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Antimicrobial resistance (AMR) is a major public health concern, and environmental bacteria have been recognized as important reservoirs of antimicrobial resistance genes (ARGs). Citrobacter, a common environmental bacterium and opportunistic pathogen in humans and other animals, has been largely understudied in terms of its diversity and AMR potential. Whole-genome (short-read) sequencing on a total of 77 Citrobacter isolates obtained from Australian silver gull (Chroicocephalus novaehollandiae) (n = 17) and influent wastewater samples (n = 60) was performed, revealing a diverse Citrobacter population, with seven different species and 33 sequence types, 17 of which were novel. From silver gull using non-selective media we isolated a broader range of species with little to no mobilised ARG carriage. Wastewater isolates (selected using Carbapenem- Resistant Enterobacterales (CRE) selective media) carried a heavy burden of ARGs (up to 21 ARGs, conferring resistance to nine classes of antibiotics), with several novel multidrug-resistant (MDR) lineages identified, including C. braakii ST1110, which carried ARGs conferring resistance to eight to nine classes of antibiotics, and C. freundii ST1105, which carried two carbapenemase genes, blaIMP-4 in class 1 integron structure, and blaKPC-2. Additionally, we identified an MDR C. portucalensis isolate carrying blaNDM-1, blaSHV-12, and mcr-9. We identified IncC, IncM2, and IncP6 plasmids as the likely vectors for many of the critically important mobilised ARGs. Phylogenetic analyses were performed to assess any epidemiological linkages between isolation sources, demonstrating low relatedness across sources beyond the ST level. However, these analyses did reveal some closer relationships between strains from disparate wastewater sources despite their collection some 13,000 km apart. These findings support the need for future surveillance of Citrobacter populations in wastewater and wildlife populations to monitor for potential

Antimicrobial resistance (AMR) is a global threat to human, animal, and environmental health. Studying AMR in environmental bacteria is crucial to understand the emergence and dissemination of resistance genes and pathogens, and to identify potential reservoirs and transmission routes. This study provides novel insights into the genomic diversity and AMR potential of environmental Acinetobacter species. By comparing the genomes of aquatic Acinetobacter isolates with clinical and non-clinical strains, we revealed that they are highly divergent yet carry pdif modules that encode resistance to antibiotics commonly used in clinical settings. We also demonstrated that an environmental A. baumannii isolate can acquire clinically relevant plasmids and carries virulence factors similar to those of hospital-associated strains. These findings suggest that environmental Acinetobacter species may serve as reservoirs and vectors of clinically important genes. Consequently, further research is warranted to comprehensively understand the ecology and evolution of this genus.

This study examines the genetic contexts and evolutionary steps responsible for the formation of the widely spread transposon Tn6925 carrying blaTEM and aacC2e, which confers resistance to beta-lactam and aminoglycoside antibiotics in Gram-negative bacteria. The blaTEM-1 and aacC2e genes were found in several transposons. They were first observed within an IS26 bounded 3.7 kb transposon (Tn6925) on several Acinetobacter baumannii plasmids located within a 4.7 kb dif module. Truncated and expanded variations of Tn6925 were found across other A. baumannii plasmids, as well as in other Gram-negative bacteria (including Vibrio cholerae). Moreover, blaTEM-1 and aacC2e were in much larger resistance-heavy transposons including the ISAba1-bounded 24.6 kb (here called Tn6927), found in an A. baumannii chromosome. A novel ISKpn12-bounded transposon was also observed to contain blaTEM and aacC2e which was found interrupting Tn5393 along with an IS26 pseudo-compound transposon to form a 24.9 kb resistance island in an Acinetobacter pittii plasmid. Multiple mobile genetic elements are involved in the formation of transposon structures that circulate blaTEM and aacC2e. Among these, IS26 and ISAba1 appear to have played a major role in the formation and spread of these elements in the Acinetobacter species.

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.

