Survival in maturation ponds of coliform bacteria with transferable drug resistance

The behaviour of drug-resistant coliform bacteriasin a series of sewage maturation ponds was investigated. The average reduction through the ponds of coliforms resistant to ampicillin, chloramphenicol, kanamycin, streptomycin or tetracycline was 6.83 per cent lower than that of drug-sensitive bacteria. This difference was mainly due to coliforms with transferable resistance (R⁺ coliforms) which increased from 0.86 per cent to 2.45 per cent through the ponds. The spectrum of transferable resistance of coliforms in the effluent did not differ notably from that of coliforms in the influent. The average incidence of Escherichia coli I among R⁺ coliforms decreased from 66.08 per cent to 62.09 per cent. This indicates that low-level transfer of R factors may occur in ponds. Possible mechanisms and the epidemiological significance of the increased survival of R⁺ coliforms in maturation ponds are discussed.     

Behaviour in conventional sewage purification processes of coliform bacteria with transferable or non-transferable drug-resistance

Transferable (R⁺) and non-transferable (R⁻) resistance of coliform bacteria in settled sewage and after biofiltration, secondary sedimentation, chlorination and sand filtration was investigated. The percentage of R⁻ coliforms resistant to ampicillin (A), chloramphenicol (C) or streptomycin (S), but not kanamycin (K) or tetracycline (T), was slightly reduced through the purification system. On the average the percentage of R⁺ coliforms resistant to one or more of the same drugs was reduced by about 50%. This reduction was mainly accomplished by biofiltration and sand filtration. The ratio of transferable to non-transferable resistance for A, C, K or T increased during secondary sedimentation and chlorination. R factor transfer in water may explain these findings. Rapid passage over stony surfaces in biological and sand filters is unfavourable for conjugation and may damage sex pilli while R factor transfer can be expected under the relatively stagnant conditions in sedimentation and chlorination tanks. The incidence of R factors conferring resistance to all five drugs simultaneously increased through the purification system. These R factors are probably large and may carry markers which determine resistance to sewage purification processes. Between 30 and 40% of all R factors mediated resistance to at least four of the five drugs studied. The incidence of Escherichia coli I among R⁺ coliforms varied but did not exceed 50%. The finding that conventional sewage purification has a limited effect on the incidence of drug resistance in bacteria supports the view that sewage should be treated by more advanced methods prior to discharge into the environment.     

Transferable drug resistance in bacteria of coastal canal water and sediment

This study was undertaken to determine the comparative incidence of drug-resistant coliforms and salmonellae in the water and bottom sediments of six coastal canal communities which are used heavily for recreational activities. Of a total of 423 coliforms, 300 fecal coliforms and 100 salmonella isolates examined, 74.9% coliforms, 61.3% fecal coliforms and 71.0% salmonellae were resistant to one or more of the 12 antibiotics tested. Multiple resistance occurred in 48.9% coliforms, 32.7% fecal coliforms and 68.0% salmonella strains. A total of 178 coliforms, 137 fecal coliforms and 51 salmonella isolates were tested for the presence of resistance transfer factors, and 53.9%, 53.3% and 56.9% isolates, respectively, were capable of transferring their resistance patterns to Escherichia coli and/or Salmonella choleraesuis recipient strains. A significantly higher number of drug-resistant bacteria carrying R-factors (R⁺) was found to occur in sediment than in surface water.     

Drug resistant coliforms call for review of water quality standards

The therapeutic value of antimicrobial drugs is diminishing due to the rapid increase of resistant bacteria. A current prominent type of resistance is mediated by R factors (extrachromosomal nucleic acid elements) which may cause high level resistance to many drugs. These factors may also provide resistance to other antibacterial agents such as u.v. light, heavy metals, bacteriocins and phages, and may enhance the virulence and infectivity of pathogens. Intestinal Gram-negative bacteria like coliforms may act as reservoirs of R factors and transfer them to pathogens. Evidence is presented that sewage polluted water may play an important role in the spread of coliform and other bacteria carrying R factors. Since coliforms have joined forces with bacteria increasingly involved in disease, they can no longer be regarded as harmless indicators of faecal pollution. This calls for a re-evaluation of water quality standards and for more advanced purification of sewage prior to discharge into the environment.     

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.     

Vertical variation in magnitude and partitioning of oxygen demand in a contaminated sediment

Antimicrobial resistance is a phenomenon of increasing importance. Sewage treatment processes are a vehicle for dissemination of resistant bacteria in both aquatic and terrestrial environments. To assess the number of antimicrobial resistant E. coli present in the wastewater inflow, effluent and sludge from urban sewage treatment plants in Portugal, 42 samples of crude inflow, treated effluent and sludge were collected in 14 municipal sewage treatment plants in Portugal. A total of 940 E. coli strains were recovered and tested, using the diffusion agar method, regarding their sensitivity to 12 different antimicrobial drugs. Resistance to tetracycline, ampicillin, trimethoprime/sulfamethoxazole, streptomycin and ciprofloxacin was found in 42.2, 32.6, 23.3, 26.4 and 11.9% of the isolates, respectively. No correlation was found with regards to medical antimicrobial use in Portugal. The highest resistance rates were found in E. coli strains isolated from the district capitals sampled. Wastewater treatment resulted in E. coli decrease between less than one log to four logs; nevertheless, an average of 1.17 × 10⁶ CFU/100 ml were present in the outflow of the plants. This investigation highlights the necessity for constant monitoring of the final effluent and sludge from these treatment facilities.     

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.     

Effect of phosphorus stripping on water chemistry and diatom ecology in an eastern lowland river

The effect of nutrient removal at Ashford sewage treatment works on the benthic diatom ecology of the River Stour was studied. This paper describes assemblages above and below the works both before and after phosphorus stripping was installed. Taxa typical of eutrophic conditions dominated all samples, including those upstream of the works, suggesting that the river was already eutrophic before receiving the sewage effluent. Once nutrient removal was installed, phosphorus concentrations measured downstream of the works were similar to those measured upstream; however, there was little change in the diatom flora. Analysis of chemical data collected from the site suggest that the river may be nitrogen-limited for part of the year, and that, even with phosphorus stripping installed, the river still exceeds the Environment Agency's proposed interim targets for phosphorus concentrations in a eutrophic river.     

Recovery of viruses from waste-water and effluent by the direct inoculation method

Chloroform or antibiotic treated sewage and effluent samples were inoculated directly onto MK₂ or Vero cell monolayers for enterovirus recovery. The results obtained showed a high enterovirus recovery. The average PFU (100 ml)⁻¹ from sewage samples was 4496 and from effluent samples was 5684. The conventional ratio of enteroviruses to coliforms in sewage is considered to be 1:65,000. By the direct inoculation method the ratio obtained was 1: 20,000 for sewage and 1: 2000 for effluent. It is believed the number of enteroviruses in sewage to be even higher than the ones obtained.     

The physicochemical speciation of Cd, Pb, Cu, Fe and Mn in the final effluent of a sewage treatment works and its impact on speciation in the receiving river

A scheme for the speciation of metals in freshwaters has been applied to the analysis of the final effluent from a sewage treatment plant and to the receiving river upstream and downstream of the effluent outfall. The treatment plant was selected because of the high influent and effluent concentrations of Cd. The metal speciation patterns in the effluent are interpreted primarily in terms of organic interactions, which appear to be exerting a solubilizing effect on Cd and Cu, but not on the Pb and Fe which are principally associated with the particulate size fraction (> 12 μm). The influx of metals with the sewage effluent alters the speciation pattern in the river. A large part of the Cd is added to the smallest size fraction (< 0.015 μm). However, the major part of each metal, with the exception of Mn, is associated with the colloidal and particulate size fractions, thus minimising the immediate toxic significance to aquatic life.     

