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The aim of the present study is to isolate, characterize and identify a novel strain ZC from the Metlaoui phosphate laundries wastewater (MPLW). The chemical characterization of this phosphate rich effluent showed an alkaline pH and is saline, highly turbid and rich in suspended matter and total solids. The MPLW samples were loaded with potentially toxic metals, presented in decreasing order as follows: magnesium (5655 mg L−1), potassium (45 mg L−1), lead (1 mg L−1), iron (0.7 mg L−1), cadmium (0.5 mg L−1), copper (0.3 mg L−1) and zinc (0.1 mg L−1). Due to the high COD/BOD5 ratio, a poorly biodegradable organic load is underlining. The newly isolated strain was identified as Lysinibacillus fusiformis using 16S rDNA sequencing analysis. The viability of this new strain was tested in presence of the zinc, lead, cadmium, manganese and copper at 1, 10 and 100 mM. The L. fusiformis survival, under metallic stress, was inversely proportional to metal ion concentrations, while lead and zinc were the most toxic ones using MTT assay. Then, the newly isolated strain was characterized in terms of enzyme production, proteomic alteration and antibiotic resistance. The strain ZC revealed some modifications in the biochemical and enzymatic profiles by either the appearance or/and the disappearance of some activities. In addition, the increase in metal ions stress and concentrations was proportional to the adherence and to the hydrophobicity. The presence of the metal ions suggested the change of sensitivity to the resistance of this strain towards tobramycin, kanamycin, neomycin, netilmicin and cefoxitin, showing an increase in the MARindex. The strain ZC, used as a biological tool for MPLW treatment, showed a reduction in the metal ion contents. This reduction was due to accumulation and/or adsorption, showing a bioprocessing performance of the newly isolated L. fusiformis.

The rapid and accurate detection of pathogenic agents represents a milestone for precocious diagnosis and personalized therapy in nosocomial infections. The issue can be addressed by the rational identification of certain bacteria markers, such as siderophores, that are highly involved in their metabolism and also in their interactions with the host. Developing sensors with a high sensitivity and selectivity towards this type of compounds could be an important starting point in the direction of a rapid differential diagnosis of bacterial infections. Herein we report the elaboration of a nanohybrid sensing platform based on graphene, polypyrrole and gold for the detection of pyoverdine, a marker for Pseudomonas aeruginosa. The composite material showed a catalytic effect towards the electronic transfer rate and enhanced active surface area, displaying a limit of detection of 0.33 μM and a linear range of 1–100 μM being obtained. The sensor was tested in the presence of common interfering molecules, found in hospital environments or in biological samples and excellent recovery rates were obtained. The selectivity of the sensor was also proved to be successful when testing on real samples such as human serum, saliva and tap water. Keywords: Nosocomial infections, Pseudomonas aeruginosa, Pyoverdine, Sensor, Electrochemical detection

Disease outbreaks related to waterborne pathogen contamination throughout the world as well as challenges that lie ahead for addressing persistent infection are of renewed interest. In this research, we studied the effects of prolonged exposure of Salmonella enterica serovar Typhimurium to the cues encountered in the extracellular environment particularly in seawater microcosm on bacterial virulence and subsequent infection in Caco-2 cells. Our data show a significant difference in biofilm formation, swimming and swarming motilities between normal and stressed cells of S. Typhimurium under differing NaCl conditions (P

© The Author(s)., [Background]: In nature, microorganisms have to adapt to long-term stressful conditions often with growth limitations. However, little is known about the evolution of the adaptability of new bacteria to such environments. Pseudomonas aeruginosa, an opportunistic pathogen, after natural evaporation of seawater, was shown to be trapped in laboratory-grown halite crystals and to remain viable after entrapment for years. However, how this bacterium persists and survives in such hypersaline conditions is not understood., [Results]: In this study, we aimed to understand the basis of survival, and to characterise the physiological changes required to develop salt tolerance using P. aeruginosa as a model. Several clones of P. aeruginosa were rescued after 14 years in naturally evaporated marine salt crystals. Incubation of samples in nutrient-rich broth allowed re-growth and subsequent plating yielded observable colonies. Whole genome sequencing of the P. aeruginosa isolates confirmed the recovery of the original strain. The re-grown strains, however, showed a new phenotype consisting of an enhanced growth in growing salt concentration compared to the ancestor strain. The intracellular accumulation of K+ was elicited by high concentration of Na+ in the external medium to maintain the homeostasis. Whole transcriptomic analysis by microarray indicated that 78 genes had differential expression between the parental strain and its derivative clones. Sixty-one transcripts were up-regulated, while 17 were down-regulated. Based on a collection of single-gene knockout mutants and gene ontology analysis, we suggest that the adaptive response in P. aeruginosa to hyper-salinity relies on multiple gene product interactions., [Conclusions]: The individual gene contributions build up the observed phenotype, but do not ease the identification of salinity-related metabolic pathways. The long-term inclusion of P. aeruginosa in salt crystals primes the bacteria, mediating a readjustment of the bacterial physiology to growth in higher salt concentrations. Our findings provide a starting point to understand how P. aeruginosa, a relevant environmental and pathogenic bacterium, survives to long-term salt stress., This work was supported by Plan Nacional de I+D+i 2013-2016 and the Instituto de Salud Carlos III, Subdireccion General de Redes y Centros de Investigacion Cooperativa, Ministerio de Economia, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0009)—co-financed by the European Development Regional Fund “A Way to Achieve Europe”—by Operative Program Intelligent Growth 2014-2020. ARR was also supported SFB 973 (Deutsche Forschungsgemeinschaft, project C5). We acknowledge support by the German Research Foundation and the OpenAccess Publication Fund of Freie Universität Berlin.

