In situ survival of Vibrio cholerae and Escherichia coli in tropical coral reefs

Vibrio cholerae and Escherichia coli were inoculated into membrane diffusion chambers and placed around two small coral reef islands in Puerto Rico and monitored for 5 days. Several chambers were also buried in the sands of one of the reefs. Both E. coli and V. cholerae densities declined by 2 orders of magnitude, as measured by direct particle counts with a Coulter Counter (Coulter Electronics, Inc., Hialeah, Fla.). However, the density of neither bacteria changed dramatically when the same samples were analyzed by epifluorescent direct counts. Differences in the two direct count methods were accounted for by changes in cell morphology that occurred in both bacteria after exposure to seawater. Morphological changes occurred more rapidly in E. coli compared with those in V. cholerae. Bacteria in chambers exposed to sediment did not show significant changes in morphology and had only a slight decline in density. Physiological activity declined by more than 40% for both bacteria within 24 h. The decline in activity was less severe in the sediments. Tropical coral reef sands and turtle grass beds were shown to be less stressful environments for V. cholerae and E. coli than would have been predicted from temperature and microcosm studies. V. cholerae can survive the in situ conditions of a tropical coral reef and could become a source of bacterial contamination for fish and shellfish in this environment. The simultaneous monitoring of E. coli levels established that this bacteria can not be used as an indicator of V. cholerae or other fecal-borne pathogens in coral reef environments because of the greater stress these environments put on E. coli. Both bacteria could be of greater public health importance in tropical marine areas than previously imagined.     

In situ survival of Vibrio cholerae and Escherichia coli in a tropical rain forest watershed.

For 12 months, Vibrio cholerae and fecal coliform densities were monitored along with nine other water quality parameters at 12 sites in a rain forest watershed in Puerto Rico. Densities of V. cholerae and fecal coliforms were not significantly correlated, even though the highest densities of both bacteria were found at a sewage outfall. High densities of V. cholerae were also found at pristine sites at the highest point in the watershed. The density of Escherichia coli and V. cholerae in membrane diffusion chambers did not change significantly during the course of two such studies. Physiological activity, as measured by electron transport system activity and relative nucleic acid composition, indicated that both E. coli and V. cholerae remained active. This study suggests that V. cholerae is indigenous to tropical fresh waters and that assays other than those that detect fecal coliforms or E. coli must be used for assessing public health risk in tropical waters.     

In situ survival of Vibrio cholerae and Escherichia coli in a tropical rain forest watershed

For 12 months, Vibrio cholerae and fecal coliform densities were monitored along with nine other water quality parameters at 12 sites in a rain forest watershed in Puerto Rico. Densities of V. cholerae and fecal coliforms were not significantly correlated, even though the highest densities of both bacteria were found at a sewage outfall. High densities of V. cholerae were also found at pristine sites at the highest point in the watershed. The density of Escherichia coli and V. cholerae in membrane diffusion chambers did not change significantly during the course of two such studies. Physiological activity, as measured by electron transport system activity and relative nucleic acid composition, indicated that both E. coli and V. cholerae remained active. This study suggests that V. cholerae is indigenous to tropical fresh waters and that assays other than those that detect fecal coliforms or E. coli must be used for assessing public health risk in tropical waters.     

Survival of Vibrio cholerae and Escherichia coli in estuarine waters and sediments.

In in vitro estuarine water and sediment chambers, the survival of Vibrio cholerae and Escherichia coli was determined by plate counting and direct counting techniques. V. cholerae strains included environmental, clinical, and serotype O1 and non-O1 isolates, whereas E. coli strains included ATCC 25922 and a freshly cultured human isolate. Recovery of V. cholerae varied significantly with incubation temperature. Growth and extended periods of survival occurred in sterile sediments, sterile waters, and nonsterile waters, but not in nonsterile sediments. In contrast to V. cholerae, viable cells of E. coli decreased rapidly in both sterile and nonsterile estuarine waters. Direct counts revealed that E. coli cells were intact in the estuarine water, but attempts to resuscitate them were unsuccessful. The data suggest that V. cholerae survives better in estuarine waters than E. coli. The results may explain the recent observations that V. cholerae levels do not correlate well with fecal coliform concentrations in estuarine waters. Furthermore, the results add increasing evidence to support the theory that V. cholerae is an autochthonous bacterium in estuaries.     

