenicity Island (HPI), a key ExPEC virulence determinant; ii) broad distribution of two novel phage elements, each carrying sitABCD and iii) carriage of the transmissible locus of stress tolerance (tLST), an element linked to sanitation resistance. Of the 169 HPI carrying isolates, 49 (48 %) represented diverse B2 isolates hosting FII-64 ColV-like plasmids that lacked iutABC and sitABC operons typical of ColV plasmids, but carried the serine protease autotransporter gene, sha. Diverse E. coli also carried archetypal ColV plasmids (52 isolates; 12 %). Clusters of closely related E. coli (<50 SNVs) from ST58, ST457 and ST746, sourced from healthy gulls, humans, and companion animals, were frequently identified. In summary, anthropogenically impacted gulls host an expansive E. coli population, including: i) putative ExPEC that carry ColV virulence gene cargo (101 isolates; 23.4 %) and HPI (169 isolates; 39 %); ii) atypical enteropathogenic E. coli (EPEC) (17 isolates; 3.9 %), and iii) E. coli that carry the tLST (20 isolates; 4.6 %). Gulls play an important role in the evolution and transmission of E. coli that impact human health.

Detecting horizontal gene transfer among microbiota: an innovative pipeline for identifying co-shared genes within the mobilome through advanced comparative analysis.

Horizontal gene transfer (HGT) is a key driver in the evolution of bacterial genomes. The acquisition of genes mediated by HGT may enable bacteria to adapt to ever-changing environmental conditions. Long-term application of antibiotics in intensive agriculture is associated with the dissemination of antibiotic resistance genes among bacteria with the consequences causing public health concern. Commensal farm-animal-associated gut microbiota are considered the reservoir of the resistance genes. Therefore, in this study, we identified known and not-yet characterized mobilized genes originating from chicken and porcine fecal samples using our innovative pipeline followed by network analysis to provide appropriate visualization to support proper interpretation.

Zinc effects on bacteria: insights from Escherichia coli by multi-omics approach.

IMPORTANCE: A long-term exposure of bacteria to zinc oxide and zinc oxide nanoparticles leads to major alterations in bacterial morphology and physiology. These included biochemical and physiological processes promoting the emergence of strains with multi-drug resistance and virulence traits. After the removal of zinc pressure, bacterial phenotype reversed back to the original state; however, certain changes at the genomic, transcriptomic, and proteomic level remained. Why is this important? The extensive and intensive use of supplements in animal feed effects the intestinal microbiota of livestock and this may negatively impact the health of animals and people. Therefore, it is crucial to understand and monitor the impact of feed supplements on intestinal microorganisms in order to adequately assess and prevent potential health risks.

Phylogenomic analysis of a global collection of Escherichia coli ST38: evidence of interspecies and environmental transmission?

IMPORTANCE: Extraintestinal pathogenic Escherichia coli (ExPEC) sequence type (ST) 38 is one of the top 10 human pandemic lineages. Although a major cause of urinary tract and blood stream infections, ST38 has been poorly characterized from a global phylogenomic perspective. A comprehensive genome-scale analysis of 925 ST38 isolate genomes identified two broad ancestral clades and linkage of discrete ST38 clusters with specific bla CTX-M variants. In addition, the clades and clusters carry important virulence genes, with diverse but poorly characterized plasmids. Numerous putative interhost and environment transmission events were identified here by the presence of ST38 clones (defined as isolates with ≤35 SNPs) within humans, companion animals, food sources, urban birds, wildlife, and the environment. A small cluster of international ST38 clones from diverse sources, likely representing progenitors of a hospital outbreak that occurred in Brisbane, Australia, in 2017, was also identified. Our study emphasizes the importance of characterizing isolate genomes derived from nonhuman sources and geographical locations, without any selection bias.

Plasmid-mediated colistin resistance from fresh meat and slaughtered animals in the Czech Republic: nation-wide surveillance 2020-2021.

