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Notes: The invasive potential of environmental and clinical strains of O1 and non-O1 Vibrio cholerae was examined. Mice injected subcutaneously (s.c.) with strains of these organisms were monitored for the development of lesions or mortality. Of 12 strains of non-O1 V. cholerae tested, 7 gave a high mortality rate. All other strains produced swelling and lesions at the site of inoculation. The injection of highly virulent strains of non-O1 V. cholerae produced bacteremia. In contrast, only 1 of the 10 strains of O1 V. cholerae tested was highly lethal, and bacteremia was not detected. The highly virulent strains of O1 and non-O1 V. cholerae failed to produce keratoconjunctivitis in the Sereny test. A qualitative enzyme study showed no differences in ability to produce enzymes associated with invasiveness.

Notes: Abstract The non-culturable state of Vibrio vulnificus, strain C7184, was studied in artificial seawater microcosms held at 5, 10, 15, 20, and 30°C. Plate counts were made on a non-selective medium, total cell counts were monitored by acridine orange epifluorescence, and direct viable counts (DVSs) by the method of Kogure et al. (Can J. Microbiol. 25, 415–420; 1986) and by the INT method. From an initial inoculum of 107 cells/ml, V. vulnificus became non-culturable within 40 days at 5°C, although both indicators of viability revealed a viable population exceeding 106 cells/ml. Cells at all higher temperatures remained culturable (at least 104/ml) throughout the study. The non-culturable states of the opaque and translucent colony variants of V. vulnificus, as well as those of six other clinical and environmental strains of V. vulnificus, were examined at 5°C; all but one strain and both colony variants also became non-culturable within 40 days. In contrast, six other Vibrio spp. (V. cholerae, V. mimicus, V. parahaemolyticus, V. natriegens, V. proteolyticus, and V. campbelli) remained culturable at 5°C. Thus, entrance of V. vulnificus into the non-culturable state appears to be highly temperature dependent and, among the vibrios, this species may be especially sensitive to low temperature. The public health aspects of these findings are discussed.

Notes: Abstract: A series of 16 buffers, differing in pH and MgCl2 concentration, were used to optimize the polymerase chain reaction (PCR) amplification of a 388 bp region of the hemolysin / cytolysin gene from cells of Vibrio vulnificus present in both the culturable and nonculturable states. Both the opaque and translucent morphotypes were examined. Using whole cell lysates, we were able to obtain amplification of DNA from as few as 28.5 cells present in the viable but nonculturable state. With one exception, all buffers that produced amplification using culturable cells also produced amplification using nonculturable cells. However, regardless of the buffer employed, 100 times more nonculturable cells than culturable cells were required to obtain a PCR product. Our data suggest that caution should be exercised when employing PCR optimized against culturable cells when this method is employed for the detection of nonculturable cells.

Notes: Purpose - The purpose of this paper is to evaluate the influence of a mixture of nutrient solution, bacteria and biofilm on the consolidation, unconfined compression and desiccation characteristics of two soils that could be used in waste containment applications. Design/methodology/approach - Experimental work was conducted to investigate the influence of biofilm on the desiccation, strength and consolidation characteristics of two barrier soils. The soils were evaluated with water alone and with a biofilm solution composed of nutrients, bacteria and exopolymeric substances (EPS). These solutions were mixed with a locally available clay ("red bull tallow" (RBT)) as well as a mix of 65 percent sand and 35 percent bentonite (65-35 Mix). Findings - Reductions in strength and increases in ductility are observed with biofilm amendment for two soil types. The shear strength was reduced from 413 to 313?kPa and from 198 to 179?kPa for RBT and 65-35 Mix, respectively. Desiccation tests reveal an increase in moisture retention for early time increments in amended specimens, while both increases and decreases are noted after extended drying. Increases in the rate of consolidation and modest decreases in the compression and swell index were observed. In particular, the consolidation coefficient was increased from 0.036 to 0.064?cm2/min and from 0.060 to 0.093?cm2/min for RBT and 65-35 Mix, respectively. Practical implications - These results are useful in establishing the broader impacts of using biofilm as an additive to increase the performance (e.g. reduce hydraulic conductivity and increase resistance to crack formation) of barrier materials in waste containment applications. Moreover, the data provide insight into the geotechnical implications of biofilm-producing methanotrophic activity that occurs naturally in the covers of municipal solid waste landfills. Originality/value - Very little research has been published on the influence of biofilm on the behavior of barrier materials in general, and on geotechnical properties in particular. This paper is unique in making the connection between methanotrophic activity, soil modification and barrier material performance.

