Evaluation of biofilm removal in hospitals environment using different cleaning solutions with and without DNase.

peer reviewedABSTRACT Background Healthcare professionals are increasingly aware of the risk for patient’s safety of microbial biofilms present in hospital environments such as medical devices, water treatment systems, surfaces, etc (Muhammad et al., 2020). In several industrial sectors (food, pharmaceutics, cosmetics, etc) cleaning solutions containing a mix of efficient enzymes, including DNase, have been shown to achieve efficient removal of different types of biofilms (Coughlan et al., 2016). This study aims to evaluate several commercial cleaning solutions with or without DNase to eradicate the main biofilms implicated in hospital environments. Method The model used to assess biofilm removal was described by Iglesias and al. (2019) and adapted to include other bacterial strains encountered in healthcare environments. Briefly, strains were grown for 24h (Staphylococcus aureus, ATCC33591), or 48h (Pseudomonas aeruginosa, ATCC27853, and Escherichia coli, ATCC25922) at 37°C in 96-well microplates. Six cleaning solutions, one of which including DNase, were then applied to selected wells according to the plate template. Crystal violet staining was finally applied to measure biomass reduction. Inside the same plate, every enzymatic solutions were tested six times and each plate was tested four times. The results obtained were processed graphically with Box Plots and statistically with nonparametric tests (Kruskal Wallis and Wilcoxon tests). Results For S. aureus, the results show that only the enzymatic cleaning solution containing DNase allows an efficient biofilm removal with a reduction of 48.96% compared to the control (p-value < 0.0001). For P. aeruginosa, the results indicate that the solution containing DNase has the best removal efficiency with a reduction of 65.49% (p-value = 0.0088). Biofilm reduction was also measured for two other cleaning solutions, but these results were not reproducible on all plates. For E. coli, the results revealed once again that the product with DNase showed significant removal values with 34.59% of reduction (p-value = 0.0046). Figure 1 illustrates a summary of the results obtained for S. aureus for each solution tested, including the biomass measurements after the treatments and the statistical test results compared to the control. Figure 1: Box plot of absorbance values (570nm) obtained with crystal violet staining assay for S. aureus biofilms treated with different cleaning solutions Conclusions This study aimed to assess several commercial cleaning solutions for the removal of biofilms encountered in hospital environments. The enzymatic cocktail containing DNase shows a systematic efficiency whatever biofilms type. Other cleaning solutions do not or only partially remove biofilms and moreover, some of them, might increase the biofilms

Molecular effective quantification of pathogens and total flora in meat products

CONSALI

Growth monitoring of Listeria monocytogenes and Salmonella spp. according to the packaging technique in pork minced meat

A high level of protection of public health is one of the fundamental objectives of European food laws, as laid down in Regulation (EC) No 2073/2005. Listeria monocytogenes is a pathogen found within the food-processing industries, mainly brought by human, and was responsible of 1,381 confirmed cases of foodborne listeriosis for 2008 in Europe. Salmonella was in 2008, the second most often reported zoonotic disease in humans, and 131,468 confirmed cases of human salmonellosis were reported in Europe. The aim of the present work was to develop and to validate a quicker and more sensitive genetic method for quantification of L. monocytogenes and Salmonella spp. in meat products by the quantitative real-time PCR. After preliminary tests for primers and probes choice, the genetic method was validated with classical microbial methods (ISO 11290-2:1998, ISO 6579) using challenge tests. A mixture of 3 strains was realized for each bacterial genus: 1 referee strain (L. monocytogenesT NTCT 11994 or S. TyphimuriumT ATCC 14028) and 2 lab isolates. Experiments were carried out on pork's irradiated and not irradiated minced meat. These meats were packaged either in expanded polystyrene trays wrapped with permeable stretch film or under modified atmosphere (70% O2/30% CO2) in sealed trays. The initial inoculum (102 cfu.g-1) was homogenized in minced meat before packaging in trays. They were incubated for 14 days, at +5, +8 and +10°C and at +10 and +12°C, for respectively L. monocytogenes and Salmonella spp. In the 2 tested meats, growth speeds as well as the final populations increased according to the temperature. In the not irradiated minced meat, growth speeds of the pathogenic flora as well as the associated final populations were lower than those observed in the irradiated matrices. As soon as the total flora reached its stable growth phase, growths of the various pathogenic stagnated. The growth of the original flora inhibited partially the growth of pathogens inoculated. Generally, growth of the total flora is partially inhibited by modified atmosphere packaging. The analytical methods of molecular biology allow faster and less heavy analyses, in terms of hand of work and execution, than the methods of classic microbiology.CONSALI

Growth monitoring of Listeria monocytogenes and Salmonella spp. according to the packaging technique in pork minced meat