The use of response surface methodology based on statistical design of experiments is becoming increasingly widespread in several sciences such as analytical and environmental chemistry. In the present study, the decolourization and the degradation efficiency of CI Reactive Violet 5 (CIRV5) was studied using a novel bacterial consortium. CIRV5 (1000 p.p.m.) biodegradation was investigated under shaking condition in mineral salt medium solution (MSM) at a 7.5 pH and a 25°C temperature. The degradation pathways were also predicted, using UV-visible spectroscopy analysis. Under optimal conditions, the bacterial consortium was able to decolourize the dye completely (>99%) within 8 h. The colour and chemical oxygen demand (COD) removal were 99.29 and 94.93%, respectively. Polymerase chain reaction (PCR) technique was utilized to detect the adhesin genes ‘icaA’ and ‘icaD.’ Our results showed that Staphylococcus aureus had a high decolourization capacity. Phytotoxicity study using Triticum turgidum ssp durum showed the no toxicity of the produced products.

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.

Even with advances in molecular cloning and DNA transformation, new or alternative methods that permit DNA penetration in Salmonella enterica subspecies enterica serovar Typhimurium are required in order to use this pathogen in biotechnological or medical applications. In this work, an adapted protocol of bacterial transformation with plasmid DNA based on the "Yoshida effect" was applied and optimized on Salmonella enterica serovar Typhimurium LT2 reference strain. The plasmid transference based on the use of sepiolite as acicular materials to promote cell piercing via friction forces produced by spreading on the surface of a hydrogel. The transforming mixture containing sepiolite nanofibers, bacterial cells to be transformed and plasmid DNA were plated directly on selective medium containing 2% agar. In order to improve the procedure, three variables were tested and the transformation of Salmonella cells was accomplished using plasmids pUC19 and pBR322. Using the optimized protocol on Salmonella LT2 strain, the efficiency was about 10(5) transformed cells per 10(9) subjected to transformation with 0.2μg plasmid DNA. In summary, the procedure is fast, offers opportune efficiency and promises to become one of the widely used transformation methods in laboratories.

Introduction: The pathogenic bacterium Salmonella enterica serovar Typhimurium elicits a variety of genetic programs to adapt to stress conditions encountered within hostile environments such as host phagocytes and preserved food. Methodology: In this work, differential display (DD) methodology was used to investigate the effect of one month starvation in a salty microcosm (0.5 M NaCl) on transcript profiling in a Salmonella Typhimurium LT2 strain. cDNA fragments resulting from differentially expressed mRNA were eluted from the gel, re-amplified, cloned, and then sequenced. Results: A total of 21 differentially expressed bands were detected by DD reverse transcription-polymerase chain reaction (RT-PCR). However, only 12 of them were successfully identified as upregulated genes in stressed cells. Based on the sequencing data and BLAST analysis, these genes were sopA , ssaD , yhhK , gmK , cspC , uspA , ompR , phoP , stcC , fimA , acrA ,and yehZ. As a confirmation of the differential expression, RT-PCR was carried out using a set of specific primers. Remarkably, the expression levels of these genes were significantly increased in starved bacteria compared to standard laboratory conditions. Conclusions: Our results indicate that the starvation of Salmonella Typhimurium over one month in a salty microcosm changes the expression of stress proteins, response regulator in a two-component system, outer membrane proteins, effector proteins translocated by Salmonella pathogenicity island SPI1 and SPI2 type III secretion systems (TTSS), several metabolic enzymes, efflux pumps, and transport proteins. This suggests that the expression of the identified genes is important for the response of this pathogen to starvation in salt.

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

Pathogenic Vibrio species are an important cause of foodborne illnesses. The aim of this study was to describe the occurrence of potentially pathogenic Vibrio species in the final effluents of a wastewater treatment plant and the risk that they may pose to public health. During the 1-year monitoring, a total of 43 Vibrio strains were isolated: 23 Vibrio alginolyticus, 1 Vibrio cholerae, 4 Vibrio vulnificus, and 15 Vibrio parahaemolyticus. The PCR investigation of V. parahaemolyticus and V. cholerae virulence genes (tlh, trh, tdh, toxR, toxS, toxRS, toxT, zot, ctxAB, tcp, ace, vpi, nanH) revealed the presence of some of these genes in a significant number of strains. Intraspecies variability and genetic relationships among the environmental isolates were analyzed by random amplified polymorphic DNA-PCR (RAPD-PCR). We report the results of the first isolation and characterization of an environmental V. cholerae non-O1 non-O139 and of a toxigenic V. parahaemolyticus strain in Tunisia. We suggest that non-pathogenic Vibrio might represent a marine reservoir of virulence genes that can be transmitted between strains by horizontal transfer.

Four strains of Staphylococcus aureus were incubated for eight months in seawater microcosms and then tested for their ability to form a biofilm on different surfaces. Our results showed that the stressed strain became safranin and increased their capacity for producing biofilm on polystyrene microplates or glass wells. These bacteria were able to form a biofilm on the surfaces tested and the cell density was the highest on the stainless steel surface followed by polystyrene and the polyethylene surfaces. These properties allow these bacteria to survive, proliferate and persist even after treatment with antibacterial agents.

International audience; The objective of this study was to evaluate the effect of 3-year starvation in seawater microcosms on adhesion to and biofilm formation of two Salmonella enterica serovar Typhimurium strains on model stainless steel 316 L and gold surfaces. The bacteria were characterized in terms of morphological alteration, electrophoretic mobility, and affinity to various solvent interfaces. Scanning electron micrographs showed the appearance of coccoid and elongated cells after starvation. All stressed cells were characterized by a hyperflagellation, a significant increase in the global surface charge, and a conservation of their hydrophilic character. Epifluorescence microscopy highlighted an increase in the levels of adhered cells to stainless steel and gold surfaces after starvation stress. Confocal laser scanning microscopy produced evidence of variability between the three-dimensional biofilm architectures of the control and stressed cells on gold compared to stainless steel. The results obtained so far led us to hypothesize that the pervasiveness of nutrient deficiency in natural environments may generate new adaptation strategies for long-term starved Salmonella Typhimurium and probably create protection against other types of stress. The stress adaptation mechanisms identified in this study may induce a genetic instability and change virulence state of starved bacteria. This fundamental study provides information which may aid in the development of sanitation programs for effective pathogen removal in the food industry or from medical devices. The task is certainly complex given that several concomitant physicochemical parameters affect the adhesion to and biofilm formation on model surfaces of stressed bacteria.

