Corynebacterium megadyptis sp. nov. with two subspecies, Corynebacterium megadyptis subsp. megadyptis subsp. nov. and Corynebacterium megadyptis subsp. dunedinense subsp. nov. isolated from yellow-eyed penguins.

Novel Corynebacterium strains, 3BT and 7BT, were isolated from the oral cavities of young chicks of yellow-eyed penguins (hoiho), Megadyptes antipodes. A polyphasic taxonomic characterization of these strains revealed chemotaxonomic, biochemical and morphological features that are consistent with those of the genus Corynebacterium. The 16S rRNA gene sequence similarity values between the strains and their closest phylogenetic neighbour, Corynebacterium ciconiae CCUG 47525T were 99.07 %, values that are in line with their phylogenomic positions within the evolutionary radiation of the genus Corynebacterium. Digital DNA-DNA hybridization values and average nucleotide identities between the genome sequences of the two strains and related Corynebacterium species were well below the defined threshold values (70 and 95-96 %, respectively) for prokaryotic species delineation. The genome size of these strains varied between 2.45-2.46 Mb with G+C content 62.7-62.9 mol%. Strains 3BT and 7BT were Gram-stain positive bacilli that were able to grow in presence of 0-10 % (w/v) NaCl and at temperature ranging between 20-37 °C. The major fatty acids (>15 %) were C16 : 0 and C18 : 1 ω9c, and the mycolic acid profile included 32-36 carbon atoms. We propose that these strains represent a novel species, Corynebacterium megadyptis sp. nov. with 3BT (=DSM 111184T=NZRM 4755T) as the type strain. Phylogenomically, strains 3BT and 7BT belong to two lineages with subtle differences in MALDI-TOF spectra, chemotaxonomic profiles and phenotypic properties. The fatty acid profile of strain 3BT contains C18 : 0 as a predominant type (>15 %), which is a minor component in strain 7BT. Strain 7BT can oxidize N-acetyl-d-glucosamine, l-serine, α-hydroxy-butyric acid, l-malic acid, l-glutamic acid, bromo-succinic acid and l-lactic acid, characteristics not observed in strain 3BT. Therefore, we propose that these strains represent two subspecies, namely Corynebacterium megadyptis subsp. megadyptis subsp. nov. (type strain, 3BT=DSM 111184T=NZRM 4755T) and Corynebacterium megadyptis subsp. dunedinense subsp. nov. (type strain, 7BT=DSM 111183T=NZRM 4756T).

Investigating the Meat Pathway as a Source of Human Nontyphoidal Salmonella Bloodstream Infections and Diarrhea in East Africa.

BACKGROUND: Salmonella Enteritidis and Salmonella Typhimurium are major causes of bloodstream infection and diarrheal disease in East Africa. Sources of human infection, including the role of the meat pathway, are poorly understood. METHODS: We collected cattle, goat, and poultry meat pathway samples from December 2015 through August 2017 in Tanzania and isolated Salmonella using standard methods. Meat pathway isolates were compared with nontyphoidal serovars of Salmonella enterica (NTS) isolated from persons with bloodstream infections and diarrheal disease from 2007 through 2017 from Kenya by core genome multi-locus sequence typing (cgMLST). Isolates were characterized for antimicrobial resistance, virulence genes, and diversity. RESULTS: We isolated NTS from 164 meat pathway samples. Of 172 human NTS isolates, 90 (52.3%) from stool and 82 (47.7%) from blood, 53 (30.8%) were Salmonella Enteritidis sequence type (ST) 11 and 62 (36.0%) were Salmonella Typhimurium ST313. We identified cgMLST clusters within Salmonella Enteritidis ST11, Salmonella Heidelberg ST15, Salmonella Typhimurium ST19, and Salmonella II 42:r:- ST1208 that included both human and meat pathway isolates. Salmonella Typhimurium ST313 was isolated exclusively from human samples. Human and poultry isolates bore more antimicrobial resistance and virulence genes and were less diverse than isolates from other sources. CONCLUSIONS: Our findings suggest that the meat pathway may be an important source of human infection with some clades of Salmonella Enteritidis ST11 in East Africa, but not of human infection by Salmonella Typhimurium ST313. Research is needed to systematically examine the contributions of other types of meat, animal products, produce, water, and the environment to nontyphoidal Salmonella disease in East Africa.

