Development of an In Vivo Extended-Spectrum Cephalosporin-Resistant Escherichia coli Model in Post-Weaned Pigs and Its Use in Assessment of Dietary Interventions.

Current interventions targeting antimicrobial resistance (AMR), a major impact on commercial pork production, focus on reducing the emergence of AMR by minimising antimicrobial usage through antimicrobial stewardship and a range of alternative control methods. Although these strategies require continued advancement, strategies that directly aim to reduce or eliminate existing antimicrobial resistant bacteria, specifically bacteria resistant to critically important antimicrobials (CIAs), need to be investigated and established. This study established an in vivo model for examining the effects of postbiotics, in the form of Lactobacillus acidophilus fermentation products (LFP) and Saccharomyces cerevisiae fermentation products (SFP), on the shedding of extended-spectrum cephalosporin (ESC)-resistant E. coli. The model was successful in demonstrating the presence of ESC-resistant E. coli as evidenced by its detection in 62 of 64 pigs. There was a strong trend (p = 0.065) for the SFP postbiotics to reduce the shedding of ESC-resistant E. coli, indicating positive impacts of this additive on reducing the carriage of bacteria resistant to CIAs. Overall, this in vivo model enables future evaluation of strategies targeting ESC-resistant E. coli while increasing our knowledge on the carriage of ESC-resistant E. coli in pigs.

Antimicrobial Resistance of and Genomic Insights into Pasteurella multocida Strains Isolated from Australian Pigs.

Infection with Pasteurella multocida represents a significant economic threat to Australian pig producers, yet our knowledge of its antimicrobial susceptibilities is lagging, and genomic characterization of P. multocida strains associated with porcine lower respiratory disease is internationally scarce. This study utilized high-throughput robotics to phenotypically and genetically characterize an industry-wide collection of 252 clinical P. multocida isolates that were recovered between 2014 and 2019. Overall, antimicrobial resistance was found to be low, with clinical resistance below 1% for all tested antimicrobials except those from the tetracycline class. Five dominant sequence types, representing 64.8% of all isolates, were identified; they were disseminated across farms and had previously been detected in various animal hosts and countries. P. multocida in Australian farms remain controllable via current antimicrobial therapeutic protocols. The identification of highly dominant, interspecies-infecting strains provides insight into the epidemiology of the opportunistic pathogen, and it highlights a biosecurity threat to the Australian livestock industry. IMPORTANCE Pasteurellosis is rated by the World Animal Health Organisation (OIE) as a high-impact disease in livestock. Although it is well understood in many host-disease contexts, our understanding of the organism in porcine respiratory disease is limited. Given its high frequency of involvement in porcine respiratory disease complex (PRDC), it is important that we are aware of its antimicrobial susceptibilities so that we can respond quickly and appropriately with antimicrobial therapy. Genetic insights about the organism can help us to better understand its epidemiology and inform our biosecurity practices and prophylactic management.

In Vitro Demonstration of Targeted Phage Therapy and Competitive Exclusion as a Novel Strategy for Decolonization of Extended-Spectrum-Cephalosporin-Resistant Escherichia coli.