Bacillus species are ubiquitous and diverse both in the terrestrial and marine ecosystems. In this study, Bacillus species were isolated from Tunisian hypersaline environments and their probiotic properties were studied. In total, 13 strains were identified as a Bacillus spp. Antagonism assay revealed an inhibitory effect of these strains against pathogenic bacteria. Cell surface hydrophobicity values ranged between 0.33 and 30%. The tested strains were able to produce extracellular enzymes such as amylase, lipase, caseinase, and lecithinase. Qualitative analysis of biofilm results show that seven strains were able to produce slime on Congo red agar. Furthermore, the investigated strains were fairly adhesive to glass and polystyrene with values ranging from 0.07 to 0.70 at 570 nm. Key words: Bacillus spp., hypersaline, antagonism, extracellular enzymes, adhesion.

The objective of this work is to elaborate an immunosensing system which will detect and quantify Staphylococcus aureus bacteria. A gold electrode was modified by electrografting of 4-nitrophenyl diazonium, in situ synthesized in acidic aqueous solution. The immunosensor was fabricated by immobilizing affinity-purified polyclonal anti S. aureus antibodies on the modified gold electrode. Cyclic voltammetry (CV) and Faradaic Electrochemical Impedance Spectroscopy (EIS) were employed to characterize the stepwise assembly of the immunosensor. The performance of the developed immunosensor was evaluated by monitoring the electron-transfer resistance detected using Faradaic EIS. The experimental results indicated a linear relationship between the relative variation of the electron transfer resistance and the logarithmic value of S. aureus concentration, with a slope of 0.40 ± 0.08 per decade of concentration. A low quantification limit of 10±2 CFU per ml and a linear range up to 107±2×106 CFU per mL were obtained. The developed immunosensors showed high selectivity to Escherichia coli and Staphylococcus saprophyticus.

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.

Tunisian Juglans regia bark ethanol extract (JREE) was examined for antibacterial, antibiofilm, and antioxidant activity. Its anti-proliferative effect was evaluated on normal (MRC-5) and cancer (HT29 and HEp-2) cell lines. The data revealed that JREE inhibited the growth of pathogenic bacteria (MICs value ranged from 32 to 128 μg.mL-1) and induced a strong antibiofilm effect (BIC50 was 23, 30, and 43 μg.mL-1 for Micrococcus luteus, Staphylococcus epidermidis, and Staphylococcus aureus, respectively). The JREE showed high scavenging property against 2,2-diphenyl-l-1-picrylhydrazil (DPPH) radical (IC50 = 3.5 μg.mL-1) and exhibited anti-proliferative effects with IC50 values ranging from 6.6 to 25.5 μg.mL-1.

Three Lactobacilli were isolated from traditional Tunisian fermented food and then characterized for its ability to inhibit Listeria monocytogenes growth. Antagonistic effect of Lactobacillus plantarum on four L. monocytogenes strains was tested in soft artisanal cheese. Anti-biofilm activity of Lactobacillus extracts was also tested. Our results demonstrate that the selected lactic acid bacteria (LAB) extract exhibited a good antibacterial effect against L. monocytogenes ATCC 19115 with low MICs values: 8.33 2.1, 20 1.2 and 23.33 1, respectively. L. plantarum also exhibited a stronger inhibitory effect against L. monocytogenes when grown in cheese. Moreover, a potential anti-biofilm effect of the three LAB extracts with BIC50 values ranging from 5% to 15% for L. monocytogenes ATCC 19115 was demonstrated. Although LAB extracts were able to eradicate significantly a preformed L. monocytogenes biofilm (P < 0.05). Growth inhibition of preformed biofilm was more difficult to achieve. LAB could be used as a bioprotective culture in cheese ripening to prevent Listeria growth.

Staphylococcus aureus , showing the greatest decolorization ability, was further investigated for Methyl Red (MR) Congo Red (CR), Crystal Violet (CV) and Malachite Green (MG) decolorization using response surface methodology (RSM). The chemometric methods use, based on statistical design of experiments (DOEs) such as RSM is becoming increasingly widespread in several sciences such as analytical chemistry, engineering and environmental chemistry. Stapphylococcus aureus ATCC 25923, Stapphylococcus aureus (S1) and Stapphylococcus aureus (S2), were isolated from textile wastewater plant located in KsarHellal, Tunisia and were tested for their decolorization capacity. PCR technique was utilized to identify the 3 bacterial strains and to detect the adhesin gene “ cna” . Biodegradation of MR, CR, CV and MG (750 ppm), were investigated under shaking condition in Mineral Salt Medium (MSM) solution at pH 7.5 and temperature 30 °C, using a 3.7 × 10 5 CFU/ml as inoculum size. Our results showed that Staphylococcus aureus had a high decolorization capacity. Nuclear magnetic resonance (NMR) spectroscopy analysis confirmed the biodegradation of dyes. The four dyes mutagenicity with the S9 metabolizing system decreased significantly after biodegradation and totally disappeared. Nuclear magnetic resonance (NMR) spectroscopy analysis confirmed the biodegradation of dyes.

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.

