Dominance of Escherichia coli sequence types ST73, ST95, ST127 and ST131 in Australian urine isolates: a genomic analysis of antimicrobial resistance and virulence linked to F plasmids.

Extraintestinal pathogenic Escherichia coli (ExPEC) are the most frequent cause of urinary tract infections (UTIs) globally. Most studies of clinical E. coli isolates are selected based on their antimicrobial resistance (AMR) phenotypes; however, this selection bias may not provide an accurate portrayal of which sequence types (STs) cause the most disease. Here, whole genome sequencing (WGS) was performed on 320 E. coli isolates from urine samples sourced from a regional hospital in Australia in 2006. Most isolates (91%) were sourced from patients with UTIs and were not selected based on any AMR phenotypes. No significant differences were observed in AMR and virulence genes profiles across age sex, and uro-clinical syndromes. While 88 STs were identified, ST73, ST95, ST127 and ST131 dominated. F virulence plasmids carrying senB-cjrABC (126/231; 55%) virulence genes were a feature of this collection. These senB-cjrABC+ plasmids were split into two categories: pUTI89-like (F29:A-:B10 and/or >95 % identity to pUTI89) (n=73) and non-pUTI89-like (n=53). Compared to all other plasmid replicons, isolates with pUTI89-like plasmids carried fewer antibiotic resistance genes (ARGs), whilst isolates with senB-cjrABC+/non-pUTI89 plasmids had a significantly higher load of ARGs and class 1 integrons. F plasmids were not detected in 89 genomes, predominantly ST73. Our phylogenomic analyses identified closely related isolates from the same patient associated with different pathologies and evidence of strain-sharing events involving isolates sourced from companion and wild animals.

Identification of genes influencing the evolution of Escherichia coli ST372 in dogs and humans.

ST372 are widely reported as the major Escherichia coli sequence type in dogs globally. They are also a sporadic cause of extraintestinal infections in humans. Despite this, it is unknown whether ST372 strains from dogs and humans represent shared or distinct populations. Furthermore, little is known about genomic traits that might explain the prominence of ST372 in dogs or presence in humans. To address this, we applied a variety of bioinformatics analyses to a global collection of 407 ST372 E. coli whole-genome sequences to characterize their epidemiological features, population structure and associated accessory genomes. We confirm that dogs are the dominant host of ST372 and that clusters within the population structure exhibit distinctive O:H types. One phylogenetic cluster, 'cluster M', comprised almost half of the sequences and showed the divergence of two human-restricted clades that carried different O:H types to the remainder of the cluster. We also present evidence supporting transmission between dogs and humans within different clusters of the phylogeny, including M. We show that multiple acquisitions of the pdu propanediol utilization operon have occurred in clusters dominated by isolates of canine source, possibly linked to diet, whereas loss of the pdu operon and acquisition of K antigen virulence genes characterize human-restricted lineages.

Companion Animals Are Spillover Hosts of the Multidrug-Resistant Human Extraintestinal Escherichia coli Pandemic Clones ST131 and ST1193.

Escherichia coli sequence types 131 (ST131) and 1193 are multidrug-resistant extraintestinal pathogens that have recently spread epidemically among humans and are occasionally isolated from companion animals. This study characterized a nationwide collection of fluoroquinolone-resistant (FQ R ) E. coli isolates from extraintestinal infections in Australian cats and dogs. For this, 59 cat and dog FQ R clinical E. coli isolates (representing 6.9% of an 855-isolate collection) underwent PCR-based phylotyping and whole-genome sequencing (WGS). Isolates from commensal-associated phylogenetic groups A (14/59, 24%) and B1 (18/59, 31%) were dominant, with ST224 (10/59, 17%), and ST744 (8/59, 14%) predominating. Less prevalent were phylogenetic groups D (12/59, 20%), with ST38 (8/59, 14%) predominating, and virulence-associated phylogenetic group B2 (7/59, 12%), with ST131 predominating (6/7, 86%) and no ST1193 isolates identified. In a WGS-based comparison of 20 cat and dog-source ST131 isolates with 188 reference human and animal ST131 isolates, the cat and dog-source isolates were phylogenetically diverse. Although cat and dog-source ST131 isolates exhibited some minor sub-clustering, most were closely related to human-source ST131 strains. Furthermore, the prevalence of ST131 as a cause of FQ R infections in Australian companion animals was relatively constant between this study and the 5-year-earlier study of Platell et al. (2010) (9/125 isolates, 7.2%). Thus, although the high degree of clonal commonality among FQ R clinical isolates from humans vs. companion animals suggests the possibility of bi-directional between-species transmission, the much higher reported prevalence of ST131 and ST1193 among FQ R clinical isolates from humans as compared to companion animals suggests that companion animals are spillover hosts rather than being a primary reservoir for these lineages.

