ABSTRACT Antimicrobial resistance is a major public health threat, as resistant infections are harder to treat and carry greater risks. Using a One Health framework, this survey of freshwater samples investigated the influence of local land use on the occurrence of waterborne third-generation cephalosporin-resistant Enterobacterales (3GC-E) mediated through extended-spectrum β-lactamases (ESBLs) or plasmid-mediated AmpC (pAmpC) genes. A cross-sectional survey of 339 freshwater samples from 49 New Zealand sites was undertaken, encompassing catchments with urban, agricultural (namely dairy; sheep and beef; and mixed dairy, sheep, and beef), avian-impact, and low-impact (exotic/native forest) dominant land uses. 3GC-E ( n = 63), including E. coli , Enterobacter , Citrobacter, and Klebsiella, were isolated from freshwater samples ( n = 35), where urban (32), dairy (1), avian (1), and sheep and beef (1) were identified as the dominant land use. 3GC-E genome assemblies ( n = 43) from freshwater were compared with human ESBL- E. coli ( n = 467) assemblies. Waterborne 3GC- E. coli were phylogenetically diverse, represented by seven phylotypes and 20 sequence types (ST131, ST38, ST68, and ST219) associated with human infections, suggesting contamination with urban wastewater. Two freshwater E. coli harbored the plasmid-associated carbapenemase-encoding bla NDM-5 - ble MBL gene cassette, which was absent from the human isolates. Two further freshwater E. coli and four human E. coli isolates carried pAA plasmids containing agg genes encoding for enteroaggregative E. coli adherence factors. By linking local land use at a national scale with the occurrence of freshwater 3GC- E. coli , we demonstrate the value of a One Health approach for understanding how human activities influence the environmental dissemination of antimicrobial resistance with implications for infectious disease risk. IMPORTANCE Antimicrobial resistance (AMR) threatens public health by making infections harder and more expensive to treat. While AMR is often studied in clinical settings, environmental pathways that spread resistant bacteria remain less understood. Using a One Health approach, we investigated how land use influences the presence of third-generation cephalosporin-resistant Enterobacterales in freshwater across New Zealand. We found that resistant Escherichia coli , including strains carrying plasmid-associated virulence factors, were most frequently detected at sites impacted by urban land use and human fecal contamination. These findings show that human activities shape the environmental distribution of clinically relevant resistance and highlight freshwater as a potential exposure pathway. By linking national-scale land-use patterns with the occurrence of resistant bacteria in the environment, this study demonstrates how integrated environmental and public health surveillance can improve our understanding of AMR dissemination and inform strategies to reduce the spread of resistant pathogens.

A total of 1347 Escherichia isolates from water, soil, sediment, biofilm, and faecal samples (n = 413) across five pristine (native forest) and five impacted (pastoral or urban) sites were subtyped into E. coli phylotypes and non-E. coli Escherichia spp. (Escherichia marmotae, Escherichia ruysiae and Escherichia whittamii). Impacted sites showed a higher prevalence of E. coli, particularly the ruminant-associated phylotype B1, across water, biofilm, sediment, and mammalian faeces. In contrast, E. marmotae (189 isolates) were more common in pristine sites and avian faeces, with a prevalence of 28.7%. Metabarcoding of the hypervariable gene gnd further revealed that Escherichia population diversity was greatest in aquatic environments (water, sediment, biofilm). Escherichia population diversity was also associated with elevated freshwater E. coli concentrations, increased prevalence of pathogenic E. coli virulence factors (stx1, stx2 and eae), and higher livestock numbers. In contrast, diversity measures for Escherichia populations were lowest in avian faeces and soil samples, and samples obtained from pristine sites with fewer faecal sources. These findings highlight the ecological role of birds as reservoirs of E. marmotae and their contribution to microbial diversity in New Zealand's freshwater ecosystems.