Pathogenic bacteria such as Salmonella have the ability to respond to a wide variety of environmental stimuli. These responses allow them to survive and withstand insults both of an external location as well as within the host. The aim of this study was to investigate the effect of preadaptation in stressful conditions encountered in seawater microcosms for different periods of time on Salmonella Typhimurium survival, antibiotic susceptibility and interactions with Caco-2 cells. These results showed that the number of bacterial cells depends from the periods of stress in culture medium, highlighting the importance of using the right culture medium for the enumeration of stressed bacteria. The antibiotic resistance of starved cells was modified and their exposure to stressful conditions in seawater during 12 months significantly increased adhesion, invasion and cytotoxic activities on Caco-2 cells. Moreover, cellular cytokines IL-6 and IL-8 secretions were up-regulated. Present results seem to suggest that the preadaptation of S. Typhimurium in seawater microcosms affect the cultural characters by the appearance of the atypical cells that may play a critical role in the intestinal infection and in the systemic spread of the disease. These findings are very important to understand bacterial responses to changing conditions and explain the persistence of these atypical in eukaryotic cells.

Shigella sonnei encounter numerous different stresses during their growth, survival and infection. In this study, the effect of stress response to pH and starvation was investigated. We studied the survival, adhesion and the morphology of Shigella after its incubation in several pH. Our results show that after 2 h of incubation, the rate of cell survival was proportional to the decrease of pH of the medium, but we also noted that the reduction of the cells viability is significant for the normal cells compared to starved cells. In addition to that, the results proved the evidence that the pH can influence significantly the hydrophobicity of S. sonnei (normal and starved) and their capacity to produce biofilm on Congo red agar and on polystyrene microplate wells. The atomic force micrographs showed a reduction of the cells size after stress. Keywords : Shigella sonnei , starvation, pH, survival, adhesion, morphology African Journal of Biotechnology Vol. 12(16), pp. 2034-2040

Salmonella is an international foodborne pathogen widely disseminated in seawater that regularly causes large outbreaks of food poisoning. In this study, we have investigated the effect of starvation on the ability of Salmonella enterica serovar Typhimurium cells to adhere to polystyrene microplate and Hep2 cells in seawater microcosms after incubation for 3 years. Cell surface hydrophobicity was evaluated. Effect of stress on fatty acids composition was also established. Our results showed that after incubation in seawater, the ability of starved cells to adhere to polystyrene microplate was decreased significantly. However, the adhesion values to Hep2 cells have increased. In addition, cells surface hydrophobicity was decreased. The fatty acids composition of starved cells was modified.

Kombucha is widely consumed as black tea fermented for 7–14 days. The aim of the present study was to compare the antimicrobial activities of two kombucha beverages originating from green and black teas fermented for 21 days and to characterize the antimicrobial compounds (heat resistance and pH stability). Green and black tea infusions were fermented with a traditional kombucha culture. The resulting kombucha antibacterial/antifungal activities against some pathogenic microorganisms, including human pathogenic bacteria and clinical Candida species, were investigated using the agar diffusion method. The results showed interesting antimicrobial potentials of both experimented kombucha teas against the tested microorganisms, except Candida krusei. The green fermented tea exhibited the highest antimicrobial potential. Indeed, it showed large inhibition zones against Staphylococcus epidermidis (22 mm), Listeria monocytogenes (22 mm) and Micrococcus luteus (21.5 mm). Furthermore, interesting anti-Candida potential was revealed by the reaction of green tea kombucha against Candida parapsilosis. PRACTICAL APPLICATIONS The black fermented tea is the original and most popular preparation. This research has focused on the investigation of the antibacterial and antifungal activities of the kombucha prepared from green and black teas against a large number of human pathogens to determine and to compare the potential of the two kombucha drinks. The results showed a broad antimicrobial spectrum of kombucha against a range of pathogenic Candida involved in several candidoses. Moreover, the data showed that the antibacterial potential of kombucha prepared from green tea was higher than that of the original kombucha tea. Considering the antimicrobial activity demonstrated against a wide range of pathogenic bacteria and clinical Candida species, kombucha may be very healthful. As resistance to antimicrobial agents has become increasingly an important global health problem, these findings would be very promising and could be useful as an alternative to current synthetic antimicrobial drugs.

The cold shock protein family consists of the transfer of the foodborne pathogen Listeria monocytogenes from 37 to 4 and −20 °C and was characterized by the sharp induction of a low molecular mass protein. This major cold shock protein ferritin-like protein (Flp) has an important role in regulation of various microbial physiological processes. Flp have a molecular mass of about 18 kDa, as observed on SDS–PAGE. The purification procedure including ammonium sulfate fractionation was used. Monospecific polyclonal antibodies raised in rabbits against the purified new Flp immunostained a single 18-kDa Flp band in extracts from different cytoplasmic proteins blotted onto nitrocellulose. A 411-bp cDNA fragment that corresponds to an internal region of an flp gene was obtained by RT-PCR. Our result indicated a surexpression of major cold shock protein and an important increase in flp mRNA amount after a downshift temperature especially at −20 °C.

The purpose of the present study was to explore the potential behavioural changes of Pseudomonas aeruginosa following growth in different aquatic environmental conditions. To achieve this, P. aeruginosa was cultured in various water microcosms for 12 months under fixed (pH, nutrients and temperature) factors. P. aeruginosa responses to these conditions were investigated using colony morphotype, biochemical and enzymatic characterisation, pyocin typing, serotyping, sensitivity to different classes of antibiotics and molecular identification. Results show that starvation in water microcosms lead to unusual phenotypes. Of interest is that the pyocin changed from 24/n in the wild type to 83/a following culture in the water microcosms, and the serotype changed from O6 in the wild type to O1 in microcosm-cultured P. aeruginosa. Furthermore, the starvation period in various aquatic microcosms enhanced the resistance of P. aeruginosa against beta-lactam antibiotics. Compared to the other aquatic environments, the seawater microcosm produced the greatest amount of variations in P. aeruginosa. Overall, data demonstrated a high adaptability of P. aeruginosa to environmental changes. This may explain the unusual antibiotic-resistant phenotypes belonging to P. aeruginosa species, and their capacity for spreading that leads to human infections.

