The effect of toluidine blue on the survival,dormancy and outer membrane porin proteins (OmpC and OmpF) of Salmonella typhimurium LT2 in seawater

AIMS: To study the relationship between changes in the composition of the outer membrane proteins and the survival of Salmonella typhimurium LT2 in filtered autoclaved seawater containing Toluidine Blue (TB) dye as a photosensitizer. METHODS AND RESULTS: In samples exposed to TB and excited by artificial visible light, the total viable (TVC) and respiring cell counts (RCC) showed that, although the TVC declined to an undetectable level in 6.5 h, the RCC showed that some cells were still capable of respiration. The porin protein composition changed gradually with OmpC and OmpF becoming undetectable by sodium dodecyl sulphate-polyacrylamide gel electrophoresis after 8 h of incubation. Hydrogen peroxide-pretreated cells survived longer compared with the control. CONCLUSIONS: Oxidative pretreatment of Salm. typhimurium protects cells from some of the effects of sunlight in the presence of photosensitizers. The changes in porin proteins may play a role in this protection. SIGNIFICANCE AND IMPACT OF THE STUDY: The study shows that the survival of bacteria under conditions of stress is the result of a linked series of reactions.     

Factors influencing survival of enterotoxigenic Escherichia coli, Salmonella enterica (serovar Typhimurium) and Vibrio parahaemolyticus in marine environments

The presence and persistence of enterotoxigenic Escherichia coli (ETEC) is poorly investigated in marine habitats. Here we compared ETEC with the more studied fecal contaminant, Salmonella enterica serotype Typhimurium ( S. enterica ) and the marine bacteria Vibrio parahaemolyticus . All three species of bacteria were culturable on agar plates during 8 weeks of incubation in seawater. However, the culturability of ETEC was positively affected by low temperature whereas V. parahaemolyticus was negatively affected. High-nutrient conditions favored the growth of ETEC but not the other bacteria. When the bacteria were fed to blue mussels, V. parahaemolyticus inhibited the filtration activity and the ingestion was lower than that of the enterobacteria. On the other hand, the mussels were less efficient in eliminating V. parahaemolyticus and an in vitro study showed that the hemocytes of three different species of bivalves were not able to kill this strain of V. parahaemolyticus . The bactericidal capacity of bivalves was seemingly an efficient elimination pathway for S. enterica and ETEC. This study showed that ETEC in endemic areas should, to the same degree as S. enterica and V. parahaemolyticus , be taken in consideration when assessing the role of marine environments as a source of enteric infection.     

Viability and release of Salmonella charity and Escherichia coli from oyster feces.

Sydney Rock oysters (Crassostrea commercialis) contaminated with Salmonella charity and Escherichia coli produced feces containing viable cells of these species. The level of these bacteria in the feces depended upon the level of oyster contamination. Both S. charity and E. coli were released from the feces into overlying seawater. The extent of release into seawater depended upon the physical state of the fecal material, water temperature, and the time of contact with the water. The viability of S. charity and E. coli associated with the feces and released into seawater decreased with time and was a function of seawater temperature. The association and release of bacteria from oyster feces has important implications in oyster purification and purification tank design and may lead to a recontamination of purified oysters.     

Effects of nutrient and temperature on degradation of petroleum hydrocarbons in sub-Antarctic coastal seawater

In an attempt to evaluate the potential of petroleum bioremediation at high latitudes environments, microcosm studies using Antarctic coastal seawater contaminated with diesel or crude oil were conducted in Kerguelen Archipelago (49°22′S, 70°12′E). Microcosms were incubated at three different temperatures (4, 10 and 20°C). During experiments, changes observed in microbial assemblages (total direct count, heterotrophic cultivable microorganisms and hydrocarbon-degrading microorganisms) were generally similar for all incubation temperatures, but chemical data showed only some slight changes in biodegradation indices [Σ(C12–C20)/Σ(C21–C32) and C17/pristane]. The complete data set provided strong evidence of the presence of indigenous hydrocarbon-degrading bacteria in Antarctic seawater and their high potential for hydrocarbon bioremediation. The rate of oil degradation could be increased by the addition of a commercial fertilizer, but water temperature had little effects on biodegradation efficiency which is in conflict with the typical temperature-related assumption predicting 50% rate reduction when temperature is reduced by 10°C. Global warming of Antarctic seawater should not increase significantly the rate of oil biodegradation in these remote regions.     