In 2017 an outbreak of Mycoplasma bovis (M. bovis), an infectious agent of cattle, was identified in Aotearoa New Zealand. This study characterizes the genomic population structure of the outbreak in New Zealand and compares it with the known global population structure using multilocus sequence typing (MLST) and genomic analysis. The New Zealand outbreak strain was MLST genotyped as ST21. A comprehensive collection of 840 genomes from the New Zealand outbreak showed a pattern of clonal expansion when characterized by MLST, core genome MLST (cgMLST) and whole genome MLST (wgMLST). A lineage of genomes was found with no in silico identifiable pta2 locus, a housekeeping gene used in the MLST scheme. We compared a sample set of 40 New Zealand genomes to 47 genomes from other countries. This group had 79 ST21 genomes and eight genomes that were single nucleotide polymorphism (SNP) variants within the MLST loci of ST21. Two of the 47 international genomes showed signs of extensive unique recombination. Unique alleles in six genes were identified as present only in the New Zealand genomes. These novel variants were in the genes; haeIIIM encoding for cytosine-specific methyltransferase, cysC encoding for cysteinyl tRNA synthetase, era encoding for GTPase Era, metK encoding for S-adenosylmethionine synthase, parE encoding for DNA topoisomerase, and hisS encoding for histidine-tRNA ligase. This finding could be due to a population bottleneck, genetic drift, or positive selection. The same sample set of 40 New Zealand genomes were compared using MLST to 404 genomes from 15 other countries and 11 genomes without a known country. A FastBAPS analysis of 455 genomes showed a global population structure with 11 clusters. Some countries, such as Canada, Denmark and Australia contained both internally closely related genomes and some genomes that were more closely related to genomes found in other countries. Our results support the need for Whole Genome Sequencing (WGS) as well as MLST genotyping in M. bovis outbreaks. They also support the importance of understanding the national and international movement patterns of cattle and their genetic material, as possible routes of transmission, when managing the spread of M. bovis.

Background/Objectives: Agricultural soils accumulate inorganic contaminants from the application of phosphate fertilisers. An airstrip located at Belmont Regional Park (BRP), near Wellington, New Zealand, has been found to have a gradient of cadmium contamination due to spillage of superphosphate fertiliser. Methods: Soil samples from the BRP airstrip with a gradient of cadmium contamination, were used as a novel source to explore bacterial communities’ resistance to heavy metals (HMs) and any co-selected antibiotic (Ab) resistance. Results: Differences between BRP soil samples with higher levels of HMs compared to those with lower HM concentrations showed significantly more bacterial isolates resistant to both HMs (40.6% versus 63.1% resistant to 0.01 mM CdCl2, p < 0.05) and Abs (23.4% versus 37.8% resistant to 20 μg/mL tetracycline, p < 0.05) in soils with higher initial levels of HMs (1.14 versus 7.20 mg kg−1 Cd). Terminal restriction fragment length polymorphism (TRFLP) and 16S rDNA next-generation sequencing profiling investigated changes in HM-induced bacterial communities. Significant differences were observed among the bacterial community structures in the selected BRP soil samples. Conjugative transfer of cadmium resistance from 23–38% of cadmium-resistant isolates to a characterised recipient bacterial strain in vitro suggested many of these genes were carried by mobile genetic elements. Transconjugants were also resistant to zinc, mercury, and Abs. Higher levels of HMs in soil correlated with increased resistance to HMs, Abs, and elevated levels of HMs thus disturbed the bacterial community structure in BRP soil significantly. Conclusions: These findings suggest that HM contamination of agricultural soil can select for Ab resistance in soil bacteria with potential risks to human and animal health.

