Reducing the lipase LIPE in mutant α-synuclein mice improves Parkinson-like deficits and reveals sex differences in fatty acid metabolism.

Impaired lipid metabolism is a risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB) and can shift the physiological α-synuclein (αS) tetramer-monomer (T:M) ratio toward aggregation prone monomers. A resultant increase in phospho-serine 129+ αS monomers associating with excess mono- and polyunsaturated fatty acids contributes to the αS aggregation. We previously reported that decreasing the release of monounsaturated fatty acids (MUFAs) by reducing or inhibiting the hormone sensitive lipase (LIPE) reversed pathologic αS phosphorylation and improved soluble αS homeostasis in cultured αS triplication PD neurons and reduced DAergic neurodegeneration in a C.elegans αS model. However, assessing LIPE as a potential therapeutic target for progressive PD motor phenotypes has not been investigated. 3K αS mice, representing a biochemical and neuropathological amplification of the E46K fPD-causing mutation, have decreased αS T:M ratios, lipidic aggregates, and a L-DOPA responsive PD-like motor syndrome. Here, we reduced LIPE by crossings of 3K mice with LIPE null mice, which attenuated motor deficits in male LIPE+/- knockdown (LKD)-3K mice. Heterozygous LIPE reduction was associated with an improved αS T:M ratio, and dopaminergic neurotransmitter levels and fiber densities. In female 3K-LKD mice, an increase in pS129+ and larger lipid droplets (LDs) likely decreased the benefits seen in males. Reducing LIPE decreased MUFA release from neutral lipid storage, thereby reducing MUFA in phospholipid membranes with which αS interacts. Our study highlights fatty acid turnover as a therapeutic target for Lewy body diseases and support LIPE as a promising target in males. LIPE regulation represents a novel approach to mitigate PD and DLB risk and treat disease.

Targeting Unique Ligand Binding Domain Structural Features Downregulates DKK1 in Y537S ESR1 Mutant Breast Cancer Cells.

Resistance to endocrine therapies remains a major clinical hurdle in breast cancer. Mutations to estrogen receptor alpha (ERα) arise after continued therapeutic pressure. Next generation selective estrogen receptor modulators and degraders/downregulators (SERMs and SERDs) show clinical efficacy, but responses are often non-durable. A tyrosine to serine point mutation at position 537 in the ERα ligand binding domain (LBD) is among the most common and most pathogenic alteration in this setting. It enables endocrine therapy resistance by superceding intrinsic structural-energetic gatekeepers of ER hormone-dependence, it enhances metastatic burden by enabling neomorphic ER-dependent transcriptional programs, and it resists SERM and SERD inhibiton by reducing their binding affinities and abilities to antagonize transcriptional coregulator binding. However, a subset of SERMs and SERDs can achieve efficacy by adopting poses that force the mutation to engage in a new interaction that favors the therapeutic receptor antagonist conformation. We previously described a chemically unconventional SERM, T6I-29, that demonstrates significant anti-proliferative activities in Y537S ERα breast cancer cells. Here, we use a comprehensive suite of structural-biochemical, in vitro, and in vivo approaches to better T6I-29's activities in breast cancer cells harboring Y537S ERα. RNA sequencing in cells treated with T6I-29 reveals a neomorphic downregulation of DKK1, a secreted glycoprotein known to play oncogenic roles in other cancers. Importantly, we find that DKK1 is significantly enriched in ER+ breast cancer plasma compared to healthy controls. This study shows how new SERMs and SERDs can identify new therapeutic pathways in endocrine-resistant ER+ breast cancers.

Targeting Unique Ligand Binding Domain Structural Features Downregulates DKK1 in Y537S ESR1 Mutant Breast Cancer Cells.