Extended-spectrum cephalosporin-resistant (ESC-R) Escherichia coli have disseminated in food-producing animals globally, attributed to horizontal transmission of blaCTX-M variants, as seen in the InCI1-blaCTX-M-1 plasmid. This ease of transmission, coupled with its demonstrated long-term persistence, presents a significant One Health antimicrobial resistance (AMR) risk. Bacteriophage (phage) therapy is a potential strategy in eliminating ESC-R E. coli in food-producing animals; however, it is hindered by the development of phage-resistant bacteria and phage biosafety concerns. Another alternative to antimicrobials is probiotics, with this study demonstrating that AMR-free commensal E. coli, termed competitive exclusion clones (CECs), can be used to competitively exclude ESC-R E. coli. This study isolated and characterized phages that lysed E. coli clones harboring the InCI1-blaCTX-M-1 plasmid, before investigation of the effect and synergy of phage therapy and competitive exclusion as a novel strategy for decolonizing ESC-resistant E. coli. In vitro testing demonstrated superiority in the combined therapy, reducing and possibly eliminating ESC-R E. coli through phage-mediated lysis coupled with simultaneous prevention of regrowth of phage-resistant mutants due to competitive exclusion with the CEC. Further investigation into this combined therapy in vivo is warranted, with on-farm application possibly reducing ESC-R prevalence, while constricting newly emergent ESC-R E. coli outbreaks prior to their dissemination throughout food-producing animals or humans. IMPORTANCE The emergence and global dissemination of resistance toward critically important antimicrobials, including extended-spectrum cephalosporins in the livestock sector, deepens the One Health threat of antimicrobial resistance. This resistance has the potential to disseminate to humans, directly or indirectly, nullifying these last lines of defense in life-threatening human infections. This study explores a novel strategy, the coadministration of bacteriophages (phages) and a competitive exclusion clone (antimicrobial-susceptible commensal E. coli), to revert an antimicrobial-resistant population to a susceptible population. While phage therapy is vulnerable to the emergence of phage-resistant bacteria, no phage-resistant bacteria emerged when a competitive exclusion clone was used in combination with the phage. Novel strategies that reduce the prevalence and slow the dissemination of extended-spectrum cephalosporin-resistant E. coli in food-producing animals have the potential to extend the time frame in which antimicrobials remain available for effective use in animal and human health.

Diversity detected in commensals at host and farm level reveals implications for national antimicrobial resistance surveillance programmes.

BACKGROUND: A key component to control of antimicrobial resistance (AMR) is the surveillance of food animals. Currently, national programmes test only limited isolates per animal species per year, an approach tacitly assuming that heterogeneity of AMR across animal populations is negligible. If the latter assumption is incorrect then the risk to humans from AMR in the food chain is underestimated. OBJECTIVES: To demonstrate the extent of phenotypic and genetic heterogeneity of Escherichia coli in swine to assess the need for improved protocols for AMR surveillance in food animals. METHODS: Eight E. coli isolates were obtained from each of 10 pigs on each of 10 farms. For these 800 isolates, AMR profiles (MIC estimates for six drugs) and PCR-based fingerprinting analysis were performed and used to select a subset (n = 151) for WGS. RESULTS: Heterogeneity in the phenotypic AMR traits of E. coli was observed in 89% of pigs, with 58% of pigs harbouring three or more distinct phenotypes. Similarly, 94% of pigs harboured two or more distinct PCR-fingerprinting profiles. Farm-level heterogeneity was detected, with ciprofloxacin resistance detected in only 60% of pigs from a single farm. Furthermore, 58 STs were identified, with the dominant STs being ST10, ST101, ST542 and ST641. CONCLUSIONS: Phenotypic and genotypic heterogeneity of AMR traits in bacteria from animal populations are real phenomena posing a barrier to correct interpretation of data from AMR surveillance. Evolution towards a more in-depth sampling model is needed to account for heterogeneity and increase the reliability of inferences.

Porcine enterotoxigenic Escherichia coli: Antimicrobial resistance and development of microbial-based alternative control strategies.

Strains of enterotoxigenic Escherichia coli (ETEC) causing post-weaning diarrhoea (PWD) in piglets have a widespread and detrimental impact on animal health and the economics of pork production. Traditional approaches to control and prevention have placed a strong emphasis on antimicrobial use (AMU) to the extent that current prevalent porcine ETEC strains have developed moderate to severe resistance. This complicates treatment of ETEC infection by limiting therapeutic options, increasing diagnostic costs and increasing mortality rates. Management factors, the use of supra-physiological levels of zinc oxide and selected feed additives have all been documented to lower the incidence of ETEC infection in pigs; however, each intervention has its own limitations and cannot solely be relied upon as an alternative to AMU. Consequently, treatment options for porcine ETEC are moving towards the use of newer antimicrobials of higher public health significance. This review focuses on microorganisms and microbial-derived products that could provide a naturally evolved solution to ETEC infection and disease. This category holds a plethora of yet to be explored possibilities, however studies based around bacteriophage therapy, probiotics and the use of probiotic fermentation products as postbiotics have demonstrated promise. Ultimately, pig producers and veterinarians need these solutions to reduce the reliance on critically important antimicrobials (CIAs), to improve economic and animal welfare outcomes, and to lessen the One Health threat potentiated by the dissemination of AMR through the food chain.