Survival and activity of Salmonella typhimurium and Escherichia coli in tropical freshwater

The survival of Salmonella typhimurium LT2 and Escherichia coli was studied in situ in a tropical rain forest watershed using membrane diffusion chambers. Numbers were determined by acridine orange staining and a Coulter counter. Population activity was determined by microautoradiography, cell respiration, frequency of dividing cells, and by nucleic acid composition. Numbers of Salm, typhimurium and E. coli decreased less than 1 log unit after 105 h as measured by direct count methods. Activity as measured by respiration, acridine orange activity, frequency of dividing cells, and microautoradiography indicated that both bacteria remained moderately active during the entire study. After 24 h, E. coli was more active than Salm. typhimurium, as measured by nucleic acid composition, and frequency of dividing cells. Both E. coli and Salm. typhimurium survived and remained active in this tropical rain forest watershed for more than 5 d, suggesting that Salm. typhimurium may be of prolonged public health significance once it is introduced into tropical surface waters. As E. coli was active and survived for a long time in this natural environment, it would seem to be unsuitable as an indicator of recent faecal contamination in tropical waters.     

Survival and activity of Salmonella typhimurium and Escherichia coli in tropical freshwater

The survival of Salmonella typhimurium LT2 and Escherichia coli was studied in situ in a tropical rain forest watershed using membrane diffusion chambers. Numbers were determined by acridine orange staining and a Coulter counter. Population activity was determined by microautoradiography, cell respiration, frequency of dividing cells, and by nucleic acid composition. Numbers of Salm. typhimurium and E. coli decreased less than 1 log unit after 105 h as measured by direct count methods. Activity as measured by respiration, acridine orange activity, frequency of dividing cells, and microautoradiography indicated that both bacteria remained moderately active during the entire study. After 24 h, E. coli was more active than Salm. typhimurium , as measured by nucleic acid composition, and frequency of dividing cells. Both E. coli and Salm. typhimurium survived and remained active in this tropical rain forest watershed for more than 5 d, suggesting that Salm. typhimurium may be of prolonged public health significance once it is introduced into tropical surface waters. As E. coli was active and survived for a long time in this natural environment, it would seem to be unsuitable as an indicator of recent faecal contamination in tropical waters.     

Uptake and retention of Vibrio cholerae O1 in the Eastern oyster, Crassostrea virginica.

Vibrio cholerae 01, the causative agent of cholera, is known to persist in estuarine environments as endogenous microflora. The recent introduction of V. cholerae 01 into estuaries of the North and South American continents has stimulated the need to determine the effect of controlled purification on reducing this pathogen in edible molluscan shellfish. Experiments defined parameters for the uptake and retention of V. cholerae 01 in tissues of Crassostrea virginica, and these parameters were compared with those for Escherichia coli and Salmonella tallahassee, bacteria which are usually eliminated from moderately contaminated shellfish within 48 h. Oysters accumulated greater concentrations of V. cholerae 01 than E. coli and S. tallahassee. When V. cholerae 01 was exposed to controlled purification at 15, 19 and 25 degrees C over 48 h, it persisted in oysters at markedly higher levels than E. coli and S. tallahassee. The concentration of a V. cholerae 01-specific agglutinin did not positively correlate with the uptake or retention of V. cholerae 01. These data show that state and federally approved controlled purification techniques are not effective at reducing V. cholerae 01 in oysters.     

Death of the Escherichia coli K-12 strain W3110 in soil and water.

Whether Escherichia coli K-12 strain W3110 can enter the "viable but nonculturable" state was studied with sterile and nonsterile water and soil at various temperatures. In nonsterile river water, the plate counts of added E. coli cells dropped to less than 10 CFU/ml in less than 10 days. Acridine orange direct counts, direct viable counts, most-probable-number estimates, and PCR analyses indicated that the added E. coli cells were disappearing from the water in parallel with the number of CFU. Similar results were obtained with nonsterile soil, although the decline of the added E. coli was slower. In sterile water or soil, the added E. coli persisted for much longer, often without any decline in the plate counts even after 50 days. In sterile river water at 37 degrees C and sterile artificial seawater at 20 and 37 degrees C, the plate counts declined by 3 to 5 orders of magnitude, while the acridine orange direct counts remained unchanged. However, direct viable counts and various resuscitation studies all indicated that the nonculturable cells were nonviable. Thus, in either sterile or nonsterile water and soil, the decline in plate counts of E. coli K-12 strain W3110 is not due to the cells entering the viable but nonculturable state, but is simply due to their death.     