Resistance to endocrine therapies remains a major clinical hurdle in breast cancer. Mutations to estrogen receptor alpha (ERα) arise after continued therapeutic pressure. Next generation selective estrogen receptor modulators and degraders/downregulators (SERMs and SERDs) show clinical efficacy, but responses are often non-durable. A tyrosine to serine point mutation at position 537 in the ERα ligand binding domain (LBD) is among the most common and most pathogenic alteration in this setting. It enables endocrine therapy resistance by superceding intrinsic structural-energetic gatekeepers of ER hormone-dependence, it enhances metastatic burden by enabling neomorphic ER-dependent transcriptional programs, and it resists SERM and SERD inhibiton by reducing their binding affinities and abilities to antagonize transcriptional coregulator binding. However, a subset of SERMs and SERDs can achieve efficacy by adopting poses that force the mutation to engage in a new interaction that favors the therapeutic receptor antagonist conformation. We previously described a chemically unconventional SERM, T6I-29, that demonstrates significant anti-proliferative activities in Y537S ERα breast cancer cells. Here, we use a comprehensive suite of structural-biochemical, in vitro, and in vivo approaches to better T6I-29's activities in breast cancer cells harboring Y537S ERα. RNA sequencing in cells treated with T6I-29 reveals a neomorphic downregulation of DKK1, a secreted glycoprotein known to play oncogenic roles in other cancers. Importantly, we find that DKK1 is significantly enriched in ER+ breast cancer plasma compared to healthy controls. This study shows how new SERMs and SERDs can identify new therapeutic pathways in endocrine-resistant ER+ breast cancers.

Unconventional isoquinoline-based SERMs elicit fulvestrant-like transcriptional programs in ER+ breast cancer cells.

Estrogen receptor alpha (ERα) is a ligand-dependent master transcriptional regulator and key driver of breast cancer pathology. Small molecule hormones and competitive antagonists favor unique ERα conformational ensembles that elicit ligand-specific transcriptional programs in breast cancer and other hormone-responsive tissues. By affecting disparate ligand binding domain structural features, unconventional ligand scaffolds can redirect ERα genomic binding patterns to engage novel therapeutic transcriptional programs. To improve our understanding of these ERα structure-transcriptional relationships, we develop a series of chemically unconventional antagonists based on the antiestrogens elacestrant and lasofoxifene. High-resolution x-ray co-crystal structures show that these molecules affect both classical and unique structural motifs within the ERα ligand binding pocket. They show moderately reduced antagonistic potencies on ERα genomic activities but are effective anti-proliferative agents in luminal breast cancer cells. Interestingly, they favor a 4-hydroxytamoxifen-like accumulation of ERα in breast cancer cells but lack uterotrophic activities in an endometrial cell line. Importantly, RNA sequencing shows that the lead molecules engage transcriptional pathways similar to the selective estrogen receptor degrader fulvestrant. This advance shows that fulvestrant-like genomic activities can be achieved without affecting ERα accumulation in breast cancer cells.

Differences in antimicrobial susceptibility testing complicating management of IMP carbapenemase-producing Enterobacterales infection.

OBJECTIVES: IMP-type carbapenemases are rarely detected in Europe and limited information is available to guide the treatment of infections caused by carbapenemase-producing Enterobacterales (CPE) producing these carbapenemases. Accurate antimicrobial susceptibility testing (AST) results are essential for optimal antibiotic management. Here we report discrepancies in AST of IMP-producing Enterobacterales (IMP-CPE) complicating the management of severe sepsis. METHODS: Antimicrobial susceptibilities were analysed by in-house VITEK® 2, Etest and broth microdilution (BMD). Carbapenemase-encoding genes were detected by PCR. Whole-genome sequencing (WGS) was performed using an Illumina MiSeq platform. RESULTS: Minimum inhibitory concentrations (MICs) determined by VITEK® 2 for Enterobacter hormaechei and Klebsiella oxytoca blood culture isolates were ≥16 mg/L for meropenem and ≤0.5 mg/L for ertapenem. In contrast, Etest analysis and BMD returned MICs of 2 mg/L and 1 mg/L, respectively. Both isolates tested positive for IMP carbapenemase-encoding genes by PCR. WGS revealed that both isolates carried the same blaIMP-4 gene. Based on VITEK® 2 susceptibilities, initial treatment was with tigecycline and amikacin. After subsequent deterioration, the patient was successfully treated with ertapenem and amikacin. CONCLUSION: This case highlights that automated AST by VITEK® 2 can over-report meropenem resistance for IMP carbapenemase-producers compared with Etest and BMD. Clinicians need to be cautious deciding against carbapenem treatment based on VITEK® 2 susceptibility testing results for IMP-positive Enterobacterales. Tigecycline was inferior to carbapenem treatment for pyelonephritis caused by isolates expressing IMP carbapenemases, however specific evidence guiding the treatment of these infections is lacking.