Robotic Antimicrobial Susceptibility Platform (RASP): a next-generation approach to One Health surveillance of antimicrobial resistance.

BACKGROUND: Surveillance of antimicrobial resistance (AMR) is critical to reducing its wide-reaching impact. Its reliance on sample size invites solutions to longstanding constraints regarding scalability. A robotic platform (RASP) was developed for high-throughput AMR surveillance in accordance with internationally recognized standards (CLSI and ISO 20776-1:2019) and validated through a series of experiments. METHODS: Experiment A compared RASP's ability to achieve consistent MICs with that of a human technician across eight replicates for four Escherichia coli isolates. Experiment B assessed RASP's agreement with human-performed MICs across 91 E. coli isolates with a diverse range of AMR profiles. Additionally, to demonstrate its real-world applicability, the RASP workflow was then applied to five faecal samples where a minimum of 47 E. coli per animal (239 total) were evaluated using an AMR indexing framework. RESULTS: For each drug-rater-isolate combination in Experiment A, there was a clear consensus of the MIC and deviation from the consensus remained within one doubling dilution (the exception being gentamicin at two dilutions). Experiment B revealed a concordance correlation coefficient of 0.9670 (95% CI: 0.9670-0.9670) between the robot- and human-performed MICs. RASP's application to the five faecal samples highlighted the intra-animal diversity of gut commensal E. coli, identifying between five and nine unique isolate AMR phenotypes per sample. CONCLUSIONS: While adhering to internationally accepted guidelines, RASP was superior in throughput, cost and data resolution when compared with an experienced human technician. Integration of robotics platforms in the microbiology laboratory is a necessary advancement for future One Health AMR endeavours.

Antimicrobial resistance and genomic insights into bovine mastitis-associated Staphylococcus aureus in Australia.

The aim of this study was to investigate antimicrobial resistance and population structure of bovine mastitis-associated Staphylococcus aureus isolates, and compare them to human isolates obtained from Western Australian hospitals and overseas strains to determine relatedness to human isolates from a zoonotic or reverse zoonotic aspect. Antimicrobial susceptibility testing was performed on 202 S. aureus isolates of which 166 isolates underwent whole genome sequencing. Only resistance to penicillin (12.4%) and erythromycin (0.5%) was identified and of note, no resistance was demonstrated to oxacillin. Genomic characterisation identified 14 multilocus sequence types (STs), with most isolates belonging to clonal complexes 97, 705, and 1. Four distinct clades based on virulence gene composition were identified. The four clades were predominantly ST based, consisting of ST352, ST97, ST81/ST1, and ST705. Core genome comparison of the bovine and human S. aureus isolates demonstrated defined clustering by ST, with the Australian bovine S. aureus isolates clustering together according to their ST separately from human isolates. In addition, a bovine specific cluster comprising Australian ST151 and ST705 isolates, and ST151 isolates from Irish dairy cattle was clearly delineated. Examination of a detailed ST352 phylogeny provided evidence for geographical clustering of Australian strains into a distinct grouping separate from international strains. This study has identified Australian S. aureus isolates have limited genetic diversity and are genetically distinct from human and international bovine S. aureus isolates. Current first line therapies for bovine mastitis in Australian dairy cattle remain appropriate.

Implications of Foraging and Interspecies Interactions of Birds for Carriage of Escherichia coli Strains Resistant to Critically Important Antimicrobials.