Genetic features of host adaptation of S. aureus to ruminants have been extensively studied, but the extent to which this adaptation occurs in nature remains unknown. In New Zealand, clonal complex 1 (CC1) is among the most common lineages in humans and the dominant lineage in cattle, enabling between-, and within-CC genomic comparisons of epidemiologically cohesive samples of isolates. We assessed the following genomic benchmarks of host adaptation to ruminants in 277 S. aureus from cattle, small ruminants, humans, and pets: 1, phylogenetic clustering of ruminant strains; 2, abundance of homo-specific ruminant-adaptive factors, and 3, scarcity of heterospecific factors. The genomic comparisons were complemented by comparative analyses of the metabolism of carbon sources that abound in ruminant milk. We identified features fulfilling the three benchmarks in virtually all ruminant isolates, including CC1. Data suggest the virulomes adapt to the ruminant niche sensu lato accross CCs. CC1 forms a ruminant-adapted clade that appears better equipped to utilise milk carbon sources than human CC1. Strain flow across the human-ruminant interface appears to only occur occasionally. Taken together, the results suggest a specialisation, rather than mere adaptation, clarifying why zoonotic and zoo-anthroponotic S. aureus transmission between ruminants and humans has hardly ever been reported.

In Aotearoa New Zealand, urinary tract infections in humans are commonly caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. This group of antimicrobial-resistant bacteria are often multidrug resistant. However, there is limited information on ESBL-producing E. coli found in the environment and their link with human clinical isolates. In this study, we examined the genetic relationship between environmental and human clinical ESBL-producing E. coli and isolates collected in parallel within the same area over 14 months. Environmental samples were collected from treated effluent, stormwater and multiple locations along an Aotearoa New Zealand river. Treated effluent, stormwater and river water sourced downstream of the treated effluent outlet were the main samples that were positive for ESBL-producing E. coli (7/14 samples, 50.0%; 3/6 samples, 50%; and 15/28 samples, 54%, respectively). Whole-genome sequence comparison was carried out on 307 human clinical and 45 environmental ESBL-producing E. coli isolates. Sequence type 131 was dominant for both clinical (147/307, 47.9%) and environmental isolates (11/45, 24.4%). Only one ESBL gene was detected in each isolate. Among the clinical isolates, the most prevalent ESBL genes were bla CTX-M-27 (134/307, 43.6%) and bla CTX-M-15 (134/307, 43.6%). Among the environmental isolates, bla CTX-M-15 (28/45, 62.2%) was the most prevalent gene. A core SNP analysis of these isolates suggested that some strains were shared between humans and the local river. These results highlight the importance of understanding different transmission pathways for the spread of ESBL-producing E. coli.

Cryptosporidiosis is a disease caused by the parasite Cryptosporidium. Globally, it is a leading cause of diarrhoea and a notifiable disease in New Zealand. Molecular analyses of Cryptosporidium isolated from notified cases do not always provide support for epidemiological links between individuals. We hypothesised this could be due to undetected diversity and the use of consensus Sanger sequence analyses. Here, we analysed 105 Cryptosporidium samples from outbreaks and sporadic cases occurring between 2010 and 2018 in New Zealand using both Next-Generation Sequencing (NGS) and Sanger sequencing of the glycoprotein 60 (gp60) locus. NGS metabarcoding at the gp60 locus uncovered significant intra- and inter-sample genotypic diversity in outbreaks and identified subtypes shared by epidemiologically linked cases, along with rare subtypes, suggesting it may be a useful tool for epidemiological investigations.

The kākāpō is a critically endangered flightless parrot which suffers from exudative cloacitis, a debilitating disease resulting in inflammation of the vent margin or cloaca. Despite this disease emerging over 20 years ago, the cause of exudative cloacitis remains elusive. We used total RNA sequencing and metatranscriptomic analysis to characterise the infectome of lesions and cloacal swabs from nine kākāpō affected with exudative cloacitis, and compared this to cloacal swabs from 45 non-diseased kākāpō. We identified three bacterial species-Streptococcus gallolyticus, Enterococcus faecalis and Escherichia coli-as significantly more abundant in diseased kākāpō compared to healthy individuals. The genetic diversity observed in both S. gallolyticus and E. faecalis among diseased kākāpō suggests that these bacteria originate from exogenous sources rather than from kākāpō-to-kākāpō transmission. The presence of extraintestinal pathogenic E. coli (ExPEC)-associated virulence factors in the diseased kākāpō population suggests that E. coli may play a critical role in disease progression by facilitating iron acquisition and causing DNA damage in host cells, possibly in association with E. faecalis. No avian viral, fungal nor other parasitic species were identified. These results, combined with the consistent presence of one E. coli gnd sequence type across multiple diseased birds, suggest that this species may be the primary cause of exudative cloacitis. These findings shed light on possible causative agents of exudative cloacitis, and offer insights into the interplay of microbial factors influencing the disease.