Genomic insights into persistence of Listeria species in the food processing environment.

AIMS: Listeria species may colonize and persist in food processing facilities for prolonged periods of time, despite hygiene interventions in place. To understand the genetic factors contributing to persistence of Listeria strains, this study undertook a comparative analysis of seven persistent and six presumed non-persistent strains, isolated from a single food processing environment, to identify genetic markers correlating to promoting persistence of Listeria strains, through whole genome sequence analysis. METHODS AND RESULTS: A diverse pool of genetic markers relevant to hygiene tolerance was identified, including disinfectant resistance markers qacH, emrC and the efflux cassette bcrABC. Both persistent and presumed non-persistent cohorts encoded a range of stress resistance markers, including heavy metal resistance, oxidative and pH stress, although trends were associated with each cohort (e.g., qacH and cadA1C resistance was more frequently found in persistent isolates). Persistent isolates were more likely to contain mutations associated with attenuated virulence, including a truncated InlA. Plasmids and transposons were widespread between cohorts. CONCLUSIONS: Results suggest that no single genetic marker identified was universally responsible for a strain's ability to persist. Persistent strains were more likely to harbour mutation associated with hypovirulence. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides additional insights into the distribution of genetic elements relevant to persistence across Listeria species, as well as strain virulence potential.

Effect of Exposure to Chlorhexidine Residues at "During Use" Concentrations on Antimicrobial Susceptibility Profile, Efflux, Conjugative Plasmid Transfer, and Metabolism of Escherichia coli.

There is no standardized protocol to predict the concentration levels of microbicides that are left on surfaces as a result of the use of these products, and there is no standardized method to predict the potential risk that such levels pose to emerging antibacterial resistance. The ability to distinguish between selection and adaption processes for antimicrobial resistance in bacteria and the impact of different concentrations of microbicide exposure have not been fully investigated to date. This study considers the effect of exposure to a low concentration of chlorhexidine digluconate (CHX) on selected phenotypes of Escherichia coli and relates the findings to the risk of emerging antimicrobial resistance. A concentration of 0.006 mg/ml CHX is a realistic "during use" exposure concentration measured on surfaces. At this concentration, it was possible for CHX-susceptible bacteria to survive, adapt through metabolic alterations, exhibit a transient decrease in antimicrobial susceptibility, and express stable clinical cross-resistance to front-line antibiotics. Efflux activity was present naturally in tested isolates, and it increased in the presence of 0.00005 mg/ml CHX but ceased with 0.002 mg/ml CHX. Phenotypic microarray assays highlighted a difference in metabolic regulation at 0.00005 mg/ml and 0.002 mg/ml CHX; more changes occurred after growth with the latter concentration. Metabolic phenotype changes were observed for substrates involved with the metabolism of some amino acids, cofactors, and secondary metabolites. It was possible for one isolate to continue transferring ampicillin resistance in the presence of 0.00005 mg/ml CHX, whilst 0.002 mg/ml CHX prevented conjugative transfer. In conclusion, E. coli phenotype responses to CHX exposure are concentration dependent, with realistic residual CHX concentrations resulting in stable clinical cross-resistance to antibiotics.