Globally, gulls have been associated with carriage of high levels of Escherichia coli strains resistant to critically important antimicrobials (CIAs), a major concern, as these antimicrobials are the sole alternative or one among only a few alternatives available to treat severe life-threatening infections in humans. Previous studies of Australian silver gulls demonstrated high levels of resistance to CIAs, particularly fluoroquinolone and extended-spectrum cephalosporins, among E. coli strains (carriage at 24% and 22%, respectively). This study aimed to identify and characterize strains from four distinct bird species inhabiting a common coastal environment, determine the frequency of carriage of CIA-resistant E. coli strains, and examine if these resistant clones and their resistance-encoding mobile genetic elements (MGEs) could be transmitted between species. CIA-resistant E. coli was detected in silver gulls (53%), little penguins (11%), and feral pigeons (10%), but not in bridled terns. In total, 37 different sequence types (STs) were identified, including clinically significant human-associated lineages, such as ST131, ST95, ST648, ST69, ST540, ST93, ST450, and ST10. Five main mobile genetic elements associated with blaCTX-M-positive E. coli strains isolated from three bird species were detected. Examination of clonal lineages and MGEs provided indirect evidence of transfer of resistance between bird species. The carriage of CIA-resistant E. coli by gulls and pigeons with proximity to humans, and in some instances food-producing animals, increases the likelihood of further bidirectional dissemination.IMPORTANCE It has been shown that 20% of Australian silver gulls carry drug-resistant Escherichia coli strains of anthropogenic origin associated with severe diseases, such as sepsis and urinary tract infections, in humans. To further characterize the dynamics of drug-resistant E. coli in wildlife populations, we investigated the carriage of critically important antimicrobial (CIA) drug-resistant E. coli in four bird species in a common environment. Our results indicated that gulls, pigeons, and penguins carried drug-resistant E. coli strains, and analysis of mobile genetic elements associated with resistance genes indicated interspecies resistance transfer. Terns, representing a bird species that forages on natural food sources at sea and distant from humans, did not test positive for drug-resistant E. coli This study demonstrates carriage of CIA-resistant bacteria in multiple bird species living in areas commonly inhabited by humans and provides further evidence for a leapfrog effect of resistance in wildlife, facilitated by feeding habits.

Emergence of Fluoroquinolone-Resistant Campylobacter jejuni and Campylobacter coli among Australian Chickens in the Absence of Fluoroquinolone Use.

In a structured survey of all major chicken-meat producers in Australia, we investigated the antimicrobial resistance (AMR) and genomic characteristics of Campylobacter jejuni (n = 108) and C. coli (n = 96) from cecal samples of chickens at slaughter (n = 200). The majority of the C. jejuni (63%) and C. coli (86.5%) samples were susceptible to all antimicrobials. Fluoroquinolone resistance was detected among both C. jejuni (14.8%) and C. coli (5.2%), although this only included three sequence types (STs) and one ST, respectively. Multidrug resistance among strains of C. jejuni (0.9%) and C. coli (4.1%) was rare, and fluoroquinolone resistance, when present, was never accompanied by resistance to any other agent. Comparative genome analysis demonstrated that Australian isolates were found dispersed on different branches/clusters within the international collection. The major fluoroquinolone-resistant STs of C. jejuni (ST7323, ST2083, and ST2343) and C. coli (ST860) present in Australian chickens were similar to those of international isolates and have been reported previously in humans and animals overseas. The detection of a subpopulation of Campylobacter isolates exclusively resistant to fluoroquinolone was unexpected since most critically important antimicrobials such as fluoroquinolones are excluded from use in Australian livestock. A number of factors, including the low level of resistance to other antimicrobials, the absence of fluoroquinolone use, the adoption of measures for preventing spread of contagion between flocks, and particularly the genomic identities of isolates, all point to humans, pest species, or wild birds as being the most plausible source of organisms. This study also demonstrates the need for vigilance in the form of surveillance for AMR based on robust sampling to manage AMR risks in the food chain.IMPORTANCECampylobacter is one of the most common causes of gastroenteritis in humans, with infections frequently resulting from exposure to undercooked poultry products. Although human illness is typically self-limiting, a minority of cases do require antimicrobial therapy. Ensuring that Campylobacter originating from meat chickens does not acquire resistance to fluoroquinolones is therefore a valuable outcome for public health. Australia has never legalized the use of fluoroquinolones in commercial chickens and until now fluoroquinolone-resistant Campylobacter has not been detected in the Australian poultry. This structured survey of meat chickens derived from all major Australian producers describes the unexpected emergence of fluoroquinolone resistance in Campylobacter jejuni and C. coli Genetic characterization suggests that these isolates may have evolved outside the Australian poultry sector and were introduced into poultry by humans, pest species, or wild birds. The findings dramatically underline the critical role of biosecurity in the overall fight against antimicrobial resistance.

