Small rodents and other mammals associated with mountain meadows as reservoirs of Giardia spp. and Campylobacter spp.

Sixty-five percent (469 of 722) of the fecal samples collected from small rodents in the central Washington Cascade mountains were positive for Giardia spp. Trapping studies showed that microtines of the genus Microtus were heavily infected with the parasite. Morphologically the cysts and trophozoites were of the Giardia duodenalis type. Small-rodent populations appear to maintain their infection throughout the year. Our data suggest that there is no difference in the percentage of positive animals in areas receiving a lot of human use as opposed to animals in those areas receiving very little or no human use. Giardia spp. were also found in elk and beaver fecal samples. Campylobacter spp. were recovered infrequently from the small rodents inhabiting alpine meadows. Of 551 specimens cultured, less than 1% were positive for the bacterium, and the isolates were identified as Campylobacter coli. Water voles were susceptible to a human isolate of Campylobacter jejuni and shed the bacterium for several weeks. C. jejuni was also isolated from a bear fecal sample collected from a protected watershed. Our studies indicate that microtines and possibly other small rodents inhabiting mountain meadows have a potential to act as a reservoir for both Giardia spp. and Campylobacter spp. Because these animals may carry human pathogens, they should be included in animal surveys designed to assess the health risks associated with mountain watersheds.     

Role of dissolution rate and solubility in biodegradation of aromatic compounds.

Strains of Moraxella sp., Pseudomonas sp., and Flavobacterium sp. able to grow on biphenyl were isolated from sewage. The bacteria produced 2.3 to 4.5 g of protein per mol of biphenyl carbon, and similar protein yields were obtained when the isolates were grown on succinate. Mineralization of biphenyl was exponential during the phase of exponential growth of Moraxella sp. and Pseudomonas sp. In biphenyl-supplemented media, Flavobacterium sp. had one exponential phase of growth apparently at the expense of contaminating dissolved carbon in the solution and a second exponential phase during which it mineralized the hydrocarbon. Phase-contrast microscopy did not show significant numbers of cells of these three species on the surface of the solid substrate as it underwent decomposition. Pseudomonas sp. did not form products that affected the solubility of biphenyl, although its excretions did increase the dissolution rate. It was calculated that Pseudomonas sp. consumed 29 nmol of biphenyl per ml in the 1 h after the end of the exponential phase of growth, but 32 nmol of substrate per ml went into solution in that period when the growth rate had declined. In a medium with anthracene as the sole added carbon source, Flavobacterium sp. converted 90% of the substrate to water-soluble products, and a slow mineralization was detected when the cell numbers were not increasing. Flavobacterium sp. and Beijerinckia sp. initially grew exponentially and then arithmetically in media with phenanthrene as the sole carbon source. Calculations based on the growth rates of these bacteria and the rates of dissolution of phenanthrene suggest that the dissolution rate of the hydrocarbon may limit the rate of its biodegradation.     

Factors Involved in Hydrolysis of Microcrystalline Cellulose by Acetivibrio cellulolyticus

Acetivibrio cellulolyticus cellulase obtained by the water elution of residual cellulose from the growth medium was compared with the cellulase activity present in culture supernatants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that water elution released most of the protein bands which adhered to undigested cellulose from the culture medium. The enzyme in the culture supernatant and that eluted from residual cellulose had specific activities for Avicel hydrolysis that were 20- to 40-fold greater than that of Trichoderma reesei cellulase. However, Ca²⁺ and a reducing agent such as dithiothreitol were required for maximum Avicel hydrolysis rates by these A. cellulolyticus enzyme preparations. The effect of these agents on p-nitrophenyl lactopyranoside hydrolysis suggested that they were required by an exoglucanase component. Supernatant enzyme preparations contained large amounts of carbohydrate which was separated from most of the cellulase protein by phenyl-Sepharose chromatography. Removal of this carbohydrate, which interfered with protein fractionations, allowed for an activity stain analysis of the supernatant enzyme.     

Protease-deficient Bacillus subtilis host strains for production of Staphylococcal protein A.

We constructed strains of Bacillus subtilis which produced very low levels of extracellular proteases. These strains carried insertion or deletion mutations in the subtilisin structural gene (apr) which were constructed in vitro by using the cloned gene. The methods used to construct the mutations involved the use of a plasmid vector which allowed the selection of chromosomal integrates and their subsequent excision by homologous recombination to effect replacement of the chromosomal apr gene by a derivative carrying an inactivating insert with a selectable marker (a cat gene conferring chloramphenicol resistance). The strains produced no subtilisin, no detectable extracellular metalloprotease activity, and residual extracellular serine protease levels as low as 0.5% of that of the standard strain from which they were derived. The strains proved to be superior host strains for the production of staphylococcal protein A, accumulating higher levels of intact protein than do previously available B. subtilis strains.     

Regulation of β-1,4-Endoglucanase Synthesis in Thermomonospora fusca

In Thermomonospora fusca YX, endocellulase synthesis varies over a 100-fold range depending on the carbon source used. This study shows that the variation is caused by two regulatory mechanisms: an induction mechanism that increases the rate of endocellulase synthesis about 20-fold and a growth rate-dependent repression mechanism that changes the rate of synthesis over a 6-fold range in both induced and noninduced cells. In T. fusca, endocellulase synthesis can be induced by cellulose, cellobiose, or cellodextrin. Cellulase is involved in inducer generation from cellulose. Growth rate-dependent repression can be reversed by limiting cultures for carbon, nitrogen, or, to a lesser extent, phosphorus. Further evidence for two separate regulatory mechanisms is provided by the isolation of mutants (CC-1 and CC-2) whose endocellulases are synthesized constitutively but are still sensitive to growth rate-dependent repression. These conclusions about total endocellulase synthesis were extended to the individual endocellulases by showing that three T. fusca endocellulases are coordinately regulated.     