This longitudinal survey followed the life cycle of a New Zealand broiler flock to investigate sources of flock colonization by Campylobacter. Samples were collected at frequent intervals from potential Campylobacter reservoirs and sources, transmission routes for Campylobacter ingress into the broiler shed, and to monitor flock colonization. Of the 738 samples, 200 (27%) tested positive for Campylobacter. Campylobacter species from sample isolates included 316 Campylobacter jejuni, 39 Campylobacter coli, and 8 Campylobacter lari isolates; only C. jejuni was isolated from chickens. C. jejuni isolates (n = 199) were sequenced and consisted of seven sequence types (STs); the most abundant was ST6964 (105 isolates). Most flock isolates were ST6964 (44 isolates) or ST50 (27 isolates). ST6964 isolates closely matched those from the previous flock and another age-matched flock on the same farm, supporting a role for an on-farm reservoir contaminating flocks. There were six STs from catching crew and equipment isolates; the most prevalent were ST6964 (19 isolates) and ST50 (21 isolates). The close genetic match, high Campylobacter prevalence in catching samples (59/130, 45%), and the timing of flock colonization occurring closely following catcher presence in the shed support that catchers and equipment might also contaminate the shed and flock from prior flocks that they visited. There was no evidence for wildlife, feed, drinking water, breeder flock, or shed litter as sources of the Campylobacter genotypes colonizing the flock. Taken together, this study identified key areas where the poultry industry might focus on-farm risk management practices to reduce colonization of broiler flocks by Campylobacter.IMPORTANCECampylobacteriosis is the most frequently notified enteric disease in New Zealand, and New Zealand has one of the highest rates of campylobacteriosis among industrialized countries. Reducing Campylobacter colonization of poultry at the farm level would reduce reliance on processing interventions for reducing Campylobacter contamination of broiler meat. This study aimed to identify on-farm sources of Campylobacter contamination in New Zealand broiler chicken flocks. No evidence was found that wildlife, chicken feed, drinking water, or parent breeder flocks were contaminating sources. Instead, carryover of Campylobacter from the previous flock or other farm flocks, and/or contamination from chicken catching crews and their equipment, may have contributed Campylobacter strains that colonized the study flock. These are key areas where the poultry industry might focus on-farm risk management practices to reduce colonization of broiler flocks by Campylobacter.

