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.

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.

Detection and Differentiation of Entomopathogenic Serratia spp. to Inform Reintroduction of the Critically Endangered Lord Howe Island Stick Insect Dryococelus australis.

Once rodents have been successfully eradicated from Lord Howe Island, Australia, the critically endangered Lord Howe Island stick insect (Dryococelus australis (Montrouzier)) may be reintroduced, a century after it was thought to have become extinct. In captive populations of D. australis, elevated mortalities have been associated with bacterial pathogens. To better define the infectious risk posed by entomopathogens to the reintroduction program, we investigated the bacteria isolated from captive D. australis kept at Melbourne Zoo and on Lord Howe Island and from environmental samples and free-living invertebrates collected on various parts of the island. At Melbourne Zoo, Serratia and Pseudomonas spp. were the bacteria most frequently isolated between 2013 and 2019. Serratia spp. were also the organisms most frequently isolated from insects sampled in April 2019 from the captive population on Lord Howe Island. In addition, Serratia spp. were isolated from a range of environmental samples collected on Lord Howe Island during March-April 2019. These environmental isolates had a broader range of biochemical and molecular characteristics than those obtained from the captive insect populations. A large proportion of these isolates were urease positive and had biochemical profiles previously not described for Serratia spp. This study highlights the need for better surveillance for potential pathogens in understudied regions and sites. We conclude that infections caused by Serratia spp. might pose a problem to the captive breeding program for D. australis but that the risk of introducing novel pathogens to Lord Howe Island through infected insects is low. Our study explores some of the potential risks involved in captive breeding and provides a valuable example of using pathogen surveillance to better inform an invertebrate conservation program.

Whole Genome Sequencing of Avian Pathogenic Escherichia coli Causing Bacterial Chondronecrosis and Osteomyelitis in Australian Poultry.

Bacterial chondronecrosis with osteomyelitis (BCO) impacts animal welfare and productivity in the poultry industry worldwide, yet it has an understudied pathogenesis. While Avian Pathogenic Escherichia coli (APEC) are known to be one of the main causes, there is a lack of whole genome sequence data, with only a few BCO-associated APEC (APECBCO) genomes available in public databases. In this study, we conducted an analysis of 205 APECBCO genome sequences to generate new baseline phylogenomic knowledge regarding the diversity of E. coli sequence types and the presence of virulence associated genes (VAGs). Our findings revealed the following: (i) APECBCO are phylogenetically and genotypically similar to APEC that cause colibacillosis (APECcolibac), with globally disseminated APEC sequence types ST117, ST57, ST69, and ST95 being predominate; (ii) APECBCO are frequent carriers of ColV-like plasmids that carry a similar set of VAGs as those found in APECcolibac. Additionally, we performed genomic comparisons, including a genome-wide association study, with a complementary collection of geotemporally-matched genomes of APEC from multiple cases of colibacillosis (APECcolibac). Our genome-wide association study found no evidence of novel virulence loci unique to APECBCO. Overall, our data indicate that APECBCO and APECcolibac are not distinct subpopulations of APEC. Our publication of these genomes substantially increases the available collection of APECBCO genomes and provides insights for the management and treatment strategies of lameness in poultry.

Obg plays a significant role in temperature sensitivity of Mycoplasma synoviae live attenuated vaccine strain MS-H.

The MS-H vaccine strain (Vaxsafe MS®; Bioproperties Pty. Ltd., Australia) is a live attenuated temperature sensitive derivative of a virulent strain of M. synoviae, 86079/7NS, and is used to prevent diseases from M. synoviae challenges in poultry farms. The genome sequence of MS-H includes 32 single nucleotide polymorphisms (SNPs) compared to that of 86079/7NS. To investigate the nature of mutations responsible for temperature sensitivity, MS-H strain was subjected to thermal adaptation in vitro and in vivo. The only observed variation detected in the MS-H culture following sequential passages with incremental incubation temperature from 33 °C to 39.5 °C was an Ala210Val variation in Obg protein, associated with loss of temperature sensitivity phenotype. An identical variation was detected in the MS-H culture reisolated from one out of five bird 28 days after inoculation with MS-H. These findings suggest that M. synoviae is capable of thermoadaptive evolution and Obg plays a significant role in this trait.

Reversion of mutations in a live mycoplasma vaccine alters its metabolism.

