Membrane Diffusion Chambers Research Articles

Impact of hypoxia on gene expression patterns by the human pathogen, Vibrio vulnificus, and bacterial community composition in a North Carolina estuary.

Estuarine environments are continuously being shaped by both natural and anthropogenic sources which directly/indirectly influence the organisms that inhabit these important niches on both individual and community levels. Human infections caused by pathogenic Vibrio species are continuing to rise, and factors associated with global climate change have been suggested to be impacting their abundance and geographical range. Along with temperature, hypoxia has also increased dramatically in the last 40 years, which has led to persistent dead zones worldwide in areas where these infections are increasing. Thus, utilizing membrane diffusion chambers, we investigated the impact of in situ hypoxia on the gene expression of one such bacterium, Vibrio vulnificus, which is an inhabitant of these vulnerable areas worldwide. By coupling these data with multiple abiotic factors, we were able to demonstrate that genes involved in numerous functions, including those involved in virulence, environmental persistence, and stressosome production, were negatively correlated with dissolved oxygen. Furthermore, comparing 16S ribosomal RNA, we found similar overall community compositions during both hypoxia and normoxia. However, unweighted beta diversity analyses revealed that although certain classes of bacteria dominate in both low‐ and high‐oxygen environments, there is the potential for quantitative shifts in lower abundant species, which may be important for effective risk assessment in areas that are becoming increasingly more hypoxic. This study emphasizes the importance of investigating hypoxia as a trigger for gene expression changes by marine Vibrio species and highlights the need for more in depth community analyses during estuarine hypoxia.

Survival of Helicobacter pylori in a natural freshwater environment.

The mode by which Helicobacter pylori, the causative agent of most gastric ulcers, is transmitted remains undetermined. Epidemiological evidence suggests these organisms are waterborne; however, H. pylori has rarely been grown from potential water sources. This may be due to the ability of this organism to rapidly enter the viable but nonculturable (VBNC) state. Our investigation examines the entrance of H. pylori into this state in laboratory cultures and a natural freshwater environment as well as the relationship between morphology and culturability. To this end, membrane diffusion chambers were utilized to expose the cells to the natural fluctuations of a freshwater stream. In both the laboratory and environment, samples were assayed for culturability using plate counts and stained using a LIVE/DEAD BacLight assay for viability and morphological determinations. Additionally, water samples were collected, six environmental parameters were measured, and resuscitation conditions were examined. H. pylori was observed to lose culturability in the laboratory and stream, although viability was maintained. While the results of our study agree with those of previous studies which suggested that there is a transition in morphology from rods to cocci as culturability is lost, the morphological distribution of cells did not change as culturability was lost in the environment. The majority of cells in the VBNC state in the laboratory are cocci; however, all morphological forms were present in the environment. The results of these studies suggest that H. pylori persists in laboratory cultures and the environment in the VBNC state and that cells in this state represent a public health hazard.

Survival and Plasmid Transfer Ability of Escherichia Coli in Wastewater

No differences were observed in the survival of plasmid-bearingand plasmid-free Escherichia coli strains in the course of a long-term survival process in wastewater, under both illuminated and non-illuminated conditions. While the CFU counts and the number of active cells decreased, the numberof nucleoid-containing cells remained constant throughout the 30 days of experimentation. Visible light efficiently contributed to the reduction in culturability, and T90 values were very different under illuminated and nonilluminatedconditions. In the latter case the time necessary to reduce theculturability of a bacterial population by 90% was 27 days, while in the former it was only 1 day. Plasmid transfer was abundant, while the survival of donor and recipient cells was extensive. After 24 hr of survival in wastewater, transfer frequency values ranged from 5.92 × 10-5 to 1.12 × 10-2, depending on mating conditions. In the absence of illumination, the potential transfer abilitiesremained for survival periods of at least 20 days. Transfer assays between free and adhered cells were carried out by means of dialysis bags and submerged membrane diffusion chambers. Transfer frequency for adhered cells was greater than for free cells (2.80 × 10-2 as opposed to 2.39 × 10-3).

Survival of Candida albicans and Pseudomonas aeruginosa in oil polluted tropical coastal waters

The effect of environmental abiotic factors on survival and activity of oil degrading isolates, Pseudomonas aeruginosa OD-1 and Candida albicans OD-2, was determined in situ using membrane diffusion chambers. The study sites were located in a tropical bay on the Atlantic Ocean with history of petroleum contamination by oil tankers. Microbial densities were measured by direct counts. 3H-thymidine uptake, microautoradiography, INT-reduction, adenosine triphosphate (ATP), and frequency of dividing cells were used to assess microbial activity. Both organisms showed a significant decrease in cell density over a three day period, although, temporal increases in densities were observed. Significant decreases in total activity were observed for both populations during the study; however, the respiration potential and ATP content per organism remained constant or even increased by the end of the study suggesting that a significant fraction of these populations were capable of withstanding in situ environmental conditions. Results suggest that increases in ambient phosphorus concentrations played a role in the prolonged in situ survival of these petroleum degrading isolates at some sites. The traffic of oil tankers might have played a significant impact on microbial survival in this bay, as violent agitation of sediments increased phosphorus concentrations available to microorganisms.