Multi-drug resistant Escherichia coli in diarrhoeagenic foals: Pulsotyping, phylotyping, serotyping, antibiotic resistance and virulence profiling.

Extraintestinal pathogenic E. coli (ExPEC) possess the ability to cause extraintestinal infections such as urinary tract infections, neonatal meningitis and sepsis. While information is readily available describing pathogenic E. coli populations in food-producing animals, studies in companion/sports animals such as horses are limited. In addition, many antimicrobial agents used in the treatment of equine infections are also utilised in human medicine, potentially contributing to the spread of antibiotic resistance determinants among pathogenic strains. The aim of this study was to phenotypically and genotypically characterise the multidrug resistance and virulence associated with 83 equine E. coli isolates recovered from foals with diarrhoeal disease. Serotyping was performed by both PCR and sequencing. Antibiotic resistance was assessed by disc diffusion. Phylogenetic groups, virulence genes, antibiotic resistance genes and integrons were determined by PCR. Thirty-nine (46%) of the isolates were classified as ExPEC and hence considered to be potentially pathogenic to humans and animals. Identified serogroups O1, O19a, O40, O101 and O153 are among previously reported human clinical ExPEC isolates. Over a quarter of the E. coli were assigned to pathogenic phylogroups B2 (6%) and D (23%). Class 1 and class 2 integrons were detected in 85% of E. coli, revealing their potential to transfer MDR to other pathogenic and non-pathogenic bacteria. With 65% of potentially pathogenic isolates harbouring one or more TEM, SHV and CTX-M-2 group ß-lactamases, in addition to the high levels of resistance to fluoroquinolones observed, our findings signal the need for increased attention to companion/sport animal reservoirs as public health threats.

Specific stereochemistry of OP-1074 disrupts estrogen receptor alpha helix 12 and confers pure antiestrogenic activity.

Complex tissue-specific and cell-specific signaling by the estrogen receptor (ER) frequently leads to the development of resistance to endocrine therapy for breast cancer. Pure ER antagonists, which completely lack tissue-specific agonist activity, hold promise for preventing and treating endocrine resistance, however an absence of structural information hinders the development of novel candidates. Here we synthesize a small panel of benzopyrans with variable side chains to identify pure antiestrogens in a uterotrophic assay. We identify OP-1074 as a pure antiestrogen and a selective ER degrader (PA-SERD) that is efficacious in shrinking tumors in a tamoxifen-resistant xenograft model. Biochemical and crystal structure analyses reveal a structure activity relationship implicating the importance of a stereospecific methyl on the pyrrolidine side chain of OP-1074, particularly on helix 12.

Salmonella in breeding pigs: Shedding pattern, transmission of infection and the role of environmental contamination in Irish commercial farrow-to-finish herds.

This study aimed to provide new insights into the epidemiology of Salmonella in pig production, focusing on potential shedding patterns in breeding pigs throughout a full production cycle and the risk of transmission of infection from the sow to her offspring. A longitudinal study was conducted on five farrow-to-finish commercial pig farms. In each herd, shedding of Salmonella in faeces was monitored in breeders through service, gestation and lactation. Swabs of the farrowing room floor and pools of faeces from piglets were collected on two occasions during lactation. Environmental pen swabs were also taken in the weaning and finisher houses. Salmonella isolates were serotyped, tested for antimicrobial resistance (AMR) and typed by Multiple-Locus Variable number tandem repeat Analysis (MLVA). Shedding by breeding pigs was low in all stages of the production cycle; 5% of sows shed at service, the production stage with highest risk of shedding (p < .01), 1.6% shed during gestation and 2.5% after farrowing. Salmonella was detected in 4% of piglet faecal pools in the second week post-farrowing and 5% in the fourth week. Serotyping and AMR profiles of Salmonella isolates revealed that strains in sows and gilts were mostly different from strains isolated in weaner and finisher facilities. MLVA typing confirmed that the source of infection in piglets was in most instances the contaminated environment rather than their dam. Based on the typing results, it appears that sows do not pose a major risk in the maintenance and transmission of Salmonella to their progeny but instead the contaminated pen environment is more significant in the perpetuation of the organism on farm.