Genomic and phenotypic comparison of two Salmonella Typhimurium strains responsible for consecutive salmonellosis outbreaks in New Zealand.

Salmonella enterica serovar Typhimurium DT160 was the predominant cause of notified human salmonellosis cases in New Zealand from 2000 to 2010, before it was superseded by another S. Typhimurium strain, DT56 variant (DT56v). Whole genome sequencing and phenotypic testing were used to compare 109 DT160 isolates with eight DT56v isolates from New Zealand animal and human sources. Phylogenetic analysis provided evidence that DT160 and DT56v strains were distantly related with an estimated date of common ancestor between 1769 and 1821. The strains replicated at different rates but had similar antimicrobial susceptibility profiles. Both strains were resistant to the phage expressed from the chromosome of the other strain, which may have contributed to the emergence of DT56v. DT160 contained the pSLT virulence plasmid, and the sseJ and sseK2 genes that may have contributed to the higher reported prevalence compared to DT56v. A linear pBSSB1-family plasmid was also found in one of the DT56v isolates, but there was no evidence that this plasmid affected bacterial replication or antimicrobial susceptibility. One of the DT56v isolates was also sequenced using long-read technology and found to contain an uncommon chromosome arrangement for a Typhimurium isolate. This study demonstrates how comparative genomics and phenotypic testing can help identify strain-specific elements and factors that may have influenced the emergence and supersession of bacterial strains of public health importance.

Transmission Dynamics of Shiga Toxin-Producing Escherichia coli in New Zealand Cattle from Farm to Slaughter.

Cattle are asymptomatic carriers of Shiga toxin-producing Escherichiacoli (STEC) strains that can cause serious illness or death in humans. In New Zealand, contact with cattle feces and living near cattle populations are known risk factors for human STEC infection. Contamination of fresh meat with STEC strains also leads to the potential for rejection of consignments by importing countries. We used a combination of PCR/matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) and whole-genome sequencing (WGS) to evaluate the presence and transmission of STEC on farms and in processing plants to better understand the potential pathways for human exposure and thus mitigate risk. Animal and environmental samples (n = 2,580) were collected from six farms and three meat processing plants in New Zealand during multiple sampling sessions in spring of 2015 and 2016. PCR/MALDI-TOF analysis revealed that 6.2% were positive for "Top 7" STEC. Top 7 STEC strains were identified in all sample sources (n = 17) tested. A marked increase in Top 7 STEC prevalence was observed between calf hides on farm (6.3% prevalence) and calf hides at processing plants (25.1% prevalence). Whole-genome sequencing was performed on Top 7 STEC bacterial isolates (n = 40). Analysis of STEC O26 (n = 25 isolates) revealed relatively low genetic diversity on individual farms, consistent with the presence of a resident strain disseminated within the farm environment. Public health efforts should focus on minimizing human contact with fecal material on farms and during handling, transport, and slaughter of calves. Meat processing plants should focus on minimizing cross-contamination between the hides of calves in a cohort during transport, lairage, and slaughter.IMPORTANCE Cattle are asymptomatic carriers of Shiga toxin-producing E. coli (STEC) strains, which can cause serious illness or death in humans. Contact with cattle feces and living near cattle are known risk factors for human STEC infection. This study evaluated STEC carriage in young calves and the farm environment with an in-depth evaluation of six farms and three meat processing plants over 2 years. An advanced molecular detection method and whole-genome sequencing were used to provide a detailed evaluation of the transmission of STEC both within and between farms. The study revealed widespread STEC contamination within the farm environment, but no evidence of recent spread between farms. Contamination of young dairy calf hides increased following transport and holding at meat processing plants. The elimination of STEC in farm environments may be very difficult given the multiple transmission routes; interventions should be targeted at decreasing fecal contamination of calf hides during transport, lairage, and processing.

Shifts in the Molecular Epidemiology of Campylobacter jejuni Infections in a Sentinel Region of New Zealand following Implementation of Food Safety Interventions by the Poultry Industry.