Genomic analysis of phylogenetic group B2 extraintestinal pathogenic E. coli causing infections in dogs in Australia.

This study investigated the prevalence of extraintestinal pathogenic E. coli (ExPEC)-associated sequence types (STs) from phylogenetic group B2 among 449 fluoroquinolone-susceptible dog clinical isolates from Australia. Isolates underwent PCR-based phylotyping and random amplified polymorphic DNA analysis to determine clonal relatedness. Of the 317 so-identified group B2 isolates, 77 underwent whole genome sequencing (WGS), whereas the remainder underwent PCR-based screening for ST complexes (STc) STc12, STc73, STc372, and ST131. The predominant ST was ST372 according to both WGS (31 % of 77) and ST-specific PCR (22 % of 240), followed by (per WGS) ST73 (17 %), ST12 (7 %), and ST80 (7 %). A WGS-based phylogenetic comparison of ST73 isolates from dogs, cats, and humans showed considerable overall phylogenetic diversity. Although most clusters were species-specific, some contained closely related human and animal (dog > cat) isolates. For dogs in Australia these findings both confirm ST372 as the predominant E. coli clonal lineage causing extraintestinal infections and clarify the importance of human-associated group B2 lineage ST73 as a cause of UTI, with some strains possibly being capable of bi-directional (i.e., dog-human and human-dog) transmission.

Genomic analysis of fluoroquinolone-susceptible phylogenetic group B2 extraintestinal pathogenic Escherichia coli causing infections in cats.

Extraintestinal pathogenic Escherichia coli (ExPEC) can cause urinary tract and other types of infection in cats, but the relationship of cat ExPEC to human ExPEC remains equivocal. This study investigated the prevalence of ExPEC-associated sequence types (STs) from phylogenetic group B2 among fluoroquinolone-susceptible cat clinical isolates. For this, 323 fluoroquinolone-susceptible cat clinical E. coli isolates from Australia underwent PCR-based phylotyping and random amplified polymorphic DNA analysis to determine clonal relatedness. Of the 274 group B2 isolates, 53 underwent whole genome sequencing (WGS), whereas 221 underwent PCR-based screening for (group B2) sequence type complexes (STc) STc12, STc73, ST131, and STc372. Group B2 was the dominant phylogenetic group (274/323, 85 %), whereas within group B2 ST73 dominated, according to both WGS (43 % of 53; followed by ST127, ST12, and ST372 [4/53, 8 % each]) and ST-specific PCR (20 % of 221). In WGS-based comparisons of cat and reference human ST73 isolates, cat isolates had a relatively conserved virulence gene profile but were phylogenetically diverse. Although in the phylogram most cat and human ST73 isolates occupied host species-specific clusters within serotype-specific clades (O2:H1, O6:H1, O25:H1, O50/O2:H1), cat and human isolates were intermingled within two serotype-specific clades: O120:H31 (3 cat and 2 human isolates) and O22:H1 (3 cat and 5 human isolates). These findings confirm the importance of human-associated group B2 lineages as a cause of urinary tract infections in cats. The close genetic relationship of some cat and human ST73 strains suggests bi-directional transmission may be possible.

Antimicrobial Susceptibility of Escherichia coli and Salmonella spp. Isolates From Healthy Pigs in Australia: Results of a Pilot National Survey.

This study investigated the frequency of antimicrobial non-susceptibility (defined as the frequency of isolates with minimum inhibitory concentrations above the CLSI susceptible clinical breakpoint) among E. coli and Salmonella spp. isolated from healthy Australian finisher pigs. E. coli (n = 201) and Salmonella spp. (n = 69) were isolated from cecal contents of slaughter-age pigs, originating from 19 farms distributed throughout Australia during July-December 2015. Isolates underwent minimum inhibitory concentration (MIC) susceptibility testing to 11 antimicrobials. The highest frequencies of non-susceptibility among respective isolates of E. coli and Salmonella spp. were to ampicillin (60.2 and 20.3%), tetracycline (68.2 and 26.1%), chloramphenicol (47.8 and 7.3%), and trimethoprim/sulfamethoxazole (33.8 and 11.6%). Four E. coli isolates had MICs above the wild-type epidemiological cut-off value for ciprofloxacin, with two isolates from the same farm classified as clinically resistant (MICs of > 4 µg/ml), a noteworthy finding given that fluoroquinolones (FQs) are not legally available for use in Australian food-producing animals. Three of these four E. coli isolates belonged to the sequence type (ST) 10, which has been isolated from both humans and production animals, whilst one isolate belonged to a new ST (7573) and possessed qnrS1. This study shows that non-susceptibility to first line antimicrobials is common among E. coli and Salmonella spp. isolates from healthy slaughter age pigs in Australia. However, very low levels of non-susceptibility to critically important antimicrobials (CIAs), namely third generation cephalosporins and fluoroquinolones were observed. Nevertheless, the isolation of two ciprofloxacin-resistant E. coli isolates from Australian pigs demonstrates that even in the absence of local antimicrobial selection pressure, fluoroquinolone-resistant E. coli clonal lineages may enter livestock production facilities despite strict biosecurity.

Antimicrobial resistance in clinical Escherichia coli isolated from companion animals in Australia.

Multidrug-resistant (MDR) Escherichia coli have become a major public health concern to both humans and animal health. While the frequency of antimicrobial resistance (AMR) in clinical E. coli is monitored regularly in human medicine, current frequency of AMR in companion animals remains unknown in Australia. In this study we conducted antimicrobial susceptibility testing (AST) and where possible, determined potential risk factors for MDR infection among 883 clinical Escherichia coli isolated from dogs (n=514), cats (n=341) and horses (n=28). AST was undertaken for 15 antimicrobial agents according to the Clinical Laboratory Standards Institute (CLSI) guidelines and interpreted using epidemiological cut-off values (ECOFFs) as well as CLSI veterinary and human clinical breakpoints. The AST revealed complete absence of resistance to carbapenems while resistance to amikacin was observed at a low level in isolates from dogs (1.6%) and cats (1.5%) compared to horses (10.7%). Among dog isolates, resistance to fluoroquinolones ranged from 9.1%-9.3% whereas among cat isolates, it ranged from 3.2%-5%. Among dog isolates, the proportion showing a 3rd generation cephalosporin (3GC) non-wild type phenotype was significantly higher (P<0.05) in skin and soft tissue infection (SSTI, n=122) isolates (17.2%-20.5%) compared to urinary tract infection (UTI, n=392) isolates (9.9%-10.2%). The frequency of multidrug resistance was 18.1%, 11.7% and 42.9% in dog, cat and horse isolates, respectively. Risk factor analysis revealed that MDR E. coli isolated from UTI were positively associated with chronicity of infection and previous antimicrobial treatment. Dogs and cats with chronic UTI that had been previously treated with antimicrobials were eight times and six times more likely to be infected with MDR E. coli compared to dogs and cats with non-chronic UTI, and no history of antimicrobial treatment, respectively. This study revealed that pre-existing disease condition and prior antimicrobial use were the major risks associated with UTI with MDR E. coli in companion animals.