Time periods of morphological stages in populations of differentiating micro-organisms.

A general method for determining the time periods of morphological stages in a population of differentiating micro-organisms is developed. The method may also be used to determine the fractional number of cells of distinct morphological types. The developmental cycles of a number of micro-organisms including budding bacteria or yeast (Saccharomyces cervisae), fissioning bacteria (Bacillus subtilis) and Caulobacter crescentus are discussed to illustrate the general method and the theoretical predictions are compared with available experimental data.     

Attached and Free-Floating Bacterioplankton in Howe Sound, British Columbia, a Coastal Marine Fjord-Embayment

Factors which influence the attachment of bacterioplankton to particles (including phytoplankton) were investigated by using (i) water samples removed from a coastal temperate fjord over an annual cycle and (ii) unialgal cultures of Prorocentrum minimum, Dunaliella tertiolecta, and Skeletonema costatum. Silt and salinity levels in this fjord seawater did not appear to influence bacterial attachment, but the percent attached bacteria was inversely related to both chlorophyll a concentrations and primary productivities. During periods of high primary productivities the percent attached bacteria was low, whereas during periods of low, increasing, and declining primary productivities the percent attached bacteria was high. A similar pattern of bacterial attachment was observed when the three phytoplankton were grown as batch cultures. The percent attached bacterial numbers increased upon the initiation of algal growth and after these cells stopped growing, but not while the algae were growing. We suggest that a major factor influencing the attachment of bacterioplankton is the physiological condition of their major nutrient source, the phytoplankton; mainly free-living bacteria are associated with growing phytoplankton, whereas a much greater proportion of the bacteria are attached among senescent phytoplankton populations.     

Antagonism Between Osmophilic Lactic Acid Bacteria and Yeasts in Brine Fermentation of Soy Sauce

Brine fermentation by osmophilic lactic acid bacteria and yeasts for long periods of time is essential to produce a good quality of shoyu (Japanese fermented soy sauce). It is well known that lactic acid fermentation by osmophilic lactic acid bacteria results in the depression of alcoholic fermentation by osmophilic yeasts, but the nature of the interaction between osmophilic lactic acid bacteria and yeasts in brine fermentation of shoyu has not been revealed. The inhibitory effect of osmophilic lactic acid bacteria on the growth of osmophilic yeasts was investigated. It was recognized that osmophilic shoyu yeasts such as Saccharomyces rouxii and Torulopsis versatilis were inhibited by a metabolite produced by osmophilic lactic acid bacteria (belonging to Pediococcus halophilus) in brine fermentation of shoyu. The primary inhibitor was considered to be acetic acid, although lactic acid was slightly inhibitory.     

Lipid Metabolism of Rumen Ciliates and Bacteria: II. Uptake of Fatty Acids and Lipid Analysis of Isotrichia intestinalis and Rumen Bacteria with Further Information on Entodinium simplex

The total lipid and free fatty acid contents of Isotricha intestinalis, Entodinium simplex, and the rumen bacterial flora of the respective protozoa were determined. Warburg manometric data showed that the sodium salts of tributyrin, oleic, and acetic acids stimulated gas production in I. intestinalis, whereas tributyrin was stimulatory with E. simplex and less active with oleic and acetic acids. Rumen bacteria provided fatty acids produced lower manometric gaseous increases when compared with the protozoa. Volatile fatty acids were produced by I. intestinalis and rumen bacteria with tributyrin, but not with tripalmitin. Sodium oleate gave little volatile fatty acid response with I. intestinalis or rumen bacteria. Washed suspensions of I. intestinalis and rumen bacteria concentrated C¹⁴-labeled oleic, palmitic, stearic, and linoleic acids within the cells during short incubation periods. Autoradiographs demonstrated the conversion of C¹⁴-labeled oleic, palmitic, stearic, linoleic, and acetic acids in the rumen protozoa and bacterial cells.     

