Tracking microbial quality, safety and environmental contamination sources in artisanal goat cheesemaking factories.

A harmonised microbiological survey was performed in two artisanal factories of raw goat milk cheeses (A and B) located in the Andalusian region (Spain). A total of 165 different control points or samples (raw materials, final products, food-contact surfaces [FCS], and air) were examined as microbial and pathogen sources of contamination of artisanal goat raw milk cheeses. For raw milk samples analysed from both producers, the concentrations of aerobic-mesophilic bacteria (AMB), total coliforms, coagulase-positive Staphylococcus spp. (CPS), lactic-acid bacteria (LAB) and moulds and yeasts ranged between 3.48 and 8.59, 2.45-5.48, 3.42-4.81, 4.99-8.59 and 3.35-6.85 log cfu/mL respectively. For the same microbial groups, the analysis of raw milk cheeses revealed concentrations ranging from 7.82 to 8.88, 2.00-6.82, 2.00-5.28, 8.11-9.57 and 2.00-5.76 log cfu/g, respectively. Although the raw material analysed from producer A presented higher microbial loads and between-batch variability, it was B the producer with the most loaded final products. Regarding the microbial air quality, the fermentation area, storage room, milk reception and packaging room were the most AMB loaded places, while the ripening chamber was the area with higher fungal loads in bioareosol from both producers. Conveyor belts, cutting machine, storage boxes and brine tank were highlighted as the most contaminated FCS evaluated. Staphylococcus aureus was the only pathogen detected within the set of 51 isolates from samples as revealed by MALDI-TOF and molecular PCR, with a prevalence of 12.5% for samples from the producer B. The public health risk attributed to the consumption of artisanal goat cheese should not be neglected, and may consider the whole cheesemaking processing chain, from microbiological quality of raw milk to the ready-to-eat final product, especially concerning the presence of S. aureus.

Microbial contamination, antimicrobial resistance and biofilm formation of bacteria isolated from a high-throughput pig abattoir.

The aim of this work was to assess the level of microbial contamination and resistance of bacteria isolated from a highthroughput heavy pig slaughterhouse (approx. 4600 pigs/day) towards antimicrobials considered as critical for human, veterinary or both chemotherapies. Samples, pre-operative and operative, were obtained in 4 different surveys. These comprised environmental sampling, i.e. air (ntotal = 192) and surfaces (ntotal = 32), in four different locations. Moreover, a total of 40 carcasses were sampled in two different moments of slaughtering following Reg. (CE) 2073/2005. Overall, 60 different colonies were randomly selected from VRBGA plates belonging to 20 species, 15 genera and 10 families being Enterobacteriaceae, Moraxellaceae and Pseudomonadaceae the most represented ones. Thirty-seven isolates presented resistance to at least one molecule and seventeen were classified as multi-drug resistant. Enterobacteriaceae, particularly E. coli, displayed high MIC values towards trimethoprim, ampicillin, tetracycline and sulphametoxazole with MICmax of 16, 32, 32 and 512 mg/L, respectively. Moreover, isolated Pseudomonas spp. showed high MIC values in critical antibiotics such as ampicillin and azithromycin with MICmax of 32 and 64 mg/L, respectively. Additionally, in vitro biofilm formation assays demonstrated that fifteen of these isolates can be classified as strong biofilm formers. Results demonstrated that a high diversity of bacteria containing antibiotic resistant and multiresistant species is present in the sampled abattoir. Considering these findings, it could be hypothesised that the processing environment could be a potential diffusion determinant of antibiotic resistant bacteria through the food chain and operators.

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.

Assessment of the Antibiotic Resistance Profile, Genetic Heterogeneity and Biofilm Production of Methicillin-Resistant Staphylococcus aureus (MRSA) Isolated from The Italian Swine Production Chain.