Pasteurella multocida causes a range of diseases in many host species throughout the world, including bovine respiratory disease (BRD) which is predominantly seen in feedlot cattle. This study assessed genetic diversity among 139 P. multocida isolates obtained from post-mortem lung swabs of BRD-affected feedlot cattle in four Australian states: New South Wales, Queensland, South Australia, and Victoria during 2014-2019. Whole-genome sequencing (WGS) was used to determine capsular serogroup, lipopolysaccharide genotypes, multi-locus sequence types and phylogenetic relationships. Two capsular types (A and D), with most isolates (132/139; 95%) belonging to type A; and three lipopolysaccharide (LPS) genotypes were identified (L1 [6/139; 4.3%], L3 [124/139; 89.2%] and L6 [9/139; 6.4%)]). Multi-locus sequence types (STs) ST9, ST13, ST17, ST20, ST36, ST50, ST58, ST79, ST124, ST125, ST132, ST167, ST185, ST327, ST394, and three novel STs [ST396, ST397, and ST398] were identified, with ST394 (59/139; 42.4%) and ST79 (44/139; 32%) the most prevalent in all four states. Isolates displaying phenotypic resistance to single, dual or multiple antibiotics (macrolide, tetracycline and aminopenicillins) were predominantly ST394 (23/139; 17%). Laterally mobile elements identified in the resistant ST394 isolates included small plasmids, encoding macrolide and/or tetracycline resistance, distributed in all states; and chromosomally located integrative conjugative elements (ICEs) (4 ST394 and 1 ST125) from the same Queensland feedlot. This study highlights the genomic diversity, epidemiological relationships and AMR associations in bovine P. multocida isolates from Australia and provides insight into the unique ST prevalence compared to other major beef-producing countries.

Avian gut microbial communities are complex and play a fundamental role in regulating biological functions within an individual. Although it is well established that diet can influence the structure and composition of the gut microbiota, foraging behaviour may also play a critical, yet unexplored role in shaping the composition, dynamics, and adaptive potential of avian gut microbiota. In this review, we examine the potential influence of coprophagic foraging behaviour on the establishment and adaptability of wild avian gut microbiomes. Coprophagy involves the ingestion of faeces, sourced from either self (autocoprophagy), conspecific animals (allocoprophagy), or heterospecific animals. Much like faecal transplant therapy, coprophagy may (i) support the establishment of the gut microbiota of young precocial species, (ii) directly and indirectly provide nutritional and energetic requirements, and (iii) represent a mechanism by which birds can rapidly adapt the microbiota to changing environments and diets. However, in certain contexts, coprophagy may also pose risks to wild birds, and their microbiomes, through increased exposure to chemical pollutants, pathogenic microbes, and antibiotic-resistant microbes, with deleterious effects on host health and performance. Given the potentially far-reaching consequences of coprophagy for avian microbiomes, and the dearth of literature directly investigating these links, we have developed a predictive framework for directing future research to understand better when and why wild birds engage in distinct types of coprophagy, and the consequences of this foraging behaviour. There is a need for comprehensive investigation into the influence of coprophagy on avian gut microbiotas and its effects on host health and performance throughout ontogeny and across a range of environmental perturbations. Future behavioural studies combined with metagenomic approaches are needed to provide insights into the function of this poorly understoo

To date, the scientific literature on health variables for Escherichia coli antimicrobial resistance (AMR) has been investigated throughout several systematic reviews, often with a focus on only one aspect of the One Health variables: human, animal, or environment. The aim of this umbrella review is to conduct a systematic synthesis of existing evidence on Escherichia coli AMR in humans in the community from a One Health perspective. PubMed, EMBASE, and CINAHL were searched on "antibiotic resistance" and "systematic review" from inception until 25 March 2022 (PROSPERO: CRD42022316431). The methodological quality was assessed, and the importance of identified variables was tabulated across all included reviews. Twenty-three reviews were included in this study, covering 860 primary studies. All reviews were of (critically) low quality. Most reviews focused on humans (20), 3 on animals, and 1 on both human and environmental variables. Antibiotic use, urinary tract infections, diabetes, and international travel were identified as the most important human variables. Poultry farms and swimming in freshwater were identified as potential sources for AMR transmission from the animal and environmental perspectives. This umbrella review highlights a gap in high-quality literature investigating the time between variable exposure, AMR testing, and animal and environmental AMR variables.

Even in the setting of optimal resuscitation in high-income countries severe sepsis and septic shock have a mortality of 20-40%, with antibiotic resistance dramatically increasing this mortality risk. To develop a reference dataset enabling the identification of common bacterial targets for therapeutic intervention, we applied a standardized genomic, transcriptomic, proteomic and metabolomic technological framework to multiple clinical isolates of four sepsis-causing pathogens: Escherichia coli, Klebsiella pneumoniae species complex, Staphylococcus aureus and Streptococcus pyogenes. Exposure to human serum generated a sepsis molecular signature containing global increases in fatty acid and lipid biosynthesis and metabolism, consistent with cell envelope remodelling and nutrient adaptation for osmoprotection. In addition, acquisition of cholesterol was identified across the bacterial species. This detailed reference dataset has been established as an open resource to support discovery and translational research.

BACKGROUND: Bovine respiratory disease (BRD) is one of the most common diseases in intensively managed cattle, often resulting in high morbidity and mortality. Although several pathogens have been isolated and extensively studied, the complete infectome of the respiratory complex consists of a more extensive range unrecognised species. Here, we used total RNA sequencing (i.e., metatranscriptomics) of nasal and nasopharyngeal swabs collected from animals with and without BRD from two cattle feedlots in Australia. RESULTS: A high abundance of bovine nidovirus, influenza D, bovine rhinitis A and bovine coronavirus was found in the samples. Additionally, we obtained the complete or near-complete genome of bovine rhinitis B, enterovirus E1, bovine viral diarrhea virus (sub-genotypes 1a and 1c) and bovine respiratory syncytial virus, and partial sequences of other viruses. A new species of paramyxovirus was also identified. Overall, the most abundant RNA virus, was the bovine nidovirus. Characterisation of bacterial species from the transcriptome revealed a high abundance and diversity of Mollicutes in BRD cases and unaffected control animals. Of the non-Mollicutes species, Histophilus somni was detected, whereas there was a low abundance of Mannheimia haemolytica. CONCLUSION: This study highlights the use of untargeted sequencing approaches to study the unrecognised range of microorganisms present in healthy or diseased animals and the need to study previously uncultured viral species that may have an important role in cattle respiratory disease. Video Abstract.

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.

Technologically enhanced surveillance systems have been proposed for the task of monitoring and responding to antimicrobial resistance (AMR) in both human, animal and environmental contexts. The use of these systems is in their infancy, although the advent of COVID-19 has progressed similar technologies in response to that pandemic. We conducted qualitative research to identify the Australian public's key concerns about the ethical, legal and social implications of an artificial intelligence (AI) and machine learning-enhanced One Health AMR surveillance system. Our study provides preliminary evidence of public support for AI/machine learning-enhanced One Health monitoring systems for AMR, provided that three main conditions are met: personal health care data must be deidentified; data use and access must be tightly regulated under strong governance; and the system must generate high-quality, reliable analyses to guide trusted health care decision-makers.

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.

We performed a comprehensive phylogenomic analysis of 925 extraintestinal pathogenic Escherichia coli (ExPEC) ST38 genomes from 38 countries and diverse hosts and sources. The phylogeny resolved two broad clades: A (593 strains; 91% human) and B (332 isolates; 42% human), each with distinct ST38 clusters linked to the carriage of specific bla CTX-M alleles, often in association with other antibiotic resistance genes, class 1 integrons and specific plasmid replicon types. Co-carriage of fyuA and irp2 virulence genes, a reliable proxy for carriage of the Yersinia high-pathogenicity island, featured in 580 (62.7%) genomes. ST38 lineages carrying combinations of ExPEC and intestinal pathogenic Escherichia coli virulence factors were also identified. The F plasmid replicon was identified in 536 (58%) genomes, and 112 of these (21%) carry cjrABC-senB, a virulence operon frequently identified in pandemic ExPEC sequence types. Most (108; 96.4%) cjrABC-senB+ ST38 isolates were from human and other sources, except food animals, and were associated with F5:A-:B10 (41 isolates), F1:A2:B20 (20 isolates), and F24:A-:B1 (15 isolates) F replicon types. ST38 genomes that were inferred to carry a ColV-F virulence plasmid (69; 7.4%) were mostly from human (12; 17.4%), avian (26; 37.7%), or poultry (10; 6.9%) sources. We identified multiple examples of putative inter-host and host-environment transmission events, where genomes differed by <35 SNPs. This work emphasizes the importance of adopting a One Health approach for phylogenomic studies that seek to improve understanding of antimicrobial resistance and pathogen evolution. 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 anc