Vertical distributions of bacteria and algae in a steeply stratified humic lake under high grazing pressure from Daphnia longispina

The vertical distributions of bacteria and algae in a steeply stratified, highly humic lake were studied during three 24 h periods in summer. The highest bacterial and algal densities and biomasses were recorded in the anoxic hypolimnion. The bacterial biomass in the hypolimnion was composed mainly of photosynthetic green sulphur bacteria (Chlorobium) which occurred at very low light intensity (< 1.5 μmol m⁻² s⁻¹). The numbers and biomasses of bacteria, both in the epilimnion and hypolimnion, were mostly higher at night than during the day, indicating possible asynchrony between the production and loss of bacteria. Because of vertical migration, the diurnal vertical distribution of algae was more variable than that of bacteria. Particularly in July and August, when cryptomonads were abundant, the biomass of algae was much higher in the epilimnion during the day than at night. The flagellated chlorophytes, Chlamydomonas spp. and Scourfieldia cordiformis, stayed mainly in the upper hypolimnion close to the oxic-anoxic boundary zone where only a small proportion of Daphnia longispina was continuously present. Unpalatable Mallomonas chrysophytes with silicified plates and bristles, and small, presumably heterotrophic, flagellates stayed in the oxic epilimnion together with a dense (up to 300 ind l⁻¹) population of D. longispina. The results indicated that, besides the physical and chemical properties of the water column, grazing pressure by Daphnia longispina strongly affected the vertical distribution of microorganisms in this polyhumic lake.     

Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Continuously Percolated Soil Columns

Although the absence of nitrate formation in grassland soils rich in organic matter has often been reported, low numbers of nitrifying bacteria are still found in these soils. To obtain more insight into these observations, we studied the competition for limiting amounts of ammonium between the chemolithotrophic ammonium-oxidizing species Nitrosomonas europaea and the heterotrophic species Arthrobacter globiformis in the presence of Nitrobacter winogradskyi with soil columns containing calcareous sandy soil. The soil columns were percolated continuously at a dilution rate of 0.007 h^-1, based on liquid volumes, with medium containing 5 mM ammonium and different amounts of glucose ranging from 0 to 12 mM.A. globiformis was the most competitive organism for limiting amounts of ammonium. The numbers of N. europaea and N. winogradskyi cells were lower at higher glucose concentrations, and the potential ammonium-oxidizing activities in the uppermost 3 cm of the soil columns were nonexistent when at least 10 mM glucose was present in the reservoir, although 10⁷ nitrifying cells per g of dry soil were still present. This result demonstrated that there was no correlation between the numbers of nitrifying bacteria and their activities. The numbers and activities of N. winogradskyi cells decreased less than those of N. europaea cells in all layers of the soil columns, probably because of heterotrophic growth of the nitrite-oxidizing bacteria on organic substrates excreted by the heterotrophic bacteria or because of nitrate reduction at reduced oxygen concentrations by the nitrite-oxidizing bacteria. Our conclusion was that the nitrifying bacteria were less competitive than the heterotrophic bacteria for ammonium in soil columns but that they survived as viable inactive cells. Inactive nitrifying bacteria may also be found in the rhizosphere of grassland plants, which is rich in organic carbon. They are possibly reactivated during periods of net mineralization.     

Ecological Dynamics of Toxic Microcystis spp. and Microcystin-Degrading Bacteria in Dianchi Lake,China

Toxic cyanobacterial blooms directly threaten both human safety and the ecosystem of surface waters. The widespread occurrence of these organisms, coupled with the tumor-promoting properties of the microcystin toxins that they produce, demands action to mitigate their potential impacts and, thus, a robust understanding of their ecological dynamics. In the present work, the abundance of toxic Microcystis spp. and microcystin (MC)-degrading bacteria in Dianchi Lake, located in Yunnan Province, China, was studied using quantitative PCR. Samples were taken at monthly intervals from June 2010 to December 2011 at three sampling stations within this freshwater lake. Results revealed that variation in the abundance of both total Microcystis spp. and toxic Microcystis spp. exhibited similar trends during the period of the algal bloom, including the reinvasion, pelagic growth, sedimentation, and overwintering periods, and that the proportion of toxic Microcystis was highest during the bloom and lowest in winter. Importantly, we observed that peaks in mlrA gene copy numbers of MC-degrading bacteria occurred in the months following observed peaks in MC concentrations. To understand this phenomenon, we added MCs to the MC-degrading bacteria (designated strains HW and SW in this study) and found that MCs significantly enhanced mlrA gene copy numbers over the number for the control by a factor of 5.2 for the microcystin-RR treatment and a factor of 3.7 for the microcystin-LR treatment. These results indicate that toxic Microcystis and MC-degrading bacteria exert both direct and indirect effects on each other and that MC-degrading bacteria also mediate a shift from toxic to nontoxic populations of Microcystis.     