Salmonella is an international food-borne pathogen disseminated widely in seawater that regularly causes large outbreaks of food poisoning. In this study, we investigated the effect of starvation on the survival ability of Salmonella enterica serovar Typhimurium rpoS + and its mutant rpoS – after a 6-month incubation in seawater microcosms using a colorimetric XTT assay and colony forming units (CFU). The effect of pre-adaptation in seawater and the rpoS gene on the tolerance to heavy metals of this microorganism was also investigated. Our results showed that the number of cells decreased by ∼ 5 log units for the strain rpoS + and by ∼ 5.5 log units for the mutant rpoS – after this period of stress. At the end of the experiments, cell viability was around of 20% and 10% for the wild type and mutant, respectively. We also revealed that the minimal inhibitory concentration values and cell viability under zinc, cobalt, mercury, chromium and cadmium heavy metals were decreased for the two starved strains.

Cells of three strains of Listeria monocytogenes, one a reference strain ATCC 19115 and two strains isolated from meat, were cold shocked at refrigeration (+4C) and freezing (−20C) temperatures during one night. Then, their fatty acids were extracted and their composition identified by gas chromatography coupled with mass spectrometry. The results showed that low thermal adaptation response of L. monocytogenes ATCC 19115 was different from that of the two recent food isolates L. monocytogenes. The three experimented strains showed a decrease of anteiso-C17:0 and an increase of anteiso-C15:0 rates. In addition, after freezing, the cellular fatty acids were detected as a signature of the membrane changes that give rise to such authenticity. The structural modification seems to be an adaptation form, allowing intracellular ice crystallization enhancement during freezing and water mobility during storage. These results demonstrated that the L. monocytogenes studied strains showed various behaviors regarding low-temperature stress maintaining membrane fluidity. PRACTICAL APPLICATIONS Listeria monocytogenes is prevalent in a variety of foods and ready-to-eat products; it may generate a serious public health problem. Nowadays, ready-to-eat, chilled and frozen food consumption is continuously increasing. Bacterial strains that can survive at extremely low temperatures adopt special physiological strategies, although the cold-regulated fatty acid (FA) adjustment is a universal adaptation to low temperatures. To counteract this phenomenon, L. monocytogenes modifies its plasmatic membrane structure to maintain its fluidity at low temperatures, allowing an optimum membrane fluidity that is crucial for bacterial survival. Our study suggested that after freezing, these bacteria underwent several modifications, which are especially critical because they negatively affect bacterial viability and physiological state and induce mechanical damage that leads to cellular death to fight against food invasion with these bacterial species. This work demonstrates the possibility of using membrane FA composition to characterize L. monocytogenes stress-state as an efficient tool for traceability.

In this study, we incubated four strains of Shigella in seawater microcosms (at room temperature and at 4°C) for eight months and we studied the alteration of their morphologic and outer membranes proteins. The starved cells showed an evolution to the filterable minicells state capable to pass membrane pore size 0.45 µm. In addition, the atomic force micrographs showed a reduction of the cells size and an evolution to coccoid-shapes. Outer membrane proteins patterns of stressed bacteria did not changed too much and these modifications were manifested by the appearance of one new band. Key words: Shigella, seawater, starvation, morphological, outer membrane proteins.

Staphylococcus aureus is an important pathogen associated to dental infection. Many antiseptic agents are used in hygienic handwash to prevent nosocomial infections by methicillin-resistant staphylococci. In this study, 22 Staphylococcus aureus strains isolated from the oral cavity of Tunisian children were investigated for their susceptibility to benzalkonium chloride (0.5–512 μg/ml) and antibiotics. The β-lactams resistance gene blaZ, the erythromycin resistance methylase genes (ermA, ermB and ermC), the macrolide efflux gene (msrA) and the disinfectant resistance genes (qacH, qacA, qacB and qacC) were also investigated. Determination of the minimal inhibitory concentration values revealed that 54.5, 54.5, 68, and 82% of isolates were resistant to benzalkonium chloride, oxacillin, tetracycline and erythromycin respectively. The frequency of strains positive for the antibiotic resistance genes tested was 100 (blaZ), 50 (ermA), 36.4 (ermB), 22.7 (ermC) and 13.6% (mrsA). The qacH and the qacA genes were found in 22.7% of isolates and qacB and qacC in 13.6%. Two strains harboured three qac (qacH, qacA and qacB or qacC) genes. These data highlight the importance of determining the susceptibility to antibiotics and disinfectants of strains isolated in dental medicine in order to monitor the epidemiology and spread of multi-drug resistant staphylococci.

In this study, we incubated four strains of Staphylococcus aureus for 8 months in seawater microcosms. The biofilm formation on microplates and on glass and the surface hydrophobicity of starved cells were investigated. Our results showed that the stressed cells increased their hydrophobicity from 12 to 44% and also their capacity for producing biofilm. Indeed, after 8 months of incubation in seawater microcosms, all strains became slime positive on CRA plates and very adherent to polystyrene microplates or glass wells.

In this study, we investigated the survival, adhesion and the morphology of Shigella after its incubation in various concentrations of salts. Our results showed that, after 48 h of incubation, the rate of cell survival is inversely proportional to the increase of salinity of the medium. In addition to that, the results prove the evidence that the concentration of salt can significantly influence the capacity of Shigella to produce biofilm on Congo red agar and on polystyrene microplate wells. Adherence and invasion assays of Shigella were performed with KB cells line. Indeed, an increase of the salts concentration enhances the ability of Shigella species to attach and to invade the tissue culture cells. The percentage of adherence was increased to 15% and the invasion to 90% at 6% of salt. The atomic force micrographs showed a reduction of the cells’ size after stress.