The growth of cladophora glomerata in a river receiving sewage effluent

A considerable improvement in the natural flora of the River Great Stour has occurred since the mid-1960s when dense growths of Cladophora glomerata dominated sections of the river with the disappearance of some rooted macrophytes. Cladophora growths in the river in response to changing river conditions and sewage treatment operations during a thirty year period are reviewed. A vegetation mapping programme undertaken from 1978 to 1982 to assess the influence of a sewage treatment works discharge on the growth of the filamentous alga has shown that previous recommendations to reduce dissolved phosphorus concentrations in the final effluent need not be implemented.     

Environmentally Persistent Oestrogen-like Substances in UK River Systems

The paper describes (a) the research into substances with oestrogenic properties in river systems, (b) the background on why and how these substances are present in river systems, (c) the potential harm which they have through endocrine disruption, and (d) the different techniques for their detection. Results are presented form a preliminary study on the overall oestrogenic potential of sewage effluent being discharged into a river. These results show high levels of oestrogenic potential in the effluent and also in stream water upstream from any sewage-treatment works. The implications for the management of rivers which contain recycled water and possible future research directions in this field are discussed.     

Multiresistant Enterobacteriaceae with class 1 and class 2 integrons in a municipal wastewater treatment plant

In this study, 1832 strains of the family Enterobacteriaceae were isolated from different stages of a municipal wastewater treatment plant, of which 221 (12.1%) were intI-positive. Among them 61.5% originated from raw sewage, 12.7% from aeration tank and 25.8% from the final effluent. All of the intI-positive strains were multiresistant, i.e. resistant to at least three unrelated antimicrobials. Although there were no significant differences in resistance range, defined as the number of antimicrobial classes to which an isolate was resistant, between strains isolated from different stages of wastewater treatment, for five β-lactams the percentage of resistant isolates was the highest in final effluent, which may reflect a selective pressure the bacteria are exposed to, and the possible route of dissemination of β-lactam resistant strains to the corresponding river. The sizes of the variable part of integrons ranged from 0.18 to 3.0 kbp and contained up to four incorporated gene cassettes. Sequence analysis identified over 30 different gene cassettes, including 24 conferring resistance to antibiotics. The highest number of different gene cassettes was found in bacteria isolated from the final effluent. The gene cassettes were arranged in 26 different resistance cassette arrays; the most often were dfrA1-aadA1, aadA1, dfrA17-aadA5 and dfrA12-orfF-aadA2. Regarding the diversity of resistance genes and the number of multiresistant bacteria in the final effluent, we concluded that municipal sewage may serve as a reservoir of integron-embedded antibiotic resistance genes.     

Qualitative and quantitative studies on the in situ adsorption, degradation and toxicity of acrylamide by the spiking of the waters of two sewage works and a river

Novel experiments which involved the dosing of acrylamide into an activated sludge sewage works, a biological filter sewage works, and a river which discharged into an estuary were performed during the period from October 1979 to July 1980. In order to assess the ability of a sewage works to degrade acrylamide, an activated sludge and a percolating biological filter plant were exposed to constant dosings of acrylamide. Little loss of acrylamide occurred in either the primary or the final settlement tanks. Approximately 50% of dosed acrylamide was lost from both the activated sludge tanks and the biological filter beds. Laboratory experiments were used to help explain the in situ results. In another study acrylamide was dosed into a stream continuously for 74 days in order to assess the initial in situ adsorption and degradation, and the possibility of initiating further in situ degradation owing to acclimatisation of microheterotrophs. Dilution effects owing to ground water infiltration and additional tributaries were allowed for by monitoring both acrylamide concentration and river flow at various stations downstream of the source of acrylamide addition. The resulting quantitative mass flow study showed that no in situ loss of acrylamide occurred for a stream retention of 4–5 h. Incubations in the laboratory of collected water samples suggested some possible increase in degradative ability of the samples which had been exposed to acrylamide. A qualitative assessment of the insect fauna before, during and after exposure to acrylamide showed a reduced species diversity to occur within 5 h when exposed to 50 μg 1⁻¹ acrylamide. Within 21 days only Hydropsyche instabilis was observed in the river. Some recolonization was noted following cessation of acrylamide dosing.     

