• Comparative analysis of Salmonella susceptibility and tolerance to the biocide chlorhexidine identifies a complex cellular defense network

      Condell, Orla; Power, Karen A.; Handler, Kristian; Finn, Sarah; Sheridan, Aine; Sergeant, Kjell; Renaut, Jenny; Burgess, Catherine M.; Hinton, Jay C.D.; Nally, Jarlath E.; Fanning, Seamus (Frontiers Media SA, 2014-08-01)
      Chlorhexidine is one of the most widely used biocides in health and agricultural settings as well as in the modern food industry. It is a cationic biocide of the biguanide class. Details of its mechanism of action are largely unknown. The frequent use of chlorhexidine has been questioned recently, amidst concerns that an overuse of this compound may select for bacteria displaying an altered susceptibility to antimicrobials, including clinically important anti-bacterial agents. We generated a Salmonella enterica serovar Typhimurium isolate (ST24CHX) that exhibited a high-level tolerant phenotype to chlorhexidine, following several rounds of in vitro selection, using sub-lethal concentrations of the biocide. This mutant showed altered suceptibility to a panel of clinically important antimicrobial compounds. Here we describe a genomic, transcriptomic, proteomic, and phenotypic analysis of the chlorhexidine tolerant S. Typhimurium compared with its isogenic sensitive progenitor. Results from this study describe a chlorhexidine defense network that functions in both the reference chlorhexidine sensitive isolate and the tolerant mutant. The defense network involved multiple cell targets including those associated with the synthesis and modification of the cell wall, the SOS response, virulence, and a shift in cellular metabolism toward anoxic pathways, some of which were regulated by CreB and Fur. In addition, results indicated that chlorhexidine tolerance was associated with more extensive modifications of the same cellular processes involved in this proposed network, as well as a divergent defense response involving the up-regulation of additional targets such as the flagellar apparatus and an altered cellular phosphate metabolism. These data show that sub-lethal concentrations of chlorhexidine induce distinct changes in exposed Salmonella, and our findings provide insights into the mechanisms of action and tolerance to this biocidal agent.
    • Complete Genome Sequence of Clostridium estertheticum DSM 8809, a Microbe Identified in Spoiled Vacuum Packed Beef

      Zhongyi, Yu; Gunn, Lynda; Brennan, Evan; Reid, Rachel; Wall, Patrick G.; O'Gaora, Peadar; Hurley, Daniel; Bolton, Declan; Fanning, Seamus (Frontiers, 2016-11-10)
      Blown pack spoilage (BPS) is a major issue for the beef industry. Etiological agents of BPS involve members of a group of Clostridium species, including Clostridium estertheticum which has the ability to produce gas, mostly carbon dioxide, under anaerobic psychotrophic growth conditions. This spore-forming bacterium grows slowly under laboratory conditions, and it can take up to 3 months to produce a workable culture. These characteristics have limited the study of this commercially challenging bacterium. Consequently information on this bacterium is limited and no effective controls are currently available to confidently detect and manage this production risk. In this study the complete genome of C. estertheticum DSM 8809 was determined by SMRT R sequencing. The genome consists of a circular chromosome of 4.7 Mbp along with a single plasmid carrying a potential tellurite resistance gene tehB and a Tn3- like resolvase-encoding gene tnpR. The genome sequence was searched for central metabolic pathways that would support its biochemical profile and several enzymes contributing to this phenotype were identified. Several putative antibiotic/biocide/metal resistance-encoding genes and virulence factors were also identified in the genome, a feature that requires further research. The availability of the genome sequence will provide a basic blueprint from which to develop valuable biomarkers that could support and improve the detection and control of this bacterium along the beef production chain.
    • Controlling Blown Pack Spoilage Using Anti-Microbial Packaging