The live attenuated temperature sensitive vaccine strain MS-H (Vaxsafe® MS, Bioproperties Pty. Ltd., Australia) is widely used to control disease associated with M. synoviae infection in commercial poultry. MS-H was derived from a field strain (86079/7NS) through N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-induced mutagenesis. Whole genomic sequence analysis of the MS-H and comparison with that of the 86079/7NS have found that MS-H contains 32 single nucleotide polymorphisms (SNPs). Three of these SNPs, found in the obgE, oppF and gapdh genes, have been shown to be prone to reversion under field condition, albeit at a low frequency. Three MS-H reisolates containing the 86079/7NS genotype in obgE (AS2), obgE and oppF (AB1), and obgE, oppF and gapdh (TS4), appeared to be more immunogenic and transmissible compared to MS-H in chickens. To investigate the influence of these reversions in the in vitro fitness of M. synoviae, the growth kinetics and steady state metabolite profiles of the MS-H reisolates, AS2, AB1 and TS4, were compared to those of the vaccine strain. Steady state metabolite profiling of the reisolates showed that changes in ObgE did not significantly influence the metabolism, while changes in OppF was associated with significant alterations in uptake of peptides and/or amino acids into the M. synoviae cell. It was also found that GAPDH plays a role in metabolism of the glycerophospholipids as well as an arginine deiminase (ADI) pathway. This study underscores the role of ObgE, OppF and GAPDH in M. synoviae metabolism, and suggests that the impaired fitness arising from variations in ObgE, OppF and GAPDH contributes to attenuation of MS-H.

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.

Virulence factors of Mycoplasma synoviae: Three genes influencing colonization, immunogenicity, and transmissibility.

Infections caused by Mycoplasma synoviae are major welfare and economic concerns in poultry industries worldwide. These infections cause chronic respiratory disease and/or synovitis in chickens and turkeys leading to reduced production and increased mortality rates. The live attenuated vaccine strain MS-H (Vaxsafe® MS), commonly used for protection against M. synoviae infection in many countries, contains 32 single nucleotide variations compared to its wildtype parent strain, 86079/7NS. Genomic analysis of vaccine strains reisolated from flocks following the administration of MS-H has identified reversions to the original 86079/7NS sequence in the obgE, oppF and gapdh genes. Here, three MS-H field reisolates containing the 86079/7NS genotype in obgE (AS2), obgE and oppF (AB1), and obgE, oppF and gapdh (TS4), as well as the vaccine MS-H and the parental strain 86079/7NS were experimentally inoculated to chickens. The strains were assessed for their ability to infect and elicit immune responses in the recipient chickens, as well as in naïve in-contact chickens. Despite the loss of temperature sensitivity phenotype and colonization of the reisolates in the lower respiratory tract, there was no significant differences detected in the microscopic mucosal thickness of the middle or lower trachea of the inoculated chickens. Concurrent reversions in ObgE, OppF and GAPDH proteins were associated with higher gross air sac lesion scores and increased microscopic upper-tracheal mucosal thickness in chickens directly inoculated with the reisolates following intratracheal administration of a virulent strain of infectious bronchitis virus. The gross air sac lesions of the chickens in-contact with those inoculated with reisolates were not significantly different to those of chickens in-contact with MS-H inoculated chickens, suggesting that horizontal transmission of the reisolates in the poultry flock will not lead to higher pathogenicity or clinical signs. These results suggest a significant role of GAPDH and/or cumulative effect of ObgE, OppF and GAPDH on M. synoviae pathogenicity. Future experiments will be required to investigate the effect of single mutations in gapdh or oppF gene on pathogenicity of M. synoviae.

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.

Genomic Diversity of a Globally Used, Live Attenuated Mycoplasma Vaccine.

