Influence of sewage treatment and urbanization on selection of multiple resistance in fecal coliform populations.

The fecal coliform populations found in the raw sewages and final sewage effluents of mechanical treatment plants, a long-term retention lagoon, shorter-term retention lagoons, a remote northern Canada river, and a heavily urbanized prairie river were examined for antibiotic resistance and the possession of R factors. It was determined that there was a decrease in the percentage of multiresistant fecal coliform populations in the mechanical sewage treatment plants and shorter-term retention lagoons; however, there was an increase in populations from the long-term retention lagoon. The percentage of the populations possessing transmissible R factors was constant in the mechanical treatment and shorter-term retention facilities; however, the ability to transmit was lost in 50% of the infective population of the long-term retention facility. A striking contrast was found between the populations of the remote northern Slave River and those of the urbanized Red River. Of the fecal coliforms in the Slave River, 7.1% were multiresistant, and only 0.79% possessed transmissible R factors. The Red River fecal coliform populations were 52.9% multiresistant, and 18.77% of the total population possessed transmissible R factors. The influence of urbanization and the type of sewage treatment have been shown to affect the selection and survival of multiresistant fecal coliforms and R+ fecal coliforms. Determination of other factors influencing the development and the survival of these populations is needed for rational wastewater management and water quality consideration.     

Incidence of R factors in coliform, fecal coliform, and Salmonella populations of the Red River in Canada.

Coliforms, fecal coliforms, and Salmonella were isolated from the Red River, Manitoba, Canada, and identified. These organisms were then examined for resistance to 12 antibiotics. Some fecal coliforms were resistant to all 12 antibiotics, and 18% of the Salmonella isolates were resistant to one or more antibiotics. A total of 52.9% of the fecal coliforms resistant to three or more antibiotics were able to transfer single or multiple resistance (R) determinants to the Salmonella recipient, and 40.7% could transfer R determinants to the Escherichia coli recipient. Of the resistant Salmonella, 57% transferred one or two determinants to the Salmonella recipient, and 39% transferred one or two determinants to the E. coli recipient. It was calculated that populations of fecal coliforms containing R factors were as high as 1,400 per 100 ml and that an accidental intake of a few milliliters of water could lead to transient or permanent colonization of the digestive tract. Consideration of data on bacteria with R factors should be made in future water quality deliberations and in discharge regulations.     

Incidence of Infectious Drug Resistance Among Fecal Coliforms Isolated from Raw Sewage

Raw sewage was examined for the incidence of antibiotic-resistant coliforms present among both total and fecal coliforms. In both groups, it was found that approximately 3% of the coliform bacteria were resistant to two or more antibiotics. Of these organisms, 48% were capable of transferring all or part of their antibiotic resistance to an antibiotic-sensitive, F⁻, derivative of Escherichia coli K-12. Among the R factors identified, those conferring resistance to streptomycin-tetracycline, ampicillin-streptomycin-tetracycline, and ampicillin or ampicillin-streptomycin accounted for 23, 20, and 15%, respectively, of the total R factors detected. The data indicate a significant level of infectious drug resistance among the fecal coliforms of the urban population. The data indicate further that because of the high incidence of coliform bacteria found to be doubly resistant to streptomycin and tetracyline, the inclusion of these antibiotics in selective media used for routine total or fecal coliform counts may serve to identify domestic sources of pollution.     

Rapid, single-step most-probable-number method for enumerating fecal coliforms in effluents from sewage treatment plants.

A single-step most-probable-number method for enumerating fecal coliforms in sewage treatment plant effluents is described. The method requires the use of only one lactose-based medium and a single incubation temperature of 44.5 degrees C, and it can be completed in 18 h or less, as compared with up to 72 h for the standard most-probable-number method. The appearance of growth is the sole criterion used for designating positives, which can be determined either by increases in the electrical impedance ratio of inoculated medium, as compared to an uninoculated control using a Bactometer model 32, or by visual examination of inoculated medium for turbidity. In trials with 53 samples of unchlorinated sewage treatment plant effluent, fecal coliform counts by the single-step most-probable-number method, throughout a range of less than 10 to almost 10(7) fecal coliforms per 100 ml of effluent, were in excellent agreement with counts abtained by the standard most-probable-number procedure. Similar agreement was obtained in comparative trials with 31 chlorinated effluent samples from two sewage treatment plants. Overall, 87% of 452 positives were confirmed as containing fecal coliforms. The applicability of the single-step most-probable-number method to automated sewage treatment plant operations is discussed.     

Rapid, single-step most-probable-number method for enumerating fecal coliforms in effluents from sewage treatment plants.

A single-step most-probable-number method for enumerating fecal coliforms in sewage treatment plant effluents is described. The method requires the use of only one lactose-based medium and a single incubation temperature of 44.5 degrees C, and it can be completed in 18 h or less, as compared with up to 72 h for the standard most-probable-number method. The appearance of growth is the sole criterion used for designating positives, which can be determined either by increases in the electrical impedance ratio of inoculated medium, as compared to an uninoculated control using a Bactometer model 32, or by visual examination of inoculated medium for turbidity. In trials with 53 samples of unchlorinated sewage treatment plant effluent, fecal coliform counts by the single-step most-probable-number method, throughout a range of less than 10 to almost 10(7) fecal coliforms per 100 ml of effluent, were in excellent agreement with counts abtained by the standard most-probable-number procedure. Similar agreement was obtained in comparative trials with 31 chlorinated effluent samples from two sewage treatment plants. Overall, 87% of 452 positives were confirmed as containing fecal coliforms. The applicability of the single-step most-probable-number method to automated sewage treatment plant operations is discussed.     

Automated electrical impedance technique for rapid enumeration of fecal coliforms in effluents from sewage treatment plants.

Fecal coliforms growing in a selective lactose-based broth medium at 44.5 degrees C generate a change in the electrical impