Role of sediment in the persistence of enteroviruses in the estuarine environment.

The survival of four enteroviruses commonly found in sewage effluents was examined when the viruses were adsorped to marine sediments in estuarine water and compared with virus survival in estuarine water alone. Echovirus 1, coxsackieviruses B3 and A9, and poliovirus 1 survived longer when associated with marine sediment. When the estuarine water was polluted with secondarily treated sewage effluent, virus survived for prolonged periods in sediments, but not in the overlaying estuarine water.     

Influence of estuarine sediment on virus survival under field conditions

The survival of poliovirus 1 (LSc) and echovirus 1 (Farouk) in estuarine water and sediment was studied in Galveston Bay, Texas. Viruses were suspended in estuarine water and sediment both in dialysis tubing and in chambers constructed with polycarbonate membrane walls. Virus inactivation rates in seawater were similar in both types of chambers. Virus adsorption to sediment greatly increased survival time. The time required to inactivate 99% (T-99) of poliovirus increased from 1.4 days in seawater alone to 6.0 days for virus adsorbed to sediment at a relatively nonpolluted site. At a more polluted site, poliovirus T-99 was increased from approximately 1 h to 4925 days by virus adsorption to sediment. This study demonstrates that under field conditions virus association with estuarine sediment acts to prolong its survival in the marine environment.     

Influence of estuarine sediment on virus survival under field conditions.

The survival of poliovirus 1 (LSc) and echovirus 1 (Farouk) in estuarine water and sediment was studied in Galveston Bay, Texas. Viruses were suspended in estuarine water and sediment both in dialysis tubing and in chambers constructed with polycarbonate membrane walls. Virus inactivation rates in seawater were similar in both types of chambers. Virus adsorption to sediment greatly increased survival time. The time required to inactivate 99% (T-99) of poliovirus increased from 1.4 days in seawater alone to 6.0 days for virus adsorbed to sediment at a relatively nonpolluted site. At a more polluted site, poliovirus T-99 was increased from approximately 1 h to 4925 days by virus adsorption to sediment. This study demonstrates that under field conditions virus association with estuarine sediment acts to prolong its survival in the marine environment.     

Development of a quantitative method for detecting enteroviruses in estuarine sediments.

Several investigators have reported on the detection of enteric viruses in marine sediments, but none determined the efficiency of their methods and only limited volumes of sediment were sampled. The purpose of this investigation was to develop a quantitative method for detecting enteroviruses in marine sediments so that their relative proportion to viruses freely suspended in estuarine water could be more accurately determined. Poliovirus was found to adsorb readily to natural marine sediments collected along the Texas Gulf coast. A number of substances were evaluated for their ability to elute adsorbed viruses. A solution of 10% fetal calf serum adjusted to pH 10.5 and 0.05M ethylenediaminetetraacetate (pH 11.0) were found to be the best eluents. Using ethylenediaminetetraacetate as an eluent, it was possible to elute virus from large volumes of sediment and reconcentrate the sediment eluate into an economically assayable volume (30 to 50 ml). Poliovirus could be recovered from the sediment with an overall efficiency of 50%. This method was found to be satisfactory for the recovery of naturally occurring animal viruses in estuarine sediments from the upper Texas Gulf coast.     

Development of a quantitative method for detecting enteroviruses in estuarine sediments.

Several investigators have reported on the detection of enteric viruses in marine sediments, but none determined the efficiency of their methods and only limited volumes of sediment were sampled. The purpose of this investigation was to develop a quantitative method for detecting enteroviruses in marine sediments so that their relative proportion to viruses freely suspended in estuarine water could be more accurately determined. Poliovirus was found to adsorb readily to natural marine sediments collected along the Texas Gulf coast. A number of substances were evaluated for their ability to elute adsorbed viruses. A solution of 10% fetal calf serum adjusted to pH 10.5 and 0.05M ethylenediaminetetraacetate (pH 11.0) were found to be the best eluents. Using ethylenediaminetetraacetate as an eluent, it was possible to elute virus from large volumes of sediment and reconcentrate the sediment eluate into an economically assayable volume (30 to 50 ml). Poliovirus could be recovered from the sediment with an overall efficiency of 50%. This method was found to be satisfactory for the recovery of naturally occurring animal viruses in estuarine sediments from the upper Texas Gulf coast.     