Defining the Niche of Vibrio parahaemolyticus During Pre- and Post-Monsoon Seasons in the Coastal Arabian Sea

The bacteria Vibrio parahaemolyticus is an important component of coastal ecosystems worldwide, and in recent years, V. parahaemolyticus has caused several cases of food-borne gastroenteritis. However, research investigating which parameters are important in regulating V. parahaemolyticus abundance in tropical areas with relatively stable temperatures and salinity are largely lacking. The objective here was to investigate which environmental forces are driving elevated abundances of V. parahaemolyticus in a tropical oligotrophic coastal area in the Arabian Sea. We analysed a large number of environmental parameters in parallel with cell densities of V. parahaemolyticus and Vibrio spp. Abundance data was obtained using real-time PCR, during two different sampling periods, representative for two distinct seasons. Water temperature and salinity were stable during and between sampling periods, but V. parahaemolyticus abundances were on average six times higher during the first sampling period in December, compared to the second period in February–March. V. parahaemolyticus abundance was found to be positively correlated to inorganic phosphate concentration and copepod abundance. We thus hypothesise that these are important factors regulating V. parahaemolyticus abundance in coastal tropical areas during these periods.     

Protozoan Cysts Act as a Survival Niche and Protective Shelter for Foodborne Pathogenic Bacteria

The production of cysts, an integral part of the life cycle of many free-living protozoa, allows these organisms to survive adverse environmental conditions. Given the prevalence of free-living protozoa in food-related environments, it is hypothesized that these organisms play an important yet currently underinvestigated role in the epidemiology of foodborne pathogenic bacteria. Intracystic bacterial survival is highly relevant, as this would allow bacteria to survive the stringent cleaning and disinfection measures applied in food-related environments. The present study shows that strains of widespread and important foodborne bacteria (Salmonella enterica, Escherichia coli, Yersinia enterocolitica, and Listeria monocytogenes) survive inside cysts of the ubiquitous amoeba Acanthamoeba castellanii, even when exposed to either antibiotic treatment (100 μg/ml gentamicin) or highly acidic conditions (pH 0.2) and resume active growth in broth media following excystment. Strain- and species-specific differences in survival periods were observed, with Salmonella enterica surviving up to 3 weeks inside amoebal cysts. Up to 53% of the cysts were infected with pathogenic bacteria, which were located in the cyst cytosol. Our study suggests that the role of free-living protozoa and especially their cysts in the persistence and epidemiology of foodborne bacterial pathogens in food-related environments may be much more important than hitherto assumed.     

Effects of Aeration Cycles on Nitrifying Bacterial Populations and Nitrogen Removal in Intermittently Aerated Reactors