Investigation of in-feed organic acids as a low cost strategy to combat Salmonella in grower pigs.

Salmonella carriage in pigs is a significant food safety issue. Dietary supplementation with organic acids has previously been shown to reduce shedding and transmission of Salmonella. Therefore, this study aimed to examine the effect of three commercially available organic acid-based products on Salmonella levels in grower pigs, using a model of experimental infection that closely mimics natural exposure to the organism. Seven week old trial pigs (n=40) with a mean weight of 14.7kg were placed in one of four pens with 10 pigs/pen. Pens had previously been contaminated with Salmonella Typhimurium 4,[5],12;i;- via seeder pigs. Trial pigs received one of four diets for 28days: 1, control diet; 2, sodium butyrate supplemented diet; 3, benzoic acid supplemented diet and 4, formic-citric acid supplemented diet. A further 10 pigs were placed in a Salmonella-free pen receiving the control diet. Pigs were weighed and blood sampled on days 0 and 28. Faeces was collected on day 0, 2, 3, 5, 7, 14, 21 and 28 and examined for Salmonella. On day 28, 5 pigs/group were euthanised and ileocaecal lymph nodes (ILN) and caecal contents sampled for culture. The remaining 5 pigs/pen were then fed the control diet and faeces were collected on days 35 and 42. On day 42 pigs were euthanised and ILN and caecal contents tested for Salmonella levels. The trial was repeated once. Within the first two days of exposure to the contaminated environment, 96% (77/80) of pigs became infected. Most pigs shed Salmonella at levels of between 100-103 CFU/g faeces for at least 7days post-exposure. A significant reduction in Salmonella faecal concentration was observed after supplementation with sodium butyrate (p=0.001) and a formic citric acid blend (p<0.0001). Average daily weight gain (ADWG) was significantly increased in all groups fed the supplemented feed when compared to the positive control group. The use of sodium butyrate or a blend of formic and citric acid in feed could be considered a cost-effective control measure to reduce Salmonella faecal shedding and improve ADWG in Salmonella infected herds.

An investigation of the effect of rapid slurry chilling on blown pack spoilage of vacuum-packaged beef primals.

The aim of this study was to investigate if rapid slurry chilling would retard or prevent blown pack spoilage (BPS) of vacuum-packaged beef primals. Beef primals were inoculated with Clostridium estertheticum subspp. estertheticum (DSMZ 8809), C. estertheticum subspp. laramenise (DSMZ 14864) and C. gasigenes (DSMZ 12272), and vacuum-packaged with and without heat shrinkage (90°C for 3 s). These packs were then subjected to immediate chilling in an ice slurry or using conventional blast chilling systems and stored at 2°C for up to 100 days. The onset and progress of BPS was monitored using the following scale; 0-no gas bubbles in drip; 1-gas bubbles in drip; 2-loss of vacuum; 3-'blown'; 4-presence of sufficient gas inside the packs to produce pack distension and 5-tightly stretched, 'overblown' packs/packs leaking. Rapid slurry chilling (as compared to conventional chilling) did not significantly affect (P > 0.05) the time to the onset or progress of BPS. It was therefore concluded that rapid chilling of vacuum-packaged beef primals, using an ice slurry system, may not be used as a control intervention to prevent or retard blown pack spoilage. SIGNIFICANCE AND IMPACT OF THE STUDY: This study adds to our growing understanding of blown pack spoilage of vacuum-packaged beef primals and suggests that rapid chilling of vacuum-packaged beef primals is not a control option for the beef industry. The results suggest that neither eliminating the heat shrinkage step nor rapid chilling of vacuum-packaged beef retard the time to blown pack spoilage.