      Reid, Rachael; Bolton, Declan; Tiuftin, Andrey; Kerry, Joe P.; Fanning, Seamus; Whyte, Paul (MDPI, 2017-08-12)
      Active (anti-microbial) packaging was prepared using three different formulations; Auranta FV; Inbac-MDA and sodium octanoate at two concentrations (2.5 and 3.5 times their minimum inhibitory concentration (MIC, the lowest concentration that will inhibit the visible growth of the organisms) against Clostridium estertheticum, DSMZ 8809). Inoculated beef samples were packaged using the active packaging and monitored for 100 days storage at 2 °C for blown pack spoilage. The time to the onset of blown pack spoilage was significantly (p < 0.01) increased using Auranta FV and sodium octanoate (caprylic acid sodium salt) at both concentrations. Moreover, sodium octanoate packs had significantly (p < 0.01) delayed blown pack spoilage as compared to Auranta FV. It was therefore concluded that Auranta FV or sodium octanoate, incorporated into the packaging materials used for vacuum packaged beef, would inhibit blown pack spoilage and in the case of the latter, well beyond the 42 days storage period currently required for beef primals
    • Development of a real-time multiplex PCR assay for the detection of multiple Salmonella serotypes in chicken samples.

      O'Regan, Edel; McCabe, Evonne; Burgess, Catherine M; McGuinness, Sheila; Barry, Thomas; Duffy, Geraldine; Whyte, Paul; Fanning, Seamus (Biomed Central, 2008-09-21)
      Background: A real-time multiplex PCR assay was developed for the detection of multiple Salmonella serotypes in chicken samples. Poultry-associated serotypes detected in the assay include Enteritidis, Gallinarum, Typhimurium, Kentucky and Dublin. The traditional cultural method according to EN ISO 6579:2002 for the detection of Salmonella in food was performed in parallel. The real-time PCR based method comprised a pre-enrichment step in Buffered Peptone Water (BPW) overnight, followed by a shortened selective enrichment in Rappaport Vasilliadis Soya Broth (RVS) for 6 hours and subsequent DNA extraction. Results: The real-time multiplex PCR assay and traditional cultural method showed 100% inclusivity and 100% exclusivity on all strains tested. The real-time multiplex PCR assay was as sensitive as the traditional cultural method in detecting Salmonella in artificially contaminated chicken samples and correctly identified the serotype. Artificially contaminated chicken samples resulted in a detection limit of between 1 and 10 CFU per 25 g sample for both methods. A total of sixty-three naturally contaminated chicken samples were investigated by both methods and relative accuracy, relative sensitivity and relative specificity of the real-time PCR method were determined to be 89, 94 and 87%, respectively. Thirty cultures blind tested were correctly identified by the real-time multiplex PCR method. Conclusion: Real-time PCR methodology can contribute to meet the need for rapid identification and detection methods in food testing laboratories.
    • Longitudinal Study of Two Irish Dairy Herds: Low Numbers of Shiga Toxin-Producing Escherichia coli O157 and O26 Super-Shedders Identified

      Murphy, Brenda P.; McCabe, Evonne; Murphy, Mary; Buckley, James F.; Crowley, Dan; Fanning, Seamus; Duffy, Geraldine (Frontiers, 2016-11-18)
      A 12-month longitudinal study was undertaken on two dairy herds to ascertain the Shiga-toxin producing Escherichia coli (STEC) O157 and O26 shedding status of the animals and its impact (if any) on raw milk. Cattle are a recognized reservoir for these organisms with associated public health and environmental implications. Animals shedding E. coli O157 at >10,000 CFU/g of feces have been deemed super-shedders. There is a gap in the knowledge regarding super-shedding of other STEC serogroups. A cohort of 40 lactating cows from herds previously identified as positive for STEC in a national surveillance project were sampled every second month between August, 2013 and July, 2014. Metadata on any potential super-shedders was documented including, e.g., age of the animal, number of lactations and days in lactation, nutritional condition, somatic cell count and content of protein in milk to assess if any were associated with risk factors for super-shedding. Recto-anal mucosal swabs (RAMS), raw milk, milk filters, and water samples were procured for each herd. The swabs were examined for E. coli O157 and O26 using a quantitative real time PCR method. Counts (CFU swab-1) were obtained from a standard calibration curve that related real-time PCR cycle threshold (Ct) values against the initial concentration of O157 or O26 in the samples. Results from Farm A: 305 animals were analyzed; 15 E. coli O157 (5%) were recovered, 13 were denoted STEC encoding either stx1 and/or stx2 virulence genes and 5 (2%) STEC O26 were recovered. One super-shedder was identified shedding STEC O26 (stx1&2). Farm B: 224 animals were analyzed; eight E. coli O157 (3.5%) were recovered (seven were STEC) and 9 (4%) STEC O26 were recovered. Three super-shedders were identified, one was shedding STEC O157 (stx2) and two STEC O26 (stx2). Three encoded the adhering and effacement gene (eae) and one isolate additionally encoded the haemolysin gene (hlyA). All four super-shedders were only super-shedding once during the 1-year sampling period. The results of this study show, low numbers of super-shedders in the herds examined, with high numbers of low and medium shedding. Although four super-shedding animals were identified, no STEC O157 or O26 were recovered from any of the raw milk, milk filter, or water samples. The authors conclude that this study highlights the need for further surveillance to assess the potential for environmental contamination and food chain security.
    • Molecular Epidemiology of Campylobacter Isolates from Poultry Production Units in Southern Ireland