In 2006, New Zealand had the highest notification rate of campylobacteriosis in the world, and poultry was considered the leading source of campylobacteriosis. Implementation of food safety interventions by the poultry industry led to a decrease in the campylobacteriosis notification rate. The aim is to examine the impact of targeted food safety interventions implemented by the New Zealand poultry industry on the source attribution of Campylobacter jejuni infections in a sentinel region. Campylobacter jejuni isolates collected from the Manawatu region of New Zealand between 2005 and 2007 ("before intervention") and 2008 and 2015 ("after intervention") from human clinical cases, chicken meat, ruminant feces, environmental water, and wild bird sources were subtyped by multilocus sequence typing. Viable counts of Campylobacter spp. from carcasses were analyzed using a zero-inflated Poisson regression model. In the period before intervention, sequence type 474 (ST-474) was the most common sequence type (ST) recovered from human cases, accounting for 28.2% of the isolates. After intervention, the proportion of human cases positive for ST-474 reduced to 9.3%. Modeling indicated that chicken meat, primarily from one supplier, was the main source of C. jejuni infection in the Manawatu region before intervention. However, after intervention poultry collectively had a similar attribution to ruminants, but more human cases were attributed to ruminants than any single chicken supplier. Viable counts on carcasses were lower in all poultry suppliers after intervention. This study provides evidence of changes in the source attribution of campylobacteriosis following targeted food safety interventions in one sector of the food supply chain.IMPORTANCE This study provides a unique insight into the effects of food safety interventions implemented in one sector of the food industry on the transmission routes of a major foodborne agent. Following the implementation of food safety interventions by the poultry industry, shifts in the molecular epidemiology of Campylobacter jejuni infections in a sentinel region of New Zealand were observed. Targeted interventions to reduce disease incidence are effective but require continued surveillance and analysis to indicate where further interventions may be beneficial.

Carriage of Extended-Spectrum-Beta-Lactamase- and AmpC Beta-Lactamase-Producing Escherichia coli Strains from Humans and Pets in the Same Households.

Extended-spectrum-beta-lactamase (ESBL)- or AmpC beta-lactamase (ACBL)-producing Escherichia coli bacteria are the most common cause of community-acquired multidrug-resistant urinary tract infections (UTIs) in New Zealand. The carriage of antimicrobial-resistant bacteria has been found in both people and pets from the same household; thus, the home environment may be a place where antimicrobial-resistant bacteria are shared between humans and pets. In this study, we sought to determine whether members (pets and people) of the households of human index cases with a UTI caused by an ESBL- or ACBL-producing E. coli strain also carried an ESBL- or ACBL-producing Enterobacteriaceae strain and, if so, whether it was a clonal match to the index case clinical strain. Index cases with a community-acquired UTI were recruited based on antimicrobial susceptibility testing of urine isolates. Fecal samples were collected from 18 non-index case people and 36 pets across 27 households. Eleven of the 27 households screened had non-index case household members (8/18 people and 5/36 animals) positive for ESBL- and/or ACBL-producing E. coli strains. Whole-genome sequence analysis of 125 E. coli isolates (including the clinical urine isolates) from these 11 households showed that within seven households, the same strain of ESBL-/ACBL-producing E. coli was cultured from both the index case and another person (5/11 households) or pet dog (2/11 households). These results suggest that transmission within the household may contribute to the community spread of ESBL- or ACBL-producing E. coliIMPORTANCEEnterobacteriaceae that produce extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases (ACBLs) are important pathogens and can cause community-acquired illnesses, such as urinary tract infections (UTIs). Fecal carriage of these resistant bacteria by companion animals may pose a risk for transmission to humans. Our work evaluated the sharing of ESBL- and ACBL-producing E. coli isolates between humans and companion animals. We found that in some households, dogs carried the same strain of ESBL-producing E. coli as the household member with a UTI. This suggests that transmission events between humans and animals (or vice versa) are likely occurring within the home environment and, therefore, the community as a whole. This is significant from a health perspective, when considering measures to minimize community transmission, and highlights that in order to manage community spread, we need to consider interventions at the household level.

Genomic Analysis of Fluoroquinolone- and Tetracycline-Resistant Campylobacter jejuni Sequence Type 6964 in Humans and Poultry, New Zealand, 2014-2016.