Vibrio cholerae O1 strains are facultative intracellular bacteria, able to survive and multiply symbiotically inside the aquatic free-living amoeba Acanthamoeba castellanii

Vibrio cholerae species are extracellular, waterborne, gram-negative bacteria that are overwhelmed by predators in aquatic environments. The unencapsulated serogroup V. cholerae O1 and encapsulated V. cholerae O139 cause epidemic and pandemic outbreaks of cholera. It has recently been shown that the aquatic and free-living amoeba Acanthamoeba castellanii is not a predator to V. cholerae O139; rather, V. cholerae O139 has shown an intracellular compatibility with this host. The aim of this study was to examine the ability of V. cholerae O1 classical and El Tor strains to grow and survive in A. castellanii. The interaction between A. castellanii and V. cholerae O1 strains was studied by means of amoeba cell counts and viable counts of the bacteria in the absence or presence of amoebae. The viable count of intracellularly growing bacteria was estimated by utilizing gentamicin assay. Confocal microscopy and electron microscopy were used to determine the intracellular localization of V. cholerae in A. castellanii. The results showed that V. cholerae O1 classical and El Tor strains grew and survived intracellularly in the cytoplasm of trophozoites, and that the bacteria were also found in the cysts of A. castellanii. The interaction showed a facultative intracellular behaviour of V. cholerae O1 classical and El Tor strains and a possible role of A. castellanii as an environmental host of V. cholerae species.     

Nearshore coral growth declining on the Mesoamerican Barrier Reef System

Anthropogenic global change and local stressors are impacting coral growth and survival worldwide, altering the structure and function of coral reef ecosystems. Here, we show that skeletal extension rates of nearshore colonies of two abundant and widespread Caribbean corals (Siderastrea siderea, Pseudodiploria strigosa) declined across the Belize Mesoamerican Barrier Reef System (MBRS) over the past century, while offshore coral conspecifics exhibited relatively stable extension rates over the same temporal interval. This decline has caused nearshore coral extension rates to converge with those of their historically slower growing offshore coral counterparts. For both species, individual mass coral bleaching events were correlated with low rates of skeletal extension within specific reef environments, but no single bleaching event was correlated with low skeletal extension rates across all reef environments. We postulate that the decline in skeletal extension rates for nearshore corals is driven primarily by the combined effects of long‐term ocean warming and increasing exposure to higher levels of land‐based anthropogenic stressors, with acute thermally induced bleaching events playing a lesser role. If these declining trends in skeletal growth of nearshore S. siderea and P. strigosa continue into the future, the structure and function of these critical nearshore MBRS coral reef systems is likely to be severely impaired.     

Assessing the ‘deep reef refugia’ hypothesis: focus on Caribbean reefs

Coral reefs in shallow-water environments (<30 m) are in decline due to local and global anthropogenic stresses. This has led to renewed interest in the ‘deep reef refugia’ hypothesis (DRRH), which stipulates that deep reef areas (1) are protected or dampened from disturbances that affect shallow reef areas and (2) can provide a viable reproductive source for shallow reef areas following disturbance. Using the Caribbean as an example, the assumptions of this hypothesis were explored by reviewing the literature for scleractinian corals—the reef framework builders on tropical reefs. Although there is evidence to support that deep reefs (>30 m) can escape the direct effects of storm-induced waves and thermal bleaching events, deep reefs are certainly not immune to disturbance. Additionally, the potential of deep reefs to provide propagules for shallow reef areas seems limited to ‘depth-generalist’ coral species, which constitute only ~25% of the total coral biodiversity. Larval connectivity between shallow and deep populations of these species may be further limited due to specific life history traits (e.g., brooding reproductive strategy and vertical symbiont acquisition mode). This review exposes how little is known about deep reefs and coral reproduction over depth. Hence, a series of urgent research priorities are proposed to determine the extent to which deep reefs may act as a refuge in the face of global reef decline.     

Multiple disturbances and the global degradation of coral reefs: are reef fishes at risk or resilient?

