Enumeration of Aeromonas hydrophila from domestic wastewater treatment plants and surface waters

R. POFFÉ AND E. OP DE BEECK. 1991. Influents, effluents and sludges from sewage purification plants and surface water samples were examined quantitatively for Aeromonas hydrophila on the mA medium of Rippey and Cabelli. Between 10⁴ and 10⁶/ml A. hydrophila were found in domestic wastewaters. On the average 99.975% were removed by activated sludge and 98.25% by trickling filters. Only 20.9% of A. hydrophila end up in the primary sludge, which contained up to 10⁷/g dry sludge. After 3 months, anaerobically (methane) fermented and partially dried sludge from trickling filters contained more than 10⁶ A. hydrophila /g dry sludge. Surface water receiving raw sewage contained several hundreds of A. hydrophila /ml, comparable with the numbers found in effluent waters, while surface water receiving no municipal wastewater and destined for the preparation of drinking water contained only small and negligible numbers. It was concluded that A. hydrophila was omnipresent in surface water.     

Cytotoxin production in 100 strains of Haemophilus ducreyi from different geographic locations

Abstract One-hundred strains of Haemophilus ducreyi , representing isolates from different parts of the world, including the reference strains, were obtained from different collections and characterized with special reference to cytotoxin production in vitro. The cytotoxic activity on cultured epithelial cells (HEp-2) was examined with two methods. The activity in bacterial sonicates was tested on freshly trypsinated cells and strains manifesting little or no cytotoxic activity in sonicates were investigated using attached living bacteria on HEp-2 cell-monolayers. Sonicates from the majority of the H. ducreyi strains (89%) produced significant cytotoxic effects on HEp-2 cells. The reciprocal cytotoxic titers of the sonicates ranged from 2.4 × 10² to 5.3 × 10⁵. Sonicates of 11 strains had low cytotoxic titers ( 1:3 to 1:81), eight of those originating from Asia and three from Africa. These 11 strains caused no damage to the cell monolayer, indicating that the 11 strains produce little or no cytotoxic activity in vitro. In summary, the majority of H. ducreyi isolates produce cytotoxic activity, which support the hypothesis that the cytotoxin may be an important virulence factor of this species.     

The relationship between physical and chemical conditions and low microbial diversity in the Blue Lagoon geothermal lake in Iceland

Abstract: The Blue Lagoon in Iceland is a shallow geothermal lake with average temperatures of 37°C, pH 7.5 and about 2.5% salinity. It was formed in 1976 from the effluents of the Svartsengi geothermal power plant and is saturated with silica which constantly precipitates in the lake. It has been colonized by a few types of specialized microorganisms which seem to proliferate in this unusual ecosystem. The average bacterial colony count in the lake was 1.3 × 10⁵ ml⁻¹ on plate count agar made with 50% Blue Lagoon fluid but 2.6 × 10⁶ ml⁻¹ when determined with the MPN method. A total of 99 isolates were purified and characterized by 54 phenotypic tests and then grouped using Numerical Taxonomy. At similarity values of 80%, one major cluster was formed containing 85% of the isolates. Four representative strains from this cluster were further characterized and all shown to be Gram-negative, obligately aerobic, non-motile rods. They were oxidase positive, catalase negative and grew optimally at 45°C and in 3.5% NaCl with doubling time of about 80 min.     

Characterization of the transferrin-iron uptake system in Neisseria meningitidis

Abstract The transferrin-iron uptake system of six Neisseria meningitidis strains was characterized using ¹²⁵I-transferrin in receptor assays and ⁵⁵Fe-loaded transferrin in uptake assays. Receptors for transferrin varied among the strains both in number (from 700 to 4700 receptors per cell) and in their affinity constants for the protein ( K _a ranged from 0.7×10⁷ to 4.0×10⁷ 1 mol⁻¹). Neither receptor numbers nor affinity constants were significantly different in carrier and invasive strains, although the K_a seem to be somewhat higher in the latter. Iron uptake from transferrin was also variable among the strains, but showed the same lack of correlation with their origin.     

