The ecology of "fecal indicator" bacteria commonly found in pulp and paper mill water systems

Coliform bacteria have long been used to indicate fecal contamination of water and thus a health hazard. In this study, the in-mill water and external effluent treatment systems of seven typical Canadian pulp and paper mills were all shown to support the growth of numerous coliforms, especially Klebsiella Spp., Escherichia coli. Enterobacter spp., and Citrobacter spp. In all mills and most sampled locations, klebsiellas were the predominant coliforms. Although all but one of the mills had no sewage input and most disinfected their feed (input) water, all contained the most typical fecal indicator bacterium, E. coli. Many of the mill coliforms were classified as fecal coliforms by standard "MPN" and metabolic tests, but this was shown to be due to their thermotolerance, not their origin. Mill coliforms were shown not to be just simple transients from feedwater or furnish (wood), but to be continuously growing, especially in some of the primary clarifiers. Isolated mill coliforms grew very well on a sterilized raw combined mill effluent. The fecal streptococci (enterococci), alternative indicators of fecal health hazards, were common in all mills in the absence of sewage. Ten strains of E. coli isolated from four mills were all shown to be non-toxigenic strains of harmless serotypes. No salmonellas were found. Therefore, the use of total coliform, fecal coliform, enterococci, or E. coli counts as indicators of fecal contamination, and thus of health hazard in pulp and paper mill effluents or biosolids (sludges) known to be free of fecal input is invalid.     

Identification of the sources of fecal coliforms in an urban watershed using antibiotic resistance analysis

Bacteria such as fecal coliforms are used as indicators of fecal pollution in natural waters. These bacteria are found in the feces of most wild and domestic animals and thus provide no information as to the source of fecal contamination, yet identification of indicator bacteria sources allows improved risk assessment, remediation, and total daily maximum load (TDML) assessment of environmental waters. This bacterial source tracking study was initiated in order to identify the dominant source(s) of fecal contamination in the urban watershed of Stevenson Creek in Clearwater, Florida. Five sites that represent areas where routine monitoring has previously shown high levels of fecal coliforms were sampled over 7 months. Fecal coliforms were enumerated by membrane filtration, and antibiotic resistance analysis was used to "fingerprint" a subset of randomly selected isolates and statistically match them to fingerprints of fecal coliforms from known sources (the library). A field test of the classification accuracy of the library was carried out by isolating fecal coliforms from the soil and waters surrounding a failing onsite wastewater treatment and disposal system (OSTDS). The vast majority of the isolates were classified into the human category. The major sources of fecal pollution in Stevenson Creek over the course of the study were wild animal, human, and, to a lesser extent, dog. Overall, wild animal feces were identified as the dominant source when fecal coliform levels were high, but when fecal coliform levels were low, the dominant source was identified as human. The results of this study demonstrate that the sources of fecal indicator bacteria within one urban watershed can vary substantially over temporal and spatial distances.     

Some effects of shoreline cottage development on lake bacteriological water quality

Eighteen cottaged lakes and one uncottaged lake in Southern Ontario, and their inflowing streams were surveyed for bacterial indicators of pollution by membrane filter techniques. Water samples were taken on 5 consecutive days in the spring, summer, and fall of the year. Inputs of fecal indicator bacteria were determined through an analysis of variance. Fecal coliform and fecal streptococcus levels in lakewater varied in a seemingly erratic manner. When the bacterial densities at different points on the lake were examined by an analysis of variance, the locations with significantly higher densities of fecal coliforms and fecal streptococci were most often found along the cottaged shoreline. Total coliform densities did not show this relationship with the cottaged shoreline. In addition, after rainfall, a large increase in numbers of fecal coliforms were found in the cottaged lakes but not in the uncottaged lake. These findings indicated that the cottaged shoreline was a greater source of fecal indicator bacteria than the uncottaged shoreline. However, the highest concentrations of fecal indicator bacteria in these lakes were found in the inflowing streams. The bacteria in these streams arose from stormwater from undeveloped forested watersheds and were probably, therefore, of soil or animal origin.     