Increased frequency of disturbances and anthropogenic activities are predicted to have a devastating impact on coral reefs that will ultimately change the composition of reef associated fish communities. We reviewed and analysed studies that document the effects of disturbance‐mediated coral loss on coral reef fishes. Meta‐analysis of 17 independent studies revealed that 62% of fish species declined in abundance within 3 years of disturbances that resulted in >10% decline in coral cover. Abundances of species reliant on live coral for food and shelter consistently declined during this time frame, while abundance of some species that feed on invertebrates, algae and/or detritus increased. The response of species, particularly those expected to benefit from the immediate loss of coral, is, however, variable and is attributed to erratic replenishment of stocks, ecological versatility of species and sublethal responses, such as changes in growth, body condition and feeding rates. The diversity of fish communities was found to be negatively and linearly correlated to disturbance‐mediated coral loss. Coral loss >20% typically resulted in a decline in species richness of fish communities, although diversity may initially increase following small declines in coral cover from high coverage. Disturbances that result in an immediate loss of habitat complexity (e.g. severe tropical storms), have a greater impact on fishes from all trophic levels, compared with disturbances that kill corals, but leave the reef framework intact (e.g. coral bleaching and outbreaks of Acanthaster planci). This is most evident among small bodied species and suggests the long‐term consequences of coral loss through coral bleaching and crown‐of‐thorn starfish outbreaks may be much more substantial than the short‐term effects currently documented.     

Effect of transport at ambient temperature on detection and isolation of Vibrio cholerae from environmental samples

It has long been assumed that prolonged holding of environmental samples at the ambient air temperature prior to bacteriological analysis is detrimental to isolation and detection of Vibrio cholerae, the causative agent of pandemic cholera. The present study was aimed at understanding the effect of transporting environmental samples at the ambient air temperature on isolation and enumeration of V. cholerae. For water and plankton samples held at ambient temperatures ranging from 31 degrees C to 35 degrees C for 20 h, the total counts did not increase significantly but the number of culturable V. cholerae increased significantly compared to samples processed within 1 h of collection, as measured by culture, acridine orange direct count, direct fluorescent-antibody-direct viable count (DFA-DVC), and multiplex PCR analyses. For total coliform counts, total bacterial counts, and DFA-DVC counts, the numbers did not increase significantly, but the culturable plate counts for V. cholerae increased significantly after samples were held at the ambient temperature during transport to the laboratory for analysis. An increase in the recovery of V. cholerae O1 and improved detection of V. cholerae O1 rfb and ctxA also occurred when samples were enriched after they were kept for 20 h at the ambient temperature during transport. Improved detection and isolation of toxigenic V. cholerae from freshwater ecosystems can be achieved by holding samples at the ambient temperature, an observation that has significant implications for tracking this pathogen in diverse aquatic environments.     

Survival and activity ofStreptococcus faecalis andEscherichia coli in tropical freshwater

The survival ofStreptococcus faecalis andEscherichia coli was studied in situ in a tropical rain forest watershed using membrane diffusion chambers. Densities were determined by acridine orange direct count and Coulter Counter. Population activity was determined by microautoradiography, cell respiration, and by nucleic acid composition. Densities ofS. faecalis andE. coli decreased less than 1 log unit after 105 hours as measured by direct count methods. Activity as measured by respiration, acridine orange activity, and microautoradiography indicated that both bacteria remained moderately active during the entire study. After 12 hours,E. coli was more active thanS. faecalis as measured by nucleic acid composition. In this tropical rain forest watershed,E. coli andS. faecalis survived and remained active for more than 5 days; consequently, both would seem to be unsuitable as indicators of recent fecal contamination in tropical waters.     

Survival and enumeration of the fecal indicators Bifidobacterium adolescentis and Escherichia coli in a tropical rain forest watershed.

The density of Bifidobacterium spp., fecal coliforms, Escherichia coli, and total anaerobic bacteria, acridine orange direct counts, percentages of total bacterial community activity and respiration, and 12 physical and chemical parameters were measured simultaneously at six sites for 12 months in the Mameyes River rain forest watershed, Puerto Rico. The densities of all bacteria were higher than those reported for uncontaminated temperate rivers, even though other water quality parameters would indicate that all uncontaminated sites were oligotrophic. The highest densities for all indicator bacteria were at the site receiving sewage effluent; however, the highest elevation site in the watershed had the next highest densities. Correlations between bacterial densities, nitrates, temperature, phosphates, and total phosphorus indicated that all viable counts were related to nutrient levels, regardless of the site sampled. In situ diffusion chamber studies at two different sites indicated that E. coli could survive, remain physiologically active, and regrow at rates that were dependent on nutrient levels of the ambient waters. Bifidobacterium adolescentis did not survive at either site but did show different rates of decline and physiological activity at the two sites. Bifidobacteria show promise as a better indicator of recent fecal contamination in tropical freshwaters than E. coli or fecal coliforms; however, the YN-6 medium did not prove to be effective for enumeration of bifidobacteria. The coliform maximum contaminant levels for assessing water usability for drinking and recreation appear to be unworkable in tropical freshwaters.     

