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.

Chlamydia psittaci is an avian pathogen with zoonotic potential. In Australia, C. psittaci has been well reported as a cause of reproductive loss in mares which subsequently have been the source of infection and illness in some in-contact humans. To date, molecular typing studies describe the predominant and clonal C. psittaci sequence type (ST)24 strains in horse, psittacine, and human infections. We sought to assess the clonality between ST24 strains and the emergence of equine ST24 with a comprehensive genomics approach. We used culture-independent probe-based and metagenomic whole-genome sequencing to investigate 13 C . psittaci genomes from horses, psittacines, and a pigeon from Australia. Published genomes of 36 C . psittaci strains were also used to contextualise our Australian dataset and investigate lineage diversity. We utilised a single-nucleotide polymorphism (SNP) based clustering and multi-locus sequence typing (MLST) approach. C. psittaci has four major phylogenetic groups (PG1-4) based on core-genome SNP-based phylogeny. PG1 contained clonal global and Australian equine, psittacine, and human ST24 genomes, with a median pairwise SNP distance of 68 SNPs. PG2, PG3, and PG4 had greater genomic diversity, including diverse STs collected from birds, livestock, human, and horse hosts from Europe and North America and a racing pigeon from Australia. We show that the clustering of C. psittaci by MLST was congruent with SNP-based phylogeny. The monophyletic ST24 clade has four major sub-lineages. The genomes of 17 Australian human, equine, and psittacine strains collected between 2008 and 2021 formed the predominant ST24 sub-lineage 1 (emerged circa 1979). Despite a temporal distribution of 13 years, the genomes within sub-lineage 1 had a median pairwise SNP distance of 32 SNPs, suggesting a recent population expansion or potential cross-host transmission. However, two C. psittaci genomes collected in 2015 from Victorian parrots clustered into distinct ST24 sub-lineage 4 (emerged circa 1965) with ovine strain C19/98 from Germany. This work describes a comprehensive phylogenomic characterisation of ST24 and identifies a timeline of potential bird-to-equine spillover events.

Chlamydia pecorum, an obligate intracellular pathogen, causes significant morbidity and mortality in livestock and the koala (Phascolarctos cinereus). A variety of C. pecorum gene-centric molecular studies have revealed important observations about infection dynamics and genetic diversity in both koala and livestock hosts. In contrast to a variety of C. pecorum molecular studies, to date, only four complete and 16 draft genomes have been published. Of those, only five draft genomes are from koalas. Here, using whole-genome sequencing and a comparative genomics approach, we describe the first two complete C. pecorum genomes collected from diseased koalas. A de novo assembly of DBDeUG_2018 and MC/MarsBar_2018 resolved the chromosomes and chlamydial plasmids each as single, circular contigs. Robust phylogenomic analyses indicate biogeographical separation between strains from northern and southern koala populations, and between strains infecting koala and livestock hosts. Comparative genomics between koala strains identified new, unique, and shared loci that accumulate single-nucleotide polymorphisms and separate between northern and southern, and within northern koala strains. Furthermore, we predicted novel type III secretion system effectors. This investigation constitutes a comprehensive genome-wide comparison between C. pecorum from koalas and provides improvements to annotations of a C. pecorum reference genome. These findings lay the foundations for identifying and understanding host specificity and adaptation behind chlamydial infections affecting koalas.

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At least 300 million urinary tract infections (UTIs) occur annually worldwide. Uropathogenic Escherichia coli (UPEC) are the leading cause of UTIs. The discovery of antibiotics has revolutionised modern medicine. Yet, overusing antibiotics has accelerated the emergence of antimicrobial resistance (AMR), with UPEC driving the dissemination of AMR globally. Resistance to broad-spectrum antibiotics like third-generation cephalosporins (3GCs) and fluoroquinolones threatens public health. Extended-spectrum b-lactamase (ESBL)-producing E. coli precipitate resistance, particularly when these antibiotics are used as empirical therapies against UPEC. In response, the Centers for Disease Control and Prevention in the United States have listed ESBL-producing Enterobacterales, such as E. coli as a severe threat. Additionally, the World Health Organization have classified 3GCs and fluoroquinolones as the highest priority (critically important antimicrobials), where these therapies are only recommended following susceptibility testing. The present report demonstrates the distributions of E. coli cases with resistance to 3GC and fluoroquinolones in Australia and New Zealand and contextualises trends with European reports. This investigation emphasises the value of epidemiology and the justification of evidence-based interventions using data as an essential resource for reducing resistance to our 'first-line' antibiotics.

