Characterizing Phage-Host Interactions in a Simplified Human Intestinal Barrier Model.

An intestinal epithelium model able to produce mucus was developed to provide an environment suitable for testing the therapeutic activity of gut bacteriophages. We show that Enterococcus faecalis adheres more effectively in the presence of mucus, can invade the intestinal epithelia and is able to translocate after damaging tight junctions. Furthermore, Enterococcus phage vB_EfaM_A2 (a member of Herelleviridae that possesses virion associated immunoglobin domains) was found to translocate through the epithelium in the presence and absence of its host bacteria. Phage A2 protected eukaryotic cells by reducing mortality and maintaining the structure of the cell layer structure. We suggest the mammalian cell model utilized within this study as an adaptable in vitro model that can be employed to enable a better understanding of phage-bacteria interactions and the protective impact of phage therapy relating to the intestinal epithelium.

The Effect of a Commercially Available Bacteriophage and Bacteriocin on Listeria monocytogenes in Coleslaw.

Changing consumer attitudes show an increased interest in non-chemical antimicrobials in food preservation and safety. This greater interest of consumers in more 'natural' or 'clean-label' food interventions is complicated by concurrent demands for minimally processed, ready-to-eat (RTE) foods with long shelf lives. Two viable interventions are bacteriophage (phage) and bacteriocins, a number of which have already been approved for use in food safety. Listeriosis is a serious foodborne infection which affects at-risk members of the population. Listeriosis incidence has increased between 2008 and 2015 and has a case fatality rate of up to 20% with antibiotic intervention. Here, we tested an intervention to attempt to control a pathogenic Listeria monocytogenes strain in a food model using two of these alternative antimicrobials. Phage P100 on its own had a significant effect on L. monocytogenes ScottA numbers in coleslaw over a 10-day period at 4 °C (p ≤ 0.001). A combination of P100 and Nisaplin® (a commercial formulation of the lantibiotic bacteriocin, nisin) had a significant effect on the pathogen (p ≤ 0.001). P100 and Nisaplin® in combination were more effective than Nisaplin® alone, but not P100 alone.

Phage therapy targeting Escherichia coli-a story with no end?

Bacteriophages (phages) or bacterial viruses have long been proposed as an alternative therapy against antibiotic-resistant bacteria such as Escherichia coli Even though poorly documented in the scientific literature, a long clinical history of phage therapy in countries such as Russia and Georgia suggests potential value in the use of phages as antibacterial agents. Escherichia coli is responsible for a wide range of diseases, intestinal (diarrhoea) and extraintestinal (UTI, septicaemia, pneumoniae, meningitis), making it an ideal target for phage therapy. This review discusses the latest research focusing on the potential of phage therapy to tackle E. coli-related illnesses. No intact phages are approved in EU or USA for human therapeutic use, but many successful in vitro and in vivo studies have been reported. However, additional research focused on in vivo multispecies models and human trials are required if phage therapy targeting E. coli pathotypes can be a story with happy end.

Tracking Foodborne Pathogenic Bacteria in Raw and Ready-to-Eat Food Illegally Sold at the Eastern EU Border.

Food illegally brought into the European Union, mainly in the personal luggage of travelers, represents a potential threat to consumers' health. The aim of this study was to investigate the presence of five pathogens in food brought into the European Union by Moldavian citizens as personal goods and illegally sold in Romania in the vicinity of the border. The occurrence of Staphylococcus aureus and Listeria monocytogenes was 7.5% and 8%, while Campylobacter spp., Escherichia coli O157:H7, and Salmonella spp. were absent in all samples. L. monocytogenes sequence type 2, 9, 121, and 155, highly prevalent among foodstuffs worldwide, was also present among isolates from ready-to-eat food illegally sold in Romania, even at the same date of sampling, indicating cross-contamination during food handling. S. aureus spa types t449, t304, and t524 were most often isolated from raw-milk cheeses contaminated with 10(3)-10(5) colony-forming units per gram, evidencing a contamination at herd level or unhygienic conditions during processing. S. aureus t011 and t3625, both included in the livestock-associated CC398, were isolated from pork lard and poultry meat. This study shows that cross-border trade from nonmember states represents a neglected route of transmission of foodborne pathogens into the European Union that could lead to sporadic or family-associated cases of disease.

Dynamics of Listeria monocytogenes colonisation in a newly-opened meat processing facility.

This study determined the colonisation scenario of Listeria monocytogenes in a newly-opened ready-to-eat meat processing facility using a combination of classical microbiology and molecular biology techniques. Samples (n=183), including food contact surfaces, non-food contact surfaces, raw materials and food samples, collected on four sampling occasions, were analysed for L. monocytogenes by the ISO 11290:1996 standard method and by real-time PCR applied to the second enrichment broth from the ISO method. No L. monocytogenes were detected on the first sampling occasion, but by the second sampling occasion a persistent clone had colonised the facility. Analysis of the second enrichment of the ISO method by real-time PCR was more sensitive for the detection of L. monocytogenes than the ISO method alone. In order to reduce the risk of cross contamination and the public health risk, awareness and proactive measures are required to control L. monocytogenes from the first days of production in a newly opened meat processing facility.

Putative Cross-Contamination Routes of Listeria monocytogenes in a Meat Processing Facility in Romania.