Survival of bacterial indicator organisms and enteric viruses in the Mediterranean coastal waters off Tel-Aviv

Field and laboratory studies were initiated on the die-away kinetics of coliforms and enteric viruses in coastal waters off Tel-Aviv. Samples were taken from the sewage boil located approx. 880 m out to sea and from three main Tel-Aviv beaches designated as sampling stations, ranging 3 km to the north and 2 km to the south. Routine bacteriological tests were made from these stations; in addition, approx. 50% of the samples were also examined for enteroviral content and occasionally, samples were taken from most of the other Tel-Aviv beaches for comparison of bacterial and viral content.The concentrations of total coliforms, fecal coliforms and fecal streptococci were correlated with the concentrations of enteroviruses. In general, the data suggests that the number of enteric bacteria in the sea was reduced relatively more rapidly than that of the enteroviruses, while fecal streptococci displayed a die-away rate similar to that of enteroviruses. The percentage of samples positive for enteroviruses was found to be lower during the summer months of the year than in the remaining months. The concentration of coliforms was also significantly lower during the summer months. This suggests that the decreased microbial level is associated with increased summertime daylight and solar radiation which affect the micro-organisms' die-away rate. About 76% of the positive enteroviral samples were found at beaches with a fecal coliform bacterial level considered safe for bathing beaches.     

Pathogenic or potentially pathogenic bacteria as contaminants of fresh water from different sources in Araraquara, Brazil

The microbiological quality of various fresh waters in the Araraquara region, state of São Paulo, Brazil was investigated. Ninety-nine water samples were taken from rivers, reservoirs, artesian and non-artesian wells, springs and tap waters, and these waters were analysed using: plate counts of heterotrophic microorganisms (per 1 ml); Most Probable Number (MPN) of fecal coliforms and E. coli (per 100 ml); tests for presence of Salmonella, Shigella, Yersinia, the E. coli pathogens of classes EPEC, ETEC and EIEC and Mycobacterium, Shigella, Yersinia and enteroinvasive E. coli (EIEC) were not isolated. The other types of microorganisms were isolated in varying proportions. We conclude that the waters investigated represent a potential microbiological health risk.     

Influence of temperature and substrate shocks on survival and succinic dehydrogenase activity of heterotrophic freshwater bacteria

The influence of temperature shocks on aquatic bacteria was studied in laboratory experiments with regard to population shifts in lake water communities of aerobic heterotrophic bacteria including coliforms. as well as in synthetic mixed cultures of psychrotolerant aquatic isolates. In addition, succinic dehydrogenase activity was used as a parameter of bioactivity. Each experiment was designed to study concomitant effects of elevated levels of organic substrates.

Simultaneous additions of complex nutrients such as 100–300 mg 1⁻¹ of meat peptone to lake water caused a synergistic action of substrate and temperature on population dynamics by depressing population densities at temperatures below 20°C. In contrast, stimulation was observed at higher temperatures particularly for the coliform group. In mixed cultures consisting of Cytophaga, Chromobacterium and Arthrobacter as important members of the heterotrophic microflora of the investigated lake water, substrate accelerated death was induced by temperature shocks beyond the maximum growth temperatures. The succinic dehydrogenase activity of a psychrotolerant strain of Cytophaga failed to show more sensitive responses to temperature shocks than did viable counts.     

In situ persistence of indicator bacteria in streams polluted with acid mine drainage

Persistence of indicator organisms (total coliforms, fecal coliforms, and fecal streptococci) associated with natural samples of raw sewage was studied following in situ exposure to five aquatic environments. Three of these streams contained significant amounts of acid mine water (AMW) while the other two were relatively uncontaminated. Indicator organisms were rapidly killed upon exposure to the acid mine systems, whereas little reduction in numbers was observed in the uncontaminated streams. Seasonal changes affected survival of indicators as reflected by prolonged persistence at colder in situ water temperatures. The fecal coliform group was most susceptible to the AMW stress, while the fecal streptococci were most persistent. An enrichment technique resulted in substantially enhanced recovery of certain species of sublethally injured survivors of acid stress. Enrichment was particularly beneficial for recovery of AMW-injured fecal coliforms. Relatively little improvement in recovery of fecal streptococci was afforded by the enrichment technique.     

