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.     

Survival, physiological response and recovery of enteric bacteria exposed to a polar marine environment.

Survival, sublethal injury, and recoverability of Escherichia coli, Enterococcus faecalis, Salmonella typhimurium, and Yersinia enterocolitica were investigated by using diffusion chambers over 54 to 56 days of in situ exposure to a polar marine environment (-1.8 degrees C; salinity, 34.5 ppt) at McMurdo Station, Antarctica. Plate counts were used to assess recoverability and injury, whereas direct viable counts (DVCs) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction were utilized to determine substrate responsiveness and respiratory activity, respectively. T90 values (times for 10-fold decreases in numbers of recoverable cells) on nonselective medium were ca. 216 to 259 h for E. coli, S. typhimurium, and Y. enterocolitica and 432 h for E. faecalis. Sublethal injury was greater in populations of indicator bacteria than in pathogens. DVCs, CTC reduction, and plate counts indicated progressive increases in viable but nonculturable cells in E. coli, S. typhimurium, and Y. enterocolitica cultures throughout the 54-day exposure. Forty-eight-day exposure of E. coli, S. typhimurium, and Y. enterocolitica resulted in decreased optimal incubation temperatures for colony formation and inability to form colonies at 37 degrees C. The detection of responsive E. coli, S. typhimurium, and Y. enterocolitica by the DVC and CTC methods remained within 1% of inoculum values during 54 days of exposure, indicating some long-term persistence in the viable-but-nonculturable state. Percentages of respiring E. coli and S. typhimurium increased significantly upon addition of nutrients at all temperatures tested, indicating that nutrient availability rather than temperature limited enteric bacterial activity in this very cold environment. Large nutrient inputs to low-temperature marine environments may thus allow for the long-term persistence of enteric bacteria in a nonrecoverable state.     

Maintenance of plasmids pBR322 and pUC8 in nonculturable Escherichia coli in the marine environment.

Maintenance of plasmids pBR322 and pUC8 in Escherichia coli that was nonculturable after exposure to seawater was studied. E. coli JM83 and JM101, which contained plasmids pBR322 and pUC8, respectively, were placed in sterile artificial seawater for 21 days. Culturability was determined by plating on both nonselective and selective agar, and plasmid maintenance was monitored by direct isolation of plasmid nucleic acid from bacteria collected on Sterivex filters. E. coli JM83 became nonculturable after incubation for 6 days in seawater yet maintained plasmid pBR322 for the entire period of the study, i.e., 21 days. E. coli JM101 was nonculturable after incubation in seawater for 21 days and also maintained plasmid pUC8 throughout the duration of the microcosm experiment. Direct counts of bacterial cells did not change significantly during exposure to seawater, even though plate counts yielded no viable (i.e., platable) cells. We concluded that E. coli cells are capable of maintaining high-copy-number plasmids, even when no longer culturable, after exposure to the estuarine or marine environment.     

Maintenance of plasmids pBR322 and pUC8 in nonculturable Escherichia coli in the marine environment

Maintenance of plasmids pBR322 and pUC8 in Escherichia coli that was nonculturable after exposure to seawater was studied. E. coli JM83 and JM101, which contained plasmids pBR322 and pUC8, respectively, were placed in sterile artificial seawater for 21 days. Culturability was determined by plating on both nonselective and selective agar, and plasmid maintenance was monitored by direct isolation of plasmid nucleic acid from bacteria collected on Sterivex filters. E. coli JM83 became nonculturable after incubation for 6 days in seawater yet maintained plasmid pBR322 for the entire period of the study, i.e., 21 days. E. coli JM101 was nonculturable after incubation in seawater for 21 days and also maintained plasmid pUC8 throughout the duration of the microcosm experiment. Direct counts of bacterial cells did not change significantly during exposure to seawater, even though plate counts yielded no viable (i.e., platable) cells. We concluded that E. coli cells are capable of maintaining high-copy-number plasmids, even when no longer culturable, after exposure to the estuarine or marine environment.     