The Occurence and Properties of Uric Acid Decomposing Anaerobic Bacteria in the Avian Caecum

S ummary . Anaerobic bacteria which decompose uric acid have been found in the caeca of chickens, turkeys, ducks, pheasants and guinea-fowl at levels between 5.4 × 10⁸ and 1.8 × 10¹⁰/g of caecal material (wet weight). A study has been made of the properties and identity of 35 strains which were present in high numbers in the chicken caecum. The isolates included strains of Bacteroides clostridiiformis var. clostridiiformis, Fusobacterium plauti, Clostridium malenominatum, Peptostreptococcus productus and a number of types which could not be identified with known species. Of these 3 were Bacteroides spp., 3 Eubacterium spp., 2 Peptostreptococcus spp. and 1 was an anaerobic Streptococcus . None of the strains had an absolute requirement for uric acid.     

Ecology, serology, and enterotoxin production of Vibrio cholerae in Chesapeake Bay.

A total of 65 isolates of Vibrio cholerae, serotypes other than O--1, have been recovered from water, sediment, and shellfish samples from the Chesapeake Bay. Isolations were not random, but followed a distinct pattern in which salinity appeared to be a controlling factor in V. cholerae distribution. Water salinity at stations yielding V. cholerae (13 out of 21 stations) was 4 to 17 0/00, whereas the salinity of water at stations from which V. cholerae organisms were not isolated was less than 4 or greater than 17 0/00. From results of statistical analyses, no correlation between incidence of fecal coliforms and V. cholerae could be detected, whereas incidence of Salmonella species, measured concurrently, was clearly correlated with fecal coliforms, with Salmonella isolated only in areas of high fecal coliform levels. A seasonal cycle could not be determined since strains of V. cholerae were detectable at low levels (ca. 1 to 10 cells/liter) throughout the year. Although none of the Chesapeake Bay isolates was agglutinable in V. cholerae O group 1 antiserum, the majority for Y-1 adrenal cells. Furthermore, rabbit ileal loop and mouse lethality tests were also positive for the Chesapeake Bay isolates, with average fluid accumulation in positive ileal loops ranging from 0.21 to 2.11 ml/cm. Serotypes of the strains of V. cholerae recovered from Chesapeake Bay were those of wide geographic distribution. It is concluded from the data assembled to date, that V. cholerae is an autochthonous estuarine bacterial species resident in Chesapeake Bay.     

Pathogenicity of live bacteria and extracellular products of motile Aeromonas isolated from eels

The pathogenic activities in vitro and in vivo of live bacteria and extracellular products (ECP) of 24 motile Aeromonas strains were investigated. Most Aer. hydrophila and Aer. jandaei isolates were pathogenic for eels (LD₅₀ 10^5·4-10^7·6 cfu fish^-1) but no Aer. sobria , Aer. caviae and Aer. allosaccharophila caused mortality in eels at doses of > 10^8·4 cfu fish^-1. Of these Aeromonas strains, Aer. hydrophila and Aer. jandaei in particular produced elastases and haemolysins against fish erythrocytes. ECP from Aer. hydrophila and Aer. jandaei caused degenerative changes in fish cell lines and were strongly toxic for eels (LD⁵⁰ 1·0–3·2 μg (g fish)^-1) reproducing the symptoms associated with natural disease. ECP from non-pathogenic species were inactive on fish cell lines as well as being poorly lethal for eels (LD⁵⁰ > 9·2 μg (g fish)^-1). All these biological activities of Aeromonas ECP were lost after heat treatment. These findings indicate differences between pathogenic and non-pathogenic Aeromonas species with respect to the expression of virulence factors, and show that elastases, haemolysins and exotoxins play a leading role in the pathogenicity of motile Aeromonas for eels.     

Biological Interactions and the Realized Niche of Euplotes vannus from the Salt Marsh Aufwuchs

SYNOPSIS. Euplotes vannus , a hypotrich ciliate. grows well over broad ranges of temperature and salinity. It requires higher densities of food (> 1 × 10⁴ cells/ml) for rapid reproduction than do the other herbivores, the foraminiferan Al-logromia laticollaris (> 1 × 10² cells/ml), and the nematode Chromadorina germanica (∼ 1 × 10³ cells/ml), to which it was compared. If food levels were initially very high (∼ 1 × 10⁸ cells/ml) the ciliates reproduced rapidly and consumed the algae faster than it could reproduce. Some balance between the algae and the ciliates was achieved at initial algal concentrations of ∼ 1 × 10⁵ cells/ml. In microcosm experiments at 25 C with equal numbers of C. germanica and A. laticollaris. E. vannus proved to be a very poor competitor; reaching only 20% of control levels when grow with C. germanica and only 13% when cultured with A. laticollaris . It was a better competitor in 2-species microcosms, at lower temperatures, and when its ratio to the other species was initially higher.
The experimental evidence suggests that E. vannus is best adapted to being a migrating initial colonizer of fresh algal blooms.     