In 2014, antimicrobial drug-resistant Campylobacter jejuni sequence type 6964 emerged contemporaneously in poultry from 3 supply companies in the North Island of New Zealand and as a major cause of campylobacteriosis in humans in New Zealand. This lineage, not previously identified in New Zealand, was resistant to tetracycline and fluoroquinolones. Genomic analysis revealed divergence into 2 major clades; both clades were associated with human infection, 1 with poultry companies A and B and the other with company C. Accessory genome evolution was associated with a plasmid, phage insertions, and natural transformation. We hypothesize that the tetO gene and a phage were inserted into the chromosome after conjugation, leaving a remnant plasmid that was lost from isolates from company C. The emergence and rapid spread of a resistant clone of C. jejuni in New Zealand, coupled with evolutionary change in the accessory genome, demonstrate the need for ongoing Campylobacter surveillance among poultry and humans.

Use of Genomics to Investigate Historical Importation of Shiga Toxin-Producing Escherichia coli Serogroup O26 and Nontoxigenic Variants into New Zealand.

Shiga toxin-producing Escherichia coli serogroup O26 is an important public health pathogen. Phylogenetic bacterial lineages in a country can be associated with the level and timing of international imports of live cattle, the main reservoir. We sequenced the genomes of 152 E. coli O26 isolates from New Zealand and compared them with 252 E. coli O26 genomes from 14 other countries. Gene variation among isolates from humans, animals, and food was strongly associated with country of origin and stx toxin profile but not isolation source. Time of origin estimates indicate serogroup O26 sequence type 21 was introduced at least 3 times into New Zealand from the 1920s to the 1980s, whereas nonvirulent O26 sequence type 29 strains were introduced during the early 2000s. New Zealand's remarkably fewer introductions of Shiga toxin-producing Escherichia coli O26 compared with other countries (such as Japan) might be related to patterns of trade in live cattle.

Campylobacter jejuni Strains Associated with Wild Birds and Those Causing Human Disease in Six High-Use Recreational Waterways in New Zealand.

Campylobacter jejuni, a leading cause of gastroenteritis worldwide, has been frequently isolated from recreational rivers and streams in New Zealand, yet the public health significance of this is unknown. This study uses molecular tools to improve our understanding of the epidemiology and sources of Campylobacter in recreational waterways, with a view to preventing human infection. Epidemiological and microbiological data were collected between 2005 and 2009 from six high-use recreational waterways in the Manawatu-Wanganui region of the North Island. Campylobacter spp. and C. jejuni were isolated from 33.2% and 20.4% of 509 samples, respectively. Isolation of Campylobacter was observed in both low and high river flows. After adjusting for the confounding effects of river flow, there was a significantly higher likelihood of isolating Campylobacter in the winter month of June compared to January. A high diversity of C. jejuni multilocus sequence types was seen, with the most commonly isolated being the water rail-associated ST-2381 (19/91 isolates [20.9%]), ST-1225 (8/91 isolates [8.8%]), and ST-45 (6/91 isolates [6.6%]). The ST-2381 was found in all rivers, while the most commonly isolated ST from human cases in New Zealand, the poultry-associated strain ST-474, was isolated only in one river. Although the majority of Campylobacter sequence types identified in river water were strains associated with wild birds that are rarely associated with human disease, poultry and ruminant-associated Campylobacter strains that are found in human infection were also identified and could present a public health risk.IMPORTANCE In 2016, there was a large-scale waterborne outbreak of campylobacteriosis in New Zealand, which was estimated to have affected over 5,000 people. This highlighted the need for a greater understanding of the sources of contamination of both surface and groundwater and risks associated with exposure to both drinking and recreational water. This study reports the prevalence and population structure of Campylobacter jejuni in six recreational waters of the Manawatu-Wanganui region of New Zealand and models the relationship between Campylobacter spp. and ruminant-associated Campylobacter and the parameters "sites," "months," and "river flow." Here, we demonstrate that both low and high river flows, month of the year, and recreational sites could influence the Campylobacter isolation from recreational waters. The presence of genotypes associated with human infection allowed us to describe potential risks associated with recreational waters.