The Mycoplasma synoviae live attenuated vaccine strain MS-H (Vaxsafe MS; Bioproperties Pty., Ltd., Australia) is commonly used around the world to prevent chronic infections caused by M. synoviae in birds and to minimize economic losses in the poultry industry. MS-H is a temperature-sensitive strain that is generated via the chemical mutagenesis of a virulent M. synoviae isolate, 86079/7NS. 32 single nucleotide polymorphisms have been found in the genome of MS-H compared to that of 86079/7NS, including 25 in predicted coding sequences (CDSs). There is limited information on the stability of these mutations in MS-H in vitro during the propagation of the vaccine manufacturing process or in vivo after the vaccination of chickens. Here, we performed a comparative analysis of MS-H genomes after in vitro and in vivo passages under different circumstances. Studying the dynamics of the MS-H population can provide insights into the factors that potentially affect the health of vaccinated birds. The genomes of 11 in vitro laboratory passages and 138 MS-H bird reisolates contained a total of 254 sequence variations. Of these, 39 variations associated with CDSs were detected in more than one genome (range = 2 to 62, median = 2.5), suggesting that these sequences are particularly prone to mutations. From the 25 CDSs containing previously characterized variations between MS-H and 86079/7NS, 7 were identified in the MS-H reisolates and progenies examined here. In conclusion, the MS-H genome contains individual regions that are prone to mutations that enable the restoration of the genotype or the phenotype of wild-type 86079/7NS in those regions. However, accumulated mutations in these regions are rare. IMPORTANCE Preventative measures, such as vaccination, are commonly used for the control of mycoplasmal infections in poultry. A live attenuated vaccine strain (Vaxsafe MS; MS-H; Bioproperties Pty. Ltd., Australia) is used for the prevention of disease caused by M. synoviae in many countries. However, information on the stability of previously characterized mutations in the MS-H genome is limited. In this study, we performed a comparative analysis of the whole-genome sequences of MS-H seeds used for vaccine manufacturing, commercial batches of the vaccine, cultures minimally passaged under small-scale laboratory and large-scale manufacturing conditions, MS-H reisolated from specific-pathogen-free (SPF) chickens that were vaccinated under controlled conditions, and MS-H reisolated from vaccinated commercial poultry flocks around the world. This study provides a comprehensive assessment of genome stability in MS-H after in vitro and in vivo passages under different circumstances and suggests that most of the mutations in the attenuated MS-H vaccine strain are stable.

Nebulization as a more efficient method than atomizer for experimental reproduction of avian colibacillosis in young chickens.

Infection and immunity studies involving genetically modified organisms (GMOs), such as gene knockout bacterial mutants, require stringent physical containment to prevent the accidental spread of these organisms into the environment. Experimental respiratory tract infection models often require the animals, for example birds, to be transported several times between a negative pressure housing isolator and a bespoke aerosol exposure chamber under positive pressure. While the exposure chamber is sealed and fitted with HEPA filters, the repeated movements of infected animals and opening of the chamber can still pose a serious risk of breaching containment of the organism in the experimental facility. In the current study, the ability of two aerosol infection protocols that expose birds to avian pathogenic E. coli (APEC) aerosols directly within the housing isolator was evaluated. Young chicks were exposed to APEC E956 within the negative pressure housing isolators using either a nebulizer or an atomizer. Birds exposed twice (days 1 and 4) to aerosols of APEC E956 produced by the nebulizer developed a rapidly progressing disease mimicking field cases of avian colibacillosis. However, birds exposed to aerosols of APEC E956 produced by an atomizer did not develop colibacillosis even after three exposures to APEC E956 on days 1, 4 and 7. Consequently, the current study reports the nebulizer was more efficacious in producing avian colibacillosis under stricter bacterial containment settings.RESEARCH HIGHLIGHTS Two aerosol exposure methods were evaluated to develop avian colibacillosis.Nebulizer method found to be more efficient in reproducing avian colibacillosis.Refined infection method can be used to study genetically modified organisms (GMOs).

Genomic and Temporal Trends in Canine ExPEC Reflect Those of Human ExPEC.

Companion animals and humans are known to share extraintestinal pathogenic Escherichia coli (ExPEC), but the extent of E. coli sequence types (STs) that cause extraintestinal diseases in dogs is not well understood. Here, we generated whole-genome sequences of 377 ExPEC collected by the University of Melbourne Veterinary Hospital from dogs over an 11-year period from 2007 to 2017. Isolates were predominantly from urogenital tract infections (219, 58.1%), but isolates from gastrointestinal specimens (51, 13.5%), general infections (72, 19.1%), and soft tissue infections (34, 9%) were also represented. A diverse collection of 53 STs were identified, with 18 of these including at least five sequences. The five most prevalent STs were ST372 (69, 18.3%), ST73 (31, 8.2%), ST127 (22, 5.8%), ST80 (19, 5.0%), and ST58 (14, 3.7%). Apart from ST372, all of these are prominent human ExPEC STs. Other common ExPEC STs identified included ST12, ST131, ST95, ST141, ST963, ST1193, ST88, and ST38. Virulence gene profiles, antimicrobial resistance carriage, and trends in plasmid carriage for specific STs were generally reflective of those seen in humans. Many of the prominent STs were observed repetitively over an 11-year time span, indicating their persistence in the dogs in the community, which is most likely driven by household sharing of E. coli between humans and their pets. The case of ST372 as a dominant canine lineage observed sporadically in humans is flagged for further investigation. IMPORTANCE Pathogenic E. coli that causes extraintestinal infections (ExPEC) in humans and canines represents a significant burden in hospital and veterinary settings. Despite the obvious interrelationship between dogs and humans favoring both zoonotic and anthropozoonotic infections, whole-genome sequencing projects examining large numbers of canine-origin ExPEC are lacking. In support of anthropozoonosis, we found that most STs from canine infections are dominant human ExPEC STs (e.g., ST73, ST127, ST131) with similar genomic traits, such as plasmid carriage and virulence gene burden. In contrast, we identified ST372 as the dominant canine ST and a sporadic cause of infection in humans, supporting zoonotic transfer. Furthermore, we highlight that, as is the case in humans, STs in canine disease are consistent over time, implicating the gastrointestinal tract as the major community reservoir, which is likely augmented by exposure to human E. coli via shared diet and proximity.