Isolation of Pectolytic clostridia from Potatoes

S ummary : A method for selective counting and isolation of pectolytic clostridia in the presence of Erwinia carotovora is described; using this method pectolytic clostridia have been found in numbers of 8 × 10⁵–1 × 10⁸/g of rotting potato tissue in the presence of 1–4 × 10⁸ E. carotovora /g.     

Quantification of pathogenic micro-organisms in the sludge from treated hospital wastewater

The sludge from hospital waste treatment facilities is a potential source of infectious organisms. The average numbers of micro-organisms in the sludge of hospital wastewater in Taiwan were as follows: total count 8·1 × 10⁷ cfu g⁻¹ (dry weight of sludge), and 1·4 × 10⁶, 3·6 × 10⁵, 1·6 × 10⁵, 2·2 × 10⁵ and 5·5 × 10⁴ cfu g⁻¹ (dry weight of sludge) for total coliforms, faecal coliforms, faecal streptococci, Pseudomonas aeruginosa and Salmonella spp., respectively . Salmonella spp. were detected in 37% (10 of 27) of the sludges from hospital wastewaters. Therefore, the treatment of such sludge to reduce pathogenic micro-organisms should be considered.     

The effect of moisture potential on growth and survival of root nodule bacteria in peat culture and on seed

Peat from three sources was dried, milled and packed separately in polyethylene bags and sterilized by irradiation. The carrier was impregnated with broth cultures of either Rhizobium leguminosarum bv. trifolii strain WU95, Bradyrhizobium japonicum strain CB1809 or B. lupini strain WU425 and sterile water to provide five moisture potentials in the range > - 1 × 10⁴ - 1 × 10⁶ Pa. The packets were stored at 26°C under conditions which restricted moisture loss. Numbers of root nodule bacteria were counted at intervals up to 12 weeks. No single moisture potential was optimum for all strains in all carriers because of a significant ( P < 0.05) interaction between moisture potential × strain × carrier × time. Where direct comparisons could be made, all strains survived best at - 1 × 10⁴ and/or −3.2 × 10⁴ Pa. Seeds of Trifolium subterraneum and polypropylene beads (used to avoid seed coat toxicity), were inoculated with WU95 prepared in two sources of peat and at each of the above moisture potentials and stored at 15°C. Seed coat toxicity significantly effected the log death rate ( k ) of WU95 on subterraneum clover seed for the period 0–0.25 d ( k 1.796) compared with k - 0.399 for polypropylene beads. In the first 24 h moisture did not affect survival but by 28 d rhizobia grown in Badenoch peat survived best at −3.2 × 10⁴ Pa. In Millicent peat, survival was equally as good at −3.2 × 10⁴ and −1 × 10⁴ Pa.     

Lysogens and free viruses in fresh, brackish, and marine waters: a Bayesian analysis

A yearlong study was conducted to determine factors that affect the abundance and distribution of lysogens and free viruses at fresh-, brackish-, and saltwater stations in Newport Bay, CA. The viral and bacterial abundance were highest in the freshwater (average 1.1 × 10⁸ and 1.1 × 10⁷ mL⁻¹, respectively) and lowest in the marine water (average 0.4 × 10⁸ and 0.5 × 10⁷ mL⁻¹, respectively). Bacterial and viral counts were also several times higher during the summer than in winter. Approximately, 35% of the 141 samples were inducible in the presence of mitomycin C. The highest percentage of inducible lysogens was observed in marine waters (42%), while the lowest percentage was observed in the warmer freshwater (23%). A statistical model for the joint occurrence of lysogens and free viruses was formulated and estimated using Bayesian techniques to understand the key environmental determinants of viruses and lysogens. Our results support the existence of significant heterogeneity between the saltwater and freshwater sites. A parsimonious model that combines the two saltwater sites performs best among the specifications that were considered. Bacteria and water temperature were significant determinants of virus counts, whereas lysogen relationships are unclear. Importantly, conditional on the covariates, viruses and lysogen fractions exhibit robust negative correlation.     

