Journal Articles
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Item New Campylobacter Lineages in New Zealand Freshwater: Pathogenesis and Public Health Implications(John Wiley and Sons, 2024-12) Cookson AL; Burgess S; Midwinter AC; Marshall JC; Moinet M; Rogers L; Fayaz A; Biggs PJ; Brightwell GThis 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.Item Metabolism of Caprine Milk Carbohydrates by Probiotic Bacteria and Caco-2:HT29⁻MTX Epithelial Co-Cultures and Their Impact on Intestinal Barrier Integrity(MDPI (Basel, Switzerland), 2018-07-23) Barnett AM; Roy NC; Cookson AL; McNabb WCThe development and maturation of the neonatal intestine is generally influenced by diet and commensal bacteria, the composition of which, in turn, can be influenced by the diet. Colonisation of the neonatal intestine by probiotic Lactobacillus strains can strengthen, preserve, and improve barrier integrity, and adherence of probiotics to the intestinal epithelium can be influenced by the available carbon sources. The goal of the present study was to examine the role of probiotic lactobacilli strains alone or together with a carbohydrate fraction (CF) from caprine milk on barrier integrity of a co-culture model of the small intestinal epithelium. Barrier integrity (as measured by trans epithelial electrical resistance (TEER)), was enhanced by three bacteria/CF combinations (Lactobacillus rhamnosus HN001, L. plantarum 299v, and L. casei Shirota) to a greater extent than CF or bacteria alone. Levels of occludin mRNA were increased for all treatments compared to untreated co-cultures, and L. plantarum 299v in combination with CF had increased mRNA levels of MUC4, MUC2 and MUC5AC mucins and MUC4 protein abundance. These results indicate that three out of the four probiotic bacteria tested, in combination with CF, were able to elicit a greater increase in barrier integrity of a co-culture model of the small intestinal epithelium compared to that for either component alone. This study provides additional insight into the individual or combined roles of microbe⁻diet interactions in the small intestine and their beneficial contribution to the intestinal barrier.Item Assessing antimicrobial resistance in pasture-based dairy farms: a 15-month surveillance study in New Zealand.(American Society for Microbiology, 2024-10-23) Collis RM; Biggs PJ; Burgess SA; Midwinter AC; Liu J; Brightwell G; Cookson ALAntimicrobial resistance is a global public and animal health concern. Antimicrobial resistance genes (ARGs) have been detected in dairy farm environments globally; however, few longitudinal studies have utilized shotgun metagenomics for ARG surveillance in pasture-based systems. This 15-month study aimed to undertake a baseline survey using shotgun metagenomics to assess the relative abundance and diversity of ARGs in two pasture-based dairy farm environments in New Zealand with different management practices. There was no statistically significant difference in overall ARG relative abundance between the two dairy farms (P = 0.321) during the study period. Compared with overseas data, the relative abundance of ARG copies per 16S rRNA gene in feces (0.08-0.17), effluent (0.03-0.37), soil (0.20-0.63), and bulk tank milk (0.0-0.12) samples was low. Models comparing the presence or absence of resistance classes found in >10% of all feces, effluent, and soil samples demonstrated no statistically significant associations (P > 0.05) with "season," and only multi-metal (P = 0.020) and tetracycline (P = 0.0003) resistance were significant at the "farm" level. Effluent samples harbored the most diverse ARGs, some with a recognized public health risk, whereas soil samples had the highest ARG relative abundance but without recognized health risks. This highlights the importance of considering the genomic context and risk of ARGs in metagenomic data sets. This study suggests that antimicrobial resistance on pasture-based dairy farms is low and provides essential baseline ARG surveillance data for such farming systems. IMPORTANCE: Antimicrobial resistance is a global threat to human and animal health. Despite the detection of antimicrobial resistance genes (ARGs) in dairy farm environments globally, longitudinal surveillance in pasture-based systems remains limited. This study assessed the relative abundance and diversity of ARGs in two New Zealand dairy farms with different management practices and provided important baseline ARG surveillance data on pasture-based dairy farms. The overall ARG relative