Faecal coliforms and faecal streptococci in streams in the new guinea highlands

Procedures used to assess faecal water contamination were developed in temperate regions and their applicability to tropical waters has been questioned. This paper reports a detailed study of faecal coliform and faecal streptococci concentrations in the streams of the Saka Valley in the Highlands of New Guinea. The Saka Valley supports large populations of humans and domestic pigs, whose faeces are washed into the streams by surface runoff following rain. Faecal coliforms and faecal streptococci, enumerated by the membrane filtration technique, were found to be excellent indicators of faecal pollution in the New Guinea Highlands. A consideration of faecal coliform to faecal streptococci ratios and other factors, leads to the conclusion that most faecal contamination derives from pigs. All waters investigated, with the exception of spring waters, were grossly contaminated with faecal material and these polluted streams provide the total domestic water needs of the New Guineans. The fluctuations in pollution levels are described and their association with water temperature, rainfall and turbidity is analysed. The interpretation of the water contamination data is made speculative by the lack of information on the coliform and streptococcal flora of fresh pig and human faeces under New Guinea Highlands conditions.     

Rainwater catchment water quality in Micronesia

Rainwater catchment systems (RWCS) in Micronesia were sampled to assess their bacteriological water quality and to determine which RWCS characteristics had a significant impact on water quality. Total and fecal coliform bacteria tests were used to evaluate 203 catchments on 10 islands. Fifty-seven percent of the RWCS had no fecal coliform bacteria and 61% had fewer than 10 total coliform bacteria per 100 ml. Catchment characteristics were found to have a statistically significant effect on total coliform bacteria levels but they did not affect faecal coliform bacteria concentrations. Rainwater catchment systems were found to provide acceptable water in most cases but disinfection prior to consumption is still highly recommended.     

The limitation of the ratio of fecal coliforms to total coliphage as a water pollution index

The fecal coliform populations of raw sewage, sewage lagoon effluent, and river water were determined using the most probable number technique. The total coliphage populations of the three water sources were determined using Escherichia coli B (ATCC 11303-1) host cells. The ratios of fecal coliforms to coliphage in the three water samples were 87:1, 4.2:1, and 0.15:1, respectively. The ratio of fecal coliforms to coliphage in stored raw sewage decreased from 87:1 to about 1:1 within 7 days at 20°C and within 28 days at 4°C. These changing ratios resulted from the greater longevity of the coliphage compared with that of the coliform bacteria. The use of the ratio of fecal coliforms to coliphage is not considered reliable as an index of when a fecal pollution event occurred because the ratio is influenced by prior contamination, presence of sediment, chlorination, and temperature.     

Evaluation of microbiological accumulation capability of the commercial sponge Spongia officinalis var. adriatica (Schmidt) (Porifera, Demospongiae)

This study was carried out to evaluate the microbiological accumulation capability of the demosponge Spongia officinalis var. adriatica. Six microbiological parameters were researched in two sampling periods in the water and in reared sponge samples coming from sites with different degrees of microbial contamination: an off-shore fish farm displaced off the Apulian coast (Southern Adriatic Sea) and a no-impacted area displaced into the Marine Protected Area of Porto Cesareo (Apulian coast-Ionian Sea). We detected the density of culturable heterotrophic bacteria by spread plate on marine agar, total culturable bacteria at 37 degrees C on Plate Count Agar and vibrios on thiosulphate-citrate-bile-sucrose-salt (TCBS) agar. Total and fecal coliforms as well as fecal streptococci concentrations were detected by the MPN method. Bacterial densities were always higher in the sponge homogenates compared with the corresponding seawater in the sampling points and in both sampling periods. As regard vibrios, total culturable bacteria at 37 degrees C and fecal streptococci concentrations, the highest values were observed in the sponge samples coming from the off-shore fish farm during the summer period. The ability of Spongia officinalis var. adriatica to accumulate the microbial pollution indicators suggests that this species can be employed as a bioindicator for monitoring water quality.     