Enterobacteriaceae, coliforms, faecal coliforms and salmonellas in raw ewes'milk

Thirty-nine samples of freshly drawn ewes' milk collected at 13 farms, and 120 samples of raw ewes' milk collected on arrival at a dairy were examined. Farm samples had geometric mean counts of 4.4 × 10² Enterobacteriaceae/ml, 3.9 × 10² coliforms/ml and 2.0 × 10² faecal coliforms/ml, whereas the respective mean counts were 6.2 × 10³/ml, 5.4 × 10³/ml and 1.3 × 10³/ml for dairy samples. Salmonellas were not detected by enrichment procedures in any of the 159 samples examined. Escherichia coli (47.5% strains), Enterobacter cloacae (17.7%), Ent. agglomerans (11.3%), Hafnia alvei (6.5%) and Klebsiella oxytoca (6.0%) were the predominant species in 434 Enterobacteriaceae strains isolated from farm samples. Levels and species of Enterobacteriaceae found in the present work in raw ewes' milk imply a considerable risk of early blowing in cheese-making from unpasteurized milk.     

OBSERVATIONS ON THE MICROFLORA OF MINCED CHICKEN MEAT IRRADIATED WITH 4 MeV CATHODE RAYS

SUMMARY: Experiments are described in which minced chicken meat, packed anaerobically, was irradiated at room temperature and in the frozen state with a wide range of doses of 4 MeV cathode rays. Sterility was achieved in 14 out of 15 samples which had received 2 × 10⁶ rads or more. Doses of 0·5 and 1·0 × 10⁶ rads allowed survival of a few bacteria/g, usually spore formers. Bacterial counts indicated an approximately logarithmic decrease in numbers at lower doses, while freezing reduced the bactericidal effect.
The storage life at 5° was prolonged only slightly by doses of 5 × 10⁴ and 10 × 10⁴ rads, and highly variable results were obtained with 17·5 × 10⁴ rads. A dose of 25 × 10⁴ rads, however, increased the storage life very considerably. The types of bacteria present initially, and after irradiation with low doses and storage at 5°, were studied. After storage for 12 days or more various types of nonsporing Gram-positive rods were predominant in almost all samples, both control and irradiated. Streptococci were also important where irradiation with 17·5 × 10⁴ and 25 × 10⁴ rads was followed by long storage.     

Identification and characterization of Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosomal DNA

Abstract Bacillus anthracis can be identified on the basis of the detection of virulence factor genes located on two plasmids, pXO1 and pXO2. Thus isolates lacking both pXO1 and pXO2 are indistinguishable from closely related B. cereus group bacteria. We developed a multiplex PCR assay for characterization of B. anthracis isolates, and simultaneous confirmation of the species identity independent of plasmid content. The assay amplifies lef, cya, pag (pXO1) and cap (pXO2) genes, and a B. anthracis specific chromosomal marker, giving an easy-to-read profile. This system unambiguously identified virulent (pXO1⁺/2⁺) and avirulent (pXO1⁺/2⁻, pXO1⁻/2⁺ and pXO1⁻/2⁻) strains of B. anthracis and distinguished 'anthrax-like' strains from other B. cereus group bacteria.     

DIRECT DETECTION OF ESCHERICHIA COLI 0157:H7 IN UNPASTEURIZED APPLE JUICE WITH AN EVANESCENT WAVE BIOSENSOR

An evanescent wave biosensor was used to detect Escherichia coli O157:H7 in unpasteurized apple juice. Light is launched from a 635 nm laser diode into silica or polystyrene optical waveguides, generating an evanescent field which extends from the waveguide surface. Fluorescent molecules within the evanescent field are excited resulting in an emission signal that the biosensor then detects and quantifies. A sandwich immunoassay was performed on the waveguides using cyanine 5 dye-labeled anti-E. coli O157:H7 antibodies for generation of the specific fluorescent signal. The lower limit of detection was between 6.0 × 10² and 6.0 × 10⁴ CFU/mL with silica waveguides and between 3.2 × 10⁴ and 3.2 × 10⁴ CFU/mL using polystyrene waveguides. One-hundred percent correct identification of true positive samples occurred at 6.0 × 10⁴ and 3.2 × 10⁴ CFU/mL for silica and polystyrene waveguides, respectively. Signals from a variety of non-E. coli O157 bacteria, including closely related enterotoxigenic strains of E. coli at concentrations of ˜ 10⁶ CFU/mL, were below the limits of detection. Assays were conducted in near real-time with results obtained within 15 min of sample processing.     