abundance on these two farms was low, which provides further evidence for consumers of the safety of New Zealand's export products. Effluent samples harbored the most diverse range of ARGs, some of which were classified with a recognized risk to public health, whereas soil samples had the highest ARG relative abundance; however, the soil ARGs were not classified with a recognized public health risk. This emphasizes the need to consider genomic context and risk as well as ARG relative abundance in resistome studies.Item Population structure and pathogen interaction of Escherichia coli in freshwater: Implications of land-use for water quality and public health in Aotearoa New Zealand.(John Wiley & Sons, Inc., 2024-08-02) Cookson AL; Devane M; Marshall JC; Moinet M; Gardner A; Collis RM; Rogers L; Biggs PJ; Pita AB; Cornelius AJ; Haysom I; Hayman DTS; Gilpin BJ; Leonard MFreshwater samples (n = 199) were obtained from 41 sites with contrasting land-uses (avian, low impact, dairy, urban, sheep and beef, and mixed sheep, beef and dairy) and the E. coli phylotype of 3980 isolates (20 per water sample enrichment) was determined. Eight phylotypes were identified with B1 (48.04%), B2 (14.87%) and A (14.79%) the most abundant. Escherichia marmotae (n = 22), and Escherichia ruysiae (n = 1), were rare (0.68%) suggesting that these environmental strains are unlikely to confound water quality assessments. Phylotypes A and B1 were overrepresented in dairy and urban sites (p < 0.0001), whilst B2 were overrepresented in low impact sites (p < 0.0001). Pathogens ((Salmonella, Campylobacter, Cryptosporidium or Giardia) and the presence of diarrhoeagenic E. coli-associated genes (stx and eae) were detected in 89.9% (179/199) samples, including 80.5% (33/41) of samples with putative non-recent faecal inputs. Quantitative PCR to detect microbial source tracking targets from human, ruminant and avian contamination were concordant with land-use type and E. coli phylotype abundance. This study demonstrated that a potential recreational health risk remains where pathogens occurred in water samples with low E. coli concentration, potential non-recent faecal sources, low impact sites and where human, ruminant and avian faecal sources were absent.Item The characterisation of antimicrobial resistant Escherichia coli from dairy calves.(Microbiology Society, 2023-08) Mwenifumbo M; Cookson AL; Zhao S; Fayaz A; Browne AS; Benschop J; Burgess SAIntroduction. Dairy calves, particularly pre-weaned calves have been identified as a common source of multidrug resistant (MDR) Escherichia coli. Gap statement. E. coli strains isolated from dairy calves and the location of their resistance genes (plasmid or chromosomal) have not been well characterised. Aim. To characterise the phenotypic and genotypic features as well as the population structure of antimicrobial-resistant E. coli isolated from calves located on dairy farms that feed waste-milk to their replacement calves. Methodology. Recto-anal swab enrichments from 40 dairy calves (≤ 14 days old) located on four dairy farms were examined for tetracycline, streptomycin, ciprofloxacin, and third-generation cephalosporin resistant E. coli. Whole genome sequencing was performed using both short- and long-read technologies on selected antimicrobial resistant E. coli. Results. Fifty-eight percent (23/40) of calves harboured antimicrobial resistant E. coli: 43 % (17/40) harboured tetracycline resistant, and 23 % (9/40) harboured chromosomal mediated AmpC producing E. coli. Whole genome sequencing of 27 isolates revealed five sequence types, with ST88 being the dominant ST (17/27, 63 % of the sequenced isolates) followed by ST1308 (3/27, 11 %), along with the extraintestinal pathogenic E. coli lineages ST69 (3/27, 11 %), ST10 (2/27, 7 %), and ST58 (2/27, 7 %). Additionally, 16 isolates were MDR, harbouring additional resistance genes that were not tested phenotypically. Oxford Nanopore long-read sequencing technologies enabled the location of multiple resistant gene cassettes in IncF plasmids to be determined. Conclusion. Our study identified a high incidence of tetracycline and streptomycin-resistant E. coli in dairy calves, and highlighted the presence of multidrug-resistant strains, emphasising the need for further investigation into potential associations with farm management practices.Item Draft genome sequences of Escherichia spp. isolates from New Zealand environmental sources.