Occurrence, molecular characterization and antibiogram of water quality indicator bacteria in river water serving a water treatment plant

Water pollution by microorganisms of fecal origin is a current world-wide public health concern. Total coliforms, fecal coliforms (Escherichia coli) and enterococci are indicators commonly used to assess the microbiological safety of water resources. In this study, influent water samples and treated water were collected seasonally from a water treatment plant and two major water wells in a Black Belt county of Alabama and evaluated for water quality indicator bacteria. Influent river water samples serving the treatment plant were positive for total coliforms, fecal coliforms (E. coli), and enterococci. The highest number of total coliform most probable number (MPN) was observed in the winter (847.5 MPN/100 mL) and the lowest number in the summer (385.6 MPN/100 mL). Similarly E. coli MPN was substantially higher in the winter (62.25 MPN/100 mL). Seasonal variation of E. coli MPN in influent river water samples was strongly correlated with color (R² = 0.998) and turbidity (R² = 0.992). Neither E. coli nor other coliform type bacteria were detected in effluent potable water from the treatment plant. The MPN of enterococci was the highest in the fall and the lowest in the winter. Approximately 99.7 and 51.5 enterococci MPN/100 mL were recorded in fall and winter seasons respectively. One-way ANOVA tests revealed significant differences in seasonal variation of total coliforms (P < 0.05), fecal coliforms (P < 0.01) and enterococci (P < 0.01). Treated effluent river water samples and well water samples revealed no enterococci contamination. Representative coliform bacteria selected by differential screening on Coliscan Easygel were identified by 16S ribosomal RNA gene sequence analysis. E. coli isolates were sensitive to gentamicin, trimethoprim/sulfamethazole, ciprofloxacin, vancomycin, tetracycline, ampicillin, cefixime, and nitrofurantoin. Nonetheless, isolate BO-54 displayed decreased sensitivity compared to other E. coli isolates. Antibiotic sensitivity pattern can be employed in microbial source tracking.     

North and south american studies on the potential of coliphage as a water quality indicator

Studies were undertaken to assess the potential of coliphages to be used universally as water quality indicators and more specifically as health hazard indicators. Data were obtained from three water bodies, a northern Canadian River, inshore water samples from Lake Ontario and from marine beaches in Brazil. Data from this two continent, three water body study indicated (a) that within location fecal coliform and coliphages are positively correlated, (b) coliphage values can be predicted by using fecal coliform MPN, fecal streptococci MF and E. coli MF data and (c) a water quality guideline of 20 coliphage/100 ml for recreational fresh waters is proposed.     

Increases in fecal coliform bacteria resulting from centrifugal dewatering of digested biosolids

In many countries, the classification of biosolids for disposal purposes can be based, in part, on fecal coliform levels, with alternative criteria also available based on the stabilization process used, such as anaerobic digestion. The assumption that these alternative criteria provide equivalent protection may be flawed. This paper demonstrates that fecal coliform levels determined after digestion do not always indicate the bacterial levels after the same biosolids have been dewatered by centrifugation. In samples from mesophilic digestion, half had significant increases in coliform numbers (P<0.05) with up to one order of magnitude increase during centrifugation, suggesting coliform regrowth. Thermophilically digested samples had significant increases of several orders of magnitude during dewatering, more likely from reactivation of viable but non-culturable coliforms than from regrowth. In other cases, centrifugation induced coliform regrowth or reactivation upon incubation and storage of dewatered samples, but not digested samples. These 2-3 order of magnitude increases occurred with both 25 and 37 degrees C incubations. Coliform increases continued for up to 5 days, then gradually declined. However, by day 20 coliform numbers were still 2 orders of magnitude greater than when originally sampled. The magnitude of the increases could be due either to regrowth or reactivation, but the nature of the longer-term increases--also seen in biosolids/soil mixtures--suggests regrowth. Differences in numbers between digested and dewatered samples could not be duplicated with high shear processing in lab-scale devices, with nitrogen purging to remove volatile or gaseous constituents, or with redilution using centrate. They could not be attributed to enumeration methods, to interference of Bacillus spp. on apparent coliform counts, or to temperature changes. The increases have practical implications in the use of fecal coliform or alternative criteria to define pathogen content in biosolids.     