International audience; Understanding the mechanism of the bacterial cell adhesion to solid surfaces is of great medical and industrial importance. Bacterial adhesion to inert surfaces, such as a catheter, and other indwelling devices can form biofilm, consequently cause severe morbidity and often fatal infections. Initial bacterial adhesion to the material surfaces is a complicated process that is affected by various physicochemical properties of both bacterial cells and substratum surfaces. The surface properties of the cells were characterized by the sessile drop technique. Moreover, the interfacial free energy of Staphylococcus aureus adhesion to the supporting materials was determined. The results showed that S. aureus examined at different pH levels could be considered hydrophilic. We noted hat the electron-donor character of S. aureus was important at intermediate pH (pH 5, pH 7, and pH 9) and it decreased at both limits acidic and basic conditions. In addition, the adhesion of Staphylococcus aureus ATCC 25923 to the hydrophilic glass and hydrophobic indium tin oxide (ITO)-coated glass surfaces at different pH values (2, 3, 5, 7, 9 and 11) was investigated using atomic force microscopy (AFM) and image analysis was assessed with the Mathlab(A (R)) program. The data analysis showed that cells (number of adhering cells to glass and ITO-coated glass surface) adhered strongly at acidic pH and weakly at alkaline pH. Also, S. aureus has the ability to attach to both hydrophobic and hydrophilic surfaces, but the adhesion was higher on hydrophobic surface.

Staphylococcus aureus is an important pathogen that forms biofilm. In this study, 22 S. aureus have been isolated from the oral cavity of Tunisian children and investigated for slime production using Congo red agar method (CRA) and semi quantitative adherence assay. The hydrophobicity of strains was evaluated by the microbial adhesion to solvent (MATS) test. The adherence of S. aureus to Hep2 cells was examined by light microscopy. The genes implicated in adhesion (icaA, icaD, fnbA, cna, clfA) were detected. Polymerase chain reaction was used. The affinity to hexadecane was low proving a hydrophilic character of all the studied strains. Qualitative biofilm production revealed that 50% of strains were slime producers. The result of OD(570) showed that four strains isolated from the caries-active children were highly biofilm positive. In addition, 50% of strains were icaA and icaD positive. The fnbA gene was present in 59.1% of isolated strains. Furthermore, 54.5% of strains harboured the cna gene, 9.1% were clfA positive and 50% were hla positive. Quantitative adherence varied considerably among the tested strains. All strains showed adherence to Hep2 cells. However, the level of adhesion varied between strains as follows. Seven strains were defined as moderately adherent, nine as strongly adherent and six as weakly adherent. The percentage of infected cells ranged from 15+/-0.0376 (B374) to 96+/-0.019 (B295) and the total number of bacteria per 100 cells ranged from 15+/-5.1 (B374) to 1824+/-30.1 (B295).

The ability of Listeria monocytogenes to survive and grow at refrigeration temperature in a wide spectrum of foods is a public health concern. Essential oil of clove (Syzium aromaticum) was investigated for its ability to inhibit the growth of this pathogen in food. Three strains of L. monocytogenes (106 cfu/g) were inoculated on fresh-cut salmon and stored (4C and 25C) for 2 weeks. The effect of clove oil (1% and 2%) on the growth of cells was determined. The results proved that all strains survived and grew at 4C and 25C. In addition, we noted that the growth was inhibited under storage conditions in the presence of clove oil (1% or 2%). Listeria counts in treated samples were 1–4 log10 cfu/g less compared with controls at different intervals during storage. The results revealed the potential of clove oil as a natural preservative in fresh salmon. PRACTICAL APPLICATIONS Listeria monocytogenes continues to be a significant threat to the safety of the food supply. It was isolated from a variety of foods and ready-to-eat products and generated a serious public health problem. Because of its growth capacity at refrigerating temperatures and its survival in diverse environments, the control of L. monocytogenes is becoming a challenge. Several agents were used to suppressing contamination by L. monocytogenes, such as the addition of biopreservatives and essential oil. Spices and herbs are commonly used in food preservation because they do not have a strong effect on food quality unlike chemical and synthetic compounds. Clove oil, contains eugenol which when tested on various agar media, has antimicrobial properties, and it was able to restrict the proliferation of L. monocytogenes in food products. For this reason, clove oil has good potential as an antilisterial substance in food preservation as it may be more acceptable to consumers.

The survival of bacteria in natural environments is of practical importance. In this study, we investigated the effect of starvation on the survival and adhesion ability of Shigella in domestic treatment plant effluent microcosms after incubation at either room temperature or 4°C for 1 month. Our results showed that the number of cells decreased considerably after this period of stress. We also revealed some modifications to the biochemical and enzymatic profiles, and antibiotic susceptibility of stressed cells. Adherence assays to the human oral cavity epidermoid carcinoma (KB) cell line revealed an increase in the number of adherent cells from 0.7 to 1.85% after 1 month of incubation in domestic treatment plant effluent.

Salmonella enterica serovar Typhimurium was stressed by incubation in seawater and soil microcosms for 20 years. Recovery of viable but nonculturable Salmonella was obtained by incubation in nutrient broth and buffered peptone water medium for 2 months, and also in a mouse model when administered orally but not by intra-peritoneal injection. The 16S rDNA gene sequences from 20-year stressed strains were homologous, with a high degree of similarity, and all were identical to the respective initial strain.

Gamma-irradiation technology sterilizes microorganisms and thereby prevents decay and improves the safety and shelf stability of food products. In this study we treated the foodborne pathogens Vibrio parahaemolyticus and Vibrio alginolyticus with gamma-irradiation (0.5 kGy) to evaluate their adaptative response. Outer membrane protein patterns of irradiated bacteria were found altered when analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. These modifications were manifested by the appearance and/or disappearance of bands as well as in the expression level of certain proteins. In addition, we searched for the presence of eight Vibrio cholerae virulence genes, toxR, toxS, toxRS, ctxA, zot, ace, toxT, and virulence pathogenicity island (VPI), in the genome of investigated strains. The expression of toxR, toxS, VPI, and ace genes in gamma-irradiated bacteria, studied by reverse transcriptase polymerase chain reaction, was altered. These variations were manifested by an increase and/or a decrease in the expression level of tested virulence genes.

