Use of fecal streptococci as indicators of pollution in soil.

The survival, recovery, and identification of Streptococcus isolates from soil was investigated by (i) examination of survival in soil under different moisture and temperature conditions, (ii) evaluation of media combinations for recovering fecal streptococci from soil, and (iii) partial identification of isolates from diverse habitats. Cool, moist conditions prolonged the survival of Streptococcus faecalis in soil for at least 12 weeks, whereas freezing was lethal, with the populations being reduced up to 95% when several freeze-thaw treatments occurred. Media evaluations indicated that both the efficiency of recovery and enumeration of the fecal streptococci from soil can be influenced by the combination of media used. Taxonomic data revealed a need to develop procedures to differentiate between isolates of fecal origin and plant-derived streptococci that possess many of the cultural reactions of S. faecalis. It was found that recent fecal isolates exhibited a much greater incidence of multiple antibiotic resistance than soil or vegetation isolates, and this characteristic, coupled with the use of enterococci as indicators of fecal contamination in soil systems, is discussed.     

Validity of fecal coliforms, total coliforms, and fecal streptococci as indicators of viruses in chlorinated primary sewage effluents.

Quantities of combined chlorine that usually destroyed more than 99.999% of the indigenous fecal coliforms, total coliforms, and fecal streptococci in primary sewage effluents destroyed only 85 to 99% of the indigenous viruses present. Viruses were recovered from five of eight chlorinated primary effluents from which fecal coliforms were not recovered by standard most-probable-number procedures. The limited volumes of such chlorinated effluents that can be tested for indicator bacteria with currently available multiple-tube and membrane filter techniques restrict the value of fecal coliforms, fecal streptococci, and even total coliforms as indicators of viruses in these effluents. Although fecal coliforms and fecal streptococci are useful indicators of viruses in effluents from which these bacteria are recovered, the absence of these bacteria and even total coliforms from disinfected effluents (in standard tests) does not assure that viruses are also absent.     

Microorganisms as possible indicators of soil pollution by dioxin-containing defoliants

The diversity of microorganisms from soils treated in the past with various dosages of dioxin-containing defoliants was studied. Population alterations dependent on dioxin concentrations were elucidated. Soil fungi and, to a smaller extent, actinomycetes were found to be the most sensitive to dioxins.     

Identification of strains isolated as total and fecal coliforms and comparison of both groups as indicators of fecal pollution in tropical climates

This study was undertaken to better characterize the groups of total coliforms (TC) and fecal coliforms (FC) and to evaluate both groups as indicators of fecal contamination of drinking well water in a tropical climate (The Ivory Coast, West Africa). Isolated colonies obtained as TC or FC on membrane filters were identified using the API-20E system. From the well water samples, 58 golden-green colonies with a metallic sheen isolated on Endo medium (TC) were identified as Escherichia coli (55%), Enterobacter (26%), Klebsiella (14%), Proteus (3%), and Citrobacter (2%). Among 132 colonies isolated on Endo medium as non-TC (not showing the characteristic golden metallic sheen), 10% were identified as E. coli. The 196 blue colonies isolated on M-FC medium at 44.5 degrees C (FC) were identified as E. coli (66%), Klebsiella (12%), Enterobacter (10%), Citrobacter (5%), Salmonella (3%), Serratia (3%), Proteus (2%), and Yersinia (0.5%). Among 24 nonblue colonies on M-FC medium, none were identified as E. coli. Of the colonies isolated from human feces, E. coli represents 92% of the TC and 89% of the FC. Although these results are limited, they tend to confirm the greater specificity of the fecal coliform technique over that of total coliform for the detection of fecal contamination of untreated well water. From the results presented here and the observations of other workers, it is suggested that the use of FC instead of TC should be considered as the method of choice for determining drinking water pollution of untreated groundwater supplies.     