The aim of this study was to determine the occurrence of plasmid-mediated colistin resistance in domestic and imported meat and slaughter animals in the Czech Republic during 2020-2021 by using selective cultivation and direct PCR testing. A total of 111 colistin-resistant Escherichia coli isolates with mcr-1 gene were obtained from 65 (9.9%, n = 659) samples and subjected to whole-genome sequencing. Isolates with mcr were frequently found in fresh meat from domestic production (14.2%) as well as from import (28.8%). The mcr-1-positive E. coli isolates predominantly originated from meat samples (16.6%), mainly poultry (27.1%), and only minor part of the isolates came from the cecum (1.7%). In contrast to selective cultivation, 205 (31.1%) samples of whole-community DNA were positive for at least one mcr variant, and other genes besides mcr-1 were detected. Analysis of whole-genome data of sequenced E. coli isolates revealed diverse sequence types (STs) including pathogenic lineages and dominance of ST1011 (15.6%) and ST162 (12.8%). Most isolates showed multidrug-resistant profile, and 9% of isolates produced clinically important beta-lactamases. The mcr-1 gene was predominantly located on one of three conjugative plasmids of IncX4 (83.5%), IncI2 (7.3%), and IncHI2 (7.3%) groups. Seventy-two percent isolates of several STs carried ColV plasmids. The study revealed high prevalence of mcr genes in fresh meat of slaughter animals. Our results confirmed previous assumptions that the livestock, especially poultry production, is an important source of colistin-resistant E. coli with the potential of transfer to humans via the food chain. IMPORTANCE We present the first data on nation-wide surveillance of plasmid-mediated colistin resistance in the Czech Republic. High occurrence of plasmid-mediated colistin resistance was found in meat samples, especially in poultry from both domestic production and import, while the presence of mcr genes was lower in the gut of slaughter animals. In contrast to culture-based approach, testing of whole-community DNA showed higher prevalence of mcr and presence of various mcr variants. Our results support the importance of combining cultivation methods with direct culture-independent techniques and highlight the need for harmonized surveillance of plasmid-mediated colistin resistance. Our study confirmed the importance of livestock as a major reservoir of plasmid-mediated colistin resistance and pointed out the risks of poultry meat for the transmission of mcr genes toward humans. We identified several mcr-associated prevalent STs, especially ST1011, which should be monitored further as they represent zoonotic bacteria circulating between different environments.

Broad-Host Dissemination of Plasmids Coharboring the fos Operon for Fructooligosaccharide Metabolism with Antibiotic Resistance Genes.

The fos operon encoding short-chain fructooligosaccharide (scFOS) utilization enables bacteria of the family Enterobacteriaceae to grow and be sustained in environments where they would struggle to survive. Despite several cases of the detection of the fos operon in isolates of avian and equine origins, its global distribution in bacterial genomes remains unknown. The presence of the plasmid-harbored fos operon among resistant bacteria may promote the spread of antibiotic resistance. A collection of 11,538 antimicrobial-resistant Enterobacteriaceae isolates from various sources was screened for the fosT gene encoding the scFOS transporter. Out of 307 fosT-positive isolates, 80% of them originated from sources not previously linked to fosT (humans, wastewater, and animals). The chromosomally harbored fos operon was detected in 163/237 isolates subjected to whole-genome sequencing. In the remaining 74 isolates, the operon was carried by plasmids. Further analyses focusing on the isolates with a plasmid-harbored fos operon showed that the operon was linked to various incompatibility (Inc) groups, including the IncHI1, IncF-type, IncK2, IncI1, and IncY families. Long-read sequencing of representative plasmids showed the colocalization of fos genes with antibiotic resistance genes (ARGs) in IncHI1 (containing a multidrug resistance region), IncK2 (blaTEM-1A), IncI1 [sul2 and tet(A)], and IncY [aadA5, dfrA17, sul2, and tet(A)] plasmids, while IncF-type plasmids had no ARGs but coharbored virulence-associated genes. Despite the differences in the locations and structures of the fos operons, all isolates except one were proven to utilize scFOSs. In this study, we show that the fos operon and its spread are not strictly bound to one group of plasmids, and therefore, it should not be overlooked. IMPORTANCE It was believed that members of the family Enterobacteriaceae are unable to grow under conditions with short-chain fructooligosaccharides as the only source of carbon. Nevertheless, the first Escherichia coli isolate from chicken intestine was able to utilize these sugars owing to the chromosomally harbored fos operon. Studies on E. coli isolates from horses discovered the horizontal transfer of the fos operon on IncHI1 plasmids along with genes for antibiotic resistance. The first plasmid detected was pEQ1, originating from the feces of a hospitalized horse in the Czech Republic. Follow-up studies also revealed the dissemination of the IncHI1 plasmid-harbored fos operon in the Netherlands, Germany, Denmark, and France among healthy horses. Despite several cases of detection of the fos operon, its global distribution in bacterial genomes remains unknown. The fos operon possibly plays a role in the adaptation of plasmids among resistant bacteria and therefore may promote the spread of antibiotic resistance.