Human activities as a driver of spatial variation in the trophic structure of fish communities on Pacific coral reefs

Anthropogenic activities such as land‐use change, pollution and fishing impact the trophic structure of coral reef fishes, which can influence ecosystem health and function. Although these impacts may be ubiquitous, they are not consistent across the tropical Pacific Ocean. Using an extensive database of fish biomass sampled using underwater visual transects on coral reefs, we modelled the impact of human activities on food webs at Pacific‐wide and regional (1,000s–10,000s km) scales. We found significantly lower biomass of sharks and carnivores, where there were higher densities of human populations (hereafter referred to as human activity); however, these patterns were not spatially consistent as there were significant differences in the trophic structures of fishes among biogeographic regions. Additionally, we found significant changes in the benthic structure of reef environments, notably a decline in coral cover where there was more human activity. Direct human impacts were the strongest in the upper part of the food web, where we found that in a majority of the Pacific, the biomass of reef sharks and carnivores were significantly and negatively associated with human activity. Finally, although human‐induced stressors varied in strength and significance throughout the coral reef food web across the Pacific, socioeconomic variables explained more variation in reef fish trophic structure than habitat variables in a majority of the biogeographic regions. Notably, economic development (measured as GDP per capita) did not guarantee healthy reef ecosystems (high coral cover and greater fish biomass). Our results indicate that human activities are significantly shaping patterns of trophic structure of reef fishes in a spatially nonuniform manner across the Pacific Ocean, by altering processes that organize communities in both “top‐down” (fishing of predators) and “bottom‐up” (degradation of benthic communities) contexts.     

Direct immunofluorescence assay for rapid environmental detection ofVibrio cholerae O1

An immunofluorescence assay for direct detection of V. cholerae O1 was developed using polyclonal antibodies raised against outer membrane proteins (OMPs) of V. cholerae O1. Production of OMPs varied with growth media used; maximum production was found in tryptic soy broth. The detection system was specific because no cross-reactivity was observed with other bacteria including V. cholerae O139, E. coli, S. dysenteriae and Salmonella enterica subsp. enterica serovar Typhi. The technique was able to detect 240 CFU/mL of V. cholerae O1 suspended in phosphate-buffered saline. The assay coupled with bacterial enrichment in APW for 6 h detected as few as 5 CFU of V. cholerae in spiked samples. Moreover, a 2-h incubation of enriched bacterial cells in 0.1% yeast extract with 10 ppm nalidixic acid enhanced the bacterial size and helped in morphological identification of V. cholerae. Among 32 potable water samples from afflicted hand pumps and wells collected from a cholera-plagued area 12 were found to be contaminated with V. cholerae by immunofluorescence assay as well as by conventional culture methods. The proposed method could thus be employed in environmental surveillance of V. cholerae O1.     

珊瑚病原微生物鉴定及其分子诊断技术进展

珊瑚礁生态系统是热带海洋最突出、最具代表性的生态系统,具有极高的生态价值和经济价值,然而由珊瑚疾病引起的珊瑚礁退化已经成为珊瑚礁生态系统的主要威胁之一。许多微生物(主要包括细菌、真菌、病毒)被认为与珊瑚疾病发生密切相关,确定珊瑚疾病的病原并建立其快速诊断的方法是开展珊瑚疾病流行病学调查和制定防控措施的必由之路。本文主要综述珊瑚疾病的病原微生物及其分子诊断技术的研究进展。     

Membrane fatty acid and virulence changes in the viable but nonculturable state of Vibrio vulnificus

The nonculturable state of Vibrio vulnificus and, for comparison, that of Escherichia coli were studied in artificial-seawater microcosms at 5 degrees C. Total cell counts were monitored by acridine orange epifluorescence, metabolic activity by direct viable counts, and culturability by plate counts on selective and nonselective media. Whereas total counts remained constant, plate counts of V. vulnificus suggested nonculturability by day 24. In contrast, direct viable counts indicated significant cell viability throughout 32 days of incubation. As an indication of the metabolic changes that occurred as cells entered the state of nonrecoverability, membrane fatty acid analyses were performed. At the point of nonculturability of V. vulnificus, the major fatty acid species (C16 and C16:1) had decreased 57% from the T0 level, concomitant with the appearance of several short-chain acids. Although the bacteria were still recoverable, a similar trend was observed with E. coli. Electron microscopy of nonculturable V. vulnificus showed that the cells were rounded and reduced in size and contained fewer ribosomes. Mouse infectivity studies conducted with these cells suggested loss of virulence.