Molecular analysis of the role of osmolyte transporters opuCA and betL in Listeria monocytogenes after cold and freezing stress.

Listeria monocytogenes is a food-borne pathogen of humans and other animals. The striking ability to survive several stresses usually used for food preservation makes L. monocytogenes one of the biggest concerns to the food industry. This ubiquity can be partly explained by the ability of the organism to grow and persist at very low temperatures, a consequence of its ability to accumulate cryoprotective compound called osmolytes. A quantitative RT-PCR assay was used to measure mRNA transcript accumulation for the stress response genes opuCA and betL (encoding carnitine and betaine transporters, respectively) and the housekeeping gene 16S rRNA. Assays were conducted on mid-exponential phase L. monocytogenes cells exposed to conditions reflecting cold and freezing stress, conditions usually used to preserve foods. We showed that expression of the two cold-adapted genes encoded the transporters of the cryoprotectants carnitine and betaine in ATCC 19115 and the food-isolated L. monocytogenes S1 is induced after cold and freezing stress exposure. Furthermore, transcriptional analysis of the genes encoding opuCA and betL revealed that each transporter is induced to different degrees upon cold shock of L. monocytogenes ATCC 19115 and S1. Our results confirm an increase in carnitine uptake at low temperatures more than in betaine after cold-shocked temperature compared to the non-stress control treatment. It was concluded the use of carnitine and betaine as cryoprotectants is essential for rapid induction of the tested stress response under conditions typically encountered during food preservation.

Antibiofilm formation and anti-adhesive property of three mediterranean essential oils against a foodborne pathogen Salmonella strain.

Plant extracts, and their essential oils (EOs) are rich in a wide variety of secondary metabolites with antimicrobial properties. Our aim was to determine the bioactive compound in three mediterranean essential oils belonging to Lamiaceae family, Satureja montana L., Thymus vulgaris L. and Rosmarinus officinalis L., and to assess their antimicrobial, antibiofilm and anti-adhesive potentials against a foodborne pathogen Salmonella strain. The antibacterial activity of EOs and its biofilm inhibition potencies were investigated on 2 reference strains Salmonella typhimurium and 12 Salmonella spp. isolated from food. Biofilm inhibition were assessed using the 2, 3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) reduction assay. The analytical data indicated that various monoterpene hydrocarbons and phenolic monoterpenes constitute the major components of the oils, but their concentrations varied greatly among the oils examined. Our results showed that S. montana L. and T. vulgaris L. essential oils possess remarkable anti biofilm, anti-adhesive and bactericidal properties, compared to R. officinalis EO. There is an indication that Rosmary EO might inhibit biofilm formation at higher concentrations. Therefore, the witer savory and thyme EOs represent a source of natural compounds that exhibit potentials for use in food systems to prevent the growth of foodborne bacteria and extend the shelf life of the processed food.

Anti-bacterial and anti-biofilm activity of probiotic bacteria against oral pathogens.

In this study, three lactic acid bacteria (LAB), isolated from barley, traditional dried meat and fermented olive were characterized and tested for their anti-bacterial and anti-biofilm activities against oral bacteria. Our results revealed that the tested LAB were γ-hemolytic and were susceptible to four antibiotics. All the strains were resistant to low pH, bile salt, pepsin and pancreatin. Furthermore, FB2 displayed a high aut-oaggregative phenotype (99.54%) while FF2 exhibited the best co-aggregation rate. Concerning the microbial adhesion to solvent, FB2 was the most hydrophobic strain (data obtained with chloroform and n-hexadecane). In addition Pediococcus pentosaceus FB2 and Lactobacillus brevis FF2 displayed a significant inhibitory effect against Streptococcus salivarius B468 (MIC = 10%). Moreover the selected strains were able to inhibit biofilm formation of Bacillus cereus ATCC14579 (MBIC50 = 28.16%) and S. salivarius B468 (MBIC50 = 42.28%). The selected LAB could be considered as candidate probiotics for further application in functional food and mainly in the prevention of oral diseases.

Use of juglone as antibacterial and potential efflux pump inhibitors in Staphylococcus aureus isolated from the oral cavity.

In this study the minimal inhibitory concentration (MICs) of tetracycline (Tet), erythromycin (Ery) and benzalkonium chloride (BC) in absence and in presence of a sub-MIC of juglone (Jug) were determined. In addition, the Ethidium bromide (EtBr) efflux assay was performed to assess the effect of Jug on EtBr cells accumulation. Our results showed a selective antimicrobial activity of Jug against the tested strains. A synergistic effect of Jug, drugs (Tet and Ery) and disinfectant (BC) was noticed with a reduction rate varied from 2 to 16-fold. In addition, the efflux of EtBr was inhibited depending on the Jug concentration. In the presence of Jug, a decrease in loss of EtBr from bacteria was observed. The concentration inducing 50 % of EtBr efflux inhibition after 15 min was about 182 µg ml-1 for S. aureus ATCC 25923, 236 µg ml-1 for S. aureus B193 and 195 µg ml-1 for S. aureus B456. It appears from this study that Jug may be used as a natural source for resistance-modifying activity in same bacteria.

Molecular detection of bacteria associated to dental caries in 4-12-year-old Tunisian children.