Chlamydia psittaci is a globally distributed veterinary pathogen with zoonotic potential. Although C. psittaci infections have been reported in various hosts, isolation and culture of Chlamydia is challenging, hampering efforts to produce contemporary global C. psittaci genomes. This is particularly evident in the lack of avian C. psittaci genomes from Australia and New Zealand. In this study, we used culture-independent probe-based whole-genome sequencing to expand the global C. psittaci genome catalogue. Here, we provide new C. psittaci genomes from two pigeons, six psittacines, and novel hosts such as the Australian bustard (Ardeotis australis) and sooty shearwater (Ardenna grisea) from Australia and New Zealand. We also evaluated C. psittaci genetic diversity using multilocus sequence typing (MLST) and major outer membrane protein (ompA) genotyping on additional C. psittaci -positive samples from various captive avian hosts and field isolates from Australasia. We showed that the first C. psittaci genomes sequenced from New Zealand parrots and pigeons belong to the clonal sequence type (ST)24 and diverse ‘pigeon-type’ ST27 clade, respectively. Australian parrot-derived strains also clustered in the ST24 group, whereas the novel ST332 strain from the Australian bustard clustered in a genetically diverse clade of strains from a fulmar, parrot, and livestock. MLST and ompA genotyping revealed ST24/ompA genotype A in wild and captive parrots and a sooty shearwater, whilst ‘pigeon-types’ (ST27/35 and ompA genotypes B/E) were found in pigeons and other atypical hosts, such as captive parrots, a little blue penguin/Kororā (Eudyptula minor) and a zebra finch (Taeniopygia guttata castanotis) from Australia and New Zealand. This study provides new insights into the global phylogenomic diversity of C. psittaci and further demonstrates the multi-host generalist capacity of this pathogen.

Fowl cholera caused by Pasteurella multocida has re-emerged in Australian poultry production since the increasing adoption of free-range production systems. Currently, autogenous killed whole-cell vaccines prepared from the isolates previously obtained from each farm are the main preventative measures used. In this study, we use whole-genome sequencing and phylogenomic analysis to investigate outbreak dynamics, as well as monitoring and comparing the variations in the lipopolysaccharide (LPS) outer core biosynthesis loci of the outbreak and vaccine strains. In total, 73 isolates from two different free-range layer farms were included. Our genomic analysis revealed that all investigated isolates within the two farms (layer A and layer B) carried LPS type L3, albeit with a high degree of genetic diversity between them. Additionally, the isolates belonged to five different sequence types (STs), with isolates belonging to ST9 and ST20 being the most prevalent. The isolates carried ST-specific mutations within their LPS type L3 outer core biosynthesis loci, including frameshift mutations in the outer core heptosyltransferase gene (htpE) (ST7 and ST274) or galactosyltransferase gene (gatG) (ST20). The ST9 isolates could be separated into three groups based on their LPS outer core biosynthesis loci sequences, with evidence for potential phase variation mechanisms identified. The potential phase variation mechanisms included a tandem repeat insertion in natC and a single base deletion in a homopolymer region of gatG. Importantly, our results demonstrated that two of the three ST9 groups shared identical rep-PCR (repetitive extragenic palindromic PCR) patterns, while carrying differences in their LPS outer core biosynthesis loci region. In addition, we found that ST9 isolates either with or without the natC tandem repeat insertion were both associated with a single outbreak, which would indicate the importance of screening more than one isolate within an outbreak. Our results strongly suggest the need for a metagenomics culture-independent approach, as well as a genetic typing scheme for LPS, to ensure an appropriate vaccine strain with a matching predicted LPS structure is used.

Increasing resistance to third-generation cephalosporins (3GCs) threatens public health, as these antimicrobials are prescribed as empirical therapies for systemic infections caused by Gram-negative bacteria. Resistance to 3GCs in urinary tract infections (UTIs) and bacteraemia is associated with the globally disseminated, multidrug-resistant, uropathogenic Escherichia coli sequence type (ST)131. This study combines the epidemiology of E.coli blood culture surveillance with whole-genome sequencing (WGS) to investigate ST131 associated with bacteraemia in Wales between 2013 and 2014. This population-based prospective genomic analysis investigated temporal, geographic, and genomic risk factors. To identify spatial clusters and lineage diversity, we contextualised 142 genomes collected from twenty hospitals, against a global ST131 population (n=181). All three major ST131 clades are represented across Wales, with clade C/H30 predominant (n=102/142, 71.8%). Consistent with global findings, Welsh strains of clade C/H30 contain β-lactamase genes from the blaCTX-M-1 group (n=65/102, 63.7%), which confers resistance to 3GCs. In Wales, the majority of clade C/H30 strains belonged to sub-clade C2/H30Rx (n=88/151, 58.3%), whereas sub-clade C1/H30R strains were less common (n=14/67, 20.9%). A sub-lineage unique to Wales was identified within the C2/H30Rx sub-clade (named GB-WLS.C2/H30Rx) and is defined by six non-recombinogenic single-nucleotide polymorphisms (SNPs), including a missense variant in febE (ferric enterobactin transport protein) and fryC (fructose-like permease IIC component), and the loss of the capsular biosynthesis genes encoding the K5 antigen. Bayesian analysis predicted that GB-WLS.C2/H30Rx diverged from a common ancestor (CA) most closely related to a Canadian strain between 1998 and 1999. Further, our analysis suggests a descendent of GB-WLS.C2/H30Rx arrived through an introduction to North Wales circa 2002, spread and persists in the geographic region, causing a cluster of cases (CA emerged circa 2009) with a maximum pair-wise distance of 30 non-recombinogenic SNPs. This limited genomic diversity likely depicts local transmission within the community in North Wales. This investigation emphasises the value of genomic epidemiology, allowing detection of suspected transmission clusters and the spread of genetically similar/identical strains in local areas. These analyses will enable targeted and timely public health interventions.