Effect of a short period of starvation in oligotrophic waters on the resistance of enteric bacterial pathogens to gastric pH conditions

Abstract Enteric bacterial pathogens may undergo severe stresses in aquatic environments. In other respects, these bacteria share an oral route of infection and have to cross the gastric barrier to cause disease in human hosts. Considering the cross-protection against environmental stress, i.e. heat, acid and oxidation, induced by osmotic shock and nutrient starvation, the ability of various enterobacteria, including species of Escherichia, Shigella, Salmonella, Klebsiella and Yersinia , to acquire resistance to the acidic conditions encountered in the stomach (pH 2.5 for 2 h) after incubation in oligotrophic fresh or seawaters was examined. Acid resistance of E. coli , shigellae and Salmonella typhimurium was enhanced by a hundred to a hundred millions times after 100 min in seawater. This effect was also observed when cells were incubated in distilled water and phosphate buffers with low or high osmolarity. It is therefore not specific to seawater. Acid resistance was, however, 2 to 4 times higher in seawater. Acquired resistance depended on the growth phase of the cells. It was lost by subculturing the cells and proved partly dependent on rpoS and on de novo protein synthesis. A similar induced acid resistance was observed in faecal coliforms from human faeces and wastewater. The increased levels of surviving acid-resistant, enteric pathogens in natural waters may have sanitary and epidemiological implications.     

The application of viable count procedures for measuring viable cells in the various marine environments

Aims: To investigate whether the use of direct viable count (DVC), quantitative viable count (qDVC), colony‐forming units and the contribution of capsule‐bearing bacteria to the total number of bacteria and esterase‐active bacteria could be used to clearly differentiate viable cells in various trophic status of seawater. Methods and Results: Hundred and four marine isolates from various marine environments in Turkey (Western Black Sea, northern part of the Sea of Marmara, Northern Aegean Sea and eastern part of the Sea of Marmara) were screened. Seawater samples were taken from the surface (the upper 0–30 cm) and deeper layers (from 5 to 500 m) of the sea at different time periods between February 2002 and June 2007. For the assessment of cell elongation, minor modifications were made on DVC procedure in order to optimize the concentration of yeast extract and incubation time for enumeration of bacteria in response to nutrient addition. The best results were obtained when the yeast extract was used at a final concentration of 250 mg l^?1 (at 35°C 24 h incubation) for bacteria isolated from eutrophic areas and a final concentration of 50 mg l^?1 for those selected from oligotrophic areas. A positive correlation was found between the trophic level and the level of metabolically active bacteria. Among these methods, the bacterial number obtained by qDVC is higher than those gained by other methods. Conclusions: The results indicate that the qDVC procedure could easily differentiate between viable cells and dormant or dead cells. We suggest that this method may be applicable to detecting the level of metabolic potential of bacterial communities in marine environments. Significance and Impact of the Study: The study resulted in increased knowledge on the applicability of the qDVC method that arranges the substrate amount and incubation time as well as on the comparison of various viable bacteria count procedures related to trophic situation of seawater samples.     

电解海水的抑菌活性及对食品加工表面材料的消毒效果

为了考察直接电解海水消除细菌污染的可能性,本文将海水及海水稀释成不同浓度后通过氧化电解水装置进行电解不同时间后,所得酸性电解海水、碱性电解海水和中性电解海水对病原菌[埃希氏大肠杆菌(Escherichina coli)、沙门氏菌(Salmonella)、单核细胞增生李斯特菌(Listeria moncytogene)、摩化摩根(Morganella morganii)、副溶血性弧菌(Vibrio parahaemolyticus)]以及食品加工表面接触材料(地板砖、不锈钢板、瓷砖、手套、抹布)的消毒效果进行分析研究.结果表明,酸性电解海水具有良好的杀菌效果,能将107 CFU/mL的病原菌悬液在1 min内几乎全部杀死.碱性电解海水和中性水无明显的杀菌效果.通过模拟食品加工过程,对食品加工表面接触材料人为染菌,研究电解海水对表面材料的消毒效果,结果表明酸性电解海水仍能将表面材料含有的107CFU/cm2病原菌在5 min之内几乎全部杀灭.由此说明电解海水对食品加工表面接触材料具有明显的消毒效果,能取代以淡水为原料的电解水杀菌效果是高效廉价和不浪费淡水资源的一种理想消毒剂.     

Fluorescent-antibody method useful for detecting viable but nonculturable Salmonella spp. in chlorinated wastewater.