Clinically relevant antibiotic resistance in Escherichia coli from black kites in southwestern Siberia: a genetic and phenotypic investigation.

Wild birds including raptors can act as vectors of clinically relevant bacteria with antibiotic resistance. The aim of this study was to investigate the occurrence of antibiotic-resistant Escherichia coli in black kites (Milvus migrans) inhabiting localities in proximity to human-influenced environments in southwestern Siberia and investigate their virulence and plasmid contents. A total of 51 E. coli isolates mostly with multidrug resistance (MDR) profiles were obtained from cloacal swabs of 35 (64%, n = 55) kites. Genomic analyses of 36 whole genome sequenced E. coli isolates showed: (i) high prevalence and diversity of their antibiotic resistance genes (ARGs) and common association with ESBL/AmpC production (27/36, 75%), (ii) carriage of mcr-1 for colistin resistance on IncI2 plasmids in kites residing in proximity of two large cities, (iii) frequent association with class one integrase (IntI1, 22/36, 61%), and (iv) presence of sequence types (STs) linked to avian-pathogenic (APEC) and extra-intestinal pathogenic E. coli (ExPEC). Notably, numerous isolates had significant virulence content. One E. coli with APEC-associated ST354 carried qnrE1 encoding fluoroquinolone resistance on IncHI2-ST3 plasmid, the first detection of such a gene in E. coli from wildlife. Our results implicate black kites in southwestern Siberia as reservoirs for antibiotic-resistant E. coli. It also highlights the existing link between proximity of wildlife to human activities and their carriage of MDR bacteria including pathogenic STs with significant and clinically relevant antibiotic resistance determinants. IMPORTANCE Migratory birds have the potential to acquire and disperse clinically relevant antibiotic-resistant bacteria (ARB) and their associated antibiotic resistance genes (ARGs) through vast geographical regions. The opportunistic feeding behavior associated with some raptors including black kites and the growing anthropogenic influence on their natural habitats increase the transmission risk of multidrug resistance (MDR) and pathogenic bacteria from human and agricultural sources into the environment and wildlife. Thus, monitoring studies investigating antibiotic resistance in raptors may provide essential data that facilitate understanding the fate and evolution of ARB and ARGs in the environment and possible health risks for humans and animals associated with the acquisition of these resistance determinants by wildlife.

Hospital and community wastewater as a source of multidrug-resistant ESBL-producing Escherichia coli.

Introduction: Hospitals and wastewater are recognized hot spots for the selection and dissemination of antibiotic-resistant bacteria to the environment, but the total participation of hospitals in the spread of nosocomial pathogens to municipal wastewater treatment plants (WWTPs) and adjacent rivers had not previously been revealed. Methods: We used a combination of culturing and whole-genome sequencing to explore the transmission routes of Escherichia coli from hospitalized patients suffering from urinary tract infections (UTI) via wastewater to the environment. Samples were collected in two periods in three locations (A, B, and C) and cultured on selective antibiotic-enhanced plates. Results: In total, 408 E. coli isolates were obtained from patients with UTI (n=81), raw hospital sewage (n=73), WWTPs inflow (n=96)/outflow (n=106), and river upstream (n=21)/downstream (n=31) of WWTPs. The majority of the isolates produced extended-spectrum beta-lactamase (ESBL), mainly CTX-M-15, and showed multidrug resistance (MDR) profiles. Seven carbapenemase-producing isolates with GES-5 or OXA-244 were obtained in two locations from wastewater and river samples. Isolates were assigned to 74 different sequence types (ST), with the predominance of ST131 (n=80) found in all sources including rivers. Extraintestinal pathogenic lineages frequently found in hospital sewage (ST10, ST38, and ST69) were also found in river water. Despite generally high genetic diversity, phylogenetic analysis of ST10, ST295, and ST744 showed highly related isolates (SNP 0-18) from different sources, providing the evidence for the transmission of resistant strains through WWTPs to surface waters. Discussion: Results of this study suggest that 1) UTI share a minor participation in hospitals wastewaters; 2) a high diversity of STs and phylogenetic groups in municipal wastewaters derive from the urban influence rather than hospitals; and 3) pathogenic lineages and bacteria with emerging resistance genotypes associated with hospitals spread into surface waters. Our study highlights the contribution of hospital and municipal wastewater to the transmission of ESBL- and carbapenemase-producing E. coli with MDR profiles to the environment.