The effects of the lengths of aeration and nonaeration periods on nitrogen removal and the nitrifying bacterial community structure were assessed in intermittently aerated (IA) reactors treating digested swine wastewater. Five IA reactors were operated in parallel with different aeration-to-nonaeration time ratios (ANA). Populations of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were monitored using 16S rRNA slot blot hybridizations. AOB species diversity was assessed using amoA gene denaturant gradient gel electrophoresis. Nitrosomonas and Nitrosococcus mobilis were the dominant AOB and Nitrospira spp. were the dominant NOB in all reactors, although Nitrosospira and Nitrobacter were also detected at lower levels. Reactors operated with the shortest aeration time (30 min) showed the highest Nitrosospira rRNA levels, and reactors operated with the longest anoxic periods (3 and 4 h) showed the lowest levels of Nitrobacter, compared to the other reactors. Nitrosomonas sp. strain Nm107 was detected in all reactors, regardless of thereactor's performance. Close relatives of Nitrosomonas europaea, Nitrosomonas sp. strain ENI-11, and Nitrosospira multiformis were occasionally detected in all reactors. Biomass fractions of AOB and effluent ammonia concentrations were not significantly different among the reactors. NOB were more sensitive than AOB to long nonaeration periods, as nitrite accumulation and lower total NOB rRNA levels were observed for an ANA of 1 h:4 h. The reactor with the longest nonaeration time of 4 h performed partial nitrification, followed by denitrification via nitrite, whereas the other reactors removed nitrogen through traditional nitrification and denitrification via nitrate. Superior ammonia removal efficiencies were not associated with levels of specific AOB species or with higher AOB species diversity.     

Bacterial Diversity within Feedlot Manure

In this study, we identified the predominant culturable anaerobic bacteria and enumerated the total culturable anaerobic bacterial population present in samples of feedlot manure from Southern Queensland. Sixteen bacterial isolates were cultured from feedlot pad material with species of Lactobacillus, Clostridium and Bacillus predominating. From a library of 123 clones, produced by the amplification, cloning and partial DNA sequencing of the 16S rRNA gene, only 3% were closely related to previously described species and 21% to known genera. Of the total clone library, 96% were apparently Gram-positive and fell within families whose members were generally anaerobes. The majority (71%) of the clone library was related to either members of the family Clostridiaceae or lactic acid-producing bacteria (Lactobacillus or Lactosphaera). It was concluded that Gram-positive clostridial and lactic acid-producing bacteria predominate in feedlot pad manure. The overwhelming majority of species are novel and have not been obtained in culture. It would appear that the most likely source of the sickly-sweet nuisance odours (particularly from butyric acid) that emanate from feedlots is the by-product of anaerobic fermentation by clostridia. Gut-inhabiting and Gram-negative bacteria do not appear to survive for lengthy periods of time under the environmental conditions present in feedlot manure.     

Effect of Abandonment on Diversity and Abundance of Free-Living Nitrogen-Fixing Bacteria and Total Bacteria in the Cropland Soils of Hulun Buir,Inner Mongolia

In Inner Mongolia, steppe grasslands face desertification or degradation because of human over activity. One of the reasons for this condition is that croplands have been abandoned after inappropriate agricultural management. The soils in these croplands present heterogeneous environments in which conditions affecting microbial growth and diversity fluctuate widely in space and time. In this study, we assessed the molecular ecology of total and free-living nitrogen-fixing bacterial communities in soils from steppe grasslands and croplands that were abandoned for different periods (1, 5, and 25 years) and compared the degree of recovery. The abandoned croplands included in the study were natural restoration areas without human activity. Denaturing gradient gel electrophoresis and quantitative PCR (qPCR) were used to analyze the nifH and 16S rRNA genes to study free-living diazotrophs and the total bacterial community, respectively. The diversities of free-living nitrogen fixers and total bacteria were significantly different between each site (P<0.001). Neither the total bacteria nor nifH gene community structure of a cropland abandoned for 25 years was significantly different from those of steppe grasslands. In contrast, results of qPCR analysis of free-living nitrogen fixers and total bacteria showed significantly high abundance levels in steppe grassland (P<0.01 and P<0.03, respectively). In this study, the microbial communities and their gene abundances were assessed in croplands that had been abandoned for different periods. An understanding of how environmental factors and changes in microbial communities affect abandoned croplands could aid in appropriate soil management to optimize the structures of soil microorganisms.     