An indirect fluorescent-antibody (IFA) technique, which employed adsorbed Behring polyvalent I O antiserum, was used to detect Salmonella spp. in environmental water systems. The IFA method used in this study detected 95% of Salmonella serotypes encountered in human infections in France, with a sensitivity threshold of 7.5 x 10(3) bacteria per ml of wastewater. Specificity was assessed by testing IFA against Salmonella-free seawater and a variety of bacteria other than Salmonella spp. When used to examine raw and chlorinated wastewater over a 2-month period, the IFA method was successful in detecting Salmonella spp. in all 12 of the samples examined, with total numbers determined to be 4.5 x 10(5) to 3.3 x 10(7) salmonellae per 100 ml. In comparison, for the same samples, enumeration by culture, using the most-probable-number technique, was effective in detecting Salmonella spp. in only four of eight raw-water samples and one of four chlorinated water samples tested. Three samples were further tested by using the direct viable count procedure combined with IFA and results showed that 5 to 31.5% of the Salmonella spp. enumerated by this method in chlorinated water were substrate responsive.     

A Simple Procedure for Screening of Salmonellae using a Semi-Solid Enrichment and a Semi-Solid Indicator Medium

The incorporation of triphenylmethane dyes and divalent cations in a semi-solid medium in conjunction with a low pH retarded the motility of most enteric bacteria except for Salmonella, Enterobacter cloacae and Citrobacter freundii . The migration of the latter two species in the semi-solid medium, however, could be selectively inhibited by the incorporation of novobiocin to the medium and by incubation at 41 °C. Based on these observations, a semi-solid enrichment medium was developed and used successfully in a Salmonella screening procedure in which a semi-solid indicator medium was used as the first and a slide agglutination test as the second screen. Clinical laboratory evaluation of the procedure showed an increase of 92% in the frequency of positive isolations, with no false positives, as compared with a conventional multi-step isolation procedure.     

Fluorescent-antibody method useful for detecting viable but nonculturable Salmonella spp. in chlorinated wastewater

An indirect fluorescent-antibody (IFA) technique, which employed adsorbed Behring polyvalent I O antiserum, was used to detect Salmonella spp. in environmental water systems. The IFA method used in this study detected 95% of Salmonella serotypes encountered in human infections in France, with a sensitivity threshold of 7.5 x 10(3) bacteria per ml of wastewater. Specificity was assessed by testing IFA against Salmonella-free seawater and a variety of bacteria other than Salmonella spp. When used to examine raw and chlorinated wastewater over a 2-month period, the IFA method was successful in detecting Salmonella spp. in all 12 of the samples examined, with total numbers determined to be 4.5 x 10(5) to 3.3 x 10(7) salmonellae per 100 ml. In comparison, for the same samples, enumeration by culture, using the most-probable-number technique, was effective in detecting Salmonella spp. in only four of eight raw-water samples and one of four chlorinated water samples tested. Three samples were further tested by using the direct viable count procedure combined with IFA and results showed that 5 to 31.5% of the Salmonella spp. enumerated by this method in chlorinated water were substrate responsive.     

Climate patterns governing the presence and permanence of salmonellae in coastal areas of Bahia de Todos Santos, Mexico

Despite the importance of salmonellae as one of the major causes of food-borne infections worldwide, data regarding the presence of these organisms in the environment are limited. We investigated the presence of Salmonella spp. in Bahia de Todos Santos (Baja California, Mexico) and evaluated the environmental factors that affect the occurrence of Salmonella spp. in this arid region. A total of 1,331 samples collected from 21 sites along the coast during a period of 3 years were analyzed for Salmonella spp. Geographical and seasonal distribution of Salmonella spp. was evaluated in association with environmental parameters and with human infections in the area. The incidence of Salmonella bacteria throughout the study was 4.8%, with the highest incidence detected in wastewater (16.2%), followed by stream water (10.6%), mollusks (7.4%), and seawater (2.3%). Twenty different serotypes were identified among the 64 Salmonella isolates. The dominant serotype was Typhimurium (23.4%), followed by Vejle (6.2%). The presence of Salmonella spp. in coastal areas was mostly confined to rainy periods and areas of stream discharges, and runoff was identified as the predominant factor influencing the transport of Salmonella bacteria from source points to the sea via streams. Isolation of Salmonella spp. was negatively and significantly associated with temperature, probably because of the effect of solar radiation in the decline of permanence of Salmonella bacteria. Conversely, human infections prevailed during the warmest months and were negatively correlated with the presence of Salmonella spp. in the marine environment.     