Bovine enteroviruses as indicators of fecal contamination

Surface waters frequently have been contaminated with human enteric viruses, and it is likely that animal enteric viruses have contaminated surface waters also. Bovine enteroviruses (BEV), found in cattle worldwide, usually cause asymptomatic infections and are excreted in the feces of infected animals in large numbers. In this study, the prevalence and genotype of BEV in a closed herd of cattle were evaluated and compared with BEV found in animals in the immediate environment and in environmental specimens. BEV was found in feces from 76% of cattle, 38% of white-tailed deer, and one of three Canada geese sharing the same pastures, as well as the water obtained from animal watering tanks, from the pasture, from streams running from the pasture to an adjacent river, and from the river, which emptied into the Chesapeake Bay. Furthermore, BEV was found in oysters collected from that river downstream from the farm. These findings suggest that BEV could be used as an indicator of fecal pollution originating from animals (cattle and/or deer). Partial sequence analysis of the viral genomes indicates that different viral variants coexist in the same area. The possibility of identifying the viral strains found in the animals and in the contaminated areas by sequencing the RNA genome, could provide a tool to find the origin of the contamination and should be useful for epidemiological and viral molecular evolution studies.     

Use of Bifidobacterium dentium as an indicator of the origin of fecal water pollution

A new, simple, and specific protocol to discriminate between human and animal fecal pollution is described. The procedure is based on the detection of certain Bifidobacterium species in the samples. Two 16S rRNA gene-targeted probes are described. One of these probes (BDE) has as its target a region of the 16S rRNA gene of Bifidobacterium dentium, a Bifidobacterium species of exclusively human origin. The other probe (BAN) is based on the sequence of a region of 16S rRNA gene for several Bifidobacterium species related with animal origins. The specificity of both probes was evaluated by using 24 Bifidobacterium species, and their threshold detection limit was established by DNA-DNA hybridization. DNA-DNA hybridization with the BDE probe showed it to be specific for B. dentium, whereas that with the BAN probe showed it to be specific for B. animalis, B. asteroides, B. coryneforme, B. cuniculi, B. globosum, B. magnum, B. minimum, and B. subtile. A simple and specific protocol was also developed for the detection of their target species in environmental samples (sewage and feces). DNA-DNA hybridization with the BAN probe was only positive for samples from cattle and goats. Thus, this probe is not suitable for the identification of any animal fecal pollution. Whereas all samples with human fecal pollution showed a positive DNA-DNA hybridization result with the BDE probe, none of those with animal fecal pollution did. Therefore, this finding supports the potential use of this probe in detecting fecal pollution of human origin.     

Sulfite-reducing clostridia in the sediment of a high mountain lake (Laguna Grande, Gredos, Spain) as indicators of fecal pollution.

We studied the vertical distribution of sulfite-reducing clostridia in the sediment of a Spanish high-mountain lagoon (Laguna Grande de Gredos, central Spain), with optimal sediment characteristics (temperature < 20 degrees C) to maintain spores without growing. This allowed us to assess the original numbers of sulfite-reducing clostridia endospores settled, without postdepositional growing. Sulfite-reducing clostridia are normal inhabitants of the intestinal microbiota of humans and other mammals. These microorganisms may form endospores, which allow the bacteria to survive in almost any habitat, either terrestrial or aquatic, waiting for favorable conditions for growth. Sulfite-reducing clostridia could be suitable indicators of past human pollution because they have a great longevity in natural habitats and they cannot multiply at temperatures below 20 degrees C or in the presence of O2. We found a great increase in the numbers of clostridia (expressed as colony-forming units per gram [CFU/g] of dry weight of sediment) since the 1970s, which reflects the rise of human pressure caused by the practice of outdoor activities. Clostridia CFU/g rose dramatically after the faulty operation of the depuration system of a mountain refuge built close to the lagoon. We compared the vertical distribution of clostridia CFU/g from Laguna Grande sediments with those from a neighbor lagoon (Laguna Cimera), which showed less tourist pressure and no direct disposal of sewage. Finally, we agree with the usefulness of the numbers of sulfite-reducing clostridia as indicators of past pollution.     

