Unveiling genome plasticity and a novel phage in Mycoplasma felis: Genomic investigations of four feline isolates.

Mycoplasma felis has been isolated from diseased cats and horses, but to date only a single fully assembled genome of this species, of an isolate from a horse, has been characterized. This study aimed to characterize and compare the completely assembled genomes of four clinical isolates of M. felis from three domestic cats, assembled with the aid of short- and long-read sequencing methods. The completed genomes encoded a median of 759 ORFs (range 743-777) and had a median average nucleotide identity of 98.2 % with the genome of the available equid origin reference strain. Comparative genomic analysis revealed the occurrence of multiple horizontal gene transfer events and significant genome reassortment. This had resulted in the acquisition or loss of numerous genes within the Australian felid isolate genomes, encoding putative proteins involved in DNA transfer, metabolism, DNA replication, host cell interaction and restriction modification systems. Additionally, a novel mycoplasma phage was detected in one Australian felid M. felis isolate by genomic analysis and visualized using cryo-transmission electron microscopy. This study has highlighted the complex genomic dynamics in different host environments. Furthermore, the sequences obtained in this work will enable the development of new diagnostic tools, and identification of future infection control and treatment options for the respiratory disease complex in cats.

Genomic characterization of Mycoplasma edwardii isolated from a dog bite induced cat wound reveals multiple horizontal gene transfer events and loss of the CRISPR/Cas system.

A domestic short hair cat (Felis catus) suffering from a purulent wound infection resulting from a dog bite was sampled for bacterial culture and isolation as the wound had been unresponsive to prolonged antimicrobial treatment. A mycoplasma was isolated from the wound. Whole genome sequencing of the isolate was performed using short-read Illumina and long-read Oxford Nanopore chemistry, and the organism was identified as Mycoplasma edwardii. Comparison of the genome sequence of the isolate to a reference M. edwardii genome sequence (canid isolate) identified the loss of several key bacterial factors involved in genome editing, as well the insertion of several novel ORFs most closely related to those found in other canine mycoplasmas, specifically Mycoplasma canis, M. cynos, M. molare and M. maculosa. This is only the second known report of disease caused by M. edwardii in a non-canid species, and the first report of it infecting and causing clinical disease in a cat.

Pasteurella multocida ST20 is widespread in Australian poultry farms and may infect wild waterbirds.

The bacterial agent that causes fowl cholera, Pasteurella multocida, was isolated from two deceased wild waterbirds in Victoria, Australia, in 2013. Whole genome sequence analysis placed the isolates into ST20, a subtype described in farmed chickens from Queensland, Australia and more recently in feedlot cattle and in pigs across a broader area of the continent. This study also found ST20 between 2009 and 2022 on three chicken farms and two turkey farms located in four Australian states. The sequences of 25 of these ST20 isolates were compared to 280 P. multocida genomes from 23 countries and to 94 ST20 Illumina datasets from Queensland that have been deposited in public databases. The ST20 isolates formed a single phylogenetic clade and were clustered into four sub-groups with highly similar genomes, possessing either LPS type 1 or type 3 loci. Various repertoires of mobile genetic elements were present in isolates from farmed, but not wild birds, suggesting complex histories of spill-over between avian populations and gene acquisition within farm environments. No major antimicrobial resistance was predicted in any of the ST20 isolates by the genomic analysis. The closest relative of these isolates was a ST394 bovine respiratory tract isolate from Queensland, which differed from ST20 by only one allele and carried beta-lactam and tetracycline resistance genes. These findings underline the importance of understanding the role of wild and commercial birds in the maintenance of fowl cholera, and of implementing regular epidemiological surveillance and biosecurity management programmes in wildlife, as well as free-range poultry farms.

Global Phylogeny and F Virulence Plasmid Carriage in Pandemic Escherichia coli ST1193.