Influence of pH, salinity, and organic matter on the adsorption of enteric viruses to estuarine sediment.

This study was designed to determine the degree of adsorption of enteric viruses to marine sediment and factors controlling this association. Adsorption and elution characteristics of several enteroviruses and one rotavirus to estuarine sediments were studied under varying conditions of pH, salinity, and presence of soluble organics. Greater than 99% of the added poliovirus type 1 (LSc), coxsackievirus type B3 (Nancy), echovirus type 7 (Wallace), and rotavirus (SA-11) adsorbed to sediment. Echovirus 1 (Farouk) and a recent isolate typed as coxsackievirus B4 adsorbed significantly less than poliovirus 1 under similar conditions of varying salinity and pH. The presence of soluble organic matter, in the form of secondary sewage effluent or humic acid, did not affect these patterns of adsorption. Only echovirus 1 (Farouk) desorbed when the pH or salinity was altered and then only to a small extent. Three recent isolates of echovirus 1 and echovirus 29 (strain JV-10) also demonstrated varying amounts of adsorption to sediment. These data indicate that enteric viruses can become readily associated with sediment in the estuarine environment and that this association may play a major role in their hydrotransportation and survival.     

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.     

Relationships Between Environmental Factors, Bacterial Indicators, and the Occurrence of Enteric Viruses in Estuarine Sediments

Current standards for evaluation of the public health safety of recreational and shellfish-harvesting waters are based upon bacteriological analysis, but do not include an evaluation of the number of viruses. The objective of this study was to determine the occurrence of enteric viruses in estuarine sediments and to find a relationship, if any, between the presence of viruses in seawater or sediment or both and various biological and physicochemical characteristics of the environment. Viruses were found in greater numbers in sediment than in overlying seawater on a volume basis. Several types of enteroviruses were isolated: coxsackievirus types A16, B1, and B5, echovirus type 1, and poliovirus type 2. On several occasions, viruses were isolated from sediments when overlying seawaters met bacteriological water quality standards for recreational use. Statistical analysis of the relationship between viruses in seawater or in sediment and other variables measured yielded only one significant association: the number of viruses in sediment was found to be positively correlated with the number of fecal coliforms in sediment. No other physical, chemical, or biological characteristic of seawater or sediment that was measured showed statistically significant association with viral numbers. No correlation was found between bacterial indicators and virus in the overlying waters. The data indicated that evaluation of the presence of bacteria and viruses in sediment may provide additional insight into long-term water quality conditions and that indicator bacteria in water are not reflective of the concentration of enteric viruses in marine waters.     

Relationships between environmental factors, bacterial indicators, and the occurrence of enteric viruses in estuarine sediments

Current standards for evaluation of the public health safety of recreational and shellfish-harvesting waters are based upon bacteriological analysis, but do not include an evaluation of the number of viruses. The objective of this study was to determine the occurrence of enteric viruses in estuarine sediments and to find a relationship, if any, between the presence of viruses in seawater or sediment or both and various biological and physicochemical characteristics of the environment. Viruses were found in greater numbers in sediment than in overlying seawater on a volume basis. Several types of enteroviruses were isolated: coxsackievirus types A16, B1, and B5, echovirus type 1, and poliovirus type 2. On several occasions, viruses were isolated from sediments when overlying seawaters met bacteriological water quality standards for recreational use. Statistical analysis of the relationship between viruses in seawater or in sediment and other variables measured yielded only one significant association: the number of viruses in sediment was found to be positively correlated with the number of fecal coliforms in sediment. No other physical, chemical, or biological characteristic of seawater or sediment that was measured showed statistically significant association with viral numbers. No correlation was found between bacterial indicators and virus in the overlying waters. The data indicated that evaluation of the presence of bacteria and viruses in sediment may provide additional insight into long-term water quality conditions and that indicator bacteria in water are not reflective of the concentration of enteric viruses in marine waters.     

