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Antibiotic G-418, a new Micromonospora-produced aminoglycoside with activity against protozoa and helminths: fermentation, isolation, and preliminary characterization.

Antibiotic G-418 is a new aminoglycoside produced as the major component by a new species of Micromonospora, M. rhodorangea NRRL 5326. The antibiotic is prepared by submerged fermentation in a soybean-dextrin medium. Antibiotic G-418 is adsorbed on a cationic-exchange resin and separated from other impurities by passing it down a Dowex (1 x 2) resin column. The antibiotic, which contains 2-deoxystreptamine, has broad-spectrum antibacterial activity and is highly active against protozoa, amoebae, tapeworm, and pinworm infections in mice. This report describes the taxonomy of the organism, and fermentation, isolation, and preliminary characterization of antibiotic G-418.

Aminoglycosides↗

Role of bacteria and protozoa in the removal of Escherichia coli from estuarine waters.

The removal of Escherichia coli from estuarine water was investigated. The survival of E. coli was dependent on the presence of protozoan predators and not on the presence of lytic bacteria. When indigenous protozoa were removed from estuarine water by filtration, the destruction of coliform populations was negligible. In studies designed to prevent the growth of indigenous bacterial populations without affecting protozoan populations, coliform destruction was significant.

Animals↗

Sequestration of holotrich protozoa in the reticulo-rumen of cattle.

Studies were carried out to determine the means by which holotrich protozoa can maintain their numbers within the rumen against the washout effect associated with the flow of ingesta. When a diet composed of 2 kg of concentrate and 1.5 kg of rice straw was fed to Holstein cows, about a fourfold increase in holotrich numbers per ml of rumen fluid was observed within 1 h after the commencement of feeding, and an abrupt decrease followed. This fluctuation in numbers was not related to the time of feeding. A sole feeding of 2 kg of concentrate had almost the same effect on the holotrichs as a sole feeding of 1.5 kg of rice straw. Administration of either 2 kg of concentrate or 1.5 kg of rice straw through the rumen fistula caused similar changes, though the extent of response to the former was greater than that to the latter. The administration of either 0.7 kg of starch or 0.2 kg of glucose through the fistula had a relatively minor effect on the holotrich population. Addition of rice straw to 0.5 kg of concentrate increased the change in numbers, but its addition had little, if any, effect when 1 kg of concentrate was fed. These results suggested that the fluctuation in holotrich numbers was related not only to the nature or component of feed but also to other factors such as the quantity or volume of a diet and the act of ingesting feed. Increasing the number of feedings up to eight times per day at 3-h intervals caused a decrease in the peak heights of holotrich numbers per milliliter of rumen fluid. A thick protozoal mass which primarily consisted of holotrichs was found on the wall of the reticulum of Holstein steers slaughtered after overnight starvation. These findings suggest that holotrichs would usually sequester on the reticulum wall and migrate into the rumen only for a few hours after feeding, and that this mode of behavior would be essential for holotrichs to maintain their population within the rumen of cattle. Possible mechanisms of the migration are also discussed.

Animal Feed↗

Factors stimulating migration of holotrich protozoa into the rumen.

The effects of feeding and various reticular infusions on ruminal holotrich concentrations were studied in an attempt to identify possible factors stimulating their migration into the rumen. It was concluded that glucose entering the reticulo-rumen shortly after feeding could stimulate migration of holotrich protozoa.

Animals↗

Isolation of protozoa from water associated with a legionellosis outbreak and demonstration of intracellular multiplication of Legionella pneumophila.

At the site of a legionellosis outbreak, amoebae and two ciliates, Tetrahymena sp. and Cyclidium sp., were isolated from cooling-tower water containing Legionella pneumophila. The Tetrahymena sp. and the amoebae repeatedly showed the ability to support intracellular multiplication of L. pneumophila. Both were isolated from cooling towers specifically implicated as the source for the spread of legionellosis. These protozoa may be reservoirs supporting the survival and multiplication of virulent legionellae in cooling-tower water.

Eukaryota↗

Ruminal ciliated protozoa in bison.