Entry into, and resuscitation from, the viable but nonculturable state by Vibrio vulnificus in an estuarine environment.

Using plate counts, total cell counts, and direct viable counts, we examined the fate of cells of Vibrio vulnificus placed into natural estuarine waters during both winter and summer months. Cells inoculated into membrane diffusion chambers and placed into estuarine waters entered into a viable but nonculturable (VBNC) state in January and February, when the water temperatures were low (average, < 15 degrees C). In contrast, when cells in the VBNC state were placed into the same waters in the warmer months of August through November (average water temperature of ca. 21 degrees C), the cells appeared to undergo a rapid (typically, within 24 h) resuscitation to the fully culturable state. These results were independent of whether the cells were in the logarithmic or stationary phase and whether they were encapsulated or not. This study indicates that the inability to isolate V. vulnificus from cold estuarine sites may be accounted for by entrance of the cells into a VBNC state and that recovery from this state in natural environments may result from a temperature upshift.

Encapsulation Technologies

Encapsulation systems have been developed in which cells are separated from the immune system of the host by permselective barriers. These systems do not require a life-long regimen of high dose immunosuppressive drugs to prevent immune rejection. Furthermore, they offer a solution to the problem of human cell procurement by permitting use of cells and tissues from animal sources. Three major types of immunoisolation devices have been studied by our group. These include perfusion devices anastomosed to the vascular system as atrioventricular (AV) shunts, tubular membrane diffusion chambers, and microreactors. This technology is applicable to treating a number of diseases by transplantation of cells that produce specific bioactive substances. In essence, this approach constitutes a living drug delivery and detoxification system. Our work has focused mainly on developing a new treatment for diabetes using encapsulated pancreatic islets. In the first type of system, canine and porcine islets were distributed in a chamber surrounding a permselective acrylic membrane (nominal M(r) exclusion of 80 kDa), and the devices implanted intraperitoneally as AV shunts into diabetic, totally pancreatectomized dogs without use of immunosuppression. In the second type of system, the islets were sealed within the acrylic membranes and the chambers implanted into the peritoneum of diabetic, pancreatectomized dogs (canine islets), streptozotocin (STZ)-induced diabetic rats (canine, bovine and porcine islets), and spontaneously diabetic BB/Wor rats (canine islets) without use of immunosuppression. In the third type of system (microreactors), the islets were implanted into the peritoneum of STZ-induced diabetic mice without use of immunosuppression, into STZ-induced diabetic rats (bovine and porcine islets) both with and without use of low dose immunosuppression, and into spontaneously diabetic dogs (canine islets) with low dose CsA. Results indicate that all three types of encapsulation systems significantly improve glucose homeostasis and can function for periods of several months to more than a year.

Survival of fecal microorganisms in marine and freshwater sediments.

The survival of culturable fecal coliforms, fecal streptococci, and Clostridium perfringens spores in freshwater and marine sediments from sites near sewage outfalls was studied. In laboratory studies, the inhibition of protozoan predators with cycloheximide allowed the fecal coliforms to grow in the sediment whereas the presence of predators resulted in a net die-off. C. perfringens spores did not appear either to be affected by predators or to die off throughout the duration of the experiments (28 days). Studies using in situ membrane diffusion chambers showed that, with the exception of C. perfringens, die-off of the test organisms to 10% of their initial numbers occurred in both marine and freshwater sediments within 85 days. The usual exponential decay model could not be applied to the sediment survival data, with the exception of the data for fecal streptococci. It was concluded that application of the usual decay model to the fecal coliform data was confounded by the complex relationship between growth and predation. The survival of seeded Escherichia coli in marine sediment was studied by using an enumeration method which detected viable but nonculturable bacteria. Throughout the duration of the experiment (68 days), the same proportion of E. coli organisms remained culturable, suggesting that sediment provides a favorable, nonstarvation environment for the bacteria.

A survival study of Escherichia coli in a south African river using membrane diffusion chambers

Studies to describe the survival of Escherichia coli were performed at two sites in a river. The one site was dominated by domestic discharge and the other by industrial inputs. E coli suspensions within membrane diffusion chambers were immersed in the river at the selected sites. An identical chamber was submerged in river water in the laboratory as a comparison. Two test runs were performed, one during winter (July) and one during summer (December). Samples to determine the survival of E coli was taken on a scheduled basis. Results obtained showed no significant difference between the survival pattern of E coli as determined during the summer and winter periods or in the different parts of the river. The same survival pattern was observed for the studies performed in the laboratory.