A high level of protection of public health is one of the fundamental objectives of European food laws, as laid down in Regulation (EC) No 2073/2005. Listeria monocytogenes is a pathogen found within the food-processing industries, mainly brought by human, and was responsible of 1,381 confirmed cases of foodborne listeriosis for 2008 in Europe. Salmonella was in 2008, the second most often reported zoonotic disease in humans, and 131,468 confirmed cases of human salmonellosis were reported in Europe. The aim of the present work was to develop and to validate a quicker and more sensitive genetic method for quantification of L. monocytogenes and Salmonella spp. in meat products by the quantitative real-time PCR. After preliminary tests for primers and probes choice, the genetic method was validated with classical microbial methods (ISO 11290-2:1998, ISO 6579) using challenge tests. A mixture of 3 strains was realized for each bacterial genus: 1 referee strain (L. monocytogenesT NTCT 11994 or S. TyphimuriumT ATCC 14028) and 2 lab isolates. Experiments were carried out on pork's irradiated and not irradiated minced meat. These meats were packaged either in expanded polystyrene trays wrapped with permeable stretch film or under modified atmosphere (70% O2/30% CO2) in sealed trays. The initial inoculum (102 cfu.g-1) was homogenized in minced meat before packaging in trays. They were incubated for 14 days, at +5, +8 and +10°C and at +10 and +12°C, for respectively L. monocytogenes and Salmonella spp. In the 2 tested meats, growth speeds as well as the final populations increased according to the temperature. In the not irradiated minced meat, growth speeds of the pathogenic flora as well as the associated final populations were lower than those observed in the irradiated matrices. As soon as the total flora reached its stable growth phase, growths of the various pathogenic stagnated. The growth of the original flora inhibited partially the growth of pathogens inoculated. Generally, growth of the total flora is partially inhibited by modified atmosphere packaging. The analytical methods of molecular biology allow faster and less heavy analyses, in terms of hand of work and execution, than the methods of classic microbiology.CONSALI

Assessment of bacterial superficial contamination in classical or ritually slaughtered cattle using metagenetics and microbiological analysis

The aim of this study was to investigate the influence of the slaughter technique (Halal vs. Classical slaughter) on the superficial contamination of cattle carcasses, by using traditional microbiological procedures and 16S rDNA metagenetics. The purpose was also to investigate the neck area to identify bacteria originating from the digestive or the respiratory tract. Twenty bovine carcasses (10 from each group) were swabbed at the slaughterhouse, where both slaughtering methods are practiced. Two swabbing areas were chosen: one “legal” zone of 1,600 cm2 (composed of zones from rump, flank, brisket and forelimb) and locally on the neck area (200 cm2). Samples were submitted to classical microbiology for aerobic Total Viable Counts (TVC) at 30°C and Enterobacteriaceae counts, while metagenetic analysis was performed on the same samples. The classical microbiological results revealed no significant differences between both slaughtering practices; with values between 3.95 and 4.87 log CFU/100 cm2 and 0.49 and 1.94 log CFU/100 cm2, for TVC and Enterobacteriaceae respectively. Analysis of pyrosequencing data showed that differences in the bacterial population abundance between slaughtering methods were mainly observed in the “legal” swabbing zone compared to the neck area. Bacterial genera belonging to the Actinobacteria phylum were more abundant in the “legal” swabbing zone in “Halal” samples, while Brevibacterium and Corynebacterium were encountered more in “Halal” samples, in all swabbing areas. This was also the case for Firmicutes bacterial populations (families of Aerococcaceae, Planococcaceae). Except for Planococcoceae, the analysis of Operational Taxonomic Unit (OTU) abundances of bacteria from the digestive or respiratory tract revealed no differences between groups. In conclusion, the slaughtering method does not influence the superficial microbiological pattern in terms of specific microbiological markers of the digestive or respiratory tract. However, precise analysis of taxonomy at the genus level taxonomy highlights differences between swabbing areas. Although not clearly proven in this study, differences in hygiene practices used during both slaughtering protocols could explain the differences in contamination between carcasses from both slaughtering groups

Development of a tertiary model for predicting the growth of Listeria monocytogenes and Salmonella spp. in pork minced meat

peer reviewedTertiary models are proposed in order to predict the growth of Listeria monocytogenes and Salmonella spp. in pork minced meat packaged under stretch film. The models have been calculated from challenge-tests at 8°C (L. monocytogenes) and 12°C (Salmonella), the meat being artificially contaminated at 2 log cfu pathogen/g. In a second step, they have been validated at 5, 8 and 10°C for L. monocytogenes (r² : 0.94, 0.98 and 0.95) and 8, 10 and 12°C for Salmonella (r³ : 0.80, 0.92 and 0.98).Des modèles tertiaires sont proposés pour prédire la croissance de Listeria monocytogenes et Salmonella spp. dans de la viande de porc hachée conditionnée sous film étirable. Les modèles ont été calculés à partir des résultats de tests de croissance réalisés à 8°C (L. monocytogenes) et 12°C (Salmonella) avec de la viande artificiellement contaminée avec 2 log ufc pathogène/g. Dans un deuxième temps, ils ont été validés à 5, 8 et 10°C pour L. monocytogenes (r² : 0,94, 0,98 et 0,95) et 8, 10 and 12°C pour Salmonella (r³ : 0,80, 0,92 et 0,98).CONSALI