Extended-Spectrum Beta-Lactamase-Producing Enterobacteriaceae in Dairy Farm Environments: A New Zealand Perspective.

Antimicrobial resistance (AMR) is a global issue for both human and animal health. Infections caused by antimicrobial-resistant bacteria present treatment option challenges and are often associated with heightened severity of infection. Antimicrobial use (AMU) in human and animal health is a main driver for the development of antimicrobial-resistant bacteria. Increasing levels of AMU and the development and spread of AMR in food-producing animals, especially in poultry and swine production, has been identified as a food safety risk, but dairy production systems have been less studied. A number of farm management practices may impact on animal disease and as a result can influence the use of antimicrobials and subsequently AMR prevalence. However, this relationship is multifactorial and complex. Several AMR transmission pathways between dairy cattle, the environment, and humans have been proposed, including contact with manure-contaminated pastures, direct contact, or through the food chain from contaminated animal-derived products. The World Health Organization has defined a priority list for selected bacterial pathogens of concern to human health according to 10 criteria relating to health and AMR. This list includes human pathogens such as the extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E), which can be associated with dairy cattle, their environment, as well as animal-derived food products. ESBL-E represent a potential risk to human and animal health and an emerging food safety concern. This review addresses two areas; first, the current understanding of the role of dairy farming in the prevalence and spread of AMR is considered, highlighting research gaps using ESBL-E as an exemplar; and second, a New Zealand perspective is taken to examine how farm management practices may contribute to on-farm AMU and AMR in dairy cattle.

Molecular Epidemiology of Shiga Toxin-Producing Escherichia coli (STEC) on New Zealand Dairy Farms: Application of a Culture-Independent Assay and Whole-Genome Sequencing.

New Zealand has a relatively high incidence of human cases of Shiga toxin-producing Escherichia coli (STEC), with 8.9 STEC cases per 100,000 people reported in 2016. Previous research showed living near cattle and contact with cattle feces as significant risk factors for STEC infections in humans in New Zealand, but infection was not linked to food-associated factors. During the 2014 spring calving season, a random, stratified, cross-sectional study of dairy farms (n = 102) in six regions across New Zealand assessed the prevalence of the "Top 7" STEC bacteria (serogroups O157, O26, O45, O103, O111, O121, and O145) in young calves (n = 1,508), using a culture-independent diagnostic test (PCR/MALDI-TOF). Twenty percent (306/1,508) of calves on 75% (76/102) of dairy farms were positive for at least one of the "Top 7" STEC bacteria. STEC carriage by calves was associated with environmental factors, increased calf age, region, and increased number of calves in a shared calf pen. The intraclass correlation coefficient (ρ) indicated strong clustering of "Top 7" STEC-positive calves for O157, O26, and O45 serogroups within the same pens and farms, indicating that if one calf was positive, others in the same environment were likely to be positive as well. This finding was further evaluated with whole-genome sequencing, which indicated that a single E. coli O26 clonal strain could be found in calves in the same pen or farm, but different strains existed on different farms. This study provides evidence that would be useful for designing on-farm interventions to reduce direct and indirect human exposure to STEC bacteria.IMPORTANCE Cattle are asymptomatic carriers of Shiga toxin-producing E. coli (STEC) bacteria that can cause bloody diarrhea and kidney failure in humans if ingested. New Zealand has relatively high numbers of STEC cases, and contact with cattle feces and living near cattle are risk factors for human infection. This study assessed the national prevalence of STEC in young dairy cattle by randomly selecting 102 farms throughout New Zealand. The study used a molecular laboratory method that has relatively high sensitivity and specificity compared to traditional methods. "Top 7" STEC was found in 20% of calves on 75% of the farms studied, indicating widespread prevalence across the country. By examining the risk factors associated with calf carriage, potential interventions that could decrease the prevalence of "Top 7" STEC bacteria at the farm level were identified, which could benefit both public health and food safety.

Long-term Colonization by Campylobacter jejuni Within a Human Host: Evolution, Antimicrobial Resistance, and Adaptation.