Ruminal contents from 79 slaughtered bison and 2 ruminally cannulated bison were collected to obtain information on total numbers and species distribution of ciliated protozoa. The bison originated from numerous herds throughout the Great Plains and were grouped into three dietary categories: (i) only forage; (ii) forage with moderate levels of supplementation; and (iii) feedlot concentrate-silage diet. Total ciliate counts were highest in bison receiving grain supplementation (210.1 x 10(4)/g) and lowest in bison consuming only forage (27.1 x 10(4)/g). All protozoan species found in bison have been reported in domestic livestock, although Ophryoscolex sp., a relatively common protozoan in cattle, was detected at low concentrations in only eight bison. The uncommon holotrich Microcetus lappus was present in five bison in concentrations reaching 8.4% of the total ciliate population. Charonina ventriculi, another infrequently observed species, was present in 18 bison, with the highest concentrations in forage-fed animals. Thirty bison possessed a type B protozoan population, characterized by Epidinium sp., Eudiplodinium maggii, and Eudiplodinium bovis. Thirty-eight bison possessed a mixed A-B population, characterized by Polyplastron sp. coexisting with low numbers of Eudiplodinium maggii or Epidinium sp. or both. Thirteen bison possessed populations lacking any remnant type B ciliate species. At least 29 of the bison possessing Polyplastron sp. were known to have been in contact with cattle, whereas all bison isolated from cattle had type B populations. The reduction of type B populations in bison becomes increasingly likely as bison production expands into areas inhabited by domestic livestock.

Animal Feed↗

Dynamics of ruminal ciliated protozoa in feedlot cattle.

Fluctuations in ciliated protozoan concentrations were monitored in 40 individually fed crossbred heifers that were stepped up to an 85% concentrate diet either slowly (12 days) or rapidly (3 days), with or without monensin (30 ppm). Ruminal fluid was withdrawn from all animals by stomach tube at the start of the study, after each group reached full feed, and at 14-day intervals thereafter throughout the finishing period until termination (day 119). Neither monensin nor speed of step-up affected (P greater than 0.10) total protozoan concentrations, ruminal pH, or lactic acid concentrations. Average protozoan concentrations peaked on day 5, progressively declined until day 56, and then increased (P less than 0.05), suggesting an adaptation to ruminal conditions. Concentrations of Isotricha spp. were higher (P less than 0.05) on the final two sampling dates than at any other time. After day 28, Entodinium, Isotricha, and Polyplastron were the only surviving genera. Protozoa were not detected in 11 heifers on day 42 and day 56, but only two animals were defaunated on day 119, indicating either exogenous or endogenous refaunation. Average protozoan concentrations were not different (P greater than 0.25) between ruminal samples collected by stomach tube the day before slaughter (2.8 x 10(5)/g) and digesta samples collected the next day (1.6 x 10(5)/g). In feedlot cattle, defaunation apparently is transitory and individual animals harbor a dynamic protozoan population that fluctuates in response to changing ruminal conditions.

Animal Feed↗

Differential rates of digestion of bacteria by freshwater and marine phagotrophic protozoa.

Differential decreases over time of two bacterial species, Escherichia coli and Enterococcus faecalis, in a freshwater and a marine ecosystem were observed and explained by a differential rate of digestion of these bacteria by phagotrophic flagellates and ciliates. For this purpose, fluorescence-labeled bacteria (FLB) were used and prepared from the two species cited above. The number of FLB was observed for 5 days in fresh and marine waters in the presence or absence (0.2-mum-pore-size-filtered water) of natural microbiota. These experiments showed a longer persistence of Enterococcus faecalis FLB as opposed to Escherichia coli FLB in the presence of natural microbiota. Removal of FLB was due to protozoan grazing because no decrease of FLB number was observed in the absence of natural microbiota. In short-term (about 40 min) ingestion experiments, we found similar clearance rates of Escherichia coli and Enterococcus faecalis FLB by assemblages of flagellates from the freshwater and the marine ecosystem and by cultured assemblages of ciliates from the marine ecosystem. Clearance rates of Enterococcus faecalis FLB were greater than those of Escherichia coli FLB for assemblages of ciliates from the freshwater ecosystem. Comparison of rates of ingestion and digestion of FLB by protozoa showed that Escherichia coli FLB were digested and ingested at similar rates. However, Enterococcus faecalis FLB were digested slower than they were ingested. These results suggest that a longer persistence of Enterococcus faecalis as opposed to Escherichia coli can be explained by a differential digestion by flagellates and ciliates in aquatic ecosystems. Moreover, rates of ingestion and digestion were strongly correlated for both FLB types.