The present work describes the evolution of a resistance phenotype to a multitargeting antimicrobial agent, namely, silver nanoparticles (nanosilver; NAg), in the globally prevalent bacterial pathogen Acinetobacter baumannii. The Gram-negative bacterium has recently been listed as a critical priority pathogen requiring novel treatment options by the World Health Organization. Through prolonged exposure to the important antimicrobial nanoparticle, the bacterium developed mutations in genes that encode the protein subunits of organelle structures that are involved in cell-to-surface attachment as well as in a cell envelope capsular polysaccharide synthesis-related gene. These mutations are potentially correlated with stable physiological changes in the biofilm growth behavior and with an evident protective effect against oxidative stress, most likely as a feature of toxicity defense. We further report a different adaptation response of A. baumannii to the cationic form of silver (Ag+). The bacterium developed a tolerance phenotype to Ag+, which was correlated with an indicative surge in respiratory activity and changes in cell morphology, of which these are reported characteristics of tolerant bacterial populations. The findings regarding adaptation phenomena to NAg highlight the risks of the long-term use of the nanoparticle on a priority pathogen. The findings urge the implementation of strategies to overcome bacterial NAg adaptation, to better elucidate the toxicity mechanisms of the nanoparticle, and preserve the efficacy of the potent alternative antimicrobial agent in this era of antimicrobial resistance. IMPORTANCE Several recent studies have reported on the development of bacterial resistance to broad-spectrum antimicrobial silver nanoparticles (nanosilver; NAg). NAg is currently one of the most important alternative antimicrobial agents. However, no studies have yet established whether Acinetobacter baumannii, a globally prevalent nosocomial pathogen, can dev

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.

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

Theileria orientalis causes losses to cattle producers in Eastern Asia, Oceania and, more recently, North America. One pathogenic genotype (Ikeda) has been sequenced to the chromosomal level, while only draft genomes exist for globally distributed Chitose and Buffeli genotypes. To provide an accurate comparative gene-level analysis and help further understand their pathogenicity, we sequenced isolates of the Chitose and Buffeli genotypes of T. orientalis using long-read sequencing technology. A combination of several long-read assembly methods and short reads produced chromosomal-level assemblies for both Fish Creek (Chitose) and Goon Nure (Buffeli) isolates, including the first complete and circular apicoplast genomes generated for T. orientalis. Comparison with the Shintoku (Ikeda) reference sequence showed both large and small translocations in T. orientalis Buffeli, between chromosomes 2 and 3 and chromosomes 1 and 4, respectively. Ortholog clustering showed expansion of ABC transporter genes in Chitose and Buffeli. However, differences in several genes of unknown function, including DUF529/FAINT-domain-containing proteins, were also identified and these genes were more prevalent in Ikeda and Chitose genotypes. Phylogenetics and similarity measures were consistent with previous short-read genomic analysis. The generation of chromosomal sequences for these highly prevalent T. orientalis genotypes will also support future studies of population genetics and mixed genotype infections.

Comamonas spp. are Gram-negative bacteria that catabolize a wide range of organic and inorganic substrates. Comamonas spp. are abundant in aquatic and soil environments, including wastewater, and can cause opportunistic infections in humans. Because of their potential in wastewater bioaugmentation and bioremediation strategies, the identification of Comamonas species harboring genes encoding carbapenemases and other clinically important antibiotic resistance genes warrant further investigation. Here, we present an analysis of 39 whole-genome sequences comprising three Comamonas species from aquatic environments in South Australia that were recovered on media supplemented with carbapenems. The analysis includes a detailed description of 33 Comamonas denitrificans isolates, some of which carried chromosomally acquired blaGES-5, blaOXA, and aminoglycoside resistance (aadA) genes located on putative genomic islands (GIs). All blaGES-5- and blaOXA-containing GIs appear to be unique to this Australian collection of C. denitrificans. Notably, most open reading frames (ORFs) within the GIs, including all antimicrobial resistance (AMR) genes, had adjacent attC sites, indicating that these ORFs are mobile gene cassettes. One C. denitrificans isolate carried an IncP-1 plasmid with genes involved in xenobiotic degradation and response to oxidative stress. Our assessment of the sequences highlights the very distant nature of C. denitrificans to the other Comamonas species and its apparent disposition to acquire antimicrobial resistance genes on putative genomic islands. IMPORTANCE Antimicrobial resistance (AMR) poses a global public health threat, and the increase in resistance to "last-resort drugs," such as carbapenems, is alarming. Wastewater has been flagged as a hot spot for AMR evolution. Comamonas spp. are among the most common bacteria in wastewater and play a role in its bioaugmentation. While the ability of Comamonas species to catabolize a wide range of organic and in

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

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 emergen

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.

