Etude du microbiote susceptible de persister sur les surfaces d'un atelier de la filière viande bovine

Abstract : The aim of this work is to acquire a better knowledge of the microbial ecology of a beef processing plant to understand bacterial persistence, e.g. the presence of a clone isolated several times on several visits in the same processing plant despite regular Cleaning and disinfection (C&D) procedures. Successive swabbing were performed on a PVC conveyor belt and skinning machines made of stainless steel before and after C&D during three surveys in minimal 6 month-intervals. Total cells (live and dead cells) were quantified using real-time quantitative PCR (qPCR). Viable cells e.g. cells with intact membrane, were assessed using Ethidium Monoazide combined with qPCR. Culturable cells (CFU) were determined from plate counts on Tryptone Soy Agar. Before C&D, total cells (5.6 log cells/cm2 and 4.7 log10 cells/cm2 on PVC and stainless steel respectively) were greater than viable cells (4.5 and 4.4 log10 cells/cm2) and CFUs (3.8 and 2.9 log10 CFU/cm2). C&D lead to less than 1 log10 reduction in bacterial populations except for CFU counts on stainless steel where a 1.5 log reduction is observed. This result is highlighted by the weak attachment strengths observed on stainless steel for CFUs. Identification of the culturable microbiota revealed that out of 51 genera identified, 13 were found at all the visits. These genera represented 75, 72 and 62% of the total isolates. The most frequently identified bacteria were Pseudomonas, Staphylococcus, Microbacterium, Acinetobacter, Chryseobacterium, Psychrobacter and Kocuria. Molecular typing of three dominant genera (Staphylococcus, Pseudomonas and Acinetobacter) showed that only one strain, Staphylococcus equorum, was persistent in the premises. Our results show that the microbial ecosystem is different from one survey to another, which reflect the various geographical origins of meat products. Contrary to widespread belief, Gram negative strains were more easily eliminated by C&D than Gram positive strains. Furthermore, the microbial diversity assessed by PCR-DGGE allowed the identification of 7 genera. This molecular approach showed that dominant species are all in a viable state: none of these species was solely detected in a dead state. Of the 7 genera identified, 6 were Gram negative, Acinetobacter, Pseudomonas and Psychrobacter being predominant. This result highlights that C&D induced the lost of culturability of Gram negative bacteria although a high proportion was not detached from the surface. The predominance of Gram negative microflora, didn’t allow the detection of staphylococcal isolates which were numerous in the culturable microflora. One genus, Propionibacterium, isolated in one survey on stainless steel before C&D was only identified by PCR-DGGE. In conclusion, the present study has demonstrated that a large proportion of bacteria can survive drastic cleaning and disinfection for a transient period.
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Elissa Khamisse. Etude du microbiote susceptible de persister sur les surfaces d'un atelier de la filière viande bovine. Sociologie. AgroParisTech, 2012. Français. ⟨NNT : 2012AGPT0036⟩. ⟨pastel-00770326⟩

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