Survival and Resuscitation of Ten Strains of Campylobacter jejuni and Campylobacter coli under Acid Conditions

The culturability of 10 strains of Campylobacter jejuni and Campylobacter coli was studied after the bacteria were exposed to acid conditions for various periods of time. Campylobacter cells could not survive 2 h under acid conditions (formic acid at pH 4). The 10 Campylobacter strains could not be recovered, even when enrichment media were used. Viable cells, however, could be detected by a double-staining (5-cyano-2,3-ditolyl tetrazolium chloride [CTC]-4′,6′-diamidino-2-phenylindole [DAPI]) technique, demonstrating that the treated bacteria changed into a viable but nonculturable (VBNC) form; the number of VBNC forms decreased over time. Moreover, some VBNC forms of Campylobacter could be successfully resuscitated in specific-free-pathogen fertilized eggs via two routes, amniotic and yolk sac injecting.     

Stability of Methanobrevibacter smithii Populations in the Microbial Flora Excreted from the Human Large Bowel

Total anaerobic bacteria and Methanobrevibacter smithii populations were enumerated in fecal specimens from two individuals over 10- and 13-month periods. The ratio of M. smithii to total anaerobic count varied between the individuals, but it was a relatively constant proportion of the large-bowel microbial flora within each individual. Neither a barium enema examination of one subject nor a radical change in the diet of the other had any long-term effect on the methanogen populations.     

THE PRESENCE OF AN ENDOGENOUS RESPIRATION IN THE AUTOTROPHIC BACTERIA

It is shown that there exists in the autotrophic bacterium Thiobacillus thiooxidans a measurable oxygen uptake in the absence of the specific nutrient (sulfur). This respiration is shown to be due to the utilization of organic materials which must have been previously synthesized by the chemosynthetic process, providing evidence that autotrophic bacteria contain a dissimilatory process which involves the breakdown of organic materials and furnishes energy for the maintenance of the cell during periods in which the specific nutrient is absent. This is entirely in accord with the work of Bömeke (1939), who provided similar types of proof for Nitrosomonas and Nitrobacter. One may conclude, therefore, that autotrophic bacteria possess an endogenous respiration which involves the utilization of previously synthesized organic materials.     

Specific Growth Rate Plays a Critical Role in Hydrogen Peroxide Resistance of the Marine Oligotrophic Ultramicrobacterium Sphingomonas alaskensis Strain RB2256

The marine oligotrophic ultramicrobacterium Sphingomonas alaskensis RB2256 has a physiology that is distinctly different from that of typical copiotrophic marine bacteria, such as Vibrio angustum S14. This includes a high level of inherent stress resistance and the absence of starvation-induced stress resistance to hydrogen peroxide. In addition to periods of starvation in the ocean, slow, nutrient-limited growth is likely to be encountered by oligotrophic bacteria for substantial periods of time. In this study we examined the effects of growth rate on the resistance of S. alaskensis RB2256 to hydrogen peroxide under carbon or nitrogen limitation conditions in nutrient-limited chemostats. Glucose-limited cultures of S. alaskensis RB2256 at a specific growth rate of 0.02 to 0.13 h⁻¹ exhibited 10,000-fold-greater viability following 60 min of exposure to 25 mM hydrogen peroxide than cells growing at a rate of 0.14 h⁻¹ or higher. Growth rate control of stress resistance was found to be specific to carbon and energy limitation in this organism. In contrast, V. angustum S14 did not exhibit growth rate-dependent stress resistance. The dramatic switch in stress resistance that was observed under carbon and energy limitation conditions has not been described previously in bacteria and thus may be a characteristic of the oligotrophic ultramicrobacterium. Catalase activity varied marginally and did not correlate with the growth rate, indicating that hydrogen peroxide breakdown was not the primary mechanism of resistance. More than 1,000 spots were resolved on silver-stained protein gels for cultures growing at rates of 0.026, 0.076, and 0.18 h⁻¹. Twelve protein spots had intensities that varied by more than twofold between growth rates and hence are likely to be important for growth rate-dependent stress resistance. These studies demonstrated the crucial role that nutrient limitation plays in the physiology of S. alaskensis RB2256, especially under oxidative stress conditions.     