Anaerobic ortho Dechlorination of Polychlorinated Biphenyls by Estuarine Sediments from Baltimore Harbor

Reductive dechlorination of the ortho moiety of polychlorinated biphenyls (PCBs) as well as of meta and para moieties is shown to occur in anaerobic enrichments of Baltimore Harbor sediments. These estuarine sediments ortho dechlorinated 2,3,5,6-chlorinated biphenyl (CB), 2,3,5-CB, and 2,3,6-CB in freshwater or estuarine media within a relatively short period of 25 to 44 days. ortho dechlorination developed within 77 days in marine medium. High levels of ortho dechlorination (>90%) occurred when harbor sediments were supplied with only 2,3,5-CB. Incubation with 2,3,4,5,6-CB or 2,3,4,5-CB resulted in the formation of the ortho dechlorination product 3,5-CB; however, para dechlorination of these congeners always preceded ortho chlorine removal. ortho dechlorination of PCBs is an exceedingly rare event that has not been reported previously for marine or estuarine conditions. The activity was reproducible and could be sustained through sequential transfers. In contrast, freshwater sediments incubated under the same conditions exhibited only meta and para dechlorinations. The results indicate that unique anaerobic dechlorinating activity is catalyzed by microorganisms in the estuarine sediments from Baltimore Harbor.     

Sediment Composition Influences Spatial Variation in the Abundance of Human Pathogen Indicator Bacteria within an Estuarine Environment

Faecal contamination of estuarine and coastal waters can pose a risk to human health, particularly in areas used for shellfish production or recreation. Routine microbiological water quality testing highlights areas of faecal indicator bacteria (FIB) contamination within the water column, but fails to consider the abundance of FIB in sediments, which under certain hydrodynamic conditions can become resuspended. Sediments can enhance the survival of FIB in estuarine environments, but the influence of sediment composition on the ecology and abundance of FIB is poorly understood. To determine the relationship between sediment composition (grain size and organic matter) and the abundance of pathogen indicator bacteria (PIB), sediments were collected from four transverse transects of the Conwy estuary, UK. The abundance of culturable Escherichia coli, total coliforms, enterococci, Campylobacter, Salmonella and Vibrio spp. in sediments was determined in relation to sediment grain size, organic matter content, salinity, depth and temperature. Sediments that contained higher proportions of silt and/or clay and associated organic matter content showed significant positive correlations with the abundance of PIB. Furthermore, the abundance of each bacterial group was positively correlated with the presence of all other groups enumerated. Campylobacter spp. were not isolated from estuarine sediments. Comparisons of the number of culturable E. coli, total coliforms and Vibrio spp. in sediments and the water column revealed that their abundance was 281, 433 and 58-fold greater in sediments (colony forming units (CFU)/100g) when compared with the water column (CFU/100ml), respectively. These data provide important insights into sediment compositions that promote the abundance of PIB in estuarine environments, with important implications for the modelling and prediction of public health risk based on sediment resuspension and transport.     

Investigation of the survival characteristics of Rhodococcus coprophilus and certain fecal indicator bacteria.

Rhodococcus coprophilus and Clostridium perfringens survived in fresh water samples held at 5, 20, and 30 degrees C for over 17 weeks, whereas Escherichia coli and fecal streptococci disappeared after 5 weeks at all three temperatures. R. coprophilus survived for more than 8 months in sterilized sewage and deionized water at all three temperatures, whereas in normal sewage held at 20 degrees C, the survival time was 12 to 26 weeks. In samples held at 30 degrees C, survival times were shorter, probably because of interbacterial competition or protozoal predation. The results indicate that R. coprophilus may be a useful indicator of the presence of remote fecal pollution of farm animal origin, but not of recent pollution, when enumerated alone in polluted waters or wastewaters.     

Comparative survival of antibiotic-resistant and -sensitive fecal indicator bacteria in estuarine water.