The main aim of the present study was to evaluate the level of antibiotic resistance, prevalence and virulence features of methicillin-resistant Staphylococcus aureus (MRSA) isolated from heavy swine at abattoir level and farming environments in Lombardy (Northern Italy). With this scope, 88 different heavy swine farms were surveyed, obtaining a total of n = 440 animal swabs and n = 150 environmental swabs. A total of n = 87 MRSA isolates were obtained, with an overall MRSA incidence of 17.50% (n = 77) among animal samples and a 6.67% (n = 10) among environmental. Molecular characterisation using multilocus sequence typing (MLST) plus spa-typing showed that sequence type ST398/t899 and ST398/t011 were the most commonly isolated genotypes, although other relevant sequence types such as ST1 or ST97 were also found. A lack of susceptibility to penicillins, tetracycline and ceftiofur was detected in >91.95, 85.05 and 48.28% of the isolates, respectively. Resistance to doxycycline (32.18%), enrofloxacin (27.59%) and gentamicin (25.29%) was also observed. Additionally, a remarkable level of antibiotic multiresistance (AMR) was observed representing a 77.01% (n = 67) of the obtained isolates. Genetic analysis revealed that 97.70% and 77.01% of the isolates harboured at least one antibiotic resistance or enterotoxin gene, respectively, pointing out a high isolate virulence potential. Lastly, 55.17% (n = 48) were able to produce measurable amounts of biofilm after 24 h. In spite of the current programmes for antibiotic reduction in intensively farming, a still on-going high level of AMR and virulence potential in MRSA was demonstrated, making this pathogen a serious risk in swine production chain, highlighting once more the need to develop efficient, pathogen-specific control strategies.

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.

Efficacy of Synthetic Furanones on Listeria monocytogenes Biofilm Formation.

Furanones are analogues of acylated homoserine lactones with proven antifouling activity in both Gram-positive and Gram-negative bacteria though the interference of various quorum sensing pathways. In an attempt to find new strategies to prevent and control Listeria monocytogenes biofilm formation on stainless steel (SS) surfaces, different concentrations of six synthetic furanones were applied on biofilms formed by strains isolated from food, environmental, and clinical sources grown onto AISI 316 SS coupons. Among the furanones tested, (Z-)-4-Bromo-5-(bromomethylene)-2(5H)-furanone and 3,4-Dichloro-2(5H)-furanone significantly (p < 0.05) reduced the adhesion capacity (>1 log CFU cm-2) in 24 h treated biofilms. Moreover, individually conducted experiments demonstrated that (Z-)-4-Bromo-5-(bromomethylene)-2(5H)-furanone was able to not only significantly (p < 0.05) prevent L. monocytogenes adhesion but also to reduce the growth rate of planktonic cells up to 48 h in a dose-dependent manner. LIVE/DEAD staining followed by epifluorescence microscopy visualisation confirmed these results show an alteration of the structure of the biofilm in furanone-treated samples. Additionally, it was demonstrated that 20 µmol L-1 of 3,4-Dichloro-2(5H)-furanone dosed at 0, 24 and 96 h was able to maintain a lower level of adhered cells (>1 log CFU cm-2; p < 0.05). Since furanones do not pose a selective pressure on bacteria, these results represent an appealing novel strategy for the prevention of L. monocytogenes biofilm grown onto SS.

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.

Quantifying the combined effects of pronase and benzalkonium chloride in removing late-stage Listeria monocytogenes-Escherichia coli dual-species biofilms.

This work presents the assessment of the effectivity of a pronase (PRN)-benzalkonium chloride (BAC) sequential treatment in removing Listeria monocytogenes-Escherichia coli dual-species biofilms grown on stainless steel (SS) using fluorescence microscopy and plate count assays. The effects of PRN-BAC on the occupied area (OA) by undamaged cells in 168 h dual-species samples were determined using a first-order factorial design. Empirical equations significantly (r2 = 0.927) described a negative individual effect of BAC and a negative interactive effect of PRN-BAC achieving OA reductions up to 46%. After treatment, high numbers of remaining attached and released viable and cultivable E. coli cells were detected in PRN-BAC combinations when low BAC concentrations were used. Therefore, at appropriate BAC doses, in addition to biofilm removal, sequential application of PRN and BAC represents an appealing strategy for pathogen control on SS surfaces while hindering the dispersion of live cells into the environment.

Tolerance development in Listeria monocytogenes-Escherichia coli dual-species biofilms after sublethal exposures to pronase-benzalkonium chloride combined treatments.