Background: Campylobacteriosis is inflammation of the gastrointestinal tract as a result of Campylobacter infection. Most campylobacteriosis cases are acute and self-limiting, with Campylobacter excretion ceasing a few weeks after symptoms cease. We identified a patient with fecal specimens positive for Campylobacter jejuni (ST45) intermittently during a 10-year period. Methods: Sixteen Campylobacter isolates were collected from the patient during 2006-2016. The isolates' genomes were sequenced to determine their relatedness, and their antimicrobial susceptibility patterns and motility were measured to determine the effects of antibiotic therapy and long-term excretion on the Campylobacter population. Results: Phylogenetic analyses estimated that the isolates shared a date of common ancestor between 1998 and 2006, coinciding with the onset of symptoms for the patient. Genomic analysis identified selection for changes in motility, and antimicrobial susceptibility testing suggested that the Campylobacter population developed resistance to several antibiotics coinciding with periods of antibiotic therapy. Conclusions: The patient was consistently colonized with organisms from a Campylobacter population that adapted to the internal environment of the patient. Genomic and phylogenetic analyses can give insight into a patient's infection history and the effect of antimicrobial treatment on Campylobacter populations in this unusual situation of long-term colonization of an individual.

Genomic Analysis of Salmonella enterica Serovar Typhimurium DT160 Associated with a 14-Year Outbreak, New Zealand, 1998-2012.

During 1998-2012, an extended outbreak of Salmonella enterica serovar Typhimurium definitive type 160 (DT160) affected >3,000 humans and killed wild birds in New Zealand. However, the relationship between DT160 within these 2 host groups and the origin of the outbreak are unknown. Whole-genome sequencing was used to compare 109 Salmonella Typhimurium DT160 isolates from sources throughout New Zealand. We provide evidence that DT160 was introduced into New Zealand around 1997 and rapidly propagated throughout the country, becoming more genetically diverse over time. The genetic heterogeneity was evenly distributed across multiple predicted functional protein groups, and we found no evidence of host group differentiation between isolates collected from human, poultry, bovid, and wild bird sources, indicating ongoing transmission between these host groups. Our findings demonstrate how a comparative genomic approach can be used to gain insight into outbreaks, disease transmission, and the evolution of a multihost pathogen after a probable point-source introduction.

Molecular Epidemiology of Campylobacter coli Strains Isolated from Different Sources in New Zealand between 2005 and 2014.

UNLABELLED: Campylobacteriosis is one of the most important foodborne diseases worldwide and a significant health burden in New Zealand. Campylobacter jejuni is the predominant species worldwide, accounting for approximately 90% of human cases, followed by Campylobacter coli Most studies in New Zealand have focused on C. jejuni; hence, the impact of C. coli strains on human health is not well understood. The aim of this study was to genotype C. coli isolates collected in the Manawatu region of New Zealand from clinical cases, fresh poultry meat, ruminant feces, and environmental water sources, between 2005 and 2014, to study their population structure and estimate the contribution of each source to the burden of human disease. Campylobacter isolates were identified by PCR and typed by multilocus sequence typing. C. coli accounted for 2.9% (n = 47/1,601) of Campylobacter isolates from human clinical cases, 9.6% (n = 108/1,123) from poultry, 13.4% (n = 49/364) from ruminants, and 6.4% (n = 11/171) from water. Molecular subtyping revealed 27 different sequence types (STs), of which 18 belonged to clonal complex ST-828. ST-1581 was the most prevalent C. coli sequence type isolated from both human cases (n = 12/47) and poultry (n = 44/110). When classified using cladistics, all sequence types belonged to clade 1 except ST-7774, which belonged to clade 2. ST-854, ST-1590, and ST-4009 were isolated only from human cases and fresh poultry, while ST-3232 was isolated only from human cases and ruminant sources. Modeling indicated ruminants and poultry as the main sources of C. coli human infection. IMPORTANCE: We performed a molecular epidemiological study of Campylobacter coli infection in New Zealand, one of few such studies globally. This study analyzed the population genetic structure of the bacterium and included a probabilistic source attribution model covering different animal and water sources. The results are discussed in a global context.

Impact of systemic antimicrobial therapy on the faecal microbiome in symptomatic dairy cows.