The survival of antibiotic-resistant and -sensitive strains of Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Streptococcus equinus, and two environmental isolates, AP17 and AQ62, was examined in estuarine water. Each strain was rendered resistant to a combination of two antibiotics by serial passage in increasing concentrations of antibiotics. Cultures were incubated in filter-sterilized estuarine water for up to 7 days. Recovery was assessed by examining colony-forming ability on media with and without antibiotics. None of the antibiotic-resistant forms survived longer than its antibiotic-sensitive counterpart in estuarine water. Three of the resistant strains died off more rapidly than the antibiotic-sensitive wild type. Survival of the test bacteria in estuarine water was as follows: sensitive and resistant AQ62, resistant Escherichia coli less than sensitive Escherichia coli less than resistant AP17 less than resistant Enterococcus faecium less than sensitive AP17, sensitive and resistant S. equinus less than sensitive and resistant Enterococcus faecalis, sensitive Enterococcus faecium. The results supported the suggestion that fecal entercocci may serve as better indicators of fecal pollution than Escherichia coli in marine ecosystems. Moreover, the results indicated that the use of antibiotic-resistant mutants to follow the fate of bacteria in the environment is inappropriate without adequate studies to ensure that resistant and wild-type strains react similarly to environmental stressors.     

Occurrence of False-Positive Most Probable Number Tests for Fecal Streptococci in Marine Waters

By the use of the most probable number technique with azide dextrose and ethyl violet azide broths for enterococci, the common occurrence of false-positive tests was noted when marine and estuarine waters were sampled. Organisms isolated included a marine bacterium, gram-positive and gram-negative nonmarine bacteria, and yeasts. All cultures were capable of growth in azide-dextrose, ethyl violet-azide, and KF broths. Representative isolates grew in media containing 0.08% NaN(3). The tentatively accepted most probable number method for fecal streptococci is thus of dubious value in assessment of sewage pollution levels in estuarine waters. All positive tubes must be examined microscopically for the presence of nonstreptococcal forms.     

The relative importance of sediment and water column supplies of nutrients to the growth and tissue nutrient content of the green macroalga Enteromorpha intestinalis along an estuarine resource gradient

Large blooms of opportunistic green macroalgae such as Enteromorpha intestinalis are of ecological concern in estuaries worldwide. Macroalgae derive their nutrients from the water column but estuarine sediments may also be an important nutrient source. We hypothesized that the importance of these nutrient sources to E. intestinalis varies along a nutrient-resource gradient within an estuary. We tested this in experimental units constructed with water and sediments collected from 3 sites in Upper Newport Bay estuary, California, US, that varied greatly in water column nutrient concentrations. For each site there were three treatments: sediments + water; sediments + water + Enteromorpha intestinalis (algae); inert sand + water + algae. Water in units was exchanged weekly simulating low turnover characteristic of poorly flushed estuaries. The importance of the water column versus sediments as a source of nutrients to E. intestinalis varied with the magnitude of the different sources. When initial water column levels of dissolved inorganic nitrogen (DIN) and soluble reactive phosphorus (SRP) were low, estuarine sediments increased E. intestinalis growth and tissue nutrient content. In experimental units from sites where initial water column DIN was high, there was no effect of estuarine sediments on E. intestinalis growth or tissue N content. Salinity, however, was low in these units and may have inhibited growth. E. intestinalis growth and tissue P content were highest in units from the site with highest initial sediment nutrient content. Water column DIN was depleted each week of the experiment. Thus, the water column was a primary source of nutrients to the algae when water column nutrient supply was high, and the sediments supplemented nutrient supply to the algae when water column nutrient sources were low. Depletion of water column DIN in sediment + water units indicated that the sediments may have acted as a nutrient sink in the absence of macroalgae. Our data provide direct experimental evidence that macroalgae utilize and ecologically benefit from nutrients stored in estuarine sediments.     

Thermostabilization of enteroviruses by estuarine sediment

The effect of estuarine sediment on the thermoinactivation of poliovirus type 1 and echovirus type 1 was evaluated. Poliovirus survival was prolonged at 24 and 37 degrees C but not at 4 degrees C in the presence of sediment over the time periods observed. Further inactivation studies were performed at 50 and 55 degrees C to maximize the thermal effects, and similar protection was observed. The supernatant fluid from a mixture of seawater and sediment lacked the protective effect against thermoinactivation, suggesting that prolonged virus survival in the presence of sediment was due to adsorption to particulates. From these observations, it appears that the adsorption of enteroviruses to estuarine sediments may play a significant role in protecting them against thermoinactivation.     