Effects of sunlight and Synechocystis sp. (picocyanobacterium) on the incidence of antibiotic resistance in wastewater enteric bacteria

Experimental study of the effects of sunlight and Synechocystis sp. (picocyanobacterium) on the incidence of antibiotic resistance in Salmonella sp. and faecal coliforms (FC), was carried out in wastewaters of Marrakesh stabilization ponds (Morocco). The effect of sunlight was found to be important on the incidence of antibiotic resistance of these enteric bacterial populations. In microcosms seeded with the mixed inoculum (40 different strains) of Salmonella or FC and exposed to sunlight, the overall resistance of these bacteria (72.5 and 57.5% respectively) was significantly higher than that noted in the dark (45 and 32.5% respectively). In microcosms seeded with a monobacterial inoculum, some FC isolates became resistant to the antibiotics cephalothin, amoxicillin or sulphamethoxazole, after their incubation in sunlight. Synechocystis appeared to have no significant effect on the incidence of bacterial antibiotic resistance. The ecological significance of sunlight and Synechocystis sp. in reducing densities and increasing antibiotic resistance of pathogenic bacteria through wastewater stabilization ponds is discussed.     

Bacterial biofilm in seawater: cell surface properties of early-attached marine bacteria

The development of antifouling strategies in seawater requires knowledge of the physico-chemical properties of the cell surfaces of early adherent bacteria. The hydrophilic, electrostatic and the Lewis acid-base cell surface properties of eleven marine bacteria were characterized. Although these bacteria adhered to a hydrophilic support immersed for 3 and 6 h, they presented various physico-chemical properties. Eleven strains possessed a hydrophilic surface and five a hydrophobic surface. Although the majority of the bacteria presented an electron-donating character, some could not generate Lewis acid-base interactions with the support. On the other hand, all strains possessed an isoelectric point ranging from 2.2 to 3.4 and were negatively charged at the pH of seawater. Hydrophilicity was a preponderant property among these bacteria, but other properties should not be ignored. The development of new antifouling paints must take account all the possible interaction levels used by the bacteria to adhere to an immersed surface.     

Bacteria associated with a marine planktonic copepod in culture. I. Bacterial genera in seawater, body surface, intestines and fecal pellets and succession during fecal pellet degradation

In laboratory experiments, the bacterial flora of the zooplanktonmicrobial environments seawater, fecal pellets and associatedwith the external and internal surfaces of the copepod Acartiatonsa(Dana) were examined. The bacteria associated with fecal pelletswere dominated by Bacillus spp., Cytophaga/Flavobacterium spp.,Vibrio spp. and Pseudomonas spp. The same genera were foundin the seawater (0.22 7mu;m filtered) in which the pellets wereincubated. The bacteria showed a characteristic growth succession,and the abundance increased several orders of magnitude in theseawater during incubation of the pellets, indicating growthand proliferation based on the disintegrating/degrading fecalpellets. A carbon budget calculation revealed that organic matterfrom degrading fecal pellets could cover the carbon demand forthe growing bacterioplankton. The composition of the bacterialcommunity in the seawater and the fecal pellets also indicateda colonization of the pellets from bacterioplankton. The compositionof the bacteria associated with the copepods showed that bacterialgenera characterized as surface associated were preferentiallyassociated with fecal pellets, animal surfaces and intestines.This suggests a specific intestinal flora in the cultivatedcopepods composed of 10³ culturable bacteria per intestine (colony-formingunits, c.f.u.) or 10⁵ bacteria per intestine (acridine orangedirect counts, AODC), possibly colonizing the intestine passivelyduring filtration of algae. The activity of the bacterial communitieswas examined by the numencal ratio c.f.u.:AODC, where 1–19%of the bacteria were found active, with no significant differencebetween microbial environments.     

Bacterial Biofilm in Seawater: Cell Surface Properties of Early-attached Marine Bacteria

The development of antifouling strategies in seawater requires knowledge of the physico-chemical properties of the cell surfaces of early adherent bacteria. The hydrophilic, electrostatic and the Lewis acid-base cell surface properties of eleven marine bacteria were characterized. Although these bacteria adhered to a hydrophilic support immersed for 3 and 6?h, they presented various physico-chemical properties. Eleven strains possessed a hydrophilic surface and five a hydrophobic surface. Although the majority of the bacteria presented an electron-donating character, some could not generate Lewis acid-base interactions with the support. On the other hand, all strains possessed an isoelectric point ranging from 2.2 to 3.4 and were negatively charged at the pH of seawater. Hydrophilicity was a preponderant property among these bacteria, but other properties should not be ignored. The development of new antifouling paints must take account all the possible interaction levels used by the bacteria to adhere to an immersed surface.     