Skin thickness was found to be moderately heritable and genetically associated with lamb survival in a previous study on Romney sheep. The aims of this study were to estimate the heritabilities of skin thickness and skin temperature at around 5 and 11 mo of age and determine genetic and phenotypic correlations between them and with production traits such as fat depth, loin-eye muscle depth and width, live weights at weaning, scanning, and 12 mo, and 12-mo fleece weight, in FocusPrime (n = 2,088), Texel (n = 732), Romney (n = 825) and Highlander (n = 1,801) sheep breeds. Heritability estimates of skin thickness at 5-mo old were moderate in FocusPrime (0.39 +/- 0.12) and low in Texel and Highlander (0.11 +/- 0.15 and 0.13 +/- 0.09, respectively). Heritability estimates of skin thickness at 11-mo old were moderate in all breeds (ranging from 0.19 +/- 0.07 to 0.29 +/- 0.15). Heritability estimates of skin temperature were high in FocusPrime (0.39 +/- 0.11), low in Texel (0.17 +/- 0.11) and Highlander (0.12 +/- 0.06) and almost zero in Romney (0.04 +/- 0.03). A tendency in all breeds for negative and favorable correlations was found between skin thickness and skin temperature at 11-mo old sheep. Skin thickness at 11-mo tended to have a positive genetic correlation with fat depth in all breeds except in Texel where the correlation tended to be negative (-0.10 +/- 0.34). Genetic correlations of skin thickness at 11-mo old with the weight traits were variate. There tended to be a positive correlation with weaning weight in Texel (0.14 +/- 0.34) and Highlander (0.29 +/- 0.22). However, there tended to be negative correlations with live weight at scanning and at 12-mo of age in FocusPrime (-0.03 +/- 0.18 and -0.13 +/- 0.22, respectively) and tended to be positive in Romney (0.09 +/- 0.25 and 0.10 +/- 0.24, respectively) and Highlander (0.26 +/- 0.22 and 0.39 +/- 0.21, respectively). Moreover, genetic correlations of skin thickness at 11-mo of age with FW12 tended to be positive in both Romney (0.20 +/- 0.22) and Highlander (0.55 +/- 0.19). Further studies on the genetic correlations of skin thickness and skin temperature with lamb survival in these breeds are warranted. This research shows that skin thickness and skin temperature are heritable in FocusPrime, Texel, and Highlander sheep breeds. This study aimed to estimate the heritabilities of skin thickness and skin temperature, while also estimating the genetic and phenotypic correlations between them and with several production traits of economic importance in 5- and 11-mo-old FocusPrime, Texel, and Highlander sheep. Skin thickness showed wide phenotypic variation with moderate heritability across breeds for 11-mo skin thickness, confirming that genetic selection in this trait is feasible. Negative favorable correlations between 11-mo skin thickness and skin temperature were observed. Skin thickness at 11 mo tended to be positively correlated with fleece weight and fat depth. Genetic association of skin thickness with Romney lamb survival has been reported in an earlier study and similar research on the association of skin thickness and skin temperature on lamb survival in these breeds is warranted. If the similar association is evinced in these breeds, skin thickness and skin temperature may potentially be used as an indicator trait for improving lamb survival.

Outbreaks caused by Salmonella Enteritidis are commonly linked to eggs and poultry meat internationally, but this serovar had never been detected in Aotearoa New Zealand (NZ) poultry prior to 2021. Locally designated genomic cluster Salmonella Enteritidis_2019_C_01, was implicated in a 2019 outbreak associated with a restaurant in Auckland. Four Enteritidis_2019_C_01 sub-clusters have since been identified, two retrospectively, in the Auckland region. Authorities initiated a formal outbreak investigation after genomically indistinguishable S. Enteritidis was isolated from the NZ poultry production environment. This study analysed 231 S. Enteritidis genomes obtained from the outbreak using Bayesian phylodynamic tools to gain insight into the outbreak's dynamics and origin. We used Bayesian integrated coalescent epoch plots to estimate the change of the Enteritidis ST11 population size over time and marginal structured coalescent approximation to estimate transmission between poultry producers. We investigated human and poultry isolates to elucidate the time and location of the most recent common ancestor of the outbreak and transmission pathways. The median most recent common ancestor was estimated to be February 2019. We found evidence of amplification and spread of strain Enteritidis_2019_C_01 within the poultry industry, as well as transmission events throughout the production chain. The intervention by the public health and food safety authorities coincided with a drop in the effective population size of the S. Enteritidis ST11 as well as notified human cases. This information is crucial for understanding and preventing the transmission of S. Enteritidis in NZ poultry to ensure poultry meat and eggs are safe for consumption.