Antimicrobial Resistance of Faecal Escherichia coli Isolates from Pig Farms with Different Durations of In-feed Antimicrobial Use.

Antimicrobial use and resistance in animal and food production are of concern to public health. The primary aims of this study were to determine the frequency of resistance to 12 antimicrobials in Escherichia coli isolates from 39 pig farms and to identify patterns of antimicrobial use on these farms. Further aims were to determine whether a categorization of farms based on the duration of in-feed antimicrobial use (long-term versus short-term) could predict the occurrence of resistance on these farms and to identify the usage of specific antimicrobial drugs associated with the occurrence of resistance. Escherichia coli were isolated from all production stages on these farms; susceptibility testing was carried out against a panel of antimicrobials. Antimicrobial prescribing data were collected, and farms were categorized as long term or short term based on these. Resistance frequencies and antimicrobial use were tabulated. Logistic regression models of resistance to each antimicrobial were constructed with stage of production, duration of antimicrobial use and the use of 5 antimicrobial classes included as explanatory variables in each model. The greatest frequencies of resistance were observed to tetracycline, trimethoprim/sulphamethoxazole and streptomycin with the highest levels of resistance observed in isolates from first-stage weaned pigs. Differences in the types of antimicrobial drugs used were noted between long-term and short-term use farms. Categorization of farms as long- or short-term use was sufficient to predict the likely occurrence of resistance to 3 antimicrobial classes and could provide an aid in the control of resistance in the food chain. Stage of production was a significant predictor variable in all models of resistance constructed and did not solely reflect antimicrobial use at each stage. Cross-selection and co-selection for resistance was evident in the models constructed, and the use of trimethoprim/sulphonamide drugs in particular was associated with the occurrence of resistance to other antimicrobials.

Phenotypic changes contributing to Enterobacter gergoviae biocide resistance.

UNLABELLED: Enterobacter gergoviae is a recurrent contaminant of cosmetic and hygiene products. To understand how this bacterium adapts to biocides, we studied Ent. gergoviae CIP 76.01 and its triclosan and Methylisothiazolinone-chloromethylisothiazolinone (MIT-CMIT) tolerant isogenic mutants. They were compared with others also isolated from contaminated cosmetics. Phenotypic differences were noted and these included changes in the bacterial envelope and flagella along with differences in motility, and biofilm growth rates. Triclosan and MIT-CMIT derivatives expressed flagella and other MIT-CMIT derivatives exhibited some external appendages. Those bacteria expressing a high-level minimal inhibitory concentration to MIT-CMIT, expressed a strong biofilm formation. No differential phenotypes were noted for carbon source utilisation. Enterobacter gergoviae demonstrated a diverse response to both of these preservatives contained in cosmetic preparations, depending on their concentrations. Interestingly, this adaptive response is associated with modifications of filament structure-related proteins contributing to increase the organism motility and the production of biofilm. SIGNIFICANCE AND IMPACT OF THE STUDY: Recurrent contaminations of cosmetics products by Ent. gergoviae, needed a better understanding concerning the bacterial adaptation to preservative agents, with particular concern to triclosan and MIT-CMIT. We demonstrated that bacteria response is associated to various mechanisms represented by expression of external appendages (pili or fimbriae) that control cell motility and biofilm formation and evolving as the concentration of biocides adaptation increased. Such mechanisms which are not chemical specific can also promote a cross-resistance to other biocidal agents. The characterization of Ent. gergoviae adaptability to biocides allows industry to adjust the ranges of concentrations and composition of preservatives in formula.

Antimicrobial Resistance in Wildlife: Implications for Public Health.