Antimicrobial resistance is a global threat to human and animal health, with the misuse and overuse of antimicrobials suggested as the main drivers of resistance. Antimicrobial therapy can alter the bacterial community composition and the faecal resistome in cattle. Little is known about the impact of systemic antimicrobial therapy on the faecal microbiome in dairy cows in the presence of disease. Therefore, this study aimed to assess the impact of systemic antimicrobial therapy on the faecal microbiome in dairy cows in the pastoral farm environment, by analysing faecal samples from cattle impacted by several different clinically-defined conditions and corresponding antimicrobial treatments. Analysis at the individual animal level showed a decrease in bacterial diversity and richness during antimicrobial treatment but, in many cases, the microbiome diversity recovered post-treatment when the cow re-entered the milking herd. Perturbations in the microbiome composition and the ability of the microbiome to recover were specific at the individual animal level, highlighting that the animal is the main driver of variation. Other factors such as disease severity, the type and duration of antimicrobial treatment and changes in environmental factors may also impact the bovine faecal microbiome. AmpC-producing Escherichia coli were isolated from faeces collected during and post-treatment with ceftiofur from one cow while no third-generation cephalosporin resistant E. coli were isolated from the untreated cow samples. This isolation of genetically similar plasmid-mediated AmpC-producing E. coli has implications for the development and dissemination of antibiotic resistant bacteria and supports the reduction in the use of critically important antimicrobials.

Extended-spectrum ß-lactamase- and AmpC ß-lactamase-producing Enterobacterales associated with urinary tract infections in the New Zealand community: a case-control study.

OBJECTIVES: To assess whether having a pet in the home is a risk factor for community-acquired urinary tract infections associated with extended-spectrum ß-lactamase (ESBL)- or AmpC ß-lactamase (ACBL)- producing Enterobacterales. METHODS: An unmatched case-control study was conducted between August 2015 and September 2017. Cases (n = 141) were people with community-acquired urinary tract infection (UTI) caused by ESBL- or ACBL-producing Enterobacterales. Controls (n = 525) were recruited from the community. A telephone questionnaire on pet ownership and other factors was administered, and associations were assessed using logistic regression. RESULTS: Pet ownership was not associated with ESBL- or ACBL-producing Enterobacterales-related human UTIs. A positive association was observed for recent antimicrobial treatment, travel to Asia in the previous year, and a doctor's visit in the last 6 months. Among isolates with an ESBL-/ACBL-producing phenotype, 126/134 (94%) were Escherichia coli, with sequence type 131 being the most common (47/126). CONCLUSIONS: Companion animals in the home were not found to be associated with ESBL- or ACBL-producing Enterobacterales-related community-acquired UTIs in New Zealand. Risk factors included overseas travel, recent antibiotic use, and doctor visits.

Nitrate enrichment does not affect enteropathogenic Escherichia coli in aquatic microcosms but may affect other strains present in aquatic habitats.

Eutrophication of the planet's aquatic systems is increasing at an unprecedented rate. In freshwater systems, nitrate-one of the nutrients responsible for eutrophication-is linked to biodiversity losses and ecosystem degradation. One of the main sources of freshwater nitrate pollution in New Zealand is agriculture. New Zealand's pastoral farming system relies heavily on the application of chemical fertilisers. These fertilisers in combination with animal urine, also high in nitrogen, result in high rates of nitrogen leaching into adjacent aquatic systems. In addition to nitrogen, livestock waste commonly carries human and animal enteropathogenic bacteria, many of which can survive in freshwater environments. Two strains of enteropathogenic bacteria found in New Zealand cattle, are K99 and Shiga-toxin producing Escherichia coli (STEC). To better understand the effects of ambient nitrate concentrations in the water column on environmental enteropathogenic bacteria survival, a microcosm experiment with three nitrate-nitrogen concentrations (0, 1, and 3 mg NO3-N /L), two enteropathogenic bacterial strains (STEC O26-human, and K99-animal), and two water types (sterile and containing natural microbiota) was run. Both STEC O26 and K99 reached 500 CFU/10 ml in both water types at all three nitrate concentrations within 24 hours and remained at those levels for the full 91 days of the experiment. Although enteropathogenic strains showed no response to water column nitrate concentrations, the survival of background Escherichia coli, imported as part of the in-stream microbiota did, surviving longer in 1 and 3 mg NO3-N/Lconcentrations (P < 0.001). While further work is needed to fully understand how nitrate enrichment and in-stream microbiota may affect the viability of human and animal pathogens in freshwater systems, it is clear that these two New Zealand strains of STEC O26 and K99 can persist in river water for extended periods alongside some natural microbiota.