Microbiological effects of wastewater effluent discharge into coastal waters of Puerto Rico

Microbiological effects of non-disinfected, combined chemical and domestic wastewater effluent discharge into coastal waters north of Barceloneta. Puerto Rico were investigated by membrane filter enumeration of fecal indicator bacteria and by enrichment isolation of specific pathogens. A wastewater plume was detected and delineated around the sewage outfall, located 800 m offshore and 30 m below the ocean surface. Fecal coliform and fecal streptococcus counts within the plume were significantly (F test) larger than comparable counts outside the plume. The shape and location of the plume was compatible with current patterns and prevailing winds and extended as far as 3.7 km west of the outfall, at which point the fecal coliform count was 146 100 ml⁻¹. The average non-plume fecal coliform concentration was 6.7 100 ml⁻¹. Specific pathogens isolated included Salmonella and Vibrio spp, including V. cholerae non-01 serovar isolated from a nearby river also impacted by the sewage plant.     

Effect of total solids on fecal coliform regrowth in anaerobically digested biosolids

Fecal coliform (FC) concentrations in anaerobically digested biosolids can increase during centrifugal dewatering and afterwards in storage of dewatered cake. The immediate increase after centrifugation (reactivation) has been demonstrated to be the revitalization of fecal coliforms that had become non-culturable. The increase during storage (regrowth) has been regarded as a subsequence of reactivated bacteria growing in a favorable environment. In this paper, however, regrowth is demonstrated without preceding reactivation, using intensive laboratory centrifugation to duplicate the levels of regrowth seen in full-scale centrifugation. Higher total solids (TS) levels of the dewatered biosolids lead to greater magnitudes of FC increase. The final TS level appears much more important than the level of shear imposed during centrifugation, based on comparison of different centrifugation/dilution procedures used to obtain similar TS levels. The greater TS levels also reduce methane production, suggesting that methanogens compete with, or inhibit, the fecal coliforms. The addition of bromoethanesulfonate as a methanogen-specific inhibitor decreased the production of methane gas, and also increased the number of fecal coliforms.     

Bacterial population changes in hospital effluent treatment plant in central India

Hospital effluent with its high content of multidrug resistant (MDR) enterobacteria and the presence of enteric pathogens could pose a grave problem for the community. It was planned at our tertiary care hospital in central India to study the population changes at various steps of effluent treatment plant (ETP) like collection, aeration, clarification, liquid sludge, dried sludge, high-pressure filter and treated wastewater. The study included viable bacterial counts, coliform counts, staphylococcal, enterococcal, Pseudomonas and multiple drug resistant (MDR) gram negative bacterial counts in the different stages of ETP. In order to study the distribution of bacteria as free floating in liquid and adherent to suspended particles, enumeration of the bacteria in the filterate and the sediment was also carried out. The effluent input showed 55% of the 8.6 x 10(6)/ml bacteria as coliforms and E. coli which was a typical of fecal flora. The prevalence of MDR coliforms was 0.26%. The substantial reduction (> 3log) was seen for the effluent coming from the clarifier. The bulk of the bacteria in the hospital effluent remains firmly adhered to solid particles; aeration and clarification removes bulk of the bacteria by physical processes like flocculation. The treated liquid effluent still contains sizeable loads of MDR bacteria and inactivation by procedure such as chlorination is required. The bacteria get concentrated in sludge and a greater concentration of chlorine is required for decontamination.     