Understanding the composition of complex Escherichia coli populations from the environment is necessary for identifying strategies to reduce the impacts of fecal contamination and protect public health. Metabarcoding targeting the hypervariable gene gnd was used to reveal the complex population diversity of E. coli and phenotypically indistinct Escherichia species in water, soil, sediment, aquatic biofilm, and fecal samples from native forest and pastoral sites. The resulting amplicons were cross-referenced against a database containing over 700 different partial gnd sequences from E. coli/non-E. coli Escherichia species. Alpha and beta measures of diversity of Escherichia populations were lowest in feces, soil and sediment compared to water and aquatic biofilm samples. Escherichia populations recovered from extensive freshwater catchments dominated by sheep, beef and dairy farming were extremely diverse but well-separated from a wetland dairy site. In contrast, Escherichia populations from the low-impact native forest site with fewer fecal sources were less diverse. Metabarcoding distinguished E. coli populations important to fecal contamination monitoring from non-E. coli Escherichia environmental populations. These data represent in-depth analysis and geographic stability of Escherichia populations from environmental samples with extensive heterogeneity, and reveal links with diverse fecal sources, land-use and the overall burden of fecal contamination at sample sites.

Levels of a predictor variable that are absent when a classification tree is grown can not be subject to an explicit splitting rule. This is an issue if these absent levels are present in a new observation for prediction. To date, there remains no satisfactory solution for absent levels in random forest models. Unlike missing data, absent levels are fully observed and known. Ordinal encoding of predictors allows absent levels to be integrated and used for prediction. Using a case study on source attribution of Campylobacter species using whole genome sequencing (WGS) data as predictors, we examine how target-agnostic versus target-based encoding of predictor variables with absent levels affects the accuracy of random forest models. We show that a target-based encoding approach using class probabilities, with absent levels designated the highest rank, is systematically biased, and that this bias is resolved by encoding absent levels according to the a priori hypothesis of equal class probability. We present a novel method of ordinal encoding predictors via principal coordinates analysis (PCO) which capitalizes on the similarity between pairs of predictor levels. Absent levels are encoded according to their similarity to each of the other levels in the training data. We show that the PCO-encoding method performs at least as well as the target-based approach and is not biased.

Escherichia coli are routine indicators of fecal contamination in water quality assessments. Contrary to livestock and human activities, brushtail possums (Trichosurus vulpecula), common invasive marsupials in Aotearoa/New Zealand, have not been thoroughly studied as a source of fecal contamination in freshwater. To investigate their potential role, Escherichia spp. isolates (n = 420) were recovered from possum gut contents and feces and were compared to those from water, soil, sediment, and periphyton samples, and from birds and other introduced mammals collected within the Mākirikiri Reserve, Dannevirke. Isolates were characterized using E. coli-specific real-time PCR targeting the uidA gene, Sanger sequencing of a partial gnd PCR product to generate a gnd sequence type (gST), and for 101 isolates, whole genome sequencing. Escherichia populations from 106 animal and environmental sample enrichments were analyzed using gnd metabarcoding. The alpha diversity of Escherichia gSTs was significantly lower in possums and animals compared with aquatic environmental samples, and some gSTs were shared between sample types, e.g., gST535 (in 85% of samples) and gST258 (71%). Forty percent of isolates gnd-typed and 75% of reads obtained by metabarcoding had gSTs shared between possums, other animals, and the environment. Core-genome single nucleotide polymorphism (SNP) analysis showed limited variation between several animal and environmental isolates (<10 SNPs). Our data show at an unprecedented scale that Escherichia clones are shared between possums, other wildlife, water, and the wider environment. These findings support the potential role of possums as contributors to fecal contamination in Aotearoa/New Zealand freshwater. Our study deepens the current knowledge of Escherichia populations in under-sampled wildlife. It presents a successful application of high-resolution genomic methods for fecal source tracking, thereby broadening the analytical toolbox available to water quality managers. Phylogenetic analysis of isolates and profiling of Escherichia populations provided useful information on the source(s) of fecal contamination and suggest that comprehensive invasive species management strategies may assist in restoring not only ecosystem health but also water health where microbial water quality is compromised.

