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14 pages, 5 figures, 5 tables.-- Under a Creative Commons license, 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, The authors are grateful to EU PRIMA program and the International Joint Programming (Project Reference PCI 2019-103453) R&D Projects 2019from the Spanish Ministry of Science and Innovation (Plan Estatal de Investigación Científica y Técnica y de Innovación 2017–2020: State R&D Program Oriented to the Challenges of the Society) for funding the ArtiSaneFood project (PRIMA/0001/2018)

Unravelling the mechanisms of action of disinfectants is essential to optimise dosing regimes and minimise the emergence of antimicrobial resistance. In this work, we examine the mechanisms of action of a commonly used disinfectant - benzalkonium chloride (BAC)-over a significant pathogen -L. monocytogenes- in the food industry. For that purpose, we use modelling at multiple scales, from the cell membrane to the cell population inactivation. Molecular modelling reveals that the integration of the BAC into the membrane requires three phases: (1) the BAC approaches the cellular membrane, (2) the BAC is adsorbed on its surface, and (3) it is rapidly integrated into the lipid bilayer, where it remains at least for several nanoseconds, probably destabilising the membrane. We hypothesise that the equilibrium of adsorption, although fast, is limiting for sufficiently large BAC concentrations, and a kinetic model is derived to describe time-kill curves of a large population of cells. The model is tested and validated with time series data of free BAC decay and time-kill curves ofL. monocytogenesat different inocula and BAC dose concentrations. The knowledge gained from the molecular simulation plus the proposed kinetic model offers the means to design novel disinfection processes rationally.

9 pages, 7 figures, 4 tables.-- Under a Creative Commons license, In nature, bacterial pathogens like L. monocytogenes, live in nature associated with other microbial species in spatially-structured communities called biofilms. In the food industry, biofilms contribute to the survival and persistence of L. monocytogenes within processing facilities, thereby enhancing its risk of cross-contaminating food products. The challenge of combating biofilms has triggered the search for new antibiofilm strategies including devising ways to interfere with cell communication mechanisms (quorum-sensing) that are known to be involved with biofilm development regulation. The aim of this study was to explore cell communication signals in a L. monocytogenes-carrying microbial community isolated from a meat processing plant (location No. 96) in order to elucidate the ecological interactions that could serve as a starting point for the development of new antibiofilm strategies. Quorum quenching (QQ) and quorum sensing (QS) activities were screened among 31 bacterial strains isolated from location No. 96. Whereas no QQ activity was detected against short-chain lactone N-hexanoyl-DL-homoserine lactone (C6-HSL), it was detected against N-dodecanoyl-DL-homoserine lactone (C12-HSL) in 7 isolates (23%), particularly in Pseudomonas monteilli, Rhodococcus sp. and Rhodococcus erythropolis. QS activity assays detected HC4, C4, C6, OC6, HC10 and C16 in all the extracts, being C4, C6 and OC6 with predominantly produced by Pseudomonas monteilli, Pseudomonas gesardii, Psychrobacter maritimus and Paracoccus sp. High production levels of C16-HSL by Paracoccus sp. and the role of this long-chain lactone as a self-inhibitor of cell aggregation led us to carry out further studies focused on the effects of a Paracoccus lactone extract (PLE) against the biofilm formation by L. monocytogenes. A quantitative microscopic analysis demonstrated a significant decrease (p < 0.05) in the area occupied by biofilms formed on stainless steel (SS) coupons by different strains of L. monocytogenes in the presence of PLEs. Conversely, no significant differences were observed in the total number of viable adhered cells on SS coupons with or without PLE. The observed effect was partially reproduced by the addition of pure C16-HSL to 24 h-biofilms of L. monocytogenes L1.96. These results demonstrate that the observed effects can be attributed, at least partially, to the HSLs contained in the PLE. Overall, the present results highlight how interspecies communication within a biofilm can open up new insights for the development of new ways to combat biofilm, This research was financially supported by the Spanish Ministry of Science and Innovation (SOLISTA, AGL2016-78459-P)

Optimal disinfection protocols are fundamental to minimize bacterial resistance to the compound applied, or cross-resistance to other antimicrobials such as antibiotics. The objective is twofold: guarantee safe levels of pathogens and minimize the excess of disinfectant after a treatment. In this work, the disinfectant dose is optimized based on a mathematical model. The model explains and predicts the interplay between disinfectant and pathogen at different initial microbial densities (inocula) and dose concentrations. The study focuses on the disinfection of Escherichia coli with benzalkonium chloride, the most common quaternary ammonium compound. Interestingly, the specific benzalkonium chloride uptake (mean uptake per cell) decreases exponentially when the inoculum concentration increases. As a consequence, the optimal disinfectant dose increases exponentially with the initial bacterial concentration.