Tayler Catherine Kent

This study aimed to determine the prevalence and associated plasmid resistant genome of extended spectrum β-lactamase (ESBL) and Ambler class C cephalosporinase (AmpC) producing commensal Escherichia coli, using a targeted pDNA approach, from lactating cows and pre-weaned calves on dairy farms in Western Australian (WA). Following culture and modified double disc diffusion, ESBL and/or AmpC producing commensal E. coli were isolated from faecal samples and phenotypic antimicrobial resistance (AMR) determined. Plasmid DNA (pDNA) was extracted, amplified, and sequenced to identify the AMR and replicon composition. Phenotypic analysis of 1,117 faecal samples from 26 Western Australian dairy farms revealed a low overall prevalence (7.3%) of ESBL and/or AmpC-producing commensal E. coli, with 3.2% in cows (n = 633) and 12.8% in calves (n = 484). All isolates were classified ‘non-wildtype’ for ampicillin and ceftiofur, with 95.1% classified as ‘non-wildtype’ multidrug resistant (MDR). While blaTEM was common in the pDNA (total: 60.6%; cow: 76.9%; calf: 57.7%), a total of 13 AMR genes were identified across 126 plasmids (cow: n = 30; calf: n = 96) isolated. Multiple correspondence analysis (with chi-square–based 95% confidence ellipses) showed variation in pDNA AMR genes and replicons by farm, with generally distinct plasmid profiles, though some clustering suggests possible farm-to-farm plasmid transmission. In conclusion, though low in prevalence, ESBL and/or AmpC producing commensal E. coli having high phenotypic AMR, MDR, and pDNA diversity were isolated from dairy cattle on 65.4% of screened farms.
•Low prevalence of commensal E. coli producing ESBL and/or AmpC enzymes.•ESBL and/or AmpC producing commensal E. coli were isolated from dairy cattle on 65.4% of the 26 farms screened.•Isolates had high phenotypic resistance, multiple drug resistance, and plasmid diversity.•Targeted pDNA approach appreciates AMR diversity with lower cost, time, and data complexity.

This study aimed to determine the prevalence of antimicrobial resistance (AMR) in commensal E. coli from healthy lactating cows and calves in the Mediterranean pasture-based feeding dairy system of Western Australia (WA). Fecal samples were collected from healthy adult lactating cows and healthy calves from dairy farms in WA. Presumptive commensal E. coli was isolated from these samples and confirmed using matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Broth microdilution was used to assess the prevalence and the phenotypic AMR profiles of the E. coli isolates to 8 antimicrobial agents of dairy industry and human importance. The minimum inhibitory concentration (MIC) for each isolate was interpreted using the Epidemiologic Cutoff (ECOFF) and Clinical and Laboratory Standards Institute (CLSI) breakpoints. Genomic characterization provided multi-locus sequence types and AMR genes for a selection of isolates categorised as non-wild type (NWT) by ECOFF values for the combination of ampicillin, trimethoprim-sulfamethoxazole, and tetracycline. From a total of 1,117 fecal samples (633 adult, 484 calf) collected across 26 randomly selected farms, 891 commensal E. coli isolates were recovered (541 adult, 350 calf). Commensal E. coli classified as NWT was highest for ampicillin for both adult (68.8%; 95% CI = 64.7 – 72.7) and calf feces (67.1%; 95% CI = 62.0 – 72.0). A large proportion of tetracycline NWT and trimethoprim-sulfamethoxazole NWT organisms were also identified from calf feces, being 44.0% (95% CI = 38.7 – 49.4) and 24.6% (95% CI = 20.2 – 29.4) respectively. Clinical resistance prevalence was low, being higher for calves than for adult feces (ampicillin (adult: 7.8% (95% CI = 5.7 – 10.3); calf: 30.0% (95% CI = 25.2 – 35.1), tetracycline (adult: 6.3% (95% CI = 4.4 – 8.7); calf: 40.3% (95% CI = 35.1 – 45.6), and trimethoprim-sulfamethoxazole (adult: 2.6% (95% CI = 1.4 – 4.3); calf: 22.0% (95% CI = 17.7 – 26.7)). Commensal E. coli originating from calf feces was significantly higher in NWT prevalence compared with adult feces for ciprofloxacin (P = 0.023), gentamicin (P = 0.02), tetracycline (P < 0.001), and trimethoprim-sulfamethoxazole (P < 0.001). The overall number of antimicrobials an isolate was classified as NWT toward varied among farms and was significantly higher for isolates originating from calf than adult feces (P < 0.001). The strain type and sampling source of the commensal E. coli investigated were both associated with the commonality of the resultant resistance genome. Clinical resistance and NWT classification were highest for ampicillin, tetracycline, and trimethoprim-sulfamethoxazole, all antimicrobials commonly used in the treatment of dairy cattle in Australia. Though highly variable across farms, commensal E. coli isolated from healthy dairy calf feces had significantly higher NWT and multidrug resistance (MDR) prevalence compared with feces from healthy adult lactating dairy cows. The resistant genome identified in MDR isolates, though not always consistent with the phenotype, included QnrS1 and genes encoding AmpC β-lactamase and aminoglycoside phosphotransferase.