Resistance to critically important antimicrobials in Australian silver gulls (Chroicocephalus novaehollandiae) and evidence of anthropogenic origins.

OBJECTIVES: Antimicrobial resistance (AMR) to critically important antimicrobials (CIAs) amongst Gram-negative bacteria can feasibly be transferred amongst wildlife, humans and domestic animals. This study investigated the ecology, epidemiology and origins of CIA-resistant Escherichia coli carried by Australian silver gulls (Chroicocephalus novaehollandiae), a gregarious avian wildlife species that is a common inhabitant of coastal areas with high levels of human contact. METHODS: Sampling locations were widely dispersed around the perimeter of the Australian continent, with sites separated by up to 3500 km. WGS was used to study the diversity and molecular characteristics of resistant isolates to ascertain their epidemiological origin. RESULTS: Investigation of 562 faecal samples revealed widespread occurrence of extended-spectrum cephalosporin-resistant (21.7%) and fluoroquinolone-resistant (23.8%) E. coli. Genome sequencing revealed that CIA-resistant E. coli isolates (n = 284) from gulls predominantly belonged to human-associated extra-intestinal pathogenic E. coli (ExPEC) clones, including ST131 (17%), ST10 (8%), ST1193 (6%), ST69 (5%) and ST38 (4%). Genomic analysis revealed that gulls carry pandemic ExPEC-ST131 clades (O25:H4 H30-R and H30-Rx) and globally emerging fluoroquinolone-resistant ST1193 identified among humans worldwide. Comparative analysis revealed that ST131 and ST1193 isolates from gulls overlapped extensively with human clinical isolates from Australia and overseas. The present study also detected single isolates of carbapenem-resistant E. coli (ST410-blaOXA-48) and colistin-resistant E. coli (ST345-mcr-1). CONCLUSIONS: The carriage of diverse CIA-resistant E. coli clones that strongly resemble pathogenic clones from humans suggests that gulls can act as ecological sponges indiscriminately accumulating and disseminating CIA-resistant bacteria over vast distances.

Genomic, Antimicrobial Resistance, and Public Health Insights into Enterococcus spp. from Australian Chickens.

Due to Australia's management of antimicrobial use in poultry, particularly the discontinued use of avoparcin for nearly 20 years, it is hypothesized that vancomycin-resistant enterococci associated with human disease are not derived from poultry isolates. This study evaluated antimicrobial resistance (AMR) of five enterococcal species isolated from Australian meat chickens, genomic features of Enterococcus faecium and Enterococcus faecalis, and the phylogenetic relationship of the poultry-derived E. faecium with isolates from human sepsis cases. All enterococcal isolates from chicken ceca were subjected to antimicrobial susceptibility testing. E. faecium and E. faecalis underwent whole-genome sequencing. E. faecium was compared at the core genome level to a collection of human isolates (n = 677) obtained from cases of sepsis over a 2-year period spanning 2015 to 2016. Overall, 205 enterococci were isolated consisting of five different species. E. faecium was the most frequently isolated species (37.6%), followed by E. durans (29.7%), E. faecalis (20%), E. hirae (12.2%), and E. gallinarum (0.5%). All isolates were susceptible to vancomycin and gentamicin, while one isolate was linezolid resistant (MIC 16 mg/liter). Core genome analysis of the E. faecium demonstrated two clades consisting predominantly of human or chicken isolates in each clade, with minimal overlap. Principal component analysis for total gene content revealed three clusters comprised of vanA-positive, vanB-positive, and both vanA- and vanB-negative E. faecium populations. The results of this study provide strong evidence that Australian chicken E. faecium isolates are unlikely to be precursor strains to the currently circulating vancomycin-resistant strains being isolated in Australian hospitals.