The occurrence of several microbial species in the oral cavity of 4-12-year-old Tunisian children was investigated. Samples were taken from 158 children (81 caries actives and 77 caries free). Genomic DNA was extracted and analyzed for the presence of 17 microbial species using a polymerase chain reaction assay. All samples were positive for at least one of the target microbial strains. Streptococcus mutans was the most prevalent species (76.5%) detected in genomic DNA collected from carious lesions. Other prevalent species were Candida spp (63%), Streptococcus salivarius (59%) and Streptococcus oralis (42%). The frequency of Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus casei-group in caries lesions was 29.5%, 34.5% and 22% respectively. Pathogenic bacteria such as Staphylococcus aureus was found in 28.5% of carious lesion samples compared to 15.5% in the control. Frequency of Porphyromonas endodontali, Actinomyces radicidentis and Treponema denticola recovery did not differ significantly between origins of samples. PCR analysis of genomic DNA detect various oral bacteria that differ between caries actives and caries-free children. In addition, the association of same aciduric bacteria (S. mutans, S. salivarius, L. acidophilus) and caries formation was noticed.

Influence of freezing stress on morphological alteration and biofilm formation by Listeria monocytogenes: relationship with cell surface hydrophobicity and membrane fluidity.

The morphological changes and adhesive property of three Listeria monocytogenes strains submitted to freezing stress (-20 °C) were studied. The atomic force micrographs showed a reduction in the cell size and an evolution to coccoid shape. The phenotypic slime production of L. monocytogenes and the expression of the adhesive gene were investigated before and after 10 months of incubation in salmon at -20°. Our results showed that after ten months, stressed stains become more adherent and able to produce slime. In addition, we noted that this pathogen presents same physiological changes to adapt to starvation conditions. The cellular fatty acids composition of adhered and floating cells of three L. monocytogenes strains was taken into consideration. The stressed strains presented different chain lengths and therefore an increase in the hydrophobicity level. Moreover, we noted that the adhesive property of L. monocytogenes strains affects the Benzalkonium chloride bacterial sensitivity which increased after biofilm formation.

Modulation of drug resistance and biofilm formation of Staphylococcus aureus isolated from the oral cavity of Tunisian children.

OBJECTIVES: This study aims to investigate the antimicrobial and the anti-biofilm activities of Lactobacillus plantarum extract (LPE) against a panel of oral Staphylococcus aureus (n=9) and S. aureus ATCC 25923. The in vitro ability of LPE to modulate bacterial resistance to tetracycline, benzalchonium chloride, and chlorhexidine were tested also. METHODS: The minimum inhibitory concentrations (MICs) and the minimal bactericidal concentrations of Lactobacillus plantarum extract, tetracycline, benzalchonium chloride and clohrhexidine were determined in absence and in presence of a sub-MIC doses of LPE (1/2 MIC). In addition, the LPE potential to inhibit biofilm formation was assessed by microtiter plate and atomic force microscopy assays. Statistical analysis was performed on SPSS v. 17.0 software using Friedman test and Wilcoxon signed ranks test. These tests were used to assess inter-group difference (p<0.05). RESULTS: Our results revealed that LPE exhibited a significant antimicrobial and anti-biofilm activities against the tested strains. A synergistic effect of LPEs and drug susceptibility was observed with a 2-8-fold reduction. CONCLUSION: LPE may be considered to have resistance-modifying activity. A more detailed investigation is necessary to determine the active compound responsible for therapeutic and disinfectant modulation.

Survival of Listeria monocytogenes cells and the effect of extended frozen storage (-20°C) on the expression of its virulence gene.

Listeria monocytogenes is the main bacteria incriminated in contaminating cold storage food and ready-to-eat products. Survival of four L. monocytogenes food isolates was examined during freezing and cold storage. Slices of artisanal cheese were inoculated with Listeria strains and incubated at -20 °C. Their survival rate was recorded during the storage period. Then, transcript levels of four virulence genes (hlyA, iap, fri, and flaA) were evaluated using a semiquantitative reverse transcriptase PCR. Our results revealed that after 6 months of frozen storage, the number of L. monocytogenes cells had declined by 2.04 ± 0.1 log10 colony-forming units (CFU) g(-1), 2.52 ± 0.1 log10 CFU g(-1), and 2.58 ± 0.1 log10 CFU g(-1) for the strains S2, S3, and S4, respectively. Our data revealed that all the studied genes (hlyA, iap, fri, and flaA) were expressed after 6 months of incubation in artisanal cheese at -20 °C, and the transcript level has been affected by the factor freezing, whether for the reference strain ATCC 19115 or for other isolates. The iap gene expression was decreased for the four strains after 6 months of storage, and hlyA expression rate was consistently slightly lower. Transcript levels of fri and flaA genes were consistently higher in L. monocytogenes cells before cold exposure than in stressed cells and were significantly affected by the freezing process.

Modulation of drug resistance and biofilm formation of Staphylococcus aureus isolated from the oral cavity of Tunisian children

Abstract Objectives: This study aims to investigate the antimicrobial and the anti-biofilm activities of Lactobacillus plantarum extract (LPE) against a panel of oral Staphylococcus aureus (n = 9) and S. aureus ATCC 25923. The in vitro ability of LPE to modulate bacterial resistance to tetracycline, benzalchonium chloride, and chlorhexidine were tested also. Methods: The minimum inhibitory concentrations (MICs) and the minimal bactericidal concentrations of Lactobacillus plantarum extract, tetracycline, benzalchonium chloride and clohrhexidine were determined in absence and in presence of a sub-MIC doses of LPE (1/2 MIC). In addition, the LPE potential to inhibit biofilm formation was assessed by microtiter plate and atomic force microscopy assays. Statistical analysis was performed on SPSS v. 17.0 software using Friedman test and Wilcoxon signed ranks test. These tests were used to assess inter-group difference (p < 0.05). Results: Our results revealed that LPE exhibited a significant antimicrobial and anti-biofilm activities against the tested strains. A synergistic effect of LPEs and drug susceptibility was observed with a 2–8-fold reduction. Conclusion: LPE may be considered to have resistance-modifying activity. A more detailed investigation is necessary to determine the active compound responsible for therapeutic and disinfectant modulation.