Lower urinary tract, renal, and bloodstream infections caused by phylogroup B2 extraintestinal pathogenic Escherichia coli (ExPEC) are a leading cause of morbidity and mortality. ST1193 is a phylogroup B2, multidrug-resistant sequence type that has risen to prominence globally, but a comprehensive analysis of the F virulence plasmids it carries is lacking. We performed a phylogenomic analysis of ST1193 (n = 707) whole-genome sequences from EnteroBase using entries with comprehensive isolation metadata. The data set comprised isolates from humans (n = 634 [90%]), including 339 (48%) from extraintestinal infection sites, and isolates from companion animals, wastewater, and wildlife. Phylogenetic analyses combined with gene detection and genotyping resolved an ST1193 clade structure segregated by serotype and F plasmid carriage. Most F plasmids fell into one of three related plasmid subtypes: F-:A1:B10 (n = 444 [65.97%]), F-:A1:B1 (n = 84 [12.48%]), and F-:A1:B20 (n = 80 [11.89%]), all of which carry the virulence genes cjrABC colocalized with senB (cjrABC-senB), a trademark signature of F29:A-:B10 subtype plasmids (pUTI89). To examine the phylogenetic relationship of these plasmids with pUTI89, complete sequences of F-:A1:B1 and F-:1:B20 plasmids were resolved. Unlike pUTI89, the most dominant and widely disseminated F plasmid that carries cjrABC-senB, F plasmids in ST1193 often carry a complex resistance region with an integron truncation (intI1Δ745) signature embedded within a structure assembled by IS26. Plasmid analysis shows that ST1193 has F plasmids that carry cjrABC-senB and ARG-encoding genes but lack tra regions and are likely derivatives of pUTI89. Further epidemiological investigation of ST1193 should seek to confirm its presence in human-associated environments and identify any potential agricultural links, which are currently lacking. IMPORTANCE We have generated an updated ST1193 phylogeny using publicly available sequences, reinforcing previous assertions that Escherichia coli ST1193 is a human-associated lineage, with many examples sourced from human extraintestinal infections. ST1193 from urban-adapted birds, wastewater, and companion animals are frequent, but isolates from animal agriculture are notably absent. Phylogenomic analysis identified several clades segregated by serogroup, all noted to carry highly similar F plasmids and antimicrobial resistance (AMR) signatures. Investigation of these plasmids revealed virulence regions with similarity to pUTI89, a key F virulence plasmid among dominant pandemic extraintestinal pathogenic E. coli lineages, and encoding a complex antibiotic resistance structure mobilized by IS26. This work has uncovered a series of F virulence plasmids in ST1193 and shows that the lineage mimics the host range and virulence attributes of other E. coli strains that carry pUTI89. These observations have significant ramifications for epidemiological source tracking of emerging and established pandemic ExPEC lineages.

Healthcare-associated infections caused by chlorhexidine-tolerant Serratia marcescens carrying a promiscuous IncHI2 multi-drug resistance plasmid in a veterinary hospital.

The bacterium Serratia marcescens can cause opportunistic infections in humans and in animals. In veterinary settings, the diversity, reservoirs and modes of transmission of this pathogen are poorly understood. The phenotypes and genotypes of Serratia spp. isolated from dogs, cats, horses, a bird and a rabbit examined at an Australian veterinary hospital between 2008 and 2019 were characterised. The isolates were identified as S. marcescens (n = 15) or S. ureilytica (n = 3) and were placed into four distinct phylogenetic groups. Nine quasi-clonal isolates associated with post-surgical complications in different patients displayed high levels of resistance to the antimicrobials fluoroquinolones, cephalosporins, aminoglycosides, and to the disinfectant chlorhexidine. A Serratia sp. with a similar resistance profile was also isolated from chlorhexidine solutions used across the Hospital, suggesting that these infections had a nosocomial origin. A genomic island encoding a homolog of the Pseudomonas MexCD-OprJ biocide efflux system was detected in the chlorhexidine-tolerant Serratia. The nine multi-drug resistant Serratia isolates also possessed a Ser-83-Ile mutation in GyrA conferring fluoroquinolone resistance, and carried a large IncHI2 conjugative plasmid encoding antimicrobial and heavy metal resistances. This replicon was highly similar to a plasmid previously detected in a strain of Enterobacter hormaechei recovered from the Hospital environment. IncHI2 plasmids are commonly found in Enterobacteriaceae, but are rarely present in Serratia spp., suggesting that this plasmid was acquired from another organism. A chlorhexidine-tolerant Serratia isolate which lacked the IncHI2 plasmid was used in mating experiments to demonstrate the transfer of multi-drug resistance from a E. hormaechei donor. This study illustrates the importance of environmental surveillance of biocide-resistance in veterinary hospitals.

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.