Alterations in Ileal Mucosa Bacteria Related to Diet Complexity and Growth Performance in Young Pigs

Background

Evaluation of the prolonged impact of weaning diet on ileal mucosa bacteria and during periods of reduced and improved growth was conducted using 454 pyrosequencing.

Methodology/Principal Findings

Weaned pigs were fed HIGH or LOW complexity diets, with or without antibiotics, for 6 weeks, followed by a common grower diet. Pigs were killed at 2 (n = 4 or 5) and 8 (n = 6) weeks post-weaning (periods of reduced and improved growth, respectively). Mucosal bacteria were removed; DNA was extracted and amplified using the V1–V3 region of the 16S rRNA gene. Mucosal bacteria clustered more closely by week post-weaning than diet but 44% of bacterial species did not change from week 2 to 8. There was no effect of diet complexity or antibiotic inclusion on indices of bacterial diversity. Firmicutes made up 91 and 96% of total reads at week 2 and 8, respectively. The proportion of Clostridium paraputrificum increased (P = 0.003) from week 2 to 8 in pigs fed LOW but didn’t change in pigs fed HIGH; whereas Clostridium leptum decreased (P = 0.02) from week 2 to 8 in pigs fed LOW but didn’t change in pigs fed HIGH. The proportion of Sarcina genus was 3-fold higher in pigs fed A+ compared to A− at week 2 and 5-fold higher at week 8 despite the lack of in-feed antibiotics at that time.

Conclusions/Significance

Shifts in mucosal bacteria populations may be related to dietary induced changes in growth performance during reduced and improved growth but further studies are required to confirm causative relationship. Weaning diet results in species specific prolonged alterations in mucosal bacteria, particularly where high levels of in-feed antibiotics are used. A considerable portion of ileal mucosal bacteria colonize early and remain stable over time despite changes in diet.     

The MG1363 and IL1403 Laboratory Strains of Lactococcus lactis and Several Dairy Strains Are Diploid

Bacteria are normally haploid, maintaining one copy of their genome in one circular chromosome. We have examined the cell cycle of laboratory strains of Lactococcus lactis, and, to our surprise, we found that some of these strains were born with two complete nonreplicating chromosomes. We determined the cellular content of DNA by flow cytometry and by radioactive labeling of the DNA. These strains thus fulfill the criterion of being diploid. Several dairy strains were also found to be diploid while a nondairy strain and several other dairy strains were haploid in slow-growing culture. The diploid and haploid strains differed in their sensitivity toward UV light, in their cell size, and in their D period, the period between termination of DNA replication and cell division.In contrast to higher eukaryotes, bacteria are haploid (6, 19); i.e., they store their genetic information in a single chromosome, which is then duplicated during the cell cycle. If the growth rate is sufficiently low, bacteria are born with a single copy of the chromosome, which will then be duplicated before the bacterium divides.There are a few reports about bacteria that have more than one genome per cell, i.e., that are polyploid. Deinococcus radiodurans has been shown to have 4 to 10 copies of its genome (13, 14). The diplococcal bacterium Neisseria gonorrhoeae was found to be diploid per coccal unit (31). Azotobacter vinelandii bacteria amplify the genome during growth in rich medium more than 40 times (20, 24, 27). The giant bacterium Epulopiscium fishelsoni has been shown to amplify its genome into a polytene chromosome of 3,000-fold ploidy (2). In addition, noncomplementing diploid bacteria have been isolated from protoplast fusions in Bacillus subtilis (11) and, as a result of zygogenesis, in Escherichia coli (10). A few other bacteria with two to six different chromosomes have been reported (15, 30).The normal cell cycle is divided into three periods: (i) the B period from cell division until initiation of replication, (ii) the C period in which the cell replicates its DNA, and (iii) the D period from termination of productive replication until cell division. The D period thus includes processes such as proofreading and deconcatenation. The B period is found only in cells whose generation times exceed the length of the combined C and D periods. If the generation times become shorter than the combined lengths of the C and D periods, then the initiations of replication move into previous cell cycles (16). Fast-growing bacteria will therefore have more than one ongoing round of DNA replication at the same time; they might have 4, 8, or even 16 origins of replication (4). Normal haploid cells are born with one chromosome, either replicating or nonreplicating, and always with one terminus of replication. Not until the replication has ended do the cells have two termini. If the D period becomes longer than the generation time, which happens at high growth rates, the cells will be born with two termini as a result of the overlapping cell cycles. Long D periods are discussed further in the Discussion.We have examined the cell cycle of Lactococcus lactis subsp. cremoris MG1363 in order to determine the cell cycle periods. To our surprise, we found that slow-growing cultures of these bacteria were born with two complete chromosomes, which were replicated into four chromosomes during the C period. This strain thus fulfills the criterion of being diploid without overlapping chromosomal replication cycles. Comparison with other L. lactis strains showed that both of the subspecies, L. lactis subsp. cremoris and L. lactis subsp. lactis, had members that were either diploid, like MG1363, or haploid, like most bacteria.     