The emergence and spread of antimicrobial-resistant (AMR) bacteria in natural environments is a major concern with serious implications for human and animal health. The aim of this study was to determine the prevalence of AMR Escherichia coli (E. coli) in wild birds and mammalian species. Thirty faecal samples were collected from each of the following wildlife species: herring gulls (Larus argentatus), black-headed gulls (Larus ridibundus), lesser black-back gulls (Larus fuscus), hybrid deer species (Cervus elaphus x Cervus nippon) and twenty-six from starlings (Sturnus vulgaris). A total of 115 E. coli isolates were isolated from 81 of 146 samples. Confirmed E. coli isolates were tested for their susceptibility to seven antimicrobial agents by disc diffusion. In total, 5.4% (8/146) of samples exhibited multidrug-resistant phenotypes. The phylogenetic group and AMR-encoding genes of all multidrug resistance isolates were determined by PCR. Tetracycline-, ampicillin- and streptomycin-resistant isolates were the most common resistant phenotypes. The following genes were identified in E. coli: bla(TEM), strA, tet(A) and tet(B). Plasmids were identified in all samples that exhibited multidrug-resistant phenotypes. This study indicates that wild birds and mammals may function as important host reservoirs and potential vectors for the spread of resistant bacteria and genetic determinants of AMR.

A longitudinal study of Staphylococcus aureus colonization in pigs in Ireland.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) in livestock has refocused attention on S. aureus colonization and transmission in pigs. This study investigated the effect of the S. aureus colonization status of a sow on the colonization status of her piglets, and whether pigs carry the same strain of S. aureus throughout production. Nasal swabs were collected from the piglets of six healthy sows two days after birth and two days before and two days after they were moved into each production stage. The average prevalence of S. aureus colonization varied between 26% and 73%. The odds of being S. aureus positive were almost 12 times higher for piglets born to nasal-positive sows than for those born to nasal-negative sows, and three times higher again for piglets born to sows that were both nasal- and vaginal-positive. Isolates recovered from piglets immediately after birth were indistinguishable from those of the dam as determined by phenotypic and molecular typing, including microarray analysis and optical mapping. All isolates belonged to clonal complex 9 and the majority exhibited a novel spa type, t10449. The findings show that the S. aureus colonization status of the sow influences the colonization status of her piglets in the early production stages but strains carried by pigs change over time. Multiresistant S. aureus was detected, in particular post-weaning. Results suggest that sow status and management practices, including mixing of pigs and antimicrobial usage at weaning, should be considered when implementing control measures for S. aureus on a farm.

A comparison of the efficiency of ELISA and selected primer sets to detect Norovirus isolates in southern Ireland over a four-year period (2002-2006): variation in detection rates and evidence for continuing predominance of NoV GII.4 genotype.

Norovirus (NoV) gastroenteritis occurs in all age groups and is the most common cause of gastroenteritis in the community. However, detection methods and rates vary widely, and few data are available to compare these, particularly in Ireland. Detection of noroviruses through antigen and molecular-based strategies was carried out on 135 suspected NoV-positive samples, collected over the course of three NoV outbreaks, from 2002 to 2006, in the southern region of Ireland. A commercially available ELISA and a panel of six primer sets were evaluated to determine their suitability for NoV detection in Irish clinical samples. The key findings of this study were the detection of both GGI and GGII noroviruses by ELISA, but the detection of only GGII noroviruses by RT-PCR. In addition to this, a variation in the levels of detection from 9.4 % to 17.3 % was observed for conventional PCR assays, while a detection rate of 46.3 % was observed for the real-time PCR assay. A proportion (17.8 %) of samples were found to be negative by all detection strategies, suggesting the possibility of reporting false positives for these samples or low-copy positives that do not often repeat. Sequencing information from selected samples also revealed nucleotide polymorphisms, compromising efficient primer binding in the case of one primer pairing. Phylogenetic analysis of the partial polymerase gene identified NoV GII.4 as the dominant genotype, in accordance with previous NoV studies in Ireland. Investigating the NoV diversity of the circulating strains and the dynamics of strain replacement is important to better assess the efficacy of future NoV vaccines and to facilitate the early detection of changes in circulating NoV strains.