Introduction: Herbal medicines are popular, despite a lack of regulation and research into safety and efficacy [1]. Supplements marketed for fertility are prevalent, but most studies regarding sperm focus on motility, with little research regarding toxicity. This study aimed to characterise the biochemical changes that occur in sperm after in vitro exposure to three common herbal supplements; green tea extract (GTE), maca root and Korean ginseng.

Methods: 12 ejaculates from three Merino rams were collected for each supplement, and sperm incubated with 0, 0.1, 0.5, 1 or 5 mg/ml of GTE, Korean ginseng (Paanax ginseng) or maca root (Lepidium meyenii). Supplements were dissolved in DMSO and PBS with a final concentration of 1% DMSO in all samples. At 0.5, 3 and 6 h post-exposure, computer-assisted sperm analysis (CASA) and flow cytometry were used to assess motility, viability, acrosome reaction, membrane lipid disorder, mitochondrial superoxide production, intracellular reactive oxygen species (ROS) and DNA fragmentation.

Results: Treatment with each supplement induced acrosome reaction in treated cells (2.02%± 0.39 n=180 p < .001), but showed no difference in viability(73.3%±1.49 n=180 p > .05). There was increased mitochondrial superoxide production across all concentrations and time points for each sup-plement (9.72%± 0.79% n=180 p < .001), as well as membrane lipid disorder across all supplements (17.28%±0.85 n=180 p < .001). All concentrations of GTE, and 0.5, 1 and 5 mg/ml ginseng and maca increased DNA fragmentation (7.71% ± 0.39% n=180 p < .001). Intracellular ROSproduction increased with 0.1, 0.5 and 5 mg/ml GTE (7.42%±0.45% p < .001).

Conclusions: Despite the reported antioxidant activity of herbal supplements, the increase in mitochondrial superoxide production and DNA fragmentation suggests that exposure to these concentrations of ginseng, maca and GTE increased oxidative stress in sperm [2]. The higher membrane fluidity and reduced acrosome integrity suggests potential oestrogenic activity of herbal supplements, as oestrogen can induce capacitation-like changes[3]. Despite the popularity and perceived safety of herbal supplements, this research indicates they should be used with caution until their effects are further elucidated.

Compounds with oestrogen-like actions are now common in both the Western diet. The long-term impacts and underlying mechanisms by which oestrogenic compounds alter male reproduction, however, are unclear. To investigate this, we used a longitudinal sheep model examining the impact of oestrogenic pasture consumption on semen quality and production, testicular size, sexual behaviour and the seminal plasma proteome of Merino rams (n = 20), over a full spermatogenic cycle and in the subsequent breeding season. Throughout the study period, sexual behaviour, sperm production and motility were similar between the exposed and non-exposed rams (P > 0.05). However, between 5 and 8 weeks of exposure to dietary phytoestrogens, rams produced a higher percentage of spermatozoa with a specific malformation of the sperm midpiece and reduced DNA integrity, compared to non-exposed rams (P < 0.001). Investigation into the seminal plasma proteome revealed 93 differentially expressed proteins between phytoestrogen-exposed and control rams (P < 0.05). Exposure to phytoestrogens increased the expression of proteins involved in cellular structure development, actin cytoskeleton reorganisation, regulation of cell function and decreased expression in those related to catabolic processes. The greatest fold changes were in proteins involved in the assembly of the sperm flagella, removal of cytoplasm, spermatid development and maintenance of DNA integrity. After returning to non-oestrogenic pasture, no differences in any measure were observed between treatment groups during the subsequent breeding season. We conclude that dietary phytoestrogens can transiently disrupt specific stages of ram spermatogenesis, causing subtle decreases in sperm quality by affecting the expression of pathways involved in the structural integrity of the spermatozoa.

Oestrogenic pastures are known to cause infertility in the ewe, due primarily to the oestrogen-like actions of the metabolite equol. Despite strong evidence that phytoestrogens and their metabolites compromise male reproductive function in many other species, there is little information concerning the effect of oestrogenic pastures on ram sperm quality and function. To investigate this, ram spermatozoa were exposed in vitro to physiologically relevant concentrations of either 0, 0.001, 0.01, 0.1 and 1 μM equol and incubated over 6 h. Sperm motility, viability, DNA integrity, membrane lipid disorder, mitochondrial superoxide production, lipid peroxidation and intracellular reactive oxygen species were assessed via computer assisted sperm analysis and flow cytometry at 0.5, 3 and 6 h post-equol exposure. Whilst sperm viability was decreased only at 1 μM equol at 0.5 h post-exposure, exposure to equol at concentrations of 0.1 and 1 μM reduced sperm total and progressive motility (P < 0.001), increased sperm membrane fluidity (P < 0.001), increased mitochondrial superoxide production (P < 0.001) and promoted lipid peroxidation (P < 0.001) across all timepoints. At 6 h post-exposure to 0.1 and 1 μM equol, DNA fragmentation was greater compared that of non-exposed spermatozoa (P = 0.045). Intracellular reactive oxygen species did not change between treatment groups throughout the study (P > 0.05). It is concluded that even low concentrations of equol negatively impact the functionality of ram spermatozoa, these effects likely driven through increased mitochondrial superoxide production. This work indicates that equol may exert oestrogen-like actions upon ram spermatozoa, bringing into question as to whether oestrogenic pastures could influence ram fertility.