Viability of Salmonella spp and indicator microorganisms in seawater using membrane diffusion chambers

Diffusion chambers with polycarbonate membrane-filter side walls were used to study the comparative survival of fecal indicators (Escherichia coli and Streptococcus faecalis) and enteric pathogens (Salmonella enteritidis, S. postdam, S. typhimurium, S. london and S. infantis) in natural seawater. It was observed that the percentages of sublethal injury increased with exposure to the marine environment, and that these environmental injuries depended on the microorganism considered. A large proportion of cells lost their ability to produce colonies on the selective media, but retained this capability on a nonselective medium. All microorganisms showed low survival percentages (less than 11%) after 48 hrs of exposure to seawater, but there is not a high difference among the microbial species studied. The results obtained in the present study showed that there were no differences in the survival rates between the serotypes of Salmonella tested. Moreover, Salmonella spp exhibited a similar persistence to E. coli in the marine environment.     

Uptake and replication of Salmonella enterica in Acanthamoeba rhysodes

The ability of salmonellae to become internalized and to survive and replicate in amoebae was evaluated by using three separate serovars of Salmonella enterica and five different isolates of axenic Acanthamoeba spp. In gentamicin protection assays, Salmonella enterica serovar Dublin was internalized more efficiently than Salmonella enterica serovar Enteritidis or Salmonella enterica serovar Typhimurium in all of the amoeba isolates tested. The bacteria appeared to be most efficiently internalized by Acanthamoeba rhysodes. Variations in bacterial growth conditions affected internalization efficiency, but this effect was not altered by inactivation of hilA, a key regulator in the expression of the invasion-associated Salmonella pathogenicity island 1. Microscopy of infected A. rhysodes revealed that S. enterica resided within vacuoles. Prolonged incubation resulted in a loss of intracellular bacteria associated with morphological changes and loss of amoebae. In part, these alterations were associated with hilA and the Salmonella virulence plasmid. The data show that Acanthamoeba spp. can differentiate between different serovars of salmonellae and that internalization is associated with cytotoxic effects mediated by defined Salmonella virulence loci.     

Influence of tumor necrosis factor on the growth of vegetative and nonculturable forms of Salmonella

The growth rate of the vegetative forms and the recultivation rate of the uncultivable forms of Salmonella isogenous strains, one of these strains carrying mutation in gene pqi, were studied. The multiplication rate of the vegetative and uncultivable forms of Salmonella control strain in the spleen of infected animals at the initial stages of the infectious process was shown (in vivo) to be considerably accelerated after the preliminary incubation of the culture with cytokine (tumor necrosis factor). The multiplication rate, in vivo and in vitro, of Salmonella vegetative and uncultivable forms with mutation in gene pqi did not change after the incubation of the cells with cytokine, which is indicative of an important role played by the product of this gene in the process of the interaction of bacteria with cytokines. The full nucleotide sequence Salmonella gene pqi was determined.     

Evidence of stress in bacteria associated with long-term preservation of food in the stomach of incubating king penguins (<Emphasis Type="Italic">Aptenodytes patagonicus</Emphasis>)

Male king penguins are able to conserve food in their stomach for several weeks during their incubation fast. Since a bacterial inhibition could be involved in the process, we investigated the bacterial population of their stomach. Two types of bird were compared, according to whether they conserved (Co) or digested (Dg) food during fasting. Total bacteria and culturable bacteria were studied. In both groups, the food contained a high number of apparently viable bacteria (⁶⁷/ml), but few could be cultured by standard techniques. Culturable bacteria originated mainly from the environment or the upper part of the digestive tract. Neither prevailing bacteria from seawater and fishes nor enteric bacteria cultured. The gastric temperature was around 38°C and the pH of Co bird samples was >4, both conditions favourable for bacterial growth. Morphological characteristics of stressed bacteria (sporulated, deformed or dead cells) were more often found in samples from Co compared to Dg birds. Altogether, these results support the idea that there might be factors inhibiting bacterial growth in king penguins conserving food in their stomach.     

Uptake of Cyclic AMP by Natural Populations of Marine Bacteria

The major objective of this study was to describe the mechanism(s) of cyclic AMP uptake by natural populations of marine bacteria. A second objective was to determine whether this uptake could contribute to the intracellular regulatory pool of cyclic AMP. Using high-specific-activity ³²P-labeled cyclic AMP, we found several high-affinity uptake systems. The highest-affinity system had a half-saturation constant of <10 pM. This system was extremely specific for cyclic nucleotides, particularly cyclic AMP. It appeared to meet the criteria for active transport. Uptake of cyclic AMP over a wide concentration range (up to 2 μM) showed multiphasic kinetics, with half-saturation constants of 1 nM and greater. These lower-affinity systems were much less specific for cyclic nucleotides. Although much of the labeled cyclic AMP taken up by the high-affinity systems was metabolized, some remained as intact cyclic AMP within the cells during 1 h of incubation. This suggests that at least some of the bacteria use cyclic AMP dissolved in seawater to augment their intracellular pools.