Plasmid-mediated colistin resistance among human clinical Enterobacterales isolates: national surveillance in the Czech Republic.

The occurrence of colistin resistance has increased rapidly among Enterobacterales around the world. We performed a national survey of plasmid-mediated colistin resistance in human clinical isolates through a retrospective analysis of samples from 2009 to 2017 and a prospective sampling in 2018-2020. The aim of this study was to identify and characterize isolates with mcr genes from various regions of the Czech Republic using whole genome sequencing (WGS). Of all 1932 colistin-resistant isolates analyzed, 73 (3.8%) were positive for mcr genes. Most isolates carried mcr-1 (48/73) and were identified as Escherichia coli (n = 44) and Klebsiella pneumoniae (n = 4) of various sequence types (ST). Twenty-five isolates, including Enterobacter spp. (n = 24) and Citrobacter freundii (n = 1) carrying the mcr-9 gene were detected; three of them (Enterobacter kobei ST54) co-harbored the mcr-4 and mcr-9 genes. Multi-drug resistance phenotype was a common feature of mcr isolates and 14% (10/73) isolates also co-harbored clinically important beta-lactamases, including two isolates with carbapenemases KPC-2 and OXA-48. Phylogenetic analysis of E. coli ST744, the dominant genotype in this study, with the global collection showed Czech isolates belonged to two major clades, one containing isolates from Europe, while the second composed of isolates from diverse geographical areas. The mcr-1 gene was carried by IncX4 (34/73, 47%), IncHI2/ST4 (6/73, 8%) and IncI2 (8/73, 11%) plasmid groups. Small plasmids belonging to the ColE10 group were associated with mcr-4 in three isolates, while mcr-9 was carried by IncHI2/ST1 plasmids (4/73, 5%) or the chromosome (18/73, 25%). We showed an overall low level of occurrence of mcr genes in colistin-resistant bacteria from human clinical samples in the Czech Republic.

Horsing around: Escherichia coli ST1250 of equine origin harbouring epidemic IncHI1/ST9 plasmid with bla CTX-M-1 and an operon for short-chain fructooligosaccharides metabolism.

The relatedness of the equine-associated Escherichia coli ST1250 and its single- and double-locus variants (ST1250-SLV/DLV), obtained from horses in Europe, was studied by comparative genome analysis. A total of 54 isolates of E. coli ST1250 and ST1250-SLV/DLV from healthy and hospitalized horses across Europe [Czech Republic (n=23), the Netherlands (n=18), Germany (n=9), Denmark (n=3) and France (n=1)] from 2008-2017 were subjected to whole-genome sequencing. An additional 25 draft genome assemblies of E. coli ST1250 and ST1250-SLV/DLV were obtained from the public databases. The isolates were compared for genomic features, virulence genes, clade structure and plasmid content. The complete nucleotide sequences of eight IncHI1/ST9 and one IncHI1/ST2 plasmids were obtained using long-read sequencing by PacBio or MinION. In the collection of 79 isolates, only 10 were phylogenetically close (<8 SNP). The majority of isolates belonged to phylogroup B1 (73/79, 92.4%) and carried bla CTX-M-1 (58/79, 73.4%). The plasmid content of the isolates was dominated by IncHI1 of ST9 (56/62, 90.3%) and ST2 (6/62, 9.7%), while 84.5% (49/58) bla CTX-M-1 genes were associated with presence of IncHI1 replicon of ST9 and 6.9% (4/58) with IncHI1 replicon of ST2 within the corresponding isolates. The operon for the utilization of short chain fructooligosaccharides (fos operon) was present in 55 (55/79, 69.6%) isolates, and all of these carried IncHI1/ST9 plasmids. The eight complete IncHI1/ST9 plasmid sequences showed the presence of bla CTX-M-1 and the fos operon within the same molecule. Sequences of IncHI1/ST9 plasmids were highly conserved (>98% similarity) regardless of country of origin and varied only in the structure and integration site of MDR region. E. coli ST1250 and ST1250-SLV/DLV are phylogenetically-diverse strains associated with horses. A strong linkage of E. coli ST1250 with epidemic multi-drug resistance plasmid lineage IncHI1/ST9 carrying bla CTX-M-1 and the fos operon was identified.