Putative routes of Listeria monocytogenes contamination, based on the workflow of the employees, were studied in a meat processing facility by investigating 226 samples collected from food contact surfaces, non-food contact surfaces, raw materials, and ready-to-eat meat products on four occasions over a 1-year period. In total, 19.7% of non-food contact surfaces, 22.9% of food contact surfaces, 45% of raw materials, and 20% of ready-to-eat meat products were positive for L. monocytogenes (analyzed by the International Organization for Standardization standard method ISO 11290). Pulsed-field gel electrophoresis (PFGE) profiles were determined for a representative subset of these isolates, and 11 distinct pulsotypes were identified, two of which were frequently isolated (T4 and T8) and considered persistent. Strains from the various pulsotypes were screened for the presence of bcrABC and qacH, the genes responsible for tolerance responses to quaternary ammonium compounds. Two strains harbored bcrABC, and these strains had a higher benzalkonium chloride tolerance; however, they were not considered persistent strains. The frequently isolated PFGE pulsotype T8 strains were highly adhesive to abiotic surfaces at 10 and 20°C; however, the pulsotype T6 strain, which was isolated only at the last sampling time, had the highest adhesion ability, and the pulsotype T4 strain (the second most persistent pulsotype) had only modest adhesion. Four putative cross-contamination routes were confirmed by mapping the persistent and other isolates. This information could allow a food safety manager to adjust the work flow to improve the hygienic conditions in a meat processing facility. This study revealed the prevalence and persistence of L. monocytogenes strains in a meat processing facility and established the importance of developing strategies to avoid cross-contamination, recalls, and outbreaks of listeriosis.

Foods from black market at EU border as a neglected route of potential methicillin-resistant Staphylococcus aureus transmission.

The illegal entrance of foods to EU through black markets at the EU borders can constitute a neglected route of dissemination of foodborne pathogens, and in particular of methicillin-resistant Staphylococcus aureus (MRSA). In this study, we have assessed the presence of MRSA in foods sold in a black market at an EU border (the southeast part of Romania, on the border with Republic of Moldavia). We performed a search for MRSA among 200 food samples collected from 2012 to 2013. All S. aureus were studied by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. MRSA isolates were further characterized by multilocus sequence typing (MLST) and SCCmec typing, and tested for the presence of Panton-Valentine leukocidin (PVL) virulence factors. Overall, 32 S. aureus isolates were recovered from 16 food samples (8%). One isolate detected in a pork lard sample was MRSA (0.5%). PFGE with the restriction enzyme SmaI revealed 12 genotypes among the 32 S. aureus isolates. The MRSA isolate belonged to sequence type 398, harbored SCCmec type V, tested negative for the presence of the PVL genes and was resistant to ciprofloxacin, tetracycline and cefazolin, besides all ß-lactams. Among 31 methicillin-sensitive S. aureus (MSSA), 29% were resistant to penicillin, 9.7% to tetracycline and 3.2% to ciprofloxacin. In conclusion, in this study we report the presence of livestock-associated MRSA in foods sold in a black market at an EU border: ST398-MRSA-V. These results confirm the potential role of food in the dissemination of MRSA lineages among population, and the potential role of illegally introduced food to EU in the prevalence and evolution of MRSA clones in the community.

Sampling the processing environment for Listeria.

This chapter describes in detail the procedures used when sampling for Listeria in food processing environments. Sampling of food contact surfaces, non-food contact surfaces, and liquids such as drain effluents are addressed. Sponge stick swabs are considered advantageous for surface sampling and tips regarding their application are given. Liquids are collected using sterile dippers and the procedure for their correct use is described. Advice on places to sample, the best time for sampling and the frequency of sampling are also given. Such details help hygienists/microbiologists to be successful in their attempts to isolate strains of Listeria, even if such bacteria are well attached to surfaces or located in niches that are difficult to reach.

Comparison of polymerase chain reaction methods and plating for analysis of enriched cultures of Listeria monocytogenes when using the ISO11290-1 method.

Analysis for Listeria monocytogenes by ISO11290-1 is time-consuming, entailing two enrichment steps and subsequent plating on agar plates, taking five days without isolate confirmation. The aim of this study was to determine if a polymerase chain reaction (PCR) assay could be used for analysis of the first and second enrichment broths, saving four or two days, respectively. In a comprehensive approach involving six European laboratories, PCR and traditional plating of both enrichment broths from the ISO11290-1 method were compared for the detection of L. monocytogenes in 872 food, raw material and processing environment samples from 13 different dairy and meat food chains. After the first and second enrichments, total DNA was extracted from the enriched cultures and analysed for the presence of L. monocytogenes DNA by PCR. DNA extraction by chaotropic solid-phase extraction (spin column-based silica) combined with real-time PCR (RTi-PCR) was required as it was shown that crude DNA extraction applying sonication lysis and boiling followed by traditional gel-based PCR resulted in fewer positive results than plating. The RTi-PCR results were compared to plating, as defined by the ISO11290-1 method. For first and second enrichments, 90% of the samples gave the same results by RTi-PCR and plating, whatever the RTi-PCR method used. For the samples that gave different results, plating was significantly more accurate for detection of positive samples than RTi-PCR from the first enrichment, but RTi-PCR detected a greater number of positive samples than plating from the second enrichment, regardless of the RTi-PCR method used. RTi-PCR was more accurate for non-food contact surface and food contact surface samples than for food and raw material samples especially from the first enrichment, probably because of sample matrix interference. Even though RTi-PCR analysis of the first enrichment showed less positive results than plating, in outbreak scenarios where a rapid result is required, RTi-PCR could be an efficient way to get a preliminary result to be then confirmed by plating. Using DNA extraction from the second enrichment broth followed by RTi-PCR was reliable and a confirmed result could be obtained in three days, as against seven days by ISO11290-1.