Methodological aspects of assessing phagocytosis of Vibrio anguillarum by leucocytes of gilthead seabream (Sparus aurata L.) by flow cytometry and electron microscopy

In this paper we optimize a flow cytometric method for evaluating the phagocytic activity of leucocytes in gilthead seabream (Sparus aurata L.) and characterize the phagocytic cells observed. Optimal conditions were established for the fluorescein-labelling and analysis of the bacterium Vibrio anguillarum by flow cytometry. Head-kidney leucocytes were incubated with the heat-killed fluorescein isothiocyanate (FITC)-labelled bacteria for different periods, during which the kinetics of phagocytosis was studied. Attached and interiorized bacteria were distinguished. Although phagocytic ability reached a maximum after 60 min, phagocytic capacity reached its maximum at 20 min. The amount of ingested bacteria per phagocyte was estimated from the mean fluorescence of the leucocytes. Cytochalasin B or colchicine was used to inhibit phagocytosis. Monocyte-macrophages and acidophilic granulocytes showed phagocytic activity as demonstrated by transmission electron microscopy. In conclusion, the technique presented allows the screening of thousands of cells, and individual cell evaluation, by quantifying interiorized particles in fish phagocytes. Our ultrastructural results demonstrate that V. anguillarum is actively phagocytized by seabream macrophages and acidophilic granulocytes.     

Distribution of Indigenous Bacterial Pathogens and Potential Pathogens Associated with Roof-Harvested Rainwater

The harvesting of rainwater is gaining acceptance among many governmental authorities in countries such as Australia, Germany, and South Africa, among others. However, conflicting reports on the microbial quality of harvested rainwater have been published. To monitor the presence of potential pathogenic bacteria during high-rainfall periods, rainwater from 29 rainwater tanks was sampled on four occasions (during June and August 2012) in a sustainable housing project in Kleinmond, South Africa. This resulted in the collection of 116 harvested rainwater samples in total throughout the sampling period. The identities of the dominant, indigenous, presumptive pathogenic isolates obtained from the rainwater samples throughout the sampling period were confirmed through universal 16S rRNA PCR, and the results revealed that Pseudomonas (19% of samples) was the dominant genus isolated, followed by Aeromonas (16%), Klebsiella (11%), and Enterobacter (9%). PCR assays employing genus-specific primers also confirmed the presence of Aeromonas spp. (16%), Klebsiella spp. (47%), Legionella spp. (73%), Pseudomonas spp. (13%), Salmonella spp. (6%), Shigella spp. (27%), and Yersinia spp. (28%) in the harvested rainwater samples. In addition, on one sampling occasion, Giardia spp. were detected in 25% of the eight tank water samples analyzed. This study highlights the diverse array of pathogenic bacteria that persist in harvested rainwater during high-rainfall periods. The consumption of untreated harvested rainwater could thus pose a potential significant health threat to consumers, especially children and immunocompromised individuals, and it is recommended that harvested rainwater be treated for safe usage as an alternative water source.