Genomic Analysis of an I1 Plasmid Hosting a sul3-Class 1 Integron and blaSHV-12 within an Unusual Escherichia coli ST297 from Urban Wildlife.

Wild birds, particularly silver gulls (Chroicocephalus novaehollandiae) that nest near anthropogenic sites, often harbour bacteria resistant to multiple antibiotics, including those considered of clinical importance. Here, we describe the whole genome sequence of Escherichia coli isolate CE1867 from a silver gull chick sampled in 2012 that hosted an I1 pST25 plasmid with blaSHV-12, a ß-lactamase gene that encodes the ability to hydrolyze oxyimino ß-lactams, and other antibiotic resistance genes. Isolate CE1867 is an ST297 isolate, a phylogroup B1 lineage, and clustered with a large ST297 O130:H11 clade, which carry Shiga toxin genes. The I1 plasmid belongs to plasmid sequence type 25 and is notable for its carriage of an atypical sul3-class 1 integron with mefB∆260, a structure most frequently reported in Australia from swine. This integron is a typical example of a Tn21-derived element that captured sul3 in place of the standard sul1 structure. Interestingly, the mercury resistance (mer) module of Tn21 is missing and has been replaced with Tn2-blaTEM-1 and a blaSHV-12 encoding module flanked by direct copies of IS26. Comparisons to similar plasmids, however, demonstrate a closely related family of ARG-carrying plasmids that all host variants of the sul3-associated integron with conserved Tn21 insertion points and a variable presence of both mer and mefB truncations, but predominantly mefB∆260.

Interspecies Transmission of CMY-2-Producing Escherichia coli Sequence Type 963 Isolates between Humans and Gulls in Australia.

Escherichia coli sequence type 963 (ST963) is a neglected lineage closely related to ST38, a globally widespread extraintestinal pathogenic ST causing urinary tract infections (UTI) as well as sepsis in humans. Our current study aimed to improve the knowledge of this understudied ST by carrying out a comprehensive comparative analysis of whole-genome sequencing data consisting of 31 isolates from silver gulls in Australia along with another 80 genomes from public resources originating from geographically scattered regions. ST963 was notable for carriage of cephalosporinase gene blaCMY-2, which was identified in 99 isolates and was generally chromosomally encoded. ST963 isolates showed otherwise low carriage of antibiotic resistance genes, in contrast with the closely related E. coli ST38. We found considerable phylogenetic variability among international ST963 isolates (up to 11,273 single nucleotide polymorphisms [SNPs]), forming three separate clades. A major clade that often differed by 20 SNPs or less consisted of Australian isolates of both human and animal origin, providing evidence of zoonotic or zooanthropogenic transmission. There was a high prevalence of virulence F29:A-:B10 pUTI89-like plasmids within E. coli ST963 (n = 88), carried especially by less variable isolates exhibiting ≤1,154 SNPs. We characterized a novel 115,443-bp pUTI89-like plasmid, pCE2050_A, that carried a traS:IS5 insertion absent from pUTI89. Since IS5 was also present in a transposition unit bearing blaCMY-2 on chromosomes of ST963 strains, IS5 insertion into pUTI89 may enable mobilization of the blaCMY-2 gene from the chromosome/transposition unit to pUTI89 via homologous recombination. IMPORTANCE We have provided the first comprehensive genomic study of E. coli ST963 by analyzing various genomic and phenotypic data sets of isolates from Australian silver gulls and comparison with genomes from geographically dispersed regions of human and animal origin. Our study suggests the emergence of a specific blaCMY-2-carrying E. coli ST963 clone in Australia that is widely spread across the continent by humans and birds. Genomic analysis has revealed that ST963 is a globally dispersed lineage with a remarkable set of virulence genes and virulence plasmids described in uropathogenic E. coli. While ST963 separated into three clusters, a unique specific clade of Australian ST963 isolates harboring a chromosomal copy of AmpC ß-lactamase encoding the gene blaCMY-2 and originating from both humans and wild birds was identified. This phylogenetically close cluster comprised isolates of both animal and human origin, thus providing evidence of interspecies zoonotic transmission. The analysis of the genetic environment of the AmpC ß-lactamase-encoding gene highlighted ongoing evolutionary events that shape the carriage of this gene in ST963.