A microcosm study of the survival of Escherichia coli and Salmonella typhimurium in brackish water

The study of the survival of Escherichia coli and Salmonella typhimurium in sewage treated by stabilization ponds, and then mixed more or less rapidly with coastal marine water, has been experimentally tested in microcosms (membrane diffusion chambers). The results obtained show that under the same experiment conditions (slow saline, marine and rapid saline stress) the survival time of the two bacteria varies considerably. In sewage treated by stabilization ponds, Salmonella typhimurium and Escherichia coli exhibit the similar survival rates. T 90 evaluated for Escherichia coli and Salmonella typhimurium in the winter period was, respectively, 125 and 132 h. In brackish water the survival rate of Salmonella typhimurium was generally higher than that of Escherichia coli. When these two bacteria were suspended in stabilization ponds effluent and rapidly mixed with brackish water, survival time was particularly short, whereas it was prolonged when the bacteria were submitted to a gradual increase in salinity. Biological factors, and in particular predation, can play an important role in eliminating enteric bacteria during pond system treatment, but their efficiency is supposed to diminish significantly when there is an environmental change such as the discharge of pond system effluent into brackish water. This can explain the increase of enteric bacteria survival.

A survival study of Escherichia coli in a South African river using membrane diffusion chambers

Studies to describe the survival of Escherichia coli were performed at two sites in a river. The one site was dominated by domestic discharge and the other by industrial inputs. E. coli suspensions within membrane diffusion chambers were immersed in the river at the selected sites. An identical chamber was submerged in river water in the laboratory as a comparison. Two test runs were performed, one during winter (July) and one during summer (December). Samples to determine the survival of E coli was taken on a scheduled basis. Results obtained showed no significant difference between the survival pattern of E coli as determined during the summer and winter periods or in the different parts of the river. The same survival pattern was observed for the studies performed in the laboratory.

Conjugal transfer of R plasmids to and from Enterobacteriaceae isolated from sewage.

The potential of the transfer of natural plasmids between sewage strains has been studied. In vitro transfer was conducted at 37 degrees C in tryptone soya broth and sterile raw sewage as mating media. In situ transfer was carried out in sterile raw sewage within membrane diffusion chambers at 10.6 degrees C. When the recipient was a laboratory strain of Escherichia coli K-12, the in situ frequency values were significantly lower (P less than 0.001) than those obtained in vitro for the same mating pair. When the laboratory recipient was replaced with recipients from the same sewage source, frequency values decreased progressively from the optimum conditions to the most adverse. However, in situ frequency values were higher than those for the same donors mated with a laboratory recipient.

Survival ofEscherichia coli andYersinia enterocolitica in stream water: Comparison of field and laboratory exposure

Experiments were done to compare the influence of three aquatic exposure methods on the behavior of pathogenic and nonpathogenic enteric bacteria (Yersinia enterocolitica andEscherichia coli). Bacterial suspensions were exposed to stream water in membrane diffusion chambers in situ as well as in the laboratory using a large vessel of stream water and in enclosed bottles. The persistence of culturability of the bacterial suspensions was dependent upon the method of aquatic exposure. This difference was most apparent during the initial six days of each experiment. A steady decline in colony forming units was seen after a short stationary period in chambers in situ, while there was an abrupt increase in bacteria within chambers exposed in the laboratory. A rapid initial decrease was observed in the experimental variation using bottles, accompanied by higher levels of injury inE. coli and reduced expression of plasmid-borne virulence phenotypes inY. enterocolitica. However, there were no changes in the plasmid profiles of either organism throughout the 21-day duration of the experiments. In addition, the survival and injury of pathogenic and nonpathogenic strains of both test bacteria was very similar with aquatic exposure. These results suggest that the response of enteric bacteria in aquatic environments is influenced by experimental design as well as other factors and that the comparison of survival data should only be attempted when similar methods are used.

Survival and injury of Escherichia coli, Campylobacter jejuni, and Yersinia enterocolitica in stream water

Experiments were done to describe the survival and injury of three strains each of enteropathogenic Escherichia coli, Yersinia enterocolitica, and Campylobacter jejuni in agricultural surface water. Bacterial suspensions within membrane diffusion chambers were immersed in two large vessels of stream water that were held at 6 and 16 degrees C and changed daily. The results of daily plate counts, using selective and nonselective media, revealed some variation among strains and genera. Injury increased rapidly in all of the bacteria examined during an initial 4-day period of population stability. Bacterial persistence was generally prolonged at 6 degrees C, while the occurrence of injury was directly related to temperature. However, both survival and injury in C. jejuni were less dependent on temperature, while Y. enterocolitica displayed the greatest survival at both 6 and 16 degrees C. These results suggest that surface water in the temperate zone might serve as a persistent vehicle for the transmission of these enteropathogenic bacteria between animals and humans.