This study was designed to assess the effects that sublethal exposures to pronase (PRN) and benzalkonium chloride (BAC) combined treatments have on Listeria monocytogenes-Escherichia coli dual-species biofilms grown on stainless steel in terms of tolerance development (TD) to these compounds. Additionally, fluorescence microscopy was used to observe the changes of the biofilm structure. PRN-BAC exposure was carried out using three different approaches and TD was evaluated treating biofilms with a final 100 µg/ml PRN followed by 50 µg/ml BAC combined treatment. Results showed that exposure to PRN-BAC significantly decreased the number of adhered L. monocytogenes (P < 0.05), while E. coli counts remained generally unaltered. It was also demonstrated that the incorporation of recovery periods during sublethal exposures increased the tolerance of both species of the mixed biofilm to the final PRN-BAC treatment. Moreover, control biofilms became more resistant to PRN-BAC if longer incubation periods were used. Regardless of the treatment used, log reduction values were generally lower in L. monocytogenes compared to E. coli. Additionally, microscopy images showed an altered morphology produced by sublethal PRN-BAC in exposed L. monocytogenes-E. coli dual-species biofilms compared to control samples. Results also demonstrated that L. monocytogenes-E. coli dual-species biofilms are able to develop tolerance to PRN-BAC combined treatments depending on way they have been previously exposed. Moreover, they suggest that the generation of bacterial tolerance should be included as a parameter for sanitation procedures design.

Removal of Listeria monocytogenes dual-species biofilms using combined enzyme-benzalkonium chloride treatments.

The effects of pronase (PRN), cellulase (CEL) or DNaseI alone or combined with benzalkonium chloride (BAC) against Listeria monocytogenes-carrying biofilms were assayed. The best removal activity against L. monocytogenes-Escherichia coli biofilms was obtained using DNaseI followed by PRN and CEL. Subsequently, a modified logistic model was used to quantify the combined effects of PRN or DNaseI with BAC. A better BAC performance after PRN compared to DNaseI eradicating L. monocytogenes was observed. In E. coli the effects were the opposite. Finally, effects of DNaseI and DNaseI-BAC treatments were compared against two different L. monocytogenes-carrying biofilms. DNaseI-BAC was more effective against L. monocytogenes when co-cultured with E. coli. Nonetheless, comparing the removal effects after BAC addition, these were higher in mixed-biofilms with Pseudomonas fluorescens. However, a high number of released viable cells was observed after combined treatments. These results open new perspectives of enzymes as an anti-biofilm strategy for environmental pathogen control.

Listeria monocytogenes-carrying consortia in food industry. Composition, subtyping and numerical characterisation of mono-species biofilm dynamics on stainless steel.

In order to find out how real Listeria monocytogenes-carrying biofilms are in industrial settings, a total of 270 environmental samples belonging to work surfaces from fish (n = 123), meat (n = 75) and dairy industries (n = 72) were analysed in order to detect L. monocytogenes. 12 samples were positive for L. monocytogenes and a total of 18 different species were identified as accompanying microbiota in fish and meat industry. No L. monocytogenes was found in samples from dairy industry. Molecular characterisation combining results of AscI and ApaI macrorestriction PFGE assays yielded 7 different subtypes of L. monocytogenes sharing in 71.43% of cases the same serogroup (1/2a-3a). Results from dynamic numerical characterisation between L. monocytogenes monospecies biofilms on stainless steel (SS) using MATLAB-based tool BIOFILMDIVER demonstrated that except in isolate A1, in which a significant increase in the percentage of covered area (CA), average diffusion distance (ADD) and maximum diffusion distance (MDD) was observed after 120 h of culture, no significant differences were observed in the dynamics of the rest of the L. monocytogenes isolates. Quantitative dual-species biofilm association experiments performed on SS indicated that L. monocytogenes cell counts presented lower values in mixed-species cultures with certain species at 24 and 48 h compared with mono-species culture. However, they remained unaltered after 72 h except when co-cultured with Serratia fonticola which presented differences in all sampling times and was also the dominant species within the dual-species biofilm. When considering frequency of appearance of accompanying species, an ecological distribution was demonstrated as Escherichia coli appeared to be the most abundant in fish industry and Carnobacterium spp. in meat industry.