(American Society for Microbiology, 2024-03-12) Biggs PJ; Moinet M; Rogers LE; Devane M; Muirhead R; Stott R; Marshall JC; Cookson AL; Dennehy JJEscherichia coli is often used as a fecal indicator bacterium for water quality monitoring. We report the draft genome sequences of 500 Escherichia isolates including newly described Escherichia species, namely Escherichia marmotae, Escherichia ruysiae, and Escherichia whittamii, obtained from diverse environmental sources to assist with improved public health risk assessments.Item Development of a multiplex droplet digital PCR assay for simultaneous detection and quantification of Escherichia coli, E. marmotae, and E. Ruysiae in water samples(Elsevier BV, 2024-03-01) Moinet M; Collis RM; Rogers L; Devane ML; Biggs PJ; Stott R; Marshall J; Muirhead R; Cookson ALEscherichia 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/μL and 0.005 pg/μl for ddPCR and qPCR respectively, and of quantification of 0.5 pg/μL 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.Item Effect of Divergent Feeding Regimes During Early Life on the Rumen Microbiota in Calves(Frontiers Media S.A., 2021-10-20) Cristobal-Carballo O; McCoard SA; Cookson AL; Laven RA; Ganesh S; Lewis SJ; Muetzel S; Morgavi DPThe objective of this study was to determine whether divergent feeding regimes during the first 41 weeks of the life of a calf are associated with long-term changes in the rumen microbiota and the associated fermentation end-products. Twenty-four calves (9 ± 5 days of age) were arranged in a 2 × 2 factorial design with two divergent treatments across three dietary phases. In phase 1 (P01), calves were offered a low-milk volume/concentrate starter diet with early weaning (CO) or high-milk volume/pasture diet and late weaning (FO). In phase 2 (P02), calves from both groups were randomly allocated to either high-quality (HQ) or low-quality (LQ) pasture grazing groups. In phase 3 (P03), calves were randomly allocated to one of two grazing groups and offered the same pasture-only diet. During each dietary phase, methane (CH4) and hydrogen (H2) emissions and dry matter intake (DMI) were measured in respiration chambers, and rumen samples for the evaluation of microbiota and short-chain fatty acid (SCFA) characterizations were collected. In P01, CO calves had a higher solid feed intake but a lower CH4 yield (yCH4) and acetate:propionate ratio (A:P) compared with FO calves. The ruminal bacterial community had lower proportions of cellulolytic bacteria in CO than FO calves. The archaeal community was dominated by Methanobrevibacter boviskoreani in CO calves and by Mbb. gottschalkii in FO calves. These differences, however, did not persist into P02. Calves offered HQ pastures had greater DMI and lower A:P ratio than calves offered LQ pastures, but yCH4 was similar between groups. The cellulolytic bacteria had lower proportions in HQ than LQ calves. In all groups, the archaeal community was dominated by Mbb. gottschalkii. No treatment interactions were observed in P02. In P03, all calves had similar DMI, CH4 and H2 emissions, SCFA proportions, and microbial compositions, and no interactions with previous treatments were observed. These results indicate that the rumen microbiota and associated fermentation end-products are driven by the diet consumed at the time of sampling and that previous dietary interventions do not lead to a detectable long-term microbial imprint or changes in rumen function.Item Effect of Methane Inhibitors on Ruminal Microbiota During Early Life and Its Relationship With Ruminal Metabolism and Growth in Calves(Frontiers Media S.A., 2021-09-16) Cristobal-Carballo O; McCoard SA; Cookson AL; Ganesh S; Lowe K; Laven RA; Muetzel S; Morgavi DPThe present study aimed to determine whether dietary supplementation with methanogen inhibitors during early life may lead to an imprint on the rumen microbial community and change the rumen function and performance of calves to 49-weeks of rearing. Twenty-four 4-day-old Friesian x Jersey cross calves were randomly assigned into a control and a treatment group. Treated calves were fed a combination of chloroform (CF) and 9,10-anthraquinone (AQ) in the solid diets during the first 12 weeks of rearing. Afterward, calves were grouped by treatments until week 14, and then managed as a single group on pasture. Solid diets and water were offered ad libitum. Methane measurements, and sample collections for rumen metabolite and microbial community composition were carried out at the end of weeks 2, 4, 6, 8, 10, 14, 24 and 49. Animal growth and dry matter intake (DMI) were regularly