Survival of Salmonella paratyphi B and Pseudomonas aeruginosa in seawater after incubation or washing in the presence of osmolytes]

The authors have compared the survival in seawater of Salmonella paratyphi B and Pseudomonas aeruginosa cells grown at low or high osmolarity, in the presence of organic osmolytes: glycine betaine, choline, proline, and glutamate. The four substrates enhanced the survival potential of S. paratyphi B while only glycine betaine protected P. aeruginosa. In addition only S. paratyphi B cells were more resistant after a preliminary growth at high osmolarity. Both bacteria were sensitive to osmotic down-shock, sensitization of S. paratyphi B being inversely proportional (p greater than or equal to 0.01) to the osmolarity of the medium used to wash cells. The transit in wastewater, at low osmolarity, can therefore modify the behavior of these pathogens in the marine environment.     

Carriage of antibiotic-resistant enteric bacteria varies among sites in Galapagos reptiles

Increased overlap between humans and wildlife populations has increased the risk for novel disease emergence. Detecting contacts with a high risk for transmission of pathogens requires the identification of dependable measures of microbial exchange. We evaluated antibiotic resistance as a molecular marker for the intensity of human-wildlife microbial connectivity in the Galápagos Islands. We isolated Escherichia coli and Salmonella enterica from the feces of land iguanas (Conolophus sp.), marine iguanas (Amblyrhynchus cristatus), giant tortoises (Geochelone nigra), and seawater, and tested these bacteria with the use of the disk diffusion method for resistance to 10 antibiotics. Antibiotic-resistant bacteria were found in reptile feces from two tourism sites (Isla Plaza Sur and La Galapaguera on Isla San Cristóbal) and from seawater close to a public use beach near Puerto Baquerizo Moreno on Isla San Cristóbal. No resistance was detected at two protected beaches on more isolated islands (El Miedo on Isla Santa Fe and Cape Douglas on Isla Fernandina) and at a coastal tourism site (La Lobería on Isla San Cristóbal). Eighteen E. coli isolates from three locations, all sites relatively proximate to a port town, were resistant to ampicillin, doxycycline, tetracycline, and trimethoprin/sulfamethoxazole. In contrast, only five S. enterica isolates showed a mild decrease in susceptibility to doxycycline and tetracycline from these same sites (i.e., an intermediate resistance phenotype), but no clinical resistance was detected in this bacterial species. These findings suggest that reptiles living in closer proximity to humans potentially have higher exposure to bacteria of human origin; however, it is not clear from this study to what extent this potential exposure translates to ongoing exchange of bacterial strains or genetic traits. Resistance patterns and bacterial exchange in this system warrant further investigation to understand better how human associations influence disease risk in endemic Galápagos wildlife.     

Effect of the Salmonella pathogenicity island 2 type III secretion system on Salmonella survival in activated chicken macrophage-like HD11 cells

In order to better identify the role of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (T3SS) in chickens, we used the well-known gentamicin protection assay with activated HD11 cells. HD11 cells are a macrophage-like chicken cell line that can be stimulated with phorbol 12-myristate 13-acetate (PMA) to exhibit more macrophage-like morphology and greater production of reactive oxygen species (ROS). Activated HD11 cells were infected with a wild-type Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) strain, a SPI-2 mutant S. Typhimurium strain, a wild-type Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) strain, a SPI-2 mutant S. Enteritidis strain, or a non-pathogenic Escherichia coli (E. coli) strain. SPI-2 mutant strains were found to survive as well as their parent strain at all time points post-uptake (PU) by the HD11 cells, up to 24 h PU, while the E. coli strain was no longer recoverable by 3 h PU. We can conclude from these observations that the SPI-2 T3SS of S. Typhimurium and S. Enteritidis is not important for survival of Salmonella in the activated macrophage-like HD11 cell line, and that Salmonella must employ other mechanisms for survival in this environment, as E. coli is effectively eliminated.     