Molecular Detection and Epidemiological Features of Selected Bacterial, Viral, and Parasitic Enteropathogens in Stool Specimens from Children with Acute Diarrhea in Thi-Qar Governorate, Iraq.

Knowledge of etiology causes of diarrheal illness is essential for development and implementation of public health measures to prevent and control this disease syndrome. There are few published studies examining diarrhea in children aged <5 years in Iraq. This study aims to investigate the occurrences and epidemiology of selected bacterial (Salmonella spp. and Campylobacter spp.), viral (adenovirus, norovirus GI and GII, and astrovirus), and parasitic (Entamoeba spp. and Giardia spp.) agents in stool samples from 155 child diarrheal cases enrolled between March and August 2017, in a hospital-based cross-sectional study in Thi-Qar, southeastern Iraq. Using molecular techniques and sequence-based characterization, adenovirus was the most frequently detected enteropathogen (53/155 (34.2%)), followed by Salmonella spp. (23/155 (14.8%)), Entamoeba spp. (21/155 (13.5%)), and Campylobacter spp. (17/155 (10.9%)). Mixed infection with Salmonella spp. and Campylobacter spp. was evident, and the same was revealed between various enteric viruses, particularly adenovirus and norovirus. The most frequent co-infection pattern was between adenovirus and Campylobacter spp., in seven cases (7/155 (4.5%)). Whole-genome sequencing-derived typing data for Salmonella isolates (n = 23) revealed that sequence type 49 was the most prevalent in this sample set (15/23 (65.2%)). To the best of our knowledge, this study provides the first report on detection and identification of floR, blaCARB-2, and mphA antimicrobial resistance genes in Salmonella isolated from children in the Middle East region. Logistic regression analysis pointed to few enteropathogen-specific correlations between child age, household water source, and breastfeeding patterns in relation to the outcome of detection of individual enteropathogens. This study presents the first published molecular investigation of multiple enteropathogens among children <5 years of age in Iraq. Our data provide supporting evidence for planning of childhood diarrhea management programs. It is important to build on this study and develop future longitudinal case-control research in order to elaborate the epidemiology of enteropathogens in childhood diarrhea in Iraq.

Examination of Australian Streptococcus suis isolates from clinically affected pigs in a global context and the genomic characterisation of ST1 as a predictor of virulence.

Streptococcus suis is a major zoonotic pathogen that causes severe disease in both humans and pigs. Australia's pig herd has been quarantined for over 30 years, however S. suis remains a significant cause of disease. In this study, we investigated S. suis from 148 cases of clinical disease in pigs from 46 pig herds over a period of seven years, to determine the level of genetic difference from international isolates that may have arisen over the 30 years of separation. Isolates underwent whole genome sequencing, genome analysis and antimicrobial susceptibility testing. Data was compared at the core genome level to clinical isolates from overseas. Results demonstrated five predominant multi-locus sequence types and two major cps gene types (cps2 and 3). At the core genome level Australian isolates clustered predominantly within one large clade consisting of isolates from the UK, Canada and North America. A small proportion of Australian swine isolates (5%) were phylogenetically associated with south-east Asian and UK isolates, many of which were classified as causing systemic disease, and derived from cases of human and swine disease. Based on this dataset we provide a comprehensive outline of the current S. suis clones associated with disease in Australian pigs and their global context, with the main finding being that, despite three decades of separation, Australian S. suis are genomically similar to overseas strains. In addition, we show that ST1 clones carry a constellation of putative virulence genes not present in other Australian STs.

Salmonella enterica isolates from Western Australian rangeland goats remain susceptible to critically important antimicrobials.