Notes: Abstract: The ability to track genetically modified bacteria released into the environment is essential for assessing their persistence and dispersal. Some bacteria can enter a ‘viable but nonculturable’ (VBNC) state in which the cells remain viable while losing the ability to grow on routine culture media. Thus, VBNC cells are not detectable by standard plating methods. In order to determine what conditions, if any, induce this state in Pseudomonas fluorescens, Pseudomonas syringae, and Escherichia coli, cells were ‘marked’ with lux genes, either chromosomally or on one of two different plasmids. Variations in temperature, but not nutrient or NaCl concentrations, affected culturability of these strains and induced the VBNC state. The temperature which induced the VBNC state in the two pseudomonads depended on whether or not the cell carried one of the two lux-marked plasmids. This effect was shown not to be due to the presence of the lux genes, as their removal from the plasmid had no effect on entry into the VBNC state. Instead, the effect appeared to depend on the location of the plasmid DNA, as a strain of P. fluorescens with the same plasmid integrated into the chromosome behaved identically to the parent strain. The fact that plasmids may have such a dramatic effect on culturability has significant implications for the monitoring of genetically modified bacteria intended for environmental release.

Notes: Vibrio vulnificus is an opportunistic human pathogen which is the causative agent of food-borne disease and wound infections. V. vulnificus is able to adapt to a variety of potentially stressful environmental changes, such as osmotic, nutrient, and temperature variations in estuarine environments, as well as oxidative, osmotic, and acidity differences following infection of a human host. After exposure to sub-lethal levels of a particular environmental stress, many bacteria become resistant to unrelated stresses, a phenomenon termed cross protection. In this study, we examined the ability of osmotic shock to cross protect V. vulnificus to high temperature as well as oxidative stress. Log phase cells of V. vulnificus strain C7184o were cross protected by prior osmotic shock to both heat and oxidative challenge, but only when exogenous nutrient was present during the osmotic upshift. Further, and unlike other bacteria, nutrient starvation alone did not result in cross protection against either stress. When small amounts of nutrient were present during osmotic shock, cross protection to an otherwise lethal heat challenge developed extremely rapidly, with significant protection seen within 10 min. Cross protection to oxidative stress was slower to develop, requiring several hours. Although stationary phase alone conferred some cross protection to heat and oxidative stress, the alternate sigma factor RpoS was required for complete cross protection of log phase cells to oxidative stress but not for resistance to heat challenge. Together these findings suggest that the cross protective response in V. vulnificus is complex and appears to involve multiple mechanisms.

Notes: This review describes the factors which are currently recognized as being central to the virulence of the human pathogen, Vibrio vulnificus. This estuarine/marine bacterium occurs in high numbers in molluscan shellfish, primarily oysters, and its ingestion in raw oysters results in a ca. 60% mortality in those persons who are susceptible to this bacterium. The organism is also able to produce life-threatening wound infections. We describe here the nature of both the wound and primary septicemia infections, the virulence factors known or believed to be involved in these infections, possible immunotherapy, and some thoughts on the possibility that not all strains of this pathogen are virulent.

Notes: Vibrio vulnificus has proven difficult to culture from water or shellfish during winter months, which is attributed to the viable but nonculturable (VBNC) state. Because reactive oxygen species were found to be involved in the low temperature-induced entrance of V. vulnificus into this state, we generated an oxyR mutant which lacks catalase activity. This strain is nonculturable on solid media even at ambient temperature, due to the presence of H2O2 in such media. Low temperature incubation of the parent resulted in loss of catalase activity, making the cells H2O2 sensitive, and paralleling the loss of culturability (entry into the VBNC state). Thus, cells of V. vulnificus in the VBNC state are likely exhibiting this response to low in situ temperature and only when the artificial condition of laboratory culture is attempted are the cells nonculturable due to cold-induced loss of catalase activity. To our knowledge, this is the first study providing direct evidence for the metabolic basis of nonculturability and the viable but nonculturable state.

Notes: Abstract Possible iron-transport mechanisms were examined for 11 Vibrio vulnificus strains from clinical and environmental sources. All strains produced hydroxamate siderophores, and 10 of 11 produced phenolate siderophores. Each strain produced at least 2 new major outer membrane proteins in response to iron limitation; however, the apparent Mr of these proteins varied between strains. While certain patterns of major iron-regulated outer membrane proteins were more common among clinical strains, there was no clear correlation between outer membrane protein profile and source of the isolate. Immune serum showed a strong antigenic response to a 66-kD outer membrane protein that was common to all strains examined and was not iron-regulated. An antigenic response to other outer membrane proteins that appear to be iron-regulated was also noted.