Edward M. Fox

Listeria monocytogenes in the Irish dairy farm environment.

Listeria monocytogenes is a potentially lethal foodborne pathogen commonly found in the environment. European Union hygiene legislation places responsibility for safety on primary production facilities, including farms, as part of a policy to introduce traceability throughout the food chain. This study aimed to determine the occurrence of L. monocytogenes in the Irish dairy farm environment and in particular the milking facility. Two hundred ninety-eight environmental samples were collected from 16 farms in the southern region of Ireland. A number of farms within the group supply raw milk to the unpasteurized milk cheese industry. The samples taken included cow feces, milk, silage, soil, water, etc. Samples were enriched in Listeria enrichment broth and incubated for 48 h, followed by plating on chromogenic agar Listeria Ottavani & Agosti and further incubation of the plates for 24 to 48 h. Presumptive L. monocytogenes isolates were purified and confirmed by PCR targeting the hly gene. Overall, 19% of the samples (57 of 298) were positive for L. monocytogenes. These were serotyped using conventional and PCR methods; serotypes 1/2a, 1/2b, and 4b made up 78% of the typeable isolates. A correlation was found between the level of hygiene standards on the farm and the occurrence of L. monocytogenes. There was little difference in the occurrence of L. monocytogenes between farms supplying milk to the unpasteurized milk cheese industry and those supplying milk for processing. This study demonstrates the prevalence of L. monocytogenes in the dairy farm environment and the need for good hygiene practices to prevent its entry into the food chain.

Physiological and Transcriptional Characterization of Persistent and Nonpersistent Listeria monocytogenes Isolates

ABSTRACT This study aimed to characterize physiological differences between persistent and presumed nonpersistent Listeria monocytogenes strains isolated at processing facilities and to investigate the molecular basis for this by transcriptomic sequencing. Full metabolic profiles of two strains, one persistent and one nonpersistent, were initially screened using Biologs Phenotype MicroArray (PM) technology. Based on these results, in which major differences from selected antimicrobial agents were detected, another persistent strain and two nonpersistent strains were characterized using two antimicrobial PMs. Resistance to quaternary ammonium compounds (QACs) was shown to be higher among persistent strains. Growth of persistent and nonpersistent strains in various concentrations of the QACs benzethonium chloride (BZT) and cetylpyridinium chloride (CPC) was determined. Transcriptomic sequencing of a persistent and a presumed nonpersistent strain was performed to compare gene expression among these strains in the presence and absence of BZT. Two strains, designated “frequent persisters” because they were the most frequently isolated at the processing facility, showed overall higher resistance to QACs. Transcriptome analysis showed that BZT induced a complex peptidoglycan (PG) biosynthesis response, which may play a key role in BZT resistance. Comparison of persistent and nonpersistent strains indicated that transcription of many genes was upregulated among persistent strains. This included three gene operons: pdu, cob-cbi, and eut. These genes may play a role in the persistence of L. monocytogenes outside the human host.

Listeria monocytogenes in Irish Farmhouse cheese processing environments.

Sixteen cheesemaking facilities were sampled during the production season at monthly intervals over a two-year period. Thirteen facilities were found to have samples positive for Listeria monocytogenes. Samples were divided into 4 categories; cheese, raw milk, processing environment and external to the processing environment (samples from the farm such as silage, bedding, and pooled water). In order to attempt to identify the source, persistence and putative transfer routes of contamination with the L. monocytogenes isolates, they were differentiated using PFGE and serotyping. Of the 250 isolates, there were 52 different pulsotypes. No pulsotype was found at more than one facility. Two facilities had persistent pulsotypes that were isolated on sampling occasions at least 6 months apart. Of the samples tested, 6.3% of milk, 13.1% of processing environment and 12.3% of samples external to the processing environment, respectively, were positive for L. monocytogenes. Pulsotypes found in raw milk were also found in the processing environment, however, one of the pulsotypes from raw milk was found in cheese on only one occasion. One of the pulsotypes isolated from the environment external to the processing facility was found on the surface of cheese, however, a number of them were found in the processing environment. The results suggest that the farm environment external to the processing environment may in some cases be the source of processing environment contamination with L. monocytogenes.

PFGE analysis of Listeria monocytogenes isolates of clinical, animal, food and environmental origin from Ireland.

Listeria monocytogenes is an important foodborne human pathogen. Human infection is associated with high mortality rates. Epidemiological investigation and molecular subtyping can be useful in linking human illness with specific sources of infection. This retrospective study describes the use of PFGE to examine relationships of 222 isolates from human and non-human sources in Ireland. Human clinical isolates from other countries were also examined. Eight small clusters of human and non-human isolates (mostly serotype 4b) that were indistinguishable from one another were detected, suggesting potential sources for human infection. For non-human isolates, some PFGE types appeared to be exclusively associated with a single source, whereas other PFGE-types appeared to be more widely disseminated. Indistinguishable, or highly related clusters of isolates of Irish and non-Irish origin suggest that some PFGE patterns may be globally distributed.

Prevalence and characterization of foodborne pathogens from Australian dairy farm environments.

