Risk factor-based clustering of Listeria monocytogenes in food processing environments using principal component analysis.

Listeria monocytogenes has a range of strategies that allow it to persist as biofilms in food processing environments (FPE), making it a pathogen of concern to the food industry. The properties of these biofilms are highly variable among strains, and this significantly affects the risk of food contamination. The present study therefore aims to conduct a proof-of-concept study to cluster strains of L. monocytogenes by risk potential using principal component analysis, a multivariate approach. A set of 22 strains, isolated from food processing environments, were typed by serogrouping and pulsed-field gel electrophoresis, showing a relatively high diversity. They were characterized in terms of several biofilm properties that might pose a potential risk of food contamination. The properties studied were tolerance to benzalkonium chloride (BAC), the structural parameters of biofilms (biomass, surface area, maximum and average thickness, surface to biovolume ratio and roughness coefficient) measured by confocal laser scanning microscopy and (3) transfer of biofilm cells to smoked salmon. The PCA correlation circle revealed that the tolerance of biofilms to BAC was positively correlated with roughness, but negatively with biomass parameters. On the contrary, cell transfers were not related to three-dimensional structural parameters, which suggests the role of other variables yet unexplored. Additionally, hierarchical clustering grouped strains into three different clusters. One of them included the strains with high tolerance to BAC and roughness. Another one consisted of strains with enhanced transfer ability, whereas the third cluster contained those that stood out for the thickness of biofilms. The present study represents a novel and effective way to classify L. monocytogenes strains according to biofilm properties that condition the potential risk of reaching the consumer through food contamination. It would thus allow the selection of strains representative of different worst-case scenarios for future studies in support of QMRA and decision-making analysis.

Architectural Features and Resistance to Food-Grade Disinfectants in Listeria monocytogenes-Pseudomonas spp. Dual-Species Biofilms.

Listeria monocytogenes is considered a foodborne pathogen of serious concern capable of forming multispecies biofilms with other bacterial species, such as Pseudomonas spp., adhered onto stainless steel (SS) surfaces. In an attempt to link the biofilms' morphology and resistance to biocides, dual-species biofilms of L. monocytogenes, in co-culture with either Pseudomonas aeruginosa, Pseudomonas fluorescens, or Pseudomonas putida, were assayed to ascertain their morphological characteristics and resistance toward benzalkonium chloride (BAC) and neutral electrolyzed water (NEW). Epifluorescence microscopy analysis revealed that each dual-species biofilm was distributed differently over the SS surface and that these differences were attributable to the presence of Pseudomonas spp. Confocal laser scanning microscopy (CLSM) assays demonstrated that despite these differences in distribution, all biofilms had similar maximum thicknesses. Along with this, colocalization analyses showed a strong trend of L. monocytogenes to share location within the biofilm with all Pseudomonas assayed whilst the latter distributed throughout the surface independently of the presence of L. monocytogenes, a fact that was especially evident in those biofilms in which cell clusters were present. Finally, a modified Gompertz equation was used to fit biofilms' BAC and NEW dose-response data. Outcomes demonstrated that L. monocytogenes was less susceptible to BAC when co-cultured with P. aeruginosa or P. fluorescens, whereas susceptibility to NEW was reduced in all three dual-species biofilms, which can be attributable to both the mechanism of action of the biocide and the architectural features of each biofilm. Therefore, the results herein provided can be used to optimize already existing and develop novel target-specific sanitation treatments based on the mechanism of action of the biocide and the biofilms' species composition and structure.

Tracking bacteriome variation over time in Listeria monocytogenes-positive foci in food industry.