Detection of Escherichia coli O157 by peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) and comparison to a standard culture method.

Despite the emergence of non-O157 Shiga toxin-producing Escherichia coli (STEC) infections, E. coli serotype O157 is still the most commonly identified STEC in the world. It causes high morbidity and mortality and has been responsible for a number of outbreaks in many parts of the world. Various methods have been developed to detect this particular serotype, but standard bacteriological methods remain the gold standard. Here, we propose a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the rapid detection of E. coli O157. Testing on 54 representative strains showed that the PNA probe is highly sensitive and specific to E. coli O157. The method then was optimized for detection in food samples. Ground beef and unpasteurized milk samples were artificially contaminated with E. coli O157 concentrations ranging from 1 × 10(-2) to 1 × 10(2) CFU per 25 g or ml of food. Samples were then preenriched and analyzed by both the traditional bacteriological method (ISO 16654:2001) and PNA-FISH. The PNA-FISH method performed well in both types of food matrices with a detection limit of 1 CFU/25 g or ml of food samples. Tests on 60 food samples have shown a specificity value of 100% (95% confidence interval [CI], 82.83 to 100), a sensitivity of 97.22% (95% CI, 83.79 to 99.85%), and an accuracy of 98.33% (CI 95%, 83.41 to 99.91%). Results indicate that PNA-FISH performed as well as the traditional culture methods and can reduce the diagnosis time to 1 day.

Potential of a commercially available water acidification product for reducing Campylobacter in broilers prior to slaughter.

1. This study investigated the potential of a commercially available acidified water treatment (PWT) for reducing the number of Campylobacter in vitro and other bacteria in the gut of live broilers. 2. In vitro tests indicated that PWT was highly effective for reducing Campylobacter jejuni and Campylobacter coli at the recommended concentration in water, reducing populations by greater than 7 log10 CFU/ml after 24 h exposure. The decrease in the number of Salmonella serovar Enteritidis and Escherichia coli was not significant. 3. Addition of PWT to the broiler drinking water for the first 7 d, 2 d before and 2 d after each feed change and at feed withdrawal prior to slaughter or only after feed withdrawal had no effect on the number of Campylobacter in caecal samples on farm before thinning and depopulation compared to untreated controls. 4. Although PWT was effective for reducing Campylobacter in water, the results suggest that it does not reduce the number of Campylobacter in the caeca of broilers prior to slaughter under the conditions used in the study.

Proteomic and phenotypic analysis of triclosan tolerant verocytotoxigenic Escherichia coli O157:H19.

Triclosan is a biocidal active agent commonly used in domestic and industrial formulations. Currently, there is limited understanding of the mechanisms involved in triclosan tolerance in Escherichia coli O157. The aim of this study was to identify the differences between a triclosan susceptible E. coli O157:H19 isolate (minimum inhibitory concentration; MIC 6.25 µg/ml) and its triclosan tolerant mutant (MIC>8000 µg/ml) at a proteomic and phenotypic level. Two dimensional DIGE was used to identify differences in protein expression between the reference strain and triclosan tolerant mutant in the presence and absence of triclosan. DIGE analysis indicates the proteome of the reference E. coli O157:H19 was significantly different to its triclosan tolerant mutant. Significant changes in protein expression levels in the triclosan tolerant mutant included the known triclosan target FabI which encodes enoyl reductase, outer membrane proteins and the filament structural protein of flagella, FliC. Phenotypic studies showed that the triclosan tolerant mutant MIC decreased in the presence of efflux inhibitor phenyl-arginine-ß-naphthylamide and biofilm formation was increased in the mutant strain. The data generated indicates that enhanced triclosan tolerance is a result of multiple mechanisms which act together to achieve high-level resistance, rather than mutation of FabI alone.