AimsTo isolate canine respiratory coronavirus (CRCoV) and canine pneumovirus (CnPnV) in cell culture and to compare partial genomic sequences of CRCoV and CnPnV from New Zealand with those from other countries.MethodsOropharyngeal swab samples from dogs affected by canine infectious respiratory disease syndrome that were positive for CnPnV (n = 15) or CRCoV (n = 1) by virus-specific reverse transcriptase quantitative PCR (RT-qPCR) in a previous study comprised the starting material. Virus isolation was performed in HRT-18 cells for CRCoV and RAW 264.7 and Vero cells for CnPnV. The entire sequence of CnPnV G protein (1,266 nucleotides) and most (8,063/9,707 nucleotides) of the 3' region of CRCoV that codes for 10 structural and accessory proteins were amplified and sequenced. The sequences were analysed and compared with other sequences available in GenBank using standard molecular tools including phylogenetic analysis.ResultsVirus isolation was unsuccessful for both CRCoV and CnPnV. Pneumovirus G protein was amplified from 3/15 (20%) samples that were positive for CnPnV RNA by RT-qPCR. Two of these (NZ-048 and NZ-049) were 100% identical to each other, and 90.9% identical to the third one (NZ-007). Based on phylogenetic analysis of the G protein gene, CnPnV NZ-048 and NZ-049 clustered with sequences from the USA, Thailand and Italy in group A, and CnPnV NZ-007 clustered with sequences from the USA in group B. The characteristics of the predicted genes (length, position) and their putative protein products (size, predicted structure, presence of N- and O-glycosylation sites) of the New Zealand CRCoV sequence were consistent with those reported previously, except for the region located between open reading frame (ORF)3 (coding for S protein) and ORF6 (coding for E protein). The New Zealand virus was predicted to encode 5.9 kDa, 27 kDa and 12.7 kDa proteins, which differed from the putative coding capacity of this region reported for CRCoV from other countries.ConclusionsThis report represents the first characterisation of partial genomic sequences of CRCoV and CnPnV from New Zealand. Our results suggest that the population of CnPnV circulating in New Zealand is not homogeneous, and that the viruses from two clades described overseas are also present here. Limited conclusions can be made based on only one CRCoV sequence, but the putative differences in the coding capacity of New Zealand CRCoV support the previously reported variability of this region. The reasons for such variability and its biological implications need to be further elucidated.

In Aotearoa New Zealand, urinary tract infections in humans are commonly caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli . This group of antimicrobial resistant bacteria are often multidrug resistant. However, there is limited information on ESBL-producing E. coli found in the environment and their link with human clinical isolates. In this study, we examined the genetic relationship of environmental and human clinical ESBL-producing E. coli and isolates collected in parallel within the same area over 14 months. Environmental samples were collected from treated effluent, stormwater and multiple locations along an Aotearoa New Zealand river. Treated effluent, stormwater and river water sourced downstream of the treated outflow point were the main sources of ESBL-producing E. coli (7/14 samples, 50.0%; 3/6 samples, 50%; and 15/28 samples, 54% respectively). Whole genome sequence comparison was carried out on 307 human clinical and 45 environmental ESBL-producing E. coli isolates. Sequence type 131 was dominant for both clinical (147/307, 47.9%) and environmental isolates (11/45, 24.4%). The most prevalent ESBL genes were both bla CTX-M-27 and bla CTX-M-15 for the clinical isolates (134/307, 43.6%) and bla CTX-M-15 for the environmental isolates (28/45, 62.2%). A core single nucleotide polymorphism analysis of these isolates suggested that some strains were shared between humans and the local river. These results highlight the importance of understanding different transmission pathways for the spread of ESBL-producing E. coli . 2. Impact statement Extended spectrum beta lactamase (ESBL)-producing E. coli frequently cause urinary tract infections that exhibit multidrug resistance. Surveillance studies have identified the predominant strains and resistance genes associated with urinary tract infections. However, there is limited information on the extent of spread beyond the patient. We describe the genetic relatedness of ESBL-producing environmental and clinical E. coli isolated during the same temporal-spatial period in Aotearoa New Zealand. Comparative genomic analyses of these bacteria provide evidence of clonal spread between humans and the environment, highlighting the need to integrate environmental surveillance into antimicrobial resistance monitoring. 3. Data summary All Illumina sequence reads for this study have been deposited in GenBank under BioProject PRJNA1032159, except for strain SB0283h1, whose data can be found under BioProject PRJNA715472. The sequence read accessions for each genome are provided in the supplementary material. The code used for the genomic and statistical analyses is available from the GitHub repository . The authors confirm all supporting data and protocols have been provided within the article or through supplementary data files. ### Competing Interest Statement The authors have declared no competing interest.