Whole genome sequence analysis of Australian avian pathogenic Escherichia coli that carry the class 1 integrase gene.

Avian pathogenic Escherichia coli (APEC) cause widespread economic losses in poultry production and are potential zoonotic pathogens. Genome sequences of 95 APEC from commercial poultry operations in four Australian states that carried the class 1 integrase gene intI1, a proxy for multiple drug resistance (MDR), were characterized. Sequence types ST117 (22/95), ST350 (10/95), ST429 and ST57 (each 9/95), ST95 (8/95) and ST973 (7/95) dominated, while 24 STs were represented by one or two strains. FII and FIB repA genes were the predominant (each 93/95, 98 %) plasmid incompatibility groups identified, but those of B/O/K/Z (25/95, 26 %) and I1 (24/95, 25 %) were also identified frequently. Virulence-associated genes (VAGs) carried by ColV and ColBM virulence plasmids, including those encoding protectins [iss (91/95, 96 %), ompT (91/95, 96 %) and traT (90/95, 95 %)], iron-acquisition systems [sitA (88/95, 93 %), etsA (87/95, 92 %), iroN (84/95, 89 %) and iucD/iutA (84/95, 89 %)] and the putative avian haemolysin hylF (91/95, 96 %), featured prominently. Notably, mobile resistance genes conferring resistance to fluoroquinolones, colistin, extended-spectrum ß-lactams and carbapenems were not detected in the genomes of these 95 APEC but carriage of the sulphonamide resistance gene, sul1 (59/95, 63 %), the trimethoprim resistance gene cassettes dfrA5 (48/95, 50 %) and dfrA1 (25/95, 27 %), the tetracycline resistance determinant tet(A) (51/95, 55 %) and the ampicillin resistance genes blaTEM-1A/B/C (48/95, 52 %) was common. IS26 (77/95, 81 %), an insertion element known to capture and mobilize a wide spectrum of antimicrobial resistance genes, was also frequently identified. These studies provide a baseline snapshot of drug-resistant APEC in Australia and their role in the carriage of ColV-like virulence plasmids.

A Novel Glaesserella sp. Isolated from Pigs with Severe Respiratory Infections Has a Mosaic Genome with Virulence Factors Putatively Acquired by Horizontal Transfer.

An unknown member of the family Pasteurellaceae was repeatedly isolated from 20- to 24-week-old pigs with severe pulmonary lesions reared on the same farm in Victoria, Australia. The etiological diagnosis of the disease was inconclusive. The complete genome sequence analysis of one strain, 15-184, revealed some phylogenic proximity to Glaesserella (Haemophilus) parasuis, the cause of Glasser's disease. However, the sequences of the 16S rRNA and housekeeping genes, as well as the average nucleotide identity scores, differed from those of all other known species in the family Pasteurellaceae The protein content of 15-184 was composite, with 60% of coding sequences matching known G. parasuis products, while more than 20% had a closer relative in the genera Actinobacillus, Mannheimia, Pasteurella, and Bibersteinia Several putative virulence genes absent from G. parasuis but present in other Pasteurellaceae were also found, including the apxIII RTX toxin gene from Actinobacillus pleuropneumoniae, ABC transporters from Actinobacillus minor, and iron transporters from various species. Three prophages and one integrative conjugative element were present in the isolate. Horizontal gene transfers might explain the mosaic genomic structure and atypical metabolic and virulence characteristics of 15-184. This organism has not been assigned a taxonomic position in the family, but this study underlines the need for a large-scale epidemiological and clinical characterization of this novel pathogen in swine populations, as a genomic analysis suggests it could have a severe impact on pig health.IMPORTANCE Several species of Pasteurellaceae cause a range of significant diseases in pigs. A novel member of this family was recently isolated from Australian pigs suffering from severe respiratory infections. Comparative whole-genome analyses suggest that this bacterium represents a new species, which possesses a number of virulence genes horizontally acquired from a diverse range of other Pasteurellaceae While the possible contribution of other coinfecting noncultivable agents to the disease has not been ruled out in this study, the repertoire of virulence genes found in this organism may nevertheless explain some aspects of the associated pathology observed on the farm. The prevalence of this novel pathogen within pig populations is currently unknown. This finding is of particular importance for the pig industry, as this organism can have a serious impact on the health of these animals.