Fitness effects of blaCTX-M-15-harbouring F2:A1:B- plasmids on their native Escherichia coli ST131 H30Rx hosts.

OBJECTIVES: To investigate the fitness effects of large blaCTX-M-15-harbouring F2:A1:B- plasmids on their native Escherichia coli ST131 H30Rx hosts. METHODS: We selected five E. coli ST131 H30Rx isolates of diverse origin, each carrying an F2:A1:B- plasmid with the blaCTX-M-15 gene. The plasmid was eliminated from each isolate by displacement using an incompatible curing plasmid, pMDP5_cureEC958. WGS was performed to obtain complete chromosome and plasmid sequences of original isolates and to detect chromosomal mutations in 'cured' clones. High-throughput competition assays were conducted to determine the relative fitness of cured clones compared with the corresponding original isolates. RESULTS: We were able to successfully eliminate the F2:A1:B- plasmids from all five original isolates using pMDP5_cureEC958. The F2:A1:B- plasmids produced non-significant fitness effects in three isolates and moderate reductions in relative fitness (3%-4%) in the two remaining isolates. CONCLUSIONS: We conclude that F2:A1:B- plasmids pose low fitness costs in their E. coli ST131 H30Rx hosts. This plasmid-host fitness compatibility is likely to promote the maintenance of antibiotic resistance in this clinically important E. coli lineage.

Genomic analysis of qnr-harbouring IncX plasmids and their transferability within different hosts under induced stress.

BACKGROUND: Conjugative plasmids play a major role in the dissemination of antibiotic resistance genes. Knowledge of the plasmid characteristics and behaviour can allow development of control strategies. Here we focus on the IncX group of plasmids carrying genes conferring quinolone resistance (PMQR), reporting their transfer and persistence within host bacteria of various genotypes under distinct conditions and levels of induced stress in form of temperature change and various concentrations of ciprofloxacin supplementation. METHODS: Complete nucleotide sequences were determined for eight qnr-carrying IncX-type plasmids, of IncX1 (3), IncX2 (3) and a hybrid IncX1-2 (2) types, recovered from Escherichia coli of various origins. This data was compared with further complete sequences of IncX1 and IncX2 plasmids carrying qnr genes (n = 41) retrieved from GenBank and phylogenetic tree was constructed. Representatives of IncX1 (pHP2) and IncX2 (p194) and their qnrS knockout mutants, were studied for influence of induced stress and genetic background on conjugative transfer and maintenance. RESULTS: A high level of IncX core-genome similarity was found in plasmids of animal, environmental and clinical origin. Significant differences were found between the individual IncX plasmids, with IncX1 subgroup plasmids showing higher conjugative transfer rates than IncX2 plasmids. Knockout of qnr modified transfer frequency of both plasmids. Two stresses applied simultaneously were needed to affect transfer rate of wildtype plasmids, whereas a single stress was sufficient to affect the IncX ΔqnrS plasmids. The conjugative transfer was shown to be biased towards the host phylogenetic proximity. A long-term cultivation experiment pointed out the persistence of IncX plasmids in the antibiotic-free environment. CONCLUSIONS: The study indicated the stimulating effect of ciprofloxacin supplementation on the plasmid transfer that can be nullified by the carriage of a single PMQR gene. The findings present the significant properties and behaviour of IncX plasmids carrying antibiotic resistance genes that are likely to play a role in their dissemination and stability in bacterial populations.

Urban Wildlife Crisis: Australian Silver Gull Is a Bystander Host to Widespread Clinical Antibiotic Resistance.