Survival of faecal indicator bacteria in tropical estuarine waters (Bangpakong River,Thailand)

AIMS: To investigate the survival of cultivable bacteria in the tropical Bangpakong estuary (Eastern Thailand) under different salinities and light conditions. METHODS AND RESULTS: Dark and light microcosm experiments using membrane diffusion chambers were carried out under three different experimental conditions, namely (i) low salinity, (ii) progressive mixing with brackish water and (iii) fast mixing with high salinity water spiked with raw urban sewage. Faecal coliforms declined faster than faecal enterococci, as shown by survival T90 values ranging from 82.2 +/- 4.2 to 14.5 +/- 0.8 h and 97.5 +/- 0.4-20.6 +/- 1.2 h, respectively. The survival of freshwater heterotrophic bacteria was higher but variable (121.2 +/- 5.0-30.1 +/- 14.3 h), whereas that of heterotrophic marine bacteria was rather stable (81.5 +/- 4.2-44.6 +/- 2.5 h). CONCLUSIONS: Overall survival was higher in low salinities. Light had a further deleterious effect, since it accelerated the decay of faecal indicators, particularly in high salinities. Faecal enterococci had a higher resistance to environmental conditions compared with faecal coliforms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is relevant to the understanding of the behaviour of different faecal indicator bacteria and the optimization of sewage treatment plants aimed at the reduction and/or elimination of faecal load discharged into estuarine waters submitted to salinity variations.     

Thermostabilization of enteroviruses by estuarine sediment.

The effect of estuarine sediment on the thermoinactivation of poliovirus type 1 and echovirus type 1 was evaluated. Poliovirus survival was prolonged at 24 and 37 degrees C but not at 4 degrees C in the presence of sediment over the time periods observed. Further inactivation studies were performed at 50 and 55 degrees C to maximize the thermal effects, and similar protection was observed. The supernatant fluid from a mixture of seawater and sediment lacked the protective effect against thermoinactivation, suggesting that prolonged virus survival in the presence of sediment was due to adsorption to particulates. From these observations, it appears that the adsorption of enteroviruses to estuarine sediments may play a significant role in protecting them against thermoinactivation.     

Distribution of the Luminous Bacterium Beneckea harveyi in a Semitropical Estuarine Environment

Bioluminescent bacteria were found in the water column, sediment, shrimp, and gastrointestinal tract of marine fishes from the semitropical estuarine environment of the East Lagoon, Galveston Island, Tex. Populations in the water column decreased during cold weather while sedimentary populations persisted. The highest percentages of luminous organisms were isolated from the gastrointestinal tract of marine fishes, where they persisted during 5 days of starvation. The presence of chitin temporarily increased intestinal populations. All isolates were Beneckea harveyi, whose natural habitat appears to be the gut of fishes and whose free-living reservoir appears to be marine sediments.     

Distribution of thermophilic endospores in a temperate estuary indicate that dispersal history structures sediment microbial communities

Endospores of thermophilic bacteria are found in cold and temperate sediments where they persist in a dormant state. As inactive endospores that cannot grow at the low ambient temperatures, they are akin to tracer particles in cold sediments, unaffected by factors normally governing microbial biogeography (e.g., selection, drift, mutation). This makes thermophilic endospores ideal model organisms for studying microbial biogeography since their spatial distribution can be directly related to their dispersal history. To assess dispersal histories of estuarine bacteria, thermophilic endospores were enriched from sediments along a freshwater‐to‐marine transect of the River Tyne in high temperature incubations (50°C). Dispersal histories for 75 different taxa indicated that the majority of estuarine endospores were of terrestrial origin; most closely related to bacteria from warm habitats associated with industrial activity. A subset of the taxa detected were marine derived, with close relatives from hot deep marine biosphere habitats. These patterns are consistent with the sources of sediment in the River Tyne being predominantly terrestrial in origin. The results point to microbial communities in estuarine and marine sediments being structured by bi‐directional currents, terrestrial run‐off and industrial effluent as vectors of passive dispersal and immigration.