      O'Mahoney, Emer; Buckley, James F.; Bolton, Declan J.; Whyte, Paul; Fanning, Seamus (PLOS, 2011-12-06)
      This study aimed to identify the sources and routes of transmission of Campylobacter in intensively reared poultry farms in the Republic of Ireland. Breeder flocks and their corresponding broilers housed in three growing facilities were screened for the presence of Campylobacter species from November 2006 through September 2007. All breeder flocks tested positive for Campylobacter species (with C. jejuni and C. coli being identified). Similarly, all broiler flocks also tested positive for Campylobacter by the end of the rearing period. Faecal and environmental samples were analyzed at regular intervals throughout the rearing period of each broiler flock. Campylobacter was not detected in the disinfected house, or in one-day old broiler chicks. Campylobacter jejuni was isolated from environmental samples including air, water puddles, adjacent broiler flocks and soil. A representative subset of isolates from each farm was selected for further characterization using flaA-SVR sub-typing and multi-locus sequence typing (MLST) to determine if same-species isolates from different sources were indistinguishable or not. Results obtained suggest that no evidence of vertical transmission existed and that adequate cleaning/disinfection of broiler houses contributed to the prevention of carryover and cross-contamination. Nonetheless, the environment appears to be a potential source of Campylobacter. The population structure of Campylobacter isolates from broiler farms in Southern Ireland was diverse and weakly clonal.
    • A Review on the Applications of Next Generation Sequencing Technologies as Applied to Food-Related Microbiome Studies

      CaoYu; Fanning, Seamus; Proos, Sinead; Jordan, Kieran; Srikumar, Shabarinath; Department of Agriculture, Food and the Marine; SMART-PIF; 13/F/423 (Frontiers, 2017)
      The development of next generation sequencing (NGS) techniques has enabled researchers to study and understand the world of microorganisms from broader and deeper perspectives. The contemporary advances in DNA sequencing technologies have not only enabled finer characterization of bacterial genomes but also provided deeper taxonomic identification of complex microbiomes which in its genomic essence is the combined genetic material of the microorganisms inhabiting an environment, whether the environment be a particular body econiche (e.g., human intestinal contents) or a food manufacturing facility econiche (e.g., floor drain). To date, 16S rDNA sequencing, metagenomics and metatranscriptomics are the three basic sequencing strategies used in the taxonomic identification and characterization of food-related microbiomes. These sequencing strategies have used different NGS platforms for DNA and RNA sequence identification. Traditionally, 16S rDNA sequencing has played a key role in understanding the taxonomic composition of a food-related microbiome. Recently, metagenomic approaches have resulted in improved understanding of a microbiome by providing a species-level/strain-level characterization. Further, metatranscriptomic approaches have contributed to the functional characterization of the complex interactions between different microbial communities within a single microbiome. Many studies have highlighted the use of NGS techniques in investigating the microbiome of fermented foods. However, the utilization of NGS techniques in studying the microbiome of non-fermented foods are limited. This review provides a brief overview of the advances in DNA sequencing chemistries as the technology progressed from first, next and third generations and highlights how NGS provided a deeper understanding of food-related microbiomes with special focus on non-fermented foods.