In this study, three adherent Staphylococcus strains able to use nitrate as electron acceptor were isolated from textile wastewater activated sludge. Based on the biochemical profiles, bacterial strains were identified as Staphylococcus lentus, Staphylococcus warneri and Staphylococcus epidermidis the PCR amplification of the nir genes reveal that S. lentus and S. epidermidis were nirK positive and that S. epidermidis was nirS positive. The three strains were also icaA/icaD positive. To obtain the optimal formulation of pure cultures of the staphylococci, the influence of the different mixtures of organisms was studied using mixture design. The regression model of microorganism composition and main metabolites was established. The most predictable reduction of nitrate was 87.2 and 12% of COD consumption. The results suggested that the predictable production of nitrite would reach a minimum of 6.1 mg/l. Based on this, the response values that satisfied all expectations were optimized using MINITAB® 14 analysis software. The most optimal proportion combination was 49.75% of S. lentus (curve value), 36.85% of S. warneri (curve value) and 13.39% S. epidermidis (curve value).

The effects of freezing at −20 °C were examined on surviving ofListeria monocytogenes contaminating food during freezing. Slices of fresh salmon were inoculated with three strains ofListeria monocytogenes (108 CFU/g). The inoculated pieces of fish were dried for 1 h at 24 °C and then stored in closed containers at −20 °C for ten months. The identification of atypical cells found after freezing was achieved by PCR. This technique was based on the amplification of two specific genes ofL. monocytogenes hlyA andiap. The results showed that after ten months of frozen storage, the concentration ofL. monocytogenes determined on Trypticase Soya agar-Yeast Extract had declined by 2.39±0.01 log CFU/g and 2.22±0.01 log CFU/g for the strains isolated from meat, S1 and S2 respectively, and by 3.69±0.03 log CFU/g for the reference strain. The effect of frozen storage ofL. monocytogenes in salmon did not decrease the potential survival of food-borne pathogens’ over a period of ten months.

The presence of the ica loci and adhesins genes in clinical Staphylococcus aureus strains were considered important factors of virulence. In this study, 46 strains of Staphylococcus aureus were isolated from auricular infection, and were investigated for slime production using Congo Red Agar method (CRA). In order to detect the adhesins genes (ica A, ica D, fnb A, cna, Clf A) Polymerase Chain Reaction was used. Qualitative biofilm production of S. aureus using CRA plates revealed that 56.5% of strains were slime producers. In addition 78.26% of strains were ica A and ica D positive. While the fnbA gene was present in 76.1% of isolated strains. Furthermore, 56.5% of strains have the cna gene and 30.4% were clfA positives. Overall this study confirms the presence of fnb A and ica A/ica D genes in the majority of studies S. aureus strains isolated from Staphylococcal sepsis.

Thirty-five Staphylococcus aureus strains from auricular infections were isolated. The identification of strains was confirmed by Api ID 32 Staph strips, the antibiotic susceptibility test was performed using ATB Staph kit. PCR assay was used to detect the oxacillin resistance gene (mecA) and the erythromycin genes (ermA, ermB, ermC, msrA and mef). The susceptibility profile of all strains revealed a low resistance level to oxacillin and erythromycin. The PCR results show that 60 % of the strains are mecA positive. The frequency of erythromycin genes was: ermA + 22.8 %, ermB + 45.7, ermC + 17.1, msrA + 28.6. The mef gene was not detected in any strain. No correlations between genotypic and phenotypic methods for the determination of oxacillin and erythromycin resistance was found. However, multiplex PCR technique was shown to be a fast, practical and economic technique for the detection of methicillin-and erythromycin-resistant staphylococci.

This study characterizes 28 Vibrio alginolyticus strains isolated from seawater from the Seacoast of Monastir (Khenis; Tunisia). V. alginolyticus were isolated using the TCBS modified agar plates and the biochemical activities were tested using RapID NF plus Strips. Proteases activities, hemolysis, antibiotics susceptibility, and adhesion to fish mucus and epithelial cell lines (Hep-2 and Caco-2) were also investigated. Eight Vibriocholerae virulence genes (toxR, toxS, toxRS, toxT, ctxA, vpi, ace, zot) were investigated by PCR in genomes of V. alginolyticus strains. Most of the studied strains were β-haemolytic and produce many proteolytic enzymes. All isolates described here were resistant to several antibiotics tested. Six strains were able to adhere strongly to both Hep-2 and Caco-2 cell lines. The PCR investigation of V. cholerae genes showed a large distribution among the genomes of all V. alginolyticus strains. The toxR operon was found in 9 V. alginolyticus strains out of 28 studied. Only one strain was positive for the toxS and toxRS respectively. Five strains showed a positive amplification for the virulence pathogenic island (vpi), seven for the toxT, 3 for the ctxA and 9 for the Zonula occludens toxin (zot). The bay of Khenis harbors different genotypes of V. alginolyticus strains who inheritated several virulence genes from autochthones bacteria such as V. cholerae. These strains were able to produce several virulence enzymes and exhibit a high power to adhere to human epithelial cells and fish mucus.