monitored over the duration of the experiment. Methane emissions decreased up to 90% whilst hydrogen emissions increased in treated compared to control calves, but only for up to 2 weeks after treatment cessation. The near complete methane inhibition did not affect calves' DMI and growth. The acetate:propionate ratio decreased in treated compared to control calves during the first 14 weeks but was similar at weeks 24 and 49. The proportions of Methanobrevibacter and Methanosphaera decreased in treated compared to control calves during the first 14 weeks; however, at week 24 and 49 the archaea community was similar between groups. Bacterial proportions at the phylum level and the abundant bacterial genera were similar between treatment groups. In summary, methane inhibition increased hydrogen emissions, altered the methanogen community and changed the rumen metabolite profile without major effects on the bacterial community composition. This indicated that the main response of the bacterial community was not a change in composition but rather a change in metabolic pathways. Furthermore, once methane inhibition ceased the methanogen community, rumen metabolites and hydrogen emissions became similar between treatment groups, indicating that perhaps using the treatments tested in this study, it is not possible to imprint a low methane microbiota into the rumen in the solid feed of pre-weaned calves.Item Whole-Genome Sequencing and Virulome Analysis of Escherichia coli Isolated from New Zealand Environments of Contrasting Observed Land Use(American Society for Microbiology, 2022-05-10) Cookson AL; Marshall JC; Biggs PJ; Rogers LE; Collis RM; Devane M; Stott R; Wilkinson DA; Kamke J; Brightwell G; Elkins CAGeneric Escherichia coli is commonly used as an indicator of fecal contamination to assess water quality and human health risk. Where measured E. coli exceedances occur, the presence of other pathogenic microorganisms, such as Shiga toxin-producing E. coli (STEC), is assumed, but confirmatory data are lacking. Putative E. coli isolates (n = 709) were isolated from water, sediment, soil, periphyton, and feces samples (n = 189) from five sites representing native forest and agricultural environments. Ten E. coli isolates (1.41%) were stx2 positive, 19 (2.7%) were eae positive, and stx1-positive isolates were absent. At the sample level, stx2-positive E. coli (5 of 189, 2.6%) and eae-positive isolates (16 of 189, 8.5%) were rare. Using real-time PCR, these STEC-associated virulence factors were determined to be more prevalent in sample enrichments (stx1, 23.9%; stx2, 31.4%; eae, 53.7%) and positively correlated with generic E. coli isolate numbers (P < 0.05) determined using culture-based methods. Whole-genome sequencing (WGS) was undertaken on a subset of 238 isolates with assemblies representing seven E. coli phylogroups (A, B1, B2, C, D, E, and F), 22 Escherichia marmotae isolates, and 1 Escherichia ruysiae isolate. Virulence factors, including those from extraintestinal pathogenic E. coli, were extremely diverse in isolates from the different locations and were more common in phylogroup B2. Analysis of the virulome from WGS data permitted the identification of gene repertoires that may be involved in environmental fitness and broadly align with phylogroup. Although recovery of STEC isolates was low, our molecular data indicate that they are likely to be widely present in environmental samples containing diverse E. coli phylogroups. IMPORTANCE This study takes a systematic sampling approach to assess the public health risk of Escherichia coli recovered from freshwater sites within forest and farmland. The New Zealand landscape is dominated by livestock farming, and previous work has demonstrated that "recreational exposure to water" is a risk factor for human infection by Shiga toxin-producing Escherichia coli (STEC). Though STEC isolates were rarely isolated from water samples, STEC-associated virulence factors were identified more commonly from water sample culture enrichments and were associated with increased generic E. coli concentrations. Whole-genome sequencing data from both E. coli and newly described Escherichia spp. demonstrated the presence of virulence factors from E. coli pathotypes, including extraintestinal pathogenic E. coli. This has significance for understanding and interpreting the potential health risk from E. coli where water quality is poor and suggests a role of virulence factors in survival and persistence of E. coli and Escherichia spp.