The variation in microbial composition over time was assessed in biofilms formed in situ on selected non-food and food contact surfaces of meat and fish industries, previously identified as Listeria monocytogenes-positive foci. First, all samples were analysed for the detection and quantification of L. monocytogenes using ISO 11290-1 and ISO 11290-2 norms, respectively. Although the pathogen was initially detected in all samples, direct quantification was not possible. Psychrotrophic bacteria counts were among resident microbiota in meat industry samples (Meanmax = 6.14 log CFU/cm2) compared to those form fish industry (Meanmax = 5.85 log CFU/cm2). Visual analysis of the biofilms using epifluorescence microscopy revealed a trend to form microcolonies in which damaged/dead cells would act as anchoring structures. 16S rRNA gene metagenetic analysis demonstrated that, although Proteobacteria (71.37%) initially dominated the bacterial communities at one meat industry location, there was a dramatic shift in composition as the biofilms matured, where Actinobacteria (79.72%) became the major phylum present in later samples. This change was largely due to an increase of Nocardiaceae, Micrococcaceae and Microbacteriaceae. Nevertheless, for the other sampling location, the relative abundance of the dominating phylum (Firmicutes) remained consistent over the entire sampling period (Mean = 63.02%). In fish industry samples, Proteobacteria also initially dominated early on (90.69%) but subsequent sampling showed a higher diversity in which Bacteroidetes and Proteobacteria were the most abundant phyla accounting for the 48.04 and 37.98%, respectively by the last sampling period. Regardless of the location, the community profiles of the endpoint samples were similar to those reported previously. This demonstrated that in a given industrial setting there is a trend to establish a determinate biofilm structure due to the environmental factors and the constant incoming microbiota. This information could be used to improve the existing sanitisation protocols or for the design of novel strategies.

Current Knowledge on Listeria monocytogenes Biofilms in Food-Related Environments: Incidence, Resistance to Biocides, Ecology and Biocontrol.

Although many efforts have been made to control Listeria monocytogenes in the food industry, growing pervasiveness amongst the population over the last decades has made this bacterium considered to be one of the most hazardous foodborne pathogens. Its outstanding biocide tolerance capacity and ability to promiscuously associate with other bacterial species forming multispecies communities have permitted this microorganism to survive and persist within the industrial environment. This review is designed to give the reader an overall picture of the current state-of-the-art in L. monocytogenes sessile communities in terms of food safety and legislation, ecological aspects and biocontrol strategies.

Incidence and characterization of Staphylococcus aureus in fishery products marketed in Galicia (Northwest Spain).

A total of 298 fishery products purchased from retail outlets in Galicia (NW Spain) between January 2008 and May 2009 were analyzed for the presence of Staphylococcus aureus. S. aureus was detected in a significant proportion of products (~25%). Incidence was highest in fresh (43%) and frozen products (30%), but it was high in all other categories: salted fish (27%), smoked fish (26%), ready-to-cook products (25%), non-frozen surimis (20%), fish roes (17%) and other ready-to-eat products (10%). A significant proportion of smoked fish, surimis, fish roes and other ready-to-eat products did not comply with legal limits in force. RAPD-PCR of 125 S. aureus isolated from fishery products was carried out using three primers (AP-7, ERIC-2 and S). Isolates displayed 33 fingerprint patterns. Each pattern was attributed to a single bacterial clone. Cluster analysis based on similarity values between RAPD fingerprints did not find relationship between any RAPD pattern and any product category. Isolates were also tested for se genes and susceptibility to a range of antibiotics (cephalothin, clindamycin, chloramphenicol, erythromycin, gentamicin, oxacillin, penicillin G, tetracycline, vancomycin, methicillin, ciprofloxacin and trimethoprim-sulfamethoxazole). Most isolates (88%) were found to be sea positive. Putative enterotoxigenic strains counts reached high risk levels in 17 products. No relationship was found between the presence of se genes and RAPD patterns. All isolates were resistant to penicillin, chloramphenicol and ciprofloxacin, and most to tetracycline (82.4%), but none was methicillin-resistant. A revision of pre-requisite programs leading to improve hygienic practices in handling and processing operations from fishing or farming to retail is recommended to ensure fishery products safety.