The ability of foodborne pathogens to gain entry into food supply systems remains an ongoing concern. In dairy products, raw milk acts as a major vehicle for this transfer; however, the sources of pathogenic bacteria that contaminate raw milk are often not clear, and environmental sources of contamination or the animals themselves may contribute to the transfer. This survey examined the occurrence of 9 foodborne pathogens in raw milk and environments of 7 dairy farms (3 bovine, 3 caprine, and 1 ovine farm) in summer and autumn, in Victoria, Australia. A total of 120 samples were taken from sampling points common to dairy farms, including pasture, soil, feed, water sources, animal feces, raw milk, and milk filters. The prevalence of the Bacillus cereus group, Campylobacter, Clostridium perfringens, Cronobacter, Shiga-toxigenic Escherichia coli, Listeria, Salmonella, coagulase-positive staphylococci (CPS), and Yersinia enterocolitica across the farms was investigated. The 2 most prevalent bacteria, which were detected on all farms, were the B. cereus group, isolated from 41% of samples, followed by Cl. perfringens, which was isolated from 38% of samples. The highest occurrence of any pathogen was the B. cereus group in soil, present in 93% of samples tested. Fecal samples showed the highest diversity of pathogens, containing 7 of the 9 pathogens tested. Salmonella was isolated from 1 bovine farm, although it was found in multiple samples on both visits. Out of the 14 occurrences where any pathogen was detected in milk filters, only 5 (36%) of the corresponding raw milk samples collected at the same time were positive for the same pathogen. All of the CPS were Staphylococcus aureus, and were found in raw milk or milk filter samples from 6 of the 7 farms, but not in other sample types. Pathogenic Listeria species were detected on 3 of the 7 farms, and included 4 L. ivanovii-positive samples, and 1 L. monocytogenes-positive water sample. Shiga-toxigenic Escherichia coli were identified in fecal samples from 3 of the 7 farms and in a single raw milk sample. Cronobacter species were identified on 4 of the 7 farms, predominantly in feed samples. No Y. enterocolitica was detected. Results of this study demonstrate high standards of pathogen safety across the 7 farms, with a low incidence of pathogens detected in raw milk samples. Monitoring feed contamination levels may help control the spread of bacterial species such as Cl. perfringens and B. cereus through the farm environment, which is a natural reservoir for these organisms.

Transcriptome analysis of Listeria monocytogenes exposed to biocide stress reveals a multi-system response involving cell wall synthesis, sugar uptake, and motility

Listeria monocytogenes is a virulent food-borne pathogen most often associated with the consumption of “ready-to-eat” foods. The organism is a common contaminant of food processing plants where it may persist for extended periods of time. A commonly used approach for the control of Listeria monocytogenes in the processing environment is the application of biocides such as quaternary ammonium compounds. In this study, the transcriptomic response of a persistent strain of L. monocytogenes (strain 6179) on exposure to a sub-lethal concentration of the quaternary ammonium compound benzethonium chloride (BZT) was assessed. Using RNA-Seq, gene expression levels were quantified by sequencing the transcriptome of L. monocytogenes 6179 in the presence (4 ppm) and absence of BZT, and mapping each data set to the sequenced genome of strain 6179. Hundreds of differentially expressed genes were identified, and subsequent analysis suggested that many biological processes such as peptidoglycan biosynthesis, bacterial chemotaxis and motility, and carbohydrate uptake, were involved in the response of L. monocyotogenes to the presence of BZT. The information generated in this study further contributes to our understanding of the response of bacteria to environmental stress. In addition, this study demonstrates the importance of using the bacteriums own genome as a reference when analysing RNA-Seq data.

Collaborative Survey on the Colonization of Different Types of Cheese-Processing Facilities with Listeria monocytogenes

Cross-contamination via equipment and the food-processing environment has been implicated as the main cause of Listeria monocytogenes transmission. The aim of this study, therefore, was to determine the occurrence and potential persistence of L. monocytogenes in 19 European cheese-processing facilities. A sampling approach in 2007-2008 included, respectively, 11 and two industrial cheese producers in Austria and the Czech Republic, as well as six Irish on-farm cheese producers. From some of the producers, isolates were available from sampling before 2007. All isolates from both periods were included in a strain collection consisting of 226 L. monocytogenes isolates, which were then typed by serotyping and pulsed-field gel electrophoresis (PFGE). In addition, metabolic fingerprints from a subset of isolates were obtained by means of Fourier-transform infrared (FTIR) spectroscopy. PFGE typing showed that six processing environments were colonized with seven persistent PFGE types of L. monocytogenes. Multilocus sequence typing undertaken on representatives of the seven persisting PFGE types grouped them into distinct clades on the basis of country and origin; however, two persistent strains from an Austrian and an Irish food processor were shown to be clonal. It was concluded that despite the fact that elaborate Hazard Analysis and Critical Control Point concepts and cleaning programs are applied, persistent occurrence of L. monocytogenes can take place during cheese making. L. monocytogenes sanitation programs could be strengthened by including rapid analytical tools, such as FTIR, which allow prescreening of potentially persistent L. monocytogenes contaminants.