An in situ method to compare the potential for periphyton productivity of lotic habitats

A simple field method for measuring relative net productivity in flowing waters is described. To assess its sensitivity and practicality, the eutrophication potential of a tertiary sewage treatment plant effluent was assayed on the Fort River of Amherst, Massachusetts. Two sets of three 10 m lexan tubes (1.25 cm i.d.) were anchored to the river bed 100 m upstream and downstream from the sewage plant outfall. Data collection began following a 2 week colonization period for the periphyton community. Substrate primary productivity (mg O₂ min⁻¹) was determined by subtracting inflow dissolved oxygen concentrations from these levels at the discharge ends of each tube and multiplying the difference by the flow rate. The downstream tubes demonstrated an enhanced response at a 0.01 level of significance to an increment of 0.005 and 0.1 ppm of phosphorous and nitrogen respectively. Data analysis revealed a highly significant regression of the net productivity on stream flow which was adjusted for by an analysis of covariance. The productivity of the downstream station was distinctly higher (0.4522 mg O₂ min⁻¹) then the value of the upstream station (0.2584 mg O₂ min⁻¹). The protocol thus affords a biological summation of the sewage effluent through comparison with the control station replicates.     

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.     

Mutagenicity in Salmonella of fluorescent dye tablets used in water tracing

Fluorescent red dye tablets containing Rhodamine B induced dose-related increases of His⁺ revertant colonies in Salmonella typhimurium strains TA1538 and TA98 in the presence of S9. Fluorescent yellow/green tablets containing Uranine (Fluorescein Sodium) were not mutagenic. These fluorescent dyes are sold for public, commercial, and field use, in liquid and tablet forms, for sewage, plumbing, and water pollution tracing.     

A method for monitoring mineralization of C-labeled compounds in aqueous samples

Previously reported laboratory methods for measuring the rate of ¹⁴CO₂ release from an aqueous sample require a series of replicate growth flasks. At chosen times, some of these are sacrificed in order to quantitate the ¹⁴CO₂ released. The method presented here requires only one culture flask containing 200 ml of sample with labeled substrate, and one control-replacement flask containing 200 ml of sterile sample with labeled substrate. At each sampling time, of the liquid and headspace gas is removed from the modified 500 ml Erlenmeyer culture flask for ¹⁴CO₂ recovery. This method was used to study the mineralization of labeled amino acids, starch, n-hexadecane and anthracene by microorganisms in river water, and of n-hexadecane and n-hexadecanoic acid by microorganisms in sewage effluent.     

Sorption of polychlorinated biphenyls (PCB) to clay particulates and effects of desorption on phytoplankton

The sorption of polychlorinated biphenyls (PCB) on illite and chlorite, two minerals of defined composition, was investigated. Illite particles adsorbed higher [¹⁴C]PCB (Aroclor 1254) concentrations than did chlorite particles, with equilibrium concentration factors between particles and water being 1.4 × 10⁴ for illite and 1.0 × 10⁴ for chlorite. Desorbed PCB inhibited photosynthesis and reduced chlorophyll-a content of natural phytoplankton assemblages within dialysis membrane bags suspended in the water of a tidal marsh. There was no significant difference in effects on the phytoplankton of PCB desorbed from the two minerals. The rate of [¹⁴C]PCB desorption was dependent on the initial concentration on the clay particles. The rate of [¹⁴C]PCB loss from bags with particles was slower than that from bags without particles.     

Mutagenic activity of fish and sediments in the Sheep River, Alberta

Using the Ames Salmonella/mammalian microsome assay the mutagenic activity of fish and benthic sediments from a section of the Sheep River. Alberta receiving a chlorinated sewage effluent were investigated. The benthic sediments gave no indication of increased mutagenic activity, however fish collected from the chlorinated sewage plume showed a significant increase in mutagenic activity. The results are discussed with respect to the effects of a chlorinated sewage effluent on the lotic ecosystem in this section of the river.