Coliforms as a measure of sewage contamination of the River Zambezi

The effect of releasing untreated sewage from Victoria Falls Town into the Zambezi river was determined by bacteriological examination of water samples collected upstream of Victoria Falls and for 22 km downstream. Most probable numbers of faecal coliforms and Escherichia coli were estimated. Water upstream of the falls, on the Zimbabwe side of the river, contained between seven and 130 E. coli per 100 ml. This section of the river was free from major sources of faecal pollution. Below the falls, but before the Victoria Falls Town sewage outfall, numbers of E. coli were between 1.8 × 10² and 1.4 × 10⁴/100 ml, indicating the existence of a sewage discharge other than that from Victoria Falls Town. The river was also highly polluted from the Victoria Falls Town sewage outfall to a point 18.6 km downstream. The highest E. coli count was 3.3 × 10⁴/100 ml and declined slowly to 1.4 × 10³/100 ml 18.6 km downstream of the outfall.     

Bacterial microflora in the gastro-intestinal tract of Dover sole (Solea solea L.), with emphasis on the possible role of bacteria in the nutrition of the host

Abstract There was a progressive increase in the size of the aerobic heterotrophic bacterial populations along the gastro-intestinal tract of farmed Dover sole. Moreover, higher counts were recorded in juvenile than in adult animals. Thus, in juvenile fish, 5.2 × 10⁵, 8.0 × 10⁵ and 9.8 × 10⁶ aerobic heterotrophs/g were recovered from the stomach/foregut, midgut and hindgut/rectum, respectively. In adult fish, comparative samples revealed the presence of only 3.0 × 10⁴, 7.0 × 10⁴ and 2.3 × 10⁵ bacteria/g, respectively. There bacteria were equated with Acinetobacter, Alcaligenes , Enterobacteriaceae representatives, Flavobacterium, Micrococcus, Photobacterium, Staphylococcus and Vibrio . Of the compounds tested, many isolates, particularly those recovered from the hindgut/rectum, degraded p -nitrophenyl- β - N -acetylglucosaminide, chitin and collagen. Consequently, it is likely that such organisms may contribute to nutritional processes within Dover sole.     

A novel terrestrial halophilic environment: The phylloplane of Atriplex halimus, a salt-excreting plant

Abstract This paper describes the microbial ecosystem found on the leaves of Atriplex halimus , a salt-excreting plant in the central Negev highlands of Israel. Because of the regular nightly occurence of dew at this location, these leaves undergo a diurnal wetting so that phylloplane microorganisms experience large fluctuations in salinity and water activity, as well as tolerate repeated desiccation. During the dry season, in the late spring and summer, a significant amount of salts and organic material coats the leaf surface. During dew events the salt concentration at the leaf surface was calculated to be > 0.4 M. Direct counts of the respiring bacteria on the leaf surface ranged from 1.06×10⁴ to 5.06×10⁵ per cm². Using a variety of media it was shown that there was limited bacterial diversity which could be cultured, with greater than 90% of the isolates being orange colored Gram-negative rods. Viable counts ranged from 0.32 to 2.32×10⁴ bacteria per cm² of A. halimus leaf surface. No bacteria capable of nucleating ice were recovered in these studies. The dominant orange pigmented bacterium, identified as a halotolerant Pseudomonas sp., grew optimally at 30°C and at 5% NaCl and was capable of growth in media containing up to 20% NaCl. This bacterium could grow on a variety of organic compounds, including some associated with plant materials. The leaf bacteria were desiccation-tolerant when on the leaf surface or when directly washed off the leaves, but much less so when in isolatd culture. A major component of the tolerance to desiccation is probably related to the compounds on the leaf surface.     

The relationship between cytotoxic effect and binding to mammalian cultured cells of Clostridium perfringens enterotoxin

Abstract The relationship between the cytotoxic effect and binding to different cell lines of Clostridium perfringens enterotoxin was investigated. The enterotoxin released ⁵¹Cr from Vero and MDCK cells labeled with Na₂-⁵¹CrO₄. The effect varied depending upon the dose of enterotoxin and the duration and temperature of the interaction. The enterotoxin gave no effect on FL, KB, or L-929 cells. [¹²⁵I]Enterotoxin bound specifically to Vero and MDCK cells via a binding site of distinct nature, but not to FL, KB, or L-929 cells. The number of the binding sites located on one MDCK cell (1.98 × 10⁶ sites/cell) was three times that on one Vero cell (5.64 × 10⁵ sites/cell), although the binding affinity of MDCK cell ( K _a/ 3.76 × 10⁷ M⁻¹) was 0.1 that of Vero cells ( K _a/ 3.23 × 10⁸ M⁻¹). Binding of the enterotoxin to susceptible cells was temperature-independent.