Occurrence and densities of fungi from northern Greek coastal bathing waters and their relation with faecal pollution indicators

The objective of this study was to determine the frequency and densities of yeasts and filamentous fungi in coastal water samples as well as their correlation with the indicator bacteria of faecal pollution. The prevalence of fungi was investigated in parallel with the standard pollution indicator microorganisms in 197 marine water samples from six northern Greek prefectures during the bathing season May–October 1999. Filamentous fungi were isolated from all the examined samples and yeasts from 29 (14.7%) of them; among the positive samples, their mean counts were 90.9 and 38.4 cfu/100 ml, respectively. A total of 23 genera of filamentous fungi and four genera of yeasts were identified. Prevailing genera of filamentous fungi were Penicillium, Aspergillus and Alternaria spp., whereas Candida spp. was the most frequently isolated yeast. Counts of yeasts were significantly (p<0.01) correlated with those of total and faecal coliforms, whereas no correlation was found between filamentous fungi and the indicator bacteria of faecal pollution. Significantly higher counts of total and faecal coliforms (p<0.05), and enterococci (p<0.001), were found during the months with the higher water temperatures and bather numbers. In the six prefectures, significant differences were observed in the counts of filamentous fungi and yeasts as well as in the counts of all the faecal pollution indicators. The results of this study indicate that coastal water can be a path for contamination of swimmers with yeasts and filamentous fungi and that the pollution indicator microorganisms cannot always predict their presence.     

Evaluation of a quantitative H2S MPN test for fecal microbes analysis of water using biochemical and molecular identification

The sensitivity and specificity of the H₂S test to detect fecal bacteria in water has been variable and uncertain in previous studies, partly due to its presence-absence results. Furthermore, in groundwater samples false-positive results have been reported, with H₂S-positive samples containing no fecal coliforms or Escherichia coli. False-negative results also have been reported in other studies, with H₂S-negative samples found to contain E. coli. Using biochemical and molecular methods and a novel quantitative test format, this research identified the types and numbers of microbial community members present in natural water samples, including fecal indicators and pathogens as well as other bacteria. Representative water sources tested in this study included cistern rainwater, a protected lake, and wells in agricultural and forest settings. Samples from quantitative H₂S tests of water were further cultured for fecal bacteria by spread plating onto the selective media for detection and isolation of Aeromonas spp., E. coli, Clostridium spp., H₂S-producers, and species of Salmonella and Shigella. Isolates were then tested for H₂S production, and identified to the genus and species level using biochemical methods. Terminal Restriction Fragment Length Polymorphisms (TRFLP) was the molecular method employed to quantitatively characterize microbial community diversity. Overall, it was shown that water samples testing positive for H₂S bacteria also had bacteria of likely fecal origin and waters containing fecal pathogens also were positive for H₂S bacteria. Of the microorganisms isolated from natural water, greater than 70 percent were identified using TRFLP analysis to reveal a relatively stable group of organisms whose community composition differed with water source and over time. These results further document the validity of the H₂S test for detecting and quantifying fecal contamination of water.     

Diversity and antifungal susceptibility of yeasts from tropical freshwater environments in Southeastern Brazil

Yeast communities were isolated from water and sediment samples of two unpolluted natural lakes, located inside Rio Doce State Park, and two rivers located outside of this Park in Southeastern Brazil. A total of 134 yeast isolates were obtained and identified as belonging to 36 species. The numbers of fecal coliforms and yeast species were higher in rivers than in lakes. The genus Candida had the highest number of species with the presence of opportunistic pathogens such as Candida krusei, C. tropicalis, C. guilliermondii and C. parapsilosis. Yeasts able to grow at 37 degrees C were tested in relation to their susceptibility to common used antifungal drugs. Yeast isolates (13%) were susceptible to ketoconazole, 79% to fluconazole, 31% to terbinafine and 78% of the strains were susceptible to amphotericin B. Seven isolates from different Candida species were resistant to all antifungals tested. The high number of fecal coliforms found in these aquatic environments and the presence of resistant yeast strains to common used antifungal drugs suggest that these environments can pose potential health risks for people utilizing the contaminated waters.