Activity and adaptation of nitrilotriacetate (NTA)-degrading bacteria: field and laboratory studies

Adaptation of bacterial activity for the degradation of nitrilotriacetate (NTA) was studied using natural sediment samples and an NTA-degrading bacterium (strain ATCC 29600). Sediment samples from a river with persistent levels of NTA had much higher NTA-degradative activity than comparable samples from a less contaminated control site. When sediment from the control site was exposed to high levels of NTA a 5 day lag preceded an abrupt increase in NTA degradation while strain 29600 colonized on sand and grown in the absence of NTA became induced within eight hours. The induction of strain 29600 was compared between bacteria in suspension and cells attached to sand. The sand-associated bacteria became induced 4 to 5 h before the planktonic suspension and displayed over threefold greater specific activity. Suspensions of strain 29600 became adapted within 8 h when placed in membrane diffusion chambers that were immersed within a municipal wastewater reactor containing NTA. These findings support the concept that induction is a part of the process of bacterial adaptation to degrade NTA and sand-associated bacteria can adapt more quickly to and have a greater degradative activity for NTA than planktonic cells.

Effects of sunlight and autochthonous microbiota onEscherichia coli survival in an estuarine environment

The effects of sunlight and the indigenous microbiota onEscherichia coli survival were examined with membrane diffusion chambers deployed in Chesapeake Bay shellfish growing waters. Chambers, fitted with an “upper” UV and visible light-transmitting copolymer film and “lower” semipermeable polycarbonate membrane, were deployed parallel to the water surface to maximize light exposure. Maximum values of a coefficient, kdens, describing changes in culturable cell densities after exposure to sunlight, were 1.7 h−1 and 0.7 h−1 in preliminary tank and in situ experiments, respectively. Mortality and sublethal stress, the latter measured with an electrochemical detection technique, were largest during the first 4 h of exposure. Owing to the light-attenuating properties of Chesapeake Bay water, light-induced cell mortality was significantly reduced at 0.25 m compared with surface exposed cells, and was undetected at 0.5–1.0 m except during seasons of maximal light penetration. Combined exposure to both sunlight and the autochthonous microbiota yielded significantly greater mortality than for either factor alone.

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.

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 and distribution ofYersinia enterocolitica in a tropical rain forest stream

The survival and activity ofYersinia enterocolitica andEscherichia coli in a tropical rain forest stream were studied in situ in membrane diffusion chambers. Direct counts ofY. enterocolitica decreased by one order of magnitude during the first 6 h and then remained constant. Densities ofE. coli increased over time, doubling after 2 days. Physiological activity ofE. coli dropped initially and then stabilized at 85%. Physiological activity forY. enterocolitica increased during the first 6 h, then declined to 50%. The percentage of respiring cells as measured by 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride reduction decreased forE. coli to 10%, whereasY. enterocolitica remained near 25%;Y. enterocolitica is a survivor in tropical freshwater, as isE. coli. Indirect and direct fluorescent antibody (FA) methods were evaluated for the direct detection ofY. enterocolitica in natural habitats. Natural densities of FA-positive cells were always less than 10 cells ml−1, and no isolates were obtained by culturing samples.

Survival and activity of Streptococcus faecalis and Escherichia coli in petroleum-contaminated tropical marine waters

The in situ survival and activity of Streptococcus faecalis and Escherichia coli were studied using membrane diffusion chambers in tropical marine waters receiving oil refinery effluents. Protein synthesis, DNA synthesis, respiration or fermentation, INT reduced per cell, and ATP per cell were used to measure physiological activity. Cell densities decreased significantly over time at both sites for both S. faecalis and E. coli; however, no significant differences in survival pattern were observed between S. faecalis and E. coli. Differences in protein synthesis between the two were only observed at a study site which was not heavily oiled. E. coli was more active in protein synthesis and respiration than S. faecalis at both oiled and unoiled sites, and the percentage of the E. coli population that was respiring was significantly higher than S. faecalis fermenting cells at both sites. However, S. faecalis cells were more active in DNA synthesis and higher in ATP content than E. coli cells at both sites. Although fecal streptococci have been suggested as a better indicator of fecal contamination than fecal coliforms in marine waters, in this study both E. coli and S. faecalis survived and remained physiologically active for extended periods of time. These results suggest that the fecal streptococci group is not a better indicator of fecal contamination in tropical marine waters than the fecal coliform group, especially when that environment is high in long-chained hydrocarbons.

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.