Efficient disruption of the function of the mnuA nuclease gene using the endogenous CRISPR/Cas system in Mycoplasma gallisepticum.

Mycoplasmas are important animal pathogens, but the functions and roles of many of their genes in pathogenesis remain unclear, in large part because of the limited tools available for targeted mutagenesis in these bacteria. In this study we used the Mycoplasma gallisepticum CRISPR/Cas system to target a nuclease gene, MGA_0637 (mnuA), which is predicted to play a role in survival and virulence. Our strategy used simultaneous targeting of the ksgA kasugamycin resistance gene, as a mutation in this gene would not interfere with replication but would confer a readily detectable and selectable phenotype in transformants. A guide RNA plasmid, pKM-CRISPR, was constructed, with spacers targeting the ksgA and mnuA genes transcribed under the control of the vlhA1.1 promoter in a backbone plasmid carrying the oriC of M. imitans, and this plasmid was introduced into electrocompetent M. gallisepticum strain S6 cells. PCR assays targeting the ksgA gene, followed by Sanger sequence analyses of the phenotypically resistant transformants, detected polymorphisms within the targeted region of ksgA, confirming the activity of the endogenous CRISPR/Cas system. The nuclease activity of the kasugamycin resistant colonies was then assessed using zymogram assays. The complete or partial loss of nuclease activity in the majority of kasugamycin resistant isolates transformed with the CRISPR plasmid confirmed that the endogenous CRISPR/Cas system had effectively interfered with the function of both ksgA and mnuA genes. Sanger sequencing and RT-qPCR analyses of the mnuA gene suggested that the M. gallisepticum CRISPR/Cas system can be programmed to cleave both DNA and RNA.

Genomic characterisation of an entomopathogenic strain of Serratia ureilytica in the critically endangered phasmid Dryococelus australis.

Between 2014 and 2019, unexpected mortalities were observed in a colony of Dryococelus australis, an endangered stick-insect kept at the Melbourne Zoo for a breeding and conservation program. Pure cultures of Serratia spp. were obtained from the haemolymph of moribund and recently deceased individuals. The combined bacteriological and histopathological observations suggested an infectious cause of these mortalities. Genotyping of Serratia sp. isolated from the insects and their environment revealed a predominant strain profile. A representative isolate, AM923, was entirely sequenced and compared to 616 publicly available Serratia spp. genomes, including 37 associated with insects. The genomes were distributed into 3 distinct groups, with 63% of the insect-associated isolates within a single clade (clade A) containing AM923, separated from most environmental/plant-associated strains (clade B) and human isolates (clade C). Average nucleotide identity and phylogenetic analyses identified AM923 as S. ureilytica and revealed similarities with putatively entomopathogenic strains. An experimental infection model in honey bees (Apis mellifera) confirmed the pathogenic potential of AM923. A urease operon was found in most insect isolates and a PCR assay, based on the ureB gene sequence, was used to confirm the presence of AM923 in experimentally infected bees. This species-specific PCR could be applied to detect entomopathogenic Serratia spp. in infected insects or their environment.

Rapid typing of infectious laryngotracheitis virus directly from tracheal tissues based on next-generation sequencing.

Infectious laryngotracheitis virus (ILTV) is the causative agent of an economically important disease of chickens causing upper respiratory tract infection. Strains of ILTV are commonly identified by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and/or PCR high resolution melt (PCR-HRM) curve analysis targeting several genes. However, these techniques examine only a limited number of mutations present inside the target regions and may generate unreliable results when the sample contains more than one strain. Here, we attempted to sequence the whole genome of ILTV with known identity (class 9) directly from tracheal scrapings to circumvent in vitro culturing, which can potentially introduce variations into the genome. Despite the large number of quality reads, mapping was compromised by poor overlapping and gaps, and assembly of the complete genome sequence was not possible. In a map-to-reference alignment, the regions with low coverage were deleted, those with high coverage were concatenated and a genome sequence of 139,465 bp was obtained, which covered 91% of the ILTV genome. Sixteen single-nucleotide polymorphisms (SNPs) were found between the ILTV isolate examined and ILTV class 9 (JN804827). Despite only 91% genome coverage, using sequence analysis and comparison with previously sequenced ILTVs, we were able to classify the isolate as class 9. Therefore, this technique has the potential to replace the current PCR-HRM technique, as it provides detailed information about the ILTV isolates.