Prevalence and distribution of extended-spectrum ß-lactamase and AmpC-producing Escherichia coli in two New Zealand dairy farm environments.

Antimicrobial resistance (AMR) is a global threat to human and animal health, with the misuse and overuse of antimicrobials being suggested as the main driver of resistance. In a global context, New Zealand (NZ) is a relatively low user of antimicrobials in animal production. However, the role antimicrobial usage on pasture-based dairy farms, such as those in NZ, plays in driving the spread of AMR within the dairy farm environment remains equivocal. Culture-based methods were used to determine the prevalence and distribution of extended-spectrum ß-lactamase (ESBL)- and AmpC-producing Escherichia coli from farm environmental samples collected over a 15-month period from two NZ dairy farms with contrasting management practices. Whole genome sequencing was utilised to understand the genomic epidemiology and antimicrobial resistance gene repertoire of a subset of third-generation cephalosporin resistant E. coli isolated in this study. There was a low sample level prevalence of ESBL-producing E. coli (faeces 1.7%; farm dairy effluent, 6.7% from Dairy 4 and none from Dairy 1) but AmpC-producing E. coli were more frequently isolated across both farms (faeces 3.3% and 8.3%; farm dairy effluent 38.4%, 6.7% from Dairy 1 and Dairy 4, respectively). ESBL- and AmpC-producing E. coli were isolated from faeces and farm dairy effluent in spring and summer, during months with varying levels of antimicrobial use, but no ESBL- or AmpC-producing E. coli were isolated from bulk tank milk or soil from recently grazed paddocks. Hybrid assemblies using short- and long-read sequence data from a subset of ESBL- and AmpC-producing E. coli enabled the assembly and annotation of nine plasmids from six E. coli, including one plasmid co-harbouring 12 antimicrobial resistance genes. ESBL-producing E. coli were infrequently identified from faeces and farm dairy effluent on the two NZ dairy farms, suggesting they are present at a low prevalence on these farms. Plasmids harbouring several antimicrobial resistance genes were identified, and bacteria carrying such plasmids are a concern for both animal and public health. AMR is a burden for human, animal and environmental health and requires a holistic "One Health" approach to address.

Detecting genes associated with antimicrobial resistance and pathogen virulence in three New Zealand rivers.

The emergence of clinically significant antimicrobial resistance (AMR) in bacteria is frequently attributed to the use of antimicrobials in humans and livestock and is often found concurrently with human and animal pathogens. However, the incidence and natural drivers of antimicrobial resistance and pathogenic virulence in the environment, including waterways and ground water, are poorly understood. Freshwater monitoring for microbial pollution relies on culturing bacterial species indicative of faecal pollution, but detection of genes linked to antimicrobial resistance and/or those linked to virulence is a potentially superior alternative. We collected water and sediment samples in the autumn and spring from three rivers in Canterbury, New Zealand; sites were above and below reaches draining intensive dairy farming. Samples were tested for loci associated with the AMR-related group 1 CTX-M enzyme production (bla CTX-M) and Shiga toxin producing Escherichia coli (STEC). The bla CTX-M locus was only detected during spring and was more prevalent downstream of intensive dairy farms. Loci associated with STEC were detected in both the autumn and spring, again predominantly downstream of intensive dairying. This cross-sectional study suggests that targeted testing of environmental DNA is a useful tool for monitoring waterways. Further studies are now needed to extend our observations across seasons and to examine the relationship between the presence of these genetic elements and the incidence of disease in humans.

Draft Genome Sequences of Seven Extended-Spectrum ß-Lactamase-Producing Escherichia coli Strains Isolated from New Zealand Waterways.

Draft genomes of seven extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli strains recovered from New Zealand waterways are described. The mean genome size was 5.1 Mb, with 4,724 coding sequences. All genomes contained the ESBL gene bla CTX-M, and one carried a plasmid-mediated AmpC gene, bla CMY-2 A multidrug-resistant genotype was detected in three isolates.