Growth arrest-specific gene 6 expression in human breast cancer

Growth arrest-specific gene 6 (Gas6), identified in 1995, acts as the ligand to the Axl/Tyro3 family of tyrosine kinase receptors and exerts mitogenic activity when bound to these receptors. Overexpression of the Axl/Tyro3 receptor family has been found in breast, ovarian and lung tumours. Gas6 is upregulated 23-fold by progesterone acting through the progesterone receptor B (PRB). Recently, Gas6 has been shown to be a target for overexpression and amplification in breast cancer. Quantitative real-time PCR analysis was used to determine the levels of Gas6 mRNA expression in 49 primary breast carcinomas. Expression of PRB protein was evaluated immunohistochemically with a commercially available PRB antibody. The results showed a positive association between PRB protein and Gas6 mRNA levels (P=0.04). Gas6 correlated positively with a number of favourable prognostic variables including lymph node negativity (P=0.0002), younger age at diagnosis (P=0.04), smaller size of tumours (P=0.02), low Nottingham prognostic index scores (P=0.03) and low nuclear morphology (P=0.03). This study verifies for the first time the association between PRB and Gas6 in breast cancer tissue.

Rapid identification and source-tracking of Listeria monocytogenes using MALDI-TOF mass spectrometry

Listeria monocytogenes is an important foodborne pathogen responsible for the sometimes fatal disease listeriosis. Public health concerns and stringent regulations associated with the presence of this pathogen in food and food processing environments underline the need for rapid and reliable detection and subtyping techniques. In the current study, the application of matrix assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS) as a single identification and source-tracking tool for a collection of L. monocytogenes isolates, obtained predominantly from dairy sources within Australia, was explored. The isolates were cultured on different growth media and analysed using MALDI-TOF MS at two incubation times (24 and 48 h). Whilst reliable genus-level identification was achieved from most media, identification at the species level was found to be dependent on culture conditions. Successful speciation was highest for isolates cultured on the chromogenic Agar Listeria Ottaviani Agosti agar (ALOA, 91% of isolates) and non-selective horse blood agar (HBA, 89%) for 24h. Chemometric statistical analysis of the MALDI-TOF MS data enabled source-tracking of L. monocytogenes isolates obtained from four different dairy sources. Strain-level discrimination was also observed to be influenced by culture conditions. In addition, t-test/analysis of variance (ANOVA) was used to identify potential biomarker peaks that differentiated the isolates according to their source of isolation. Source-tracking using MALDI-TOF MS was compared and correlated with the gold standard pulsed-field gel electrophoresis (PFGE) technique. The discriminatory index and the congruence between both techniques were compared using the Simpsons Diversity Index and adjusted Rand and Wallace coefficients. Overall, MALDI-TOF MS based source-tracking (using data obtained by culturing the isolates on HBA) and PFGE demonstrated good congruence with a Wallace coefficient of 0.71 and comparable discriminatory indices of 0.89 and 0.86, respectively. MALDI-TOF MS thus represents a rapid and cost-effective source-tracking technique for L. monocytogenes.

Surveillance of Verocytotoxigenic Escherichia coli in Irish Bovine Dairy Herds

Verocytotoxigenic Escherichia coli (VTEC) are highly significant zoonotic threats to public health, and have been the causative agent implicated in numerous high‐profile outbreaks affecting large numbers of people. Serovar O157 is most frequently linked with human illness; however, other serovars, such as O26, O103, O111 and O145, have also been implicated. This study aimed to characterize the prevalence and virulence determinants of these five serovars in Irish dairy farm herds, and their milk. Using real‐time PCR (RTi‐PCR), bovine rectal faecal swabs and raw milk samples, along with milk filters, were screened for the presence of vt genes. Positive samples were then screened for the five serovars using sero‐specific PCR. Serovar‐positive samples were subjected to immunomagnetic separation, to isolate viable VTEC strains. These isolates were subsequently screened for four virulence factors: vt1, vt2, eaeA and hlyA. Three hundred and eighty six of the 600 rectal faecal swabs, 85 of the 117 milk‐filters and 43 of the 120 bulk‐tank milk samples, were positive for vt genes. From these 514 total vt‐positive samples, 58 O26, 162 O103, 1 O111, 324 O145 and 26 O157 positives were detected by sero‐specific RTi‐PCR. Immunomagnetic separation yielded 12 O26, 26 O103, 0 O111, 19 O145 and 10 O157 isolates. Ten of these isolates contained at least one of the four virulence determinants screened for (i.e. vt1, vt2, eaeA and hlyA). Of these 10 isolates, pulsed‐field gel electrophoresis showed that two of the O26 isolates from different farms were indistinguishable. Two O157 isolates were also indistinguishable. This study found serovars O103 and O145 to be the most prevalent in samples tested. Apart from the occurrence of VTEC in dairy herds, this study shows a high occurrence of vt genes in the environment, creating the possibility of horizontal gene transfer and emergence of new VTEC strains.