This study investigated faecal carriage and antimicrobial resistance (AMR) of Salmonella enterica recovered from rangeland goats. Faecal samples (n = 400) were collected at slaughter from four consignments of goats (n = 100 samples per consignment), each from one of four localities in Western Australia. Carriage of Salmonella spp. was detected in 106 samples (26.5%; 95% CI 22.4-31.0%). The rate of faecal carriage for each consignment ranged between 23-30%. PCR assays targeting the STM2755 and STM4497 genes revealed 84.9% (90/106) of the isolates were of serovar Typhimurium. Salmonella Chester (11/106, 10.4%) and S. Saintpaul (5/106, 4.7%) were characterised at invA and ompF genes. Antimicrobial susceptibility testing demonstrated that 84.0% of isolates were susceptible to all tested (n = 13) antimicrobials. Resistance was identified to azithromycin (14.2%), tetracycline (10.4%), ampicillin (5.7%), amoxicillin-clavulanate and cefoxitin (3.8%), trimethoprim/sulfamethoxazole (1.9%), gentamicin and streptomycin (0.9%). No isolate was resistant to four or more antimicrobials, or to critically important antimicrobials such as fluoroquinolones and extended spectrum cephalosporins. This is the first study reporting AMR in Salmonella isolates from Australian rangeland goats. The rate of detection of AMR was very low, some resistance to low-importance drugs was present in the Salmonella population, despite the absence of active selection pressure.

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

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

Occurrence, antimicrobial resistance and whole-genome sequencing analysis of Salmonella isolates from chicken carcasses imported into Iraq from four different countries.

Salmonella is a major cause of human foodborne illnesses worldwide; however, little is known about its occurrence and genomic characteristics in food sources in many developing countries. This study investigates the occurrence, serotypes distribution, antimicrobial resistance, and multilocus sequence types (ST) of Salmonella isolated from 400 imported frozen chicken carcasses sold in the markets of Thi-Qar, south-eastern Iraq. Salmonella was detected in 46 out of 400 tested samples [11.5% (95% confidence interval: 8.5%-15.0%)]. S. Typhimurium was the most abundant (30.4%) among 14 different serotypes recovered from the tested frozen carcasses. Antimicrobial resistance was most frequently detected against tetracycline (84.4%), nalidixic acid (80.4%), streptomycin (69.6%) and trimethoprim/sulfamethoxazole (65.2%). Whole-genome sequencing (WGS) analysis revealed that 18 isolates harbored four ß-lactamase resistance genes, with blaCARB-2 was the most commonly (14/18) detected. It was possible to identify 8 multilocus sequence types from the WGS analysis of 40 out of the 46 Salmonella isolates; with ST-11 (among S. Enteritidis) and ST-19 (among S. Typhimurium) were the most frequently detected. These results add to our understanding of the global epidemiology of Salmonella. Our work stands as one of the first reports on WGS analysis of Salmonella from retail chicken in a Middle-Eastern country. Results from this study could be valuable for guiding an informed import risk analysis aiming at reducing the exposure risk from Salmonella through imported chicken carcasses into Iraq. This work demonstrates the value of WGS as a promising tool for supporting evidence-based food safety hazard characterization.

Dissemination and persistence of extended-spectrum cephalosporin-resistance encoding IncI1-blaCTXM-1 plasmid among Escherichia coli in pigs.

This study investigated the ecology, epidemiology and plasmid characteristics of extended-spectrum cephalosporin (ESC)-resistant E. coli in healthy pigs over a period of 4 years (2013-2016) following the withdrawal of ESCs. High carriage rates of ESC-resistant E. coli were demonstrated in 2013 (86.6%) and 2014 (83.3%), compared to 2015 (22%) and 2016 (8.5%). ESC resistance identified among E. coli isolates was attributed to the carriage of an IncI1 ST-3 plasmid (pCTXM1-MU2) encoding blaCTXM-1. Genomic characterisation of selected E. coli isolates (n = 61) identified plasmid movement into multiple commensal E. coli (n = 22 STs). Major STs included ST10, ST5440, ST453, ST2514 and ST23. A subset of the isolates belong to the atypical enteropathogenic E. coli (aEPEC) pathotype that harboured multiple LEE pathogenic islands. pCTXM1-MU2 was similar (99% nt identity) to IncI1-ST3 plasmids reported from Europe, encoded resistance to aminoglycosides, sulphonamides and trimethoprim, and carried colicin Ib. pCTXM1-MU2 appears to be highly stable and readily transferable. This study demonstrates that ESC resistance may persist for a protracted period following removal of direct selection pressure, resulting in the emergence of ESC-resistance in both commensal E. coli and aEPEC isolates of potential significance to human and animal health.