Use of the water quality index and dissolved oxygen deficit as simple indicators of watersheds pollution

The use of the water quality index (WQI) and the dissolved oxygen deficit (D) as simple indicators of the watersheds pollution was investigated and compared in the Municipality of Las Rozas (north-west of Madrid, Spain). The quality of the water in Guadarrama and Manzanares rivers and Paris Park ponds, the main watersheds of this area was investigated during 2 years (from September 2001 to September 2003). It was found that the WQI was very useful for the classification of the waters monitored. The WQI was 70, which corresponds to “good” quality water at the sampling point 1 (entrance of Las Rozas) and decreased to around 64 (medium quality) at the sampling point 6 (outlet of Las Rozas) in the case of Guadarrama River. The WQI was around 65 in the influents of Manzanares River. Finally, in Paris Park the WQI ranged from around 72–55, which corresponded to a classification from “good” to “medium” quality, respectively. A high linear relationship between the WQI and the dissolved oxygen deficit (D) was found. Therefore, a fast determination of WQI may be carried out knowing the values of D, which are easily obtainable by field measurements. It was found an influence of the climate conditions on the values of WQI and D.     

Gentamicin-thallous-carbonate medium for isolation of fecal streptococci from foods.

Gentamicin-thallous-carbonate (GTC) agar was formulated by Donnelly and Hartman (Appl. Environ. Microbiol. 35:576-581, 1978) to select for fecal streptococci in sewage and water samples. The present study was conducted to determine the usefulness of GTC agar for the enumeration of fecal streptococci in foods. Comparisons were made with KF streptococcal (KF), Pfizer selective enterococcus (PSE), and thallous acetate (TA) agars. Samples of ground beef pork sausage, frozen broccoli, frozen fish, and ice cream were examined. Presumptive streptococcal counts obtained on GTC agar were significantly higher than those obtained on KF and PSE agars and were comparable to those obtained on TA agar. GTC was more sensitive than KF or PSE agars primarily because of the recovery of greater numbers of Streptococcus bovis and Streptococcus equinus strains. Percentages of confirmed fecal streptococci obtained on GTC, KF, PSE, and TA agars were 70, 95, 80, and 74, respectively. Differences between these percentages were not statistically significant, but they indicated that selectivity of GTC agar could be improved. Advantages of using GTC agar to isolate fecal streptococci from foods include a short incubation time (16 to 18 h) and large, distinct colonies that facilitate rapid enumeration and subsequent confirmation.     

The use of grasses as indicators of environmental pollution

Summary An exploratory examination of the use of rye grass as an indicator of environmental accumulation of minor elements confirmed that natural grass is contaminated by soil or airborne dust, and that this contamination cannot be washed off sampled grass with water without also removing elements from within the leaves, to an extent that varies with the age and/or environment of the grass. This reduces the suitability of natural grasses as indicators of pollution, and alternative procedures are required and proposed.     

Soil macroinvertebrates as indicators of pollution by heavy metals

A broad range of soil pollutants were found to decrease with distance from a zinc smelter from 35,000 to 77, 8270 to 40 and from 190 to less than 1 ppm for zinc, lead and cadmium, respectively. Along this gradient, observed species richness of soil macro-organisms seemed to be more affected by the land-use type than by soil pollution--minimum in crops (21), maximum in woody sites (126). IndVal index allowed isolation of 21 indicator species from the 339 morphospecies identified. Most of these indicator species were characteristic of the unpolluted sites: only two diplopods and one gastropod from polluted poplar plantations, and none from the most polluted site. Since soil invertebrates respond to different environmental factors, including direct effect of heavy metals, we suggest there may be some confounding factors generating spurious relationships between the values of species as bioindicators and the pollution status they are supposed to indicate.     