Salmonella enterica serovar typhimurium was stressed by incubation in seawater microcosms for seventeen years. The microcosms were prepared in such a way as to allow progressive evaporation of the water. Despite being introduced into the sterile seawater at very high concentrations, the Salmonella rapidly declined to levels undetectable by plate counts on nutrient agar. After two years of starvation, about half of the seawater volume had evaporated from each microcosm, and salt crystals appeared. Inoculation of the salty suspension did not result in any culturable strains in selective and non-selective media. However, incubation of samples in nutrient-rich broth, without supplemental growth factors, allowed resuscitation of stressed cells, yielding colonies that remained viable for extended periods of time. After three years the total volume of water had evaporated from each microcosm, and only salt crystals remained. These microcosms were then incubated at room temperature for seventeen years. Resuscitation of VBNC Salmonella from the salt crystals was conducted in vitro and in vivo; recovery occurring after incubation in both nutrient broth and in mice. Recovery occurred in the Salmonella administered orally into the mice, but not in those administered by intra-peritoneal injection. The identity of initial, stressed and revived strains was confirmed by DGGE analysis and sequencing of 16S RNA gene fragments.

Salmonella bovismorbificans, rare serovar, isolated from patient in Tunisia were incubated during 13 years in soil. After its resuscitation, the cells showed a biochemical profile completely inactive on Api 20E system. These cells recuperated their initial characters after 6 months of incubation in Tryptic Soy broth. The atomic force micrographs showed a reduction of the cells size and an evolution to coccoid-shapes. After resuscitation S. bovismorbificans cells recuperated their original rod shape. These cells showed an altered envelope. The resuscitate cells were identified by PCR following the amplification of the internal transcribed spacer (ITS) region of 16S–23S rRNA gene.

The antibacterial activity of the ethyl acetate extracts of seven marine sponges collected from Tunisian Mediterranean coast (Monastir) was tested against eight human pathogenic bacteria and six human pathogenic fungi using the agar disk diffusion method. The results showed that 90% of the sponge extracts presented significant activity against at least one bacterial strain and only 20% were active against fungi. Sponge extracts of Agelas oroides and Axinella damicornis appeared to be quite promising due to their capacity to inhibit the growth of Pseudomonas aeruginosa and gentamycin resistant strains Listeria monocytogenes and Enterococcus feacalis as well as a broad-spectrum activity against all the other bacteria. The antifungal activity of these sponge extracts is not so interesting, in fact only three among seven sponge extracts showed moderate activity against fungal strains. Agelas oroides showed an interesting antibacterial activity and had also a moderate activity against fungal strains tested in this study. These promising results in relation with antibacterial and antifungal activity in vitro open the way for complementary investigation in order to purify and identify active molecules.

International audience; In this report, we describe a new immunosensor designed for the detection and the quantification of Pseudomonas aeruginosa bacteria in water. The developed biosensing system was based on the immobilization of purified polyclonal anti P. aeruginosa antibodies on electropolymerized poly(pyrrole-3-carboxylic acid)/glassy carbon electrode. The building of the immunosensor step by step was evaluated by electrochemical measurements such as cyclic voltammetry (CV) and impedance spectroscopy (EIS). The electrochemical signature of the immunosensor was established by the change of the charge transfer resistance when the bacteria suspended in solution became attached to the immobilized antibodies. As a result, stable and high sensitive impedimetric immunosensor was obtained with a sensitivity of 0.19 k Omega/decade defined in the linear range from 10(1) to 10(7) CFU/mL of cellular concentrations. A low detection limit was obtained for the P. aeruginosa bacteria and a high selectivity when other bacteria were occasioned as well as Escherichia coli. The developed immunosensor was applied in detecting pathogenic P. aeruginosa in well-water.

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.

International audience; In this work, we report the adaptation of bacteria to stress conditions that induce instability of their cultural, morphological, and enzymatic characters, on which the identification of pathogenic bacteria is based. These can raise serious issues during the characterization of bacteria. The timely detection of pathogens is also a subject of great importance. For this reason, our objective is oriented towards developing an immunosensing system for rapid detection and quantification of Staphylococcus aureus. Polyclonal anti-S. aureus are immobilized onto modified gold electrode by self-assembled molecular monolayer (SAM) method. The electrochemical performances of the developed immunosensor were evaluated by impedance spectroscopy through the monitoring of the charge transfer resistance at the modified solid/liquid interface using ferri-/ferrocyanide as redox probe. The developed immunosensor was applied to detect stressed and resuscitate bacteria. As a result, a stable and reproducible immunosensor with sensitivity of 15 k Omega/decade and a detection limit of 10 CFU/mL was obtained for the S. aureus concentrations ranging from 10(1) to 10(7) CFU/mL. A low deviation in the immunosensor response (+/- 10 %) was signed when it is exposed to stressed and not stressed bacteria.

In clinicalStaphylococcus aureus strains, the presence of theica genes, biofilm formation and susceptibility to antibiotics are considered important factors of virulence. In this study, 35 strains ofS. aureus, isolated from auricular infection, were investigated for slime production using Congo red agar (CRA) method, antibiotic susceptibility, presence ofmecA gene, and presence oficaA andicaD gene. The results show that 60% of strains weremecA positive when tested by PCR although 25.7% of strains were oxacillin resistant when tested with ATB STAPH. Qualitative slime production ofS. aureus using CRA revealed that 74.3% ofS. aureus were slime producers. All the strains carried theica gene.

The effects of seawater stress on persistence of necrotizing Escherichia coli (NTEC) pathogenicity were examined in this study using an uropathogenic strain, Escherichia coli J96, previously incubated for 24 hours in waste water at stationary phase ofgrowth.Using PCR for detecting cnfl gene, we have found that E. coli J96 strains exposed for long periods in seawater retained cnfl gene. We demonstrated that this gene was also expressed during the viable state in laboratory microcosms through detection of E. coli cnfl mRNA by reverse transcription-PCR over a 5- month period. Maintenance of E. coli J96 pathogenicity was shown even when the culture returned to active growth in restored optimal conditions. The presence of cnfl mRNA at viable state (culturable and VBNC) and in resuscitated cells correlating with cytotoxic activity on HEp2 cells and necrotic activity in rabbit skin, indicated the viability and the maintenance of pathogenicity during E. coli J96 incubation in seawater microcosms.