Carriage of critically important antimicrobial resistant bacteria and zoonotic parasites amongst camp dogs in remote Western Australian indigenous communities.

Camp dogs in indigenous communities in the Western Australian Kimberley Region, share the domestic environment with humans and have the potential to act as carriers of, and sentinels for, a wide range of zoonotic agents, including intestinal parasites and antimicrobial resistant bacteria. In this study, we investigated the carriage of extended-spectrum-cephalosporin-resistant (ESC-resistant) Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA) and species of hookworm and Giardia among camp dogs in remote Western Australian Aboriginal communities. A total of 141 canine faecal samples and 156 nasal swabs were collected from dogs in four communities of the Western Australian Kimberley region. Overall, ESC-resistant E. coli was detected in 16.7% of faecal samples and MRSA was isolated from 2.6% of nasal swabs. Of most significance was the presence of the community-associated Panton-Valentine leucocidin (PVL)-positive MRSA ST93 and ST5 clones and ESC-resistant E. coli ST38 and ST131. The most prevalent zoonotic intestinal parasite infection was Ancylostoma caninum (66%). The prevalence of Giardia was 12.1%, with the main genotypes of Giardia detected being dog specific assemblages C and D, which are unlikely to cause disease in humans.

Complete genomic characterisation of two novel poxviruses (WKPV and EKPV) from western and eastern grey kangaroos.

Poxviruses have previously been detected in macropods with cutaneous papillomatous lesions, however to date, no comprehensive analysis of a poxvirus from kangaroos has been performed. Here we report the genome sequences of a western grey kangaroo poxvirus (WKPV) and an eastern grey kangaroo poxvirus (EKPV), named for the host species from which they were isolated, western grey (Macropus fuliginosus) and eastern grey (Macropus giganteus) kangaroos. Poxvirus DNA from WKPV and EKPV was isolated and entire coding genome regions determined through Roche GS Junior and Illumina Miseq sequencing, respectively. Viral genomes were assembled using MIRA and SPAdes, and annotations performed using tools available from the Viral Bioinformatics Resource Centre. Histopathology and transmission electron microscopy analysis was also performed on WKPV and its associated lesions. The WKPV and EKPV genomes show 96% identity (nucleotide) to each other and phylogenetic analysis places them on a distinct branch between the established Molluscipoxvirus and Avipoxvirus genera. WKPV and EKPV are 170 kbp and 167 kbp long, containing 165 and 162 putative genes, respectively. Together, their genomes encode up to 47 novel unique hypothetical proteins, and possess virulence proteins including a major histocompatibility complex class II inhibitor, a semaphorin-like protein, a serpin, a 3-ß-hydroxysteroid dehydrogenase/δ 5→4 isomerase, and a CD200-like protein. These viruses also encode a large putative protein (WKPV-WA-039 and EKPV-SC-038) with a C-terminal domain that is structurally similar to the C-terminal domain of a cullin, suggestive of a role in the control of host ubiquitination. The relationship of these viruses to members of the Molluscipoxvirus and Avipoxvirus genera is discussed in terms of sequence similarity, gene content and nucleotide composition. A novel genus within subfamily Chordopoxvirinae is proposed to accommodate these two poxvirus species from kangaroos; we suggest the name, Thylacopoxvirus (thylaco-: [Gr.] thylakos meaning sac or pouch).