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.

Close genetic linkage between human and companion animal extraintestinal pathogenic Escherichia coli ST127.

Escherichia coli ST127, a recently emerged global pathogen noted for high virulence gene carriage, is a leading cause of urinary tract and blood stream infections. ST127 is frequently isolated from humans and companion animals; however, it is unclear if they are distinct or related populations of ST127. We performed a phylogenomic analysis of 299 E. coli ST127 of diverse epidemiological origin to characterize their population structure, genetic determinants of virulence, antimicrobial resistance, and repertoire of mobile genetic elements with a focus on plasmids. The core gene phylogeny was divided into 13 clusters, the largest of which (BAP4) contained the majority of human and companion animal origin isolates. This dominant cluster displayed genetic differences to the remainder of the phylogeny, most notably alternative gene alleles encoding important virulence factors including lipid A, flagella, and K capsule. Furthermore, numerous close genetic linkages (<30 SNPs) between human and companion animal isolates were observed within the cluster. Carriage of antimicrobial resistance genes in the collection was limited, but virulence gene carriage was extensive. We found evidence of pUTI89-like virulence plasmid carriage in over a third of isolates, localised to four of the major phylogenetic clusters. Our study supports global scale repetitive transfer of E. coli ST127 lineages between humans and companion animals, particularly within the dominant BAP4 cluster.

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 based strain identification of fowlpox virus directly from cutaneous tissue and propagated virus.

Fowlpox (FP) is an economically important viral disease of commercial poultry. The fowlpox virus (FPV) is primarily characterised by immunoblotting, restriction enzyme analysis in combination with PCR, and/or nucleotide sequencing of amplicons. Whole-genome sequencing (WGS) of FPV directly from clinical specimens prevents the risk of potential genome modifications associated with in vitro culturing of the virus. Only one study has sequenced FPV genomes directly from clinical samples using Nanopore sequencing, however, the study didn't compare the sequences against Illumina sequencing or laboratory propagated sequences. Here, the suitability of WGS for strain identification of FPV directly from cutaneous tissue was evaluated, using a combination of Illumina and Nanopore sequencing technologies. Sequencing results were compared with the sequence obtained from FPV grown in chorioallantoic membranes (CAMs) of chicken embryos. Complete genome sequence of FPV was obtained directly from affected comb tissue using a map to reference approach. FPV sequence from cutaneous tissue was highly similar to that of the virus grown in CAMs with a nucleotide identity of 99.8%. Detailed polymorphism analysis revealed the presence of a highly comparable number of single nucleotide polymorphisms (SNPs) in the two sequences when compared to the reference genome, providing essentially the same strain identification information. Comparative genome analysis of the map to reference consensus sequences from the two genomes revealed that this field isolate had the highest nucleotide identity of 99.5% with an FPV strain from the USA (Fowlpox virus isolate, FWPV-MN00.2, MH709124) and 98.8% identity with the Australian FPV vaccine strain (FWPV-S, MW142017). Sequencing results showed that WGS directly from cutaneous tissues is not only rapid and cost-effective but also provides essentially the same strain identification information as in-vitro grown virus, thus circumventing in vitro culturing.

Characterisation of the whole genome sequence of an avian hepatitis E virus directly from clinical specimens reveals possible recombination events between European and USA strains.

Avian hepatitis E virus (aHEV) is the causative agent of an important disease of broiler breeders and layers. aHEV cannot be readily propagated in cell culture and is characterised primarily by sequencing of amplicons generated through several RT-PCRs that target individual genes. This study aims to uncover the origin of current Australian aHEV isolates based on whole genome sequencing using clinical liver tissues. Complete genome sequences of the two aHEV isolates were assembled using Nanopore and Illumina reads. The two isolates possessed only four single nucleotide polymorphisms to each other. Comparison of the sequences with aHEV genome sequences available in the GenBank showed the highest nucleotide sequence identity of 88% with the prototype USA strain (AY535004), 82% with the European (AM943647) and genotype 1 Australian strains (AM943647). Recombination analysis suggested that aHEV isolates characterised in this study are progeny of a cross between a US and a Hungarian strain. Phylogenetic tree and phylogenetic networks constructed using complete genome and individual coding sequences revealed that Australian aHEV isolates formed a distinct clade closer to the USA strains and classified as genotype 2 whereas genotype 1 Australian strain clustered together with South Korean strains.