This study investigated the diversity of thermophilic Campylobacter species isolated from three New Zealand freshwater catchments affected by pastoral and urban activities. Utilising matrix-assisted laser desorption ionisation-time of flight and whole genome sequence analysis, the study identified Campylobacter jejuni (n = 46, 46.0%), C. coli (n = 39, 39%), C. lari (n = 4, 4.0%), and two novel Campylobacter species lineages (n = 11, 11%). Core genome sequence analysis provided evidence of prolonged persistence or continuous faecal shedding of closely related strains. The C. jejuni isolates displayed distinct sequence types (STs) associated with human, ruminant, and environmental sources, whereas the C. coli STs included waterborne ST3302 and ST7774. Recombination events affecting loci implicated in human pathogenesis and environmental persistence were observed, particularly in the cdtABC operon (encoding the cytolethal distending toxin) of non-human C. jejuni STs. A low diversity of antimicrobial resistance genes (aadE-Cc in C. coli), with genotype/phenotype concordance for tetracycline resistance (tetO) in three ST177 isolates, was noted. The data suggest the existence of two types of naturalised waterborne Campylobacter: environmentally persistent strains originating from waterbirds and new environmental species not linked to human campylobacteriosis. Identifying and understanding naturalised Campylobacter species is crucial for accurate waterborne public health risk assessments and the effective allocation of resources for water quality management.

Escherichia coli are widely used by water quality managers as Fecal Indicator Bacteria, but current quantification methods do not differentiate them from benign, environmental Escherichia species such as E. marmotae (formerly named cryptic clade V) or E. ruysiae (cryptic clades III and IV). Reliable and specific techniques for their identification are required to avoid confounding microbial water quality assessments. To address this, a multiplex droplet digital PCR (ddPCR) assay targeting lipB (E. coli and E. ruysiae) and bglC (E. marmotae) was designed. The ddPCR performance was assessed using in silico analysis; genomic DNA from 40 local, international, and reference strains of target and non-target coliforms; and spiked water samples in a range relevant to water quality managers (1 to 1000 cells/100 mL). Results were compared to an analogous quantitative PCR (qPCR) and the Colilert method. Both PCR assays showed excellent sensitivity with a limit of detection of 0.05 pg/mu L and 0.005 pg/mu l for ddPCR and qPCR respectively, and of quantification of 0.5 pg/mu L of genomic DNA. The ddPCR allowed differentiation and quantification of three Escherichia species per run by amplitude multiplexing and showed a high concordance with concentrations measured by Colilert once proportional bias was accounted for. In silico specificity testing underlined the possibility to further detect and distinguish Escherichia cryptic clade VI. Finally, the applicability of the ddPCR was successfully tested on environmental water samples where E. marmotae and E. ruysiae potentially confound E. coli counts based on the Most Probable Number method, highlighting the utility of this novel ddPCR as an efficient and rapid discriminatory test to improve water quality assessments.

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.

Background/Objectives: We present hidecan, an R package for generating visualisations that summarise the results of one or more genome-wide association studies (GWAS) and differential expression analyses, as well as manually curated candidate genes, e.g., extracted from the literature. This tool is applicable to all ploidy levels; we notably provide functionalities to facilitate the visualisation of GWAS results obtained for autotetraploid organisms with the GWASpoly package. Results: We illustrate the capabilities of hidecan with examples from two autotetraploid potato datasets. Conclusions: The hidecan package is implemented in R and is publicly available on the CRAN repository and on GitHub. A description of the package, as well as a detailed tutorial, is made available alongside the package. It is also part of the VIEWpoly tool for the visualisation and exploration of results from polyploids computational tools.