Survival and transport of bacteria in egg washwater.

An evaluation of methods for monitoring the quality of water used to wash eggs at grading stations was undertaken to improve maintenance of bacterial viability during overnight sample transport. Bacterial content of samples at analysis would then better reflect conditions at the time eggs were washed. The interactive effects of temperature and the highly alkaline water conditions upon viability were the subjects of this study. Nine transport methods were examined for their efficacy in recovering total and coliform bacteria from recycled water used to wash eggs, and these were compared with samples analyzed at two commercial egg grading stations. Samples were shipped under test to the laboratory for analysis the following day. The survival of Staphylococcus aureus and Escherichia coli was also examined, but in a synthetic washwater matrix under various combinations of temperature (6 to 32 degrees C) and pH (9.5 to 10.5) to determine whether there was likely to be a different response to variations in transport treatment among gram-positive and -negative bacteria. S. aureus was much more resistant to the lethal effects of high pH and moderate temperature than E. coli. These results indicated that samples of high pH should be held (transported) at less than or equal to 13 degrees C to optimize bacterial survival. Considering cost, ease of manipulation, and the ability to protect both coliforms and the bacterial population as a whole, the method of choice for transport of industrial samples was the direct addition of washwater to containers in which powdered KH2PO4 and Na2S2O3 had been placed to yield final concentrations, when dissolved, of 0.2 and 0.05% (wt/vol), respectively.     

Enhanced Survival of the Cyanobacterium Oscillatoria terebriformis in Darkness under Anaerobic Conditions

Oscillatoria terebriformis, a thermophilic cyanobacterium, maintained viability in darkness under anaerobic conditions by fermenting exogenous glucose or fructose to lactic acid. The time period of survival was greatly extended when the environmental redox potential was lowered by the addition of sodium thioglycolate or titanium(III) citrate. When exposed to aerobic conditions in darkness, many trichomes underwent lysis in 6 h, and death of all cells occurred in 2 to 3 days. The endogenous aerobic respiration rate was high, and the limited dark aerobic survival period appeared to be due to depletion of stored glycogen. Fructose or glucose did not support or increase aerobic respiration in darkness or lengthen aerobic survival time. Enhanced survival of O. terebriformis in darkness under anaerobic, reducing conditions correlates well with the natural nighttime position of this species within sulfide-rich microbial mats associated with hot springs of western North America.     

Antimicrobial activity of lysozyme against bacteria involved in food spoilage and food-borne disease.

Egg white lysozyme was demonstrated to have antibacterial activity against organisms of concern in food safety, including Listeria monocytogenes and certain strains of Clostridium botulinum. We also found that the food spoilage thermophile Clostridium thermosaccharolyticum was highly susceptible to lysozyme and confirmed that the spoilage organisms Bacillus stearothermophilus and Clostridium tyrobutyricum were also extremely sensitive. Several gram-positive and gram-negative pathogens isolated from food poisoning outbreaks, including Bacillus cereus, Clostridium perfringens, Staphylococcus aureus, Campylobacter jejuni, Escherichia coli O157:H7, Salmonella typhimurium, and Yersinia enterocolitica, were all resistant. The results of this study suggest that lysozyme may have selected applications in food preservation, especially when thermophilic sporeformers are problems, and as a safeguard against food poisoning caused by C. botulinum and L. monocytogenes.     

A more accurate method for measurement of tuberculocidal activity of disinfectants.

The current Association of Official Analytical Chemists method for testing tuberculocidal activity of disinfectants has been shown to be inaccurate and to have a high degree of variability. An alternate test method is proposed which is more accurate, more precise, and quantitative. A suspension of Mycobacterium bovis BCG was exposed to a variety of disinfectant chemicals and a kill curve was constructed from quantitative data. Data are presented that show the discrepancy between current claims, determined by the Association of Official Analytical Chemists method, of selected commercially available products and claims generated by the proposed method. The effects of different recovery media were examined. The data indicated that Mycobacteria 7H11 and Middlebrook 7H10 agars were equal in recovery of the different chemically treated cells, with Lowenstein-Jensen agar having approximately the same recovery rate but requiring incubation for up to 3 weeks longer for countability. The kill curves generated for several different chemicals were reproducible, as indicated by the standard deviations of the slopes and intercepts of the linear regression curves.     

Abundance and distribution of Legionellaceae in Puerto Rican waters.

Waters in marine and freshwater areas of Puerto Rico were analyzed for the presence of Legionella spp. by direct fluorescent antibody assay with guinea pig confirmation. Several species, including L. bozemanii, L. dumoffii, L. gormanii, L. longbeachae, L. micdadei, and L. pneumophila, were widely distributed among all sites. Legionellaceae, including L. pneumophila, were found in high densities in water collected in the rain forest from epiphytes in trees 30 ft. (about 9.25 m) above the ground. Both interspecific and intersite variations were significant. L. pneumophila was the most abundant species at all sites, with average densities of 10(4) cells ml-1, very close to the range which is potentially pathogenic for humans. Densities of L. pneumophila were highest in sewage-contaminated coastal waters. These are the highest densities of Legionella spp. ever reported for marine habitats. Densities of L. pneumophila were positively correlated with concentrations of sulfates, phosphates, and pH. A survey of 88 fatal atypical pneumonia cases at a Puerto Rico hospital showed that 15% of the patients had L. pneumophila infections. This study establishes L. pneumophila as a relatively common cause of atypical pneumonia in Puerto Rico and suggests natural aquatic habitats as possible sources or reservoirs of pathogenic Legionella spp. in the tropics.