Sorbitol-fermenting bifidobacteria as specific indicators of human faecal pollution

Bifidobacteria were consistently present in the faeces of both man and pigs but only occasionally in the faeces of cattle and sheep, and they were not isolated from faecal samples from other animals; total counts of bifidobacteria were obtained by membrane filtration with YN-17 medium, a modification of Resnick and Levin's YN-6 medium. Mannitol-fermenting strains of bifidobacteria were isolated from both human and animal faeces, but sorbitol-fermenting strains were obtained only from human samples. These sorbitol-fermenting strains were identified as either Bifidobacterium adolescentis or B. breve and their numbers were obtained by membrane filtration on Human Bifid Sorbitol agar (HBSA). Sorbitol-fermenting bifidobacteria are specific indicators of human faecal pollution of waters and wastewaters.     

Sponge-associated marine bacteria as indicators of heavy metal pollution

Sponges invariably filter a large volume of seawater and potentially accumulate heavy metals and other contaminants from the environment. Sponges, being sessile marine invertebrates and modular in body organization, can live many years in the same location and therefore have the capability to accumulate anthropogenic pollutants such as metals over a long period. Almost all marine sponges harbor large number of microorganisms within their tissues where they reside in the extra- and intra-cellular spaces. Bacteria in seawater have already been established as biological indicators of contamination. The present study was intended to find out the heavy metal resistance pattern of sponge-associated bacteria so as to develop suitable biological indicators. The bacteria associated with a marine sponge Fasciospongia cavernosa were evaluated as potential indicator organisms. The associated bacteria including Streptomyces sp. (MSI01), Salinobacter sp. (MSI06), Roseobacter sp. (MSI09), Pseudomonas sp. (MSI016), Vibrio sp. (MSI23), Micromonospora sp. (MSI28), Saccharomonospora sp. (MSI36) and Alteromonas sp. (MSI42) showed resistance against tested heavy metals. Based on the present findings, Cd and Hg emerged as the highly resistant heavy metal pollutants in the Gulf of Mannar biosphere reserve. Plasmids in varied numbers and molecular weights were found in all the isolates. Particularly the isolates MSI01 and MSI36 harbored as many as three plasmids each. The results envisaged that the plasmids might have carried the resistance factor. No correlation was observed in number of plasmids and level of resistance. The literature evidenced that the sponge-associated bacteria were seldom exploited for pollution monitoring though they have been extensively used for bioprospecting. In this background, the present findings come up with a new insight into the development of indicator models.     

Lysosomal perturbations in fish liver as indicators for toxic effects of environmental pollution.

1. Lysomones play a key role in liver injury in fish caused by organic and inorganic xenobiotics. The lysosomal stability test was transferred to fish liver with the aim of testing responsive and practicable methods for biological-effects monitoring. 2. A two-step response of lysosomes in fish liver could be discerned, reflected by the activity (number and size of lysosomes) and the injury (membrane destabilisation) of the lysosomal detoxifying system. 3. Significant differences, with respect to lysosomal enlargement, membrane stability and pathological lipid accumulation, were found along a pollution gradient throughout the year. 4. The lysosomal tests clearly reflect the breakdown of the adaptive capacity of the fish liver to toxic injury. Therefore, a test battery measuring lysosomal perturbations should be recommended for the biological-effects monitoring.     

Methanobrevibacter ruminantium as an indicator of domesticated-ruminant fecal pollution in surface waters

A PCR-based assay (Mrnif) targeting the nifH gene of Methanobrevibacter ruminantium was developed to detect fecal pollution from domesticated ruminants in environmental water samples. The assay produced the expected amplification product only when the reaction mixture contained DNA extracted from M. ruminantium culture, bovine (80%), sheep (100%), and goat (75%) feces, and water samples from a bovine waste lagoon (100%) and a creek contaminated with bovine lagoon waste (100%). The assay appears to be specific and sensitive and can distinguish between domesticated- and nondomesticated-ruminant fecal pollution in environmental samples.