Escherichia coli sequence type 95 (ST95) is an extraintestinal pathogenic E. coli (ExPEC) renowned for its ability to cause significant morbidity and mortality in humans and poultry. A core genome analysis of 668 ST95 isolates generated 10 clades (A to J), 5 of which are reported here for the first time. F plasmid replicon sequence typing showed that almost a third (178/668 [27%]) of the collection carry pUTI89 (F29:B10) and were restricted to clade A and a sublineage of clade B. In contrast, almost half (328/668 [49%]) of the collection across multiple clades harbor ColV plasmids (multiple F types). Strikingly, ST95 lineages with pUTI89 were almost exclusively from humans, while ColV+ ST95 lineages were sourced from poultry and humans. Clade I was notable because it comprises temporally and geographically matched ColV+ isolates sourced from human and retail poultry meat, suggesting interspecies transmission via food. Clade F contained ST95 isolates of bovine origin, none of which carried ColV or pUTI89 plasmids. Remarkably, an analysis of a cohort of 34,176 E. coli isolates comprising 2,570 sequence types mirrored what was observed in ST95: (i) pUTI89 was overwhelmingly linked to E. coli sourced from humans but almost entirely absent from 13,027 E. coli isolates recovered from poultry, pigs, and cattle, and (ii) E. coli isolates harboring ColV plasmids were from multiple sources, including humans, poultry, and swine. Overall, our data suggest that F plasmids influence E. coli host range, clade structure, and zoonotic potential in ST95 and ExPEC more broadly. IMPORTANCE E. coli ST95 is one of five dominant ExPEC lineages globally and noted for causing urinary tract and bloodstream infections and neonatal meningitis in humans and colibacillosis in poultry. Using high-resolution phylogenomics, we show that F replicon sequence type is linked to ST95 clade structure and zoonotic potential. Specifically, human centric ST95 clades overwhelmingly harbor F29:B10 (pUTI89) pl

Antimicrobial resistance (AMR) is a growing threat to human and animal health. Progress in molecular biology has revealed new and significant challenges for AMR mitigation given the immense diversity of antibiotic resistance genes (ARGs), the complexity of ARG transfer, and the broad range of omnipresent factors contributing to AMR. Municipal, hospital and abattoir wastewater are collected and treated in wastewater treatment plants (WWTPs), where the presence of diverse selection pressures together with a highly concentrated consortium of pathogenic/commensal microbes create favourable conditions for the transfer of ARGs and proliferation of antibiotic resistant bacteria (ARB). The rapid emergence of antibiotic resistant pathogens of clinical and veterinary significance over the past 80 years has re-defined the role of WWTPs as a focal point in the fight against AMR. By reviewing the occurrence of ARGs in wastewater and sludge and the current technologies used to quantify ARGs and identify ARB, this paper provides a research roadmap to address existing challenges in AMR control via wastewater treatment. Wastewater treatment is a double-edged sword that can act as either a pathway for AMR spread or as a barrier to reduce the environmental release of anthropogenic AMR. State of the art ARB identification technologies, such as metagenomic sequencing and fluorescence-activated cell sorting, have enriched ARG/ARB databases, unveiled keystone species in AMR networks, and improved the resolution of AMR dissemination models. Data and information provided in this review highlight significant knowledge gaps. These include inconsistencies in ARG reporting units, lack of ARG/ARB monitoring surrogates, lack of a standardised protocol for determining ARG removal via wastewater treatments, and the inability to support appropriate risk assessment. This is due to a lack of standard monitoring targets and agreed threshold values, and paucity of information on the ARG-pathogen host re