The Australian silver gull is an urban-adapted species that frequents anthropogenic waste sites. The enterobacterial flora of synanthropic birds often carries antibiotic resistance genes. Whole-genome sequence analyses of 425 Escherichia coli isolates from cloacal swabs of chicks inhabiting three coastal sites in New South Wales, Australia, cultured on media supplemented with meropenem, cefotaxime, or ciprofloxacin are reported. Phylogenetically, over 170 antibiotic-resistant lineages from 96 sequence types (STs) representing all major phylogroups were identified. Remarkably, 25 STs hosted the carbapenemase gene blaIMP-4, sourced only from Five Islands. Class 1 integrons carrying blaIMP and blaOXA alongside blaCTX-M and qnrS were notable. Multiple plasmid types mobilized blaIMP-4 and blaOXA-1, and 121 isolates (28%) carried either a ColV-like (18%) or a pUTI89-like (10%) F virulence plasmid. Phylogenetic comparisons to human isolates provided evidence of interspecies transmission. Our study underscores the importance of bystander species in the transmission of antibiotic-resistant and pathogenic E. coli. IMPORTANCE By compiling various genomic and phenotypic data sets, we have provided one of the most comprehensive genomic studies of Escherichia coli isolates from the Australian silver gull, on media containing clinically relevant antibiotics. The analysis of genetic structures capturing antimicrobial resistance genes across three gull breeding colonies in New South Wales, Australia, and comparisons to clinical data have revealed a range of trackable genetic signatures that highlight the broad distribution of clinical antimicrobial resistance in more than 170 different lineages of E. coli. Conserved truncation sizes of the class 1 integrase gene, a key component of multiple-drug resistance structures in the Enterobacteriaceae, represent unique deletion events that are helping to link seemingly disparate isolates and highlight epidemiologically relevant data between wildlife and clinical sources. Notably, only the most anthropogenically affected of the three sites (Five Islands) was observed to host carbapenem resistance, indicating a potential reservoir among the sites sampled.

A role for ColV plasmids in the evolution of pathogenic Escherichia coli ST58.

Escherichia coli ST58 has recently emerged as a globally disseminated uropathogen that often progresses to sepsis. Unlike most pandemic extra-intestinal pathogenic E. coli (ExPEC), which belong to pathogenic phylogroup B2, ST58 belongs to the environmental/commensal phylogroup B1. Here, we present a pan-genomic analysis of a global collection of 752 ST58 isolates from diverse sources. We identify a large ST58 sub-lineage characterized by near ubiquitous carriage of ColV plasmids, which carry genes encoding virulence factors, and by a distinct accessory genome including genes typical of the Yersiniabactin High Pathogenicity Island. This sub-lineage includes three-quarters of all ExPEC sequences in our study and has a broad host range, although poultry and porcine sources predominate. By contrast, strains isolated from cattle often lack ColV plasmids. Our data indicate that ColV plasmid acquisition contributed to the divergence of the major ST58 sub-lineage, and different sub-lineages inhabit poultry, swine and cattle.

Genomic comparisons of Escherichia coli ST131 from Australia.

Escherichia coli ST131 is a globally dispersed extraintestinal pathogenic E. coli lineage contributing significantly to hospital and community acquired urinary tract and bloodstream infections. Here we describe a detailed phylogenetic analysis of the whole genome sequences of 284 Australian ST131 E. coli isolates from diverse sources, including clinical, food and companion animals, wildlife and the environment. Our phylogeny and the results of single nucleotide polymorphism (SNP) analysis show the typical ST131 clade distribution with clades A, B and C clearly displayed, but no niche associations were observed. Indeed, interspecies relatedness was a feature of this study. Thirty-five isolates (29 of human and six of wild bird origin) from clade A (32 fimH41, 2 fimH89, 1 fimH141) were observed to differ by an average of 76 SNPs. Forty-five isolates from clade C1 from four sources formed a cluster with an average of 46 SNPs. Within this cluster, human sourced isolates differed by approximately 37 SNPs from isolates sourced from canines, approximately 50 SNPs from isolates from wild birds, and approximately 52 SNPs from isolates from wastewater. Many ST131 carried resistance genes to multiple antibiotic classes and while 41 (14 %) contained the complete class one integron-integrase intI1, 128 (45 %) isolates harboured a truncated intI1 (462-1014 bp), highlighting the ongoing evolution of this element. The module intI1-dfrA17-aadA5-qacEΔ1-sul1-ORF-chrA-padR-IS1600-mphR-mrx-mphA, conferring resistance to trimethoprim, aminoglycosides, quaternary ammonium compounds, sulphonamides, chromate and macrolides, was the most common structure. Most (73 %) Australian ST131 isolates carry at least one extended spectrum ß-lactamase gene, typically blaCTX-M-15 and blaCTX-M-27. Notably, dual parC-1aAB and gyrA-1AB fluoroquinolone resistant mutations, a unique feature of clade C ST131 isolates, were identified in some clade A isolates. The results of this study indicate that the the ST131 population in Australia carries diverse antimicrobial resistance genes and plasmid replicons and indicate cross-species movement of ST131 strains across diverse reservoirs.