Influence of the RpoS (KatF) sigma factor on maintenance of viability and culturability of Escherichia coli and Salmonella typhimurium in seawater.

The sigma factor RpoS is essential for stationary-phase-specific, multiple-stress resistance. We compared the viabilities (direct viable counts) and culturabilities (colony counts) in seawater of Escherichia coli and Salmonella typhimurium strains and those in which rpoS was deleted or which were deficient in guanosine 3',5'-bispyrophosphate (ppGpp) synthesis (relA spoT). RpoS, possibly via ppGpp regulation, positively influenced the culturability of these bacteria in oligotrophic seawater. This influence closely depended, however, upon the growth state of the cells and the conditions under which they were grown prior to their transfer to seawater. The protective effect of RpoS was observed only in stationary-phase cells grown at low osmolarity. A previous exposure of cells to high osmolarity (0.5 M NaCl) also had a strong influence on the effect of RpoS on cell culturability in seawater. Both E. coli and S. typhimurium RpoS mutants lost the ability to acquire a high resistance to seawater, as observed in both logarithmic-phase and stationary-phase RpoS+ cells grown at high osmolarity. A previous growth of S. typhimurium cells under anoxic conditions also modulated the incidence of RpoS on their culturability. When grown anaerobically at high osmolarity, logarithmic-phase S. typhimurium RpoS+ cells partly lost their resistance to seawater through preadaptation to high osmolarity. When grown anaerobically at high osmolarity until stationary phase, both RpoS+ and RpoS- cells retained very high levels of both viability and culturability and then did not enter the viable but nonculturable state for over 8 days in seawater because of an RpoS-independent, unknown mechanism.     

Uptake and retention of Vibrio cholerae non-O1, Salmonella typhi, Escherichia coli and Vibrio harvey by mussels in seawater.

Vibrio cholerae non O1 is known to persist in estuarine and freshwater environments. Experiments evaluated the amount of microorganisms accumulated in mussels maintained in static seawater, contaminated with 10(4) to 10(6) cells/ml and the depuration time required in circulating water. Accumulation and retention times were compared with those for Escherichia coli, Salmonella typhi and Vibrio harvey. E. coli and S. typhi accumulated to a greater extent and were released from mussels more quickly than vibrios which became undetectable 2 to 3 days later than E. coli. Seasonal seawater temperatures (14 to 21 degrees C) had a limited influence on depuration but vibrios appear to be retained with more efficacy over 16 degrees C while E. coli and S. typhi were eliminated to a greater extent. When mussels were contaminated with mixed culture, vibrios appeared to predominate on E. coli, while no interference was observed between E. coli and S. typhi.     

Comparative analysis of extreme acid survival in Salmonella typhimurium, Shigella flexneri, and Escherichia coli.

Several members of the family Enterobacteriaceae were examined for differences in extreme acid survival strategies. A surprising degree of variety was found between three related genera. The minimum growth pH of Salmonella typhimurium was shown to be significantly lower (pH 4.0) than that of either Escherichia coli (pH 4.4) or Shigella flexneri (pH 4.8), yet E. coli and S. flexneri both survive exposure to lower pH levels (2 to 2.5) than S. typhimurium (pH 3.0) in complex medium. S. typhimurium and E. coli but not S. flexneri expressed low-pH-inducible log-phase and stationary-phase acid tolerance response (ATR) systems that function in minimal or complex medium to protect cells to pH 3.0. All of the organisms also expressed a pH-independent general stress resistance system that contributed to acid survival during stationary phase. E. coli and S. flexneri possessed several acid survival systems (termed acid resistance [AR]) that were not demonstrable in S. typhimurium. These additional AR systems protected cells to pH 2.5 and below but required supplementation of minimal medium for either induction or function. One acid-inducible AR system required oxidative growth in complex medium for expression but successfully protected cells to pH 2.5 in unsupplemented minimal medium, while two other AR systems important for fermentatively grown cells required the addition of either glutamate or arginine during pH 2.5 acid challenge. The arginine AR system was only observed in E. coli and required stationary-phase induction in acidified complex medium. The product of the adi locus, arginine decarboxylase, was responsible for arginine-based acid survival.     

Long-term survival and plasmid maintenance of Escherichia coli in marine microcosms

Abstract The survival pattern and plasmid maintenance of Escherichia coli was examined in an artificial seawater microcosm. It was found that the three strains of E. coli (EK3C, H10407 and 34309) included in the study were able to maintain a portion of cells in the culturable phase for at least 3 years in artificial seawater. Along with retaining culturability, that portion of the cell population also maintained their indigenous plasmids over the 3-year period. It is concluded that cells of E. coli maintaining culturability in seawater are selectively adapted to the salinity of seawater, remaining in a culturable state. The results of the study are significant in that it has been assumed by many public health authorities that E. coli cannot survive, without nutrient addition, in seawater for long periods of time, i.e., years of exposure to seawater.     

Intracellular accumulation of potassium and glutamate specifically enhances survival of Escherichia coli in seawater.

The high resistance of Escherichia coli grown in saline media to seawater was suppressed by an osmotic down-shock. The shock released several molecules into the medium, including potassium, glutamate, and glycine betaine when cells were previously grown in the presence of this osmolyte. Incubation of such sensitized cells in a solution containing K+ (80 mM) and glutamate (50 mM) at pH 7.4 restored their resistance to seawater up to a level close to that observed initially. The protective effect was partly due to the rapid accumulation of K+; a significant exponential relationship between intracellular concentration of K+ and resistance to seawater was observed. Glutamate was accumulated more slowly and progressively completed the action of K+. These data emphasize the specific influence of potassium glutamate on osmotically stressed E. coli cells. They confirm that regulation of osmotic pressure and, probably, of intracellular pH strongly enhances survival of E. coli in seawater. Osmotic fluctuations in waters carrying enteric bacteria from intestines to seawater, together with variations in their K+ and amino acid contents, could modify the ability of cells to survive in marine environments. These results demonstrate the need to strictly control conditions (K+ content, temperature) used to wash cells before their transfer to seawater microcosms. They suggest that the K+ and glutamate contents of media in which E. coli cells are transported to the sea can influence their subsequent survival in marine environments.     

Intracellular accumulation of potassium and glutamate specifically enhances survival of Escherichia coli in seawater.

The high resistance of Escherichia coli grown in saline media to seawater was suppressed by an osmotic down-shock. The shock released several molecules into the medium, including potassium, glutamate, and glycine betaine when cells were previously grown in the presence of this osmolyte. Incubation of such sensitized cells in a solution containing K+ (80 mM) and glutamate (50 mM) at pH 7.4 restored their resistance to seawater up to a level close to that observed initially. The protective effect was partly due to the rapid accumulation of K+; a significant exponential relationship between intracellular concentration of K+ and resistance to seawater was observed. Glutamate was accumulated more slowly and progressively completed the action of K+. These data emphasize the specific influence of potassium glutamate on osmotically stressed E. coli cells. They confirm that regulation of osmotic pressure and, probably, of intracellular pH strongly enhances survival of E. coli in seawater. Osmotic fluctuations in waters carrying enteric bacteria from intestines to seawater, together with variations in their K+ and amino acid contents, could modify the ability of cells to survive in marine environments. These results demonstrate the need to strictly control conditions (K+ content, temperature) used to wash cells before their transfer to seawater microcosms. They suggest that the K+ and glutamate contents of media in which E. coli cells are transported to the sea can influence their subsequent survival in marine environments.     

Evidence that Escherichia coli accumulates glycine betaine from marine sediments

Escherichia coli grew faster in autoclaved marine sediment than in seawater alone. When E. coli was cultivated in sediment diluted with minimal medium M63 at 0.6 M NaCl, supplemented or not supplemented with glucose or with seawater, the osmoprotector glycine betaine was accumulated in the cells. The best growth occurred on glucose. Accumulation of glycine betaine was not observed with E. coli was grown in sterile seawater alone. The fact that E. coli grew better in the sediments than in seawater is attributed somewhat to the high content of organic matter in the sediment but mainly to the accumulation of glycine betaine. Thus, osmoprotection should be considered to be an additional factor in bacterial survival in estuarine sediments.     

Evidence that Escherichia coli accumulates glycine betaine from marine sediments.

Escherichia coli grew faster in autoclaved marine sediment than in seawater alone. When E. coli was cultivated in sediment diluted with minimal medium M63 at 0.6 M NaCl, supplemented or not supplemented with glucose or with seawater, the osmoprotector glycine betaine was accumulated in the cells. The best growth occurred on glucose. Accumulation of glycine betaine was not observed with E. coli was grown in sterile seawater alone. The fact that E. coli grew better in the sediments than in seawater is attributed somewhat to the high content of organic matter in the sediment but mainly to the accumulation of glycine betaine. Thus, osmoprotection should be considered to be an additional factor in bacterial survival in estuarine sediments.     

A comparison of Salmonella serotypes isolated from New Zealand sea lions and feral pigs on the Auckland Islands by pulsed-field gel electrophoresis

The Salmonella serotypes S. Cerro and S. Newport were isolated from New Zealand sea lions (Phocarctos hookeri) and feral pigs on the Auckland Islands in the New Zealand subantarctic region. The isolates were typed by pulsed-field gel electrophoresis using Xba1 as the restriction enzyme. The isolates were indistinguishable, which suggests that Salmonella infection cycles between sea lions and pigs in this environment. Apart from a previous isolation from a single New Zealand fur seal (Arctocephalus forsteri), S. Newport has not been recorded in any animals from New Zealand, but it is associated with gastroenteritis in humans. Contamination of the marine environment by human waste is a possible source of infection for marine mammals and warrants further investigation.     

Survival strategy of Escherichia coli and Enterococcus faecalis in illuminated fresh and marine systems

Some effects of visible light on Escherichia coli and Enterococcus faecalis in natural freshwater and seawater were studied by plate counts, colony area measurements, and direct counts. A large number of somnicells (non-culturable cells) were noted in illuminated systems as compared with non-illuminated ones. Colony areas were significantly smaller in illuminated systems. Indirect activity measurements were used to test the effects of visible light on the ability of E. coli and Ent. faecalis to metabolize substrates ([14C]glucose) in natural waters. In illuminated systems, a decrease of glucose uptake was observed. When percentages of assimilation and respiration with respect to the total glucose uptake were analysed a decrease of assimilation percentages and an increase of respiration percentages were observed. In addition, differences in glucose uptake, assimilation and respiration by enteric bacteria were detected for E. coli at the beginning of the experiments between fresh- and seawater and these were interpreted as a toxic effect exerted by seawater on E. coli cells. Differences between species, natural waters and parameters studied (excepting glucose assimilation) were detected in the illuminated systems. We concluded, however, that enteric bacteria under visible light illumination show a general survival strategy characterized by reaching progressively a somnicell stage which can be defined in terms of their (1) inability to form colonies on standard bacteriological media, (2) inability to incorporate substrates, and (3) inactivation of biosynthetic processes.     

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.     

The use of marine bacteria in mutagenicity assays

Mutagenic pollution of environment is a global and important problem. This includes marine environment. Although many mutagenicity assays have been developed, there are specific problems with testing marine water and sediments for mutagenic contamination. One of them is the fact that most of genetically modified strains used in commonly available microbiological mutagenicity assays, like Escherichia coli or Salmonella, survive relatively poorly in marine waters, especially those of higher salinity. Thus, alternative assays have been developed, in which bacteria occurring naturally in marine habitats are employed. These assays, reviewed in this article, appear to be useful in testing not only marine samples but also can be used in other approaches, which involve detection and estimation of the amount of mutagenic compounds.