The spread of antimicrobial resistance (AMR) is a rapidly growing threat to humankind on both regional and global scales. As countries worldwide prepare to embrace a One Health approach to AMR management, which is one that recognizes the interconnectivity between human, animal, and environmental health, increasing attention is being paid to identifying and monitoring key contributing factors and critical control points. Presently, AMR sensing technologies have significantly progressed phenotypic antimicrobial susceptibility testing (AST) and genotypic antimicrobial resistance gene (ARG) detection in human healthcare. For effective AMR management, an evolution of innovative sensing technologies is needed for tackling the unique challenges of interconnected AMR across various and different health domains. This review comprehensively discusses the modern state-of-play for innovative commercial and emerging AMR sensing technologies, including sequencing, microfluidic, and miniaturized point-of-need platforms. With a unique view toward the future of One Health, we also provide our perspectives and outlook on the constantly changing landscape of AMR sensing technologies beyond the human health domain.

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Results

Conclusions

Mycoplasma pneumoniae is a significant cause of pneumonia and post infection sequelae affecting organ sites distant to the respiratory tract are common. It is also a model organism where extensive 'omics' studies have been conducted to gain insight into how minimal genome self-replicating organisms function. An N-terminome study undertaken here identified 4898 unique N-terminal peptides that mapped to 391 (56%) predicted M. pneumoniae proteins. True N-terminal sequences beginning with the initiating methionine (iMet) residue from the predicted Open Reading Frame (ORF) were identified for 163 proteins. Notably, almost half (317; 46%) of the ORFS derived from M. pneumoniae strain M129 are post-translationally modified, presumably by proteolytic processing, because dimethyl labelled neo-N-termini were characterised that mapped beyond the predicted N-terminus. An analysis of the N-terminome describes endoproteolytic processing events predominately targeting tryptic-like sites, though cleavages at negatively charged residues in P1' (D and E) with lysine or serine/alanine in P2' and P3' positions also occurred frequently. Surfaceome studies identified 160 proteins (23% of the proteome) to be exposed on the extracellular surface of M. pneumoniae. The two orthogonal methodologies used to characterise the surfaceome each identified the same 116 proteins, a 72% (116/160) overlap. Apart from lipoproteins, transporters, and adhesins, 93/160 (58%) of the surface proteins lack signal peptides and have well characterised, canonical functions in the cell. Of the 160 surface proteins identified, 134 were also targets of endo-proteolytic processing. These processing events are likely to have profound implications for how the host immune system recognises and responds to M. pneumoniae.

Bovine respiratory disease (BRD) causes high morbidity and mortality in beef cattle worldwide. Antimicrobial resistance (AMR) monitoring of BRD pathogens is critical to promote appropriate antimicrobial stewardship in veterinary medicine for optimal treatment and control. Here, the susceptibility of Mannheimia haemolytica and Pasteurella multicoda isolates obtained from BRD clinical cases (deep lung swabs at post-mortem) among feedlots in four Australian states (2014-2019) was determined for 19 antimicrobial agents. The M. haemolytica isolates were pan-susceptible to all tested agents apart from a single macrolide-resistant isolate (1/88; 1.1%) from New South Wales (NSW). Much higher frequencies of P. multocida isolates were resistant to tetracycline (18/140; 12.9%), tilmicosin (19/140; 13.6%), tulathromycin/gamithromycin (17/140; 12.1%), and ampicillin/penicillin (6/140; 4.6%). Five P. multocida isolates (3.6%), all obtained from NSW in 2019, exhibited dual resistance to macrolides and tetracycline, and a further two Queensland isolates from 2019 (1.4%) exhibited a multidrug-resistant phenotype to ampicillin/penicillin, tetracycline, and tilmicosin. Random-amplified polymorphic DNA (RAPD) typing identified a high degree of genetic homogeneity among the M. haemolytica isolates, whereas P. multocida isolates were more heterogeneous. Illumina whole genome sequencing identified the genes msr(E) and mph(E)encoding macrolide resistance, tet(R)-tet(H) or tet(Y) encoding tetracycline resistance, and blaROB-1 encoding ampicillin/penicillin resistance in all isolates exhibiting a corresponding resistant phenotype. The exception was the tilmicosin-resistant, tulathromycin/gamithromycin-susceptible phenotype identified in two Queensland isolates, the genetic basis of which could not be determined. These results confirm the first emergence of AMR in M. haemolytica and