Journal Article↗

Contribution of anaerobic protozoa and methanogens to hindgut metabolic activities of the American cockroach, Periplaneta americana.

The ciliate Nyctotherus ovalis occurs in high numbers in the hindgut of the American cockroach (Periplaneta americana) and harbors methanogenic bacteria as endosymbionts. The contribution of these hindgut microorganisms to metabolic and developmental processes of P. americana was studied by comparing cultures of cockroaches in which the composition of the hindgut microbial population was altered in various ways. Rearing the insects protozoan free resulted in increased insect generation time, decreased adult body weight, and absence of methane production. After feeding of protozoan-free adult cockroaches with a hindgut suspension containing N. ovalis and methanogens, methane increased to normal values and insect body weight was restored during the development of the second generation of insects. Feeding the protozoan-free cockroaches a hindgut suspension which was made free of N. ovalis resulted in an increase in methane production to only about 20% of the normal methane production level. This suggests that the methanogenic endosymbionts of N. ovalis are the major source of methane production in the hindgut. Inhibition of methanogens by addition of bromoethanesulfonic acid to the drinking water of a normal cockroach culture resulted in a reduction of methane production to about 2% of the normal level. No effects on insect body weight or the number of N. ovalis organisms were observed, but the fermentation pattern in the hindgut was shifted towards a relative increase in propionate levels. Similar results were obtained for in vitro cultures of hindgut microorganisms treated with bromoethanesulfonic acid. The results suggest a major role for hindgut protozoa in cockroach metabolic activities, especially during the insect growth period. The relatively large amounts of methane produced by cockroaches and by other methane-producing xylophagous insects suggest a major contribution by insects to global methane production.

Anaerobiosis↗

Quantitative centrifugation to extract benthic protozoa from freshwater sediments.

TWO METHODS FOR EXTRACTING PROTISTS FROM FRESHWATER SEDIMENT ARE DESCRIBED: (i) an adapted isopycnic centrifugation technique for sandy and gyttja-like sediments and (ii) a rate zonal centrifugation technique for sediments rich in particulate organic material (litter-like sediments). The recoveries of protists during isopycnic centrifugation in media of several densities were compared. No significant losses in sodium diatrizoate and Percoll were recorded. After known amounts of nanoflagellates were added to azoic sediments, the protists were extracted and counted. For sandy sediments, we found 100% recovery, and for the gyttja-like sediments we found a maximum recovery of 94%. The recovery of protozoa extracted from litter-like sediments, characteristic of littoral systems, depends on a given centrifugal force, on time, and on the dimensions of the flagellates. A recovery model which takes into account cell dimensions and centrifugation characteristics gives the minimum expected recovery.

Journal Article↗

Effects of ruminal protozoa on cellulose degradation and the growth of an anaerobic ruminal fungus, Piromyces sp. strain OTS1, in vitro.

An anaerobic rumen fungus, Piromyces sp. strain OTS1, was incubated in the presence or absence of a mixed, A-type, protozoal population obtained from a goat, in a medium containing filter paper cellulose as energy source and antibiotics to suppress bacterial growth. Fermentation end products, cellulose degradation, and chitin as an indicator of fungal biomass were examined. In the presence of protozoa, total volatile fatty acids, notably propionate and butyrate, increased, and lactate decreased. In fungus-protozoan coincubations, formate was not detected at the end of the experiment and the amount of reducing sugars remained low throughout the incubation period. The fungal growth in the coincubations was negatively affected. While protozoal predation on zoospores was one mechanism of inhibition, mature fungal cells were also affected. Total cellulose degradation was greater in fungal monocultures, but the amount of cellulose degraded per unit of fungal biomass was 25% larger in the coincubations. The negative effects that the protozoal predatory activity had on the fungal growth and subsequently on the amount of cellulose degraded by Piromyces sp. strain OTS1 were partially attenuated by the protozoal fibrolytic activity or by an enhanced fungal activity due to a more favorable environment.

Anaerobiosis↗

Transport behavior of groundwater protozoa and protozoan-sized microspheres in sandy aquifer sediments.

Transport behaviors of unidentified flagellated protozoa (flagellates) and flagellate-sized carboxylated microspheres in sandy, organically contaminated aquifer sediments were investigated in a small-scale (1 to 4-m travel distance) natural-gradient tracer test on Cape Cod and in flow-through columns packed with sieved (0.5-to 1.0-mm grain size) aquifer sediments. The minute (average in situ cell size, 2 to 3 (mu)m) flagellates, which are relatively abundant in the Cape Cod aquifer, were isolated from core samples, grown in a grass extract medium, labeled with hydroethidine (a vital eukaryotic stain), and coinjected into aquifer sediments along with bromide, a conservative tracer. The 2-(mu)m flagellates appeared to be near the optimal size for transport, judging from flowthrough column experiments involving a polydispersed (0.7 to 6.2 (mu)m in diameter) suspension of carboxylated microspheres. However, immobilization within the aquifer sediments accounted for a log unit reduction over the first meter of travel compared with a log unit reduction over the first 10 m of travel for indigenous, free-living groundwater bacteria in earlier tests. High rates of flagellate immobilization in the presence of aquifer sediments also was observed in the laboratory. However, immobilization rates for the laboratory-grown flagellates (initially 4 to 5 (mu)m) injected into the aquifer were not constant and decreased noticeably with increasing time and distance of travel. The decrease in propensity for grain surfaces was accompanied by a decrease in cell size, as the flagellates presumably readapted to aquifer conditions. Retardation and apparent dispersion were generally at least twofold greater than those observed earlier for indigenous groundwater bacteria but were much closer to those observed for highly surface active carboxylated latex microspheres. Field and laboratory results suggest that 2-(mu)m carboxylated microspheres may be useful as analogs in investigating several abiotic aspects of flagellate transport behavior in groundwater.

Journal Article↗

Relative contributions of bacteria, protozoa, and fungi to in vitro degradation of orchard grass cell walls and their interactions.

To assess the relative contributions of microbial groups (bacteria, protozoa, and fungi) in rumen fluids to the overall process of plant cell wall digestion in the rumen, representatives of these groups were selected by physical and chemical treatments of whole rumen fluid and used to construct an artificial rumen ecosystem. Physical treatments involved homogenization, centrifugation, filtration, and heat sterilization. Chemical treatments involved the addition of antibiotics and various chemicals to rumen fluid. To evaluate the potential activity and relative contribution to degradation of cell walls by specific microbial groups, the following fractions were prepared: a positive system (whole ruminal fluid), a bacterial (B) system, a protozoal (P) system, a fungal (F) system, and a negative system (cell-free rumen fluid). To assess the interactions between specific microbial fractions, mixed cultures (B+P, B+F, and P+F systems) were also assigned. Patterns of degradation due to the various treatments resulted in three distinct groups of data based on the degradation rate of cell wall material and on cell wall-degrading enzyme activities. The order of degradation was as follows: positive and F systems > B system > negative and P systems. Therefore, fungal activity was responsible for most of the cell wall degradation. Cell wall degradation by the anaerobic bacterial fraction was significantly less than by the fungal fraction, and the protozoal fraction failed to grow under the conditions used. In general, in the mixed culture systems the coculture systems demonstrated a decrease in cellulolysis compared with that of the monoculture systems. When one microbial fraction was associated with another microbial fraction, two types of results were obtained. The protozoal fraction inhibited cellulolysis of cell wall material by both the bacterial and the fungal fractions, while in the coculture between the bacterial fraction and the fungal fraction a synergistic interaction was detected.

Animals↗

Simple method of remove completely ciliate protozoa of adult ruminants.

Dioctyl sodium sulfosuccinate (Aerosol OT) has been used in a remarkably short period to obtain rumen ciliate-free cows. Two 30-g doses of Aerosol OT given on consecutive days appeared to effectively eliminate all types of rumen ciliate protozoa without harming the health of the host animal. Measurement of the rumen metabolic parameters of gas production, carbon dioxide to methane ratio, pH value, volatile fatty acids, ammonia, and in vitro cellulose digestion, along with total amylolytic streptococci counts of the rumen contents, showed that conditions in rumens of defaunated animals are normal 4 days after such treatment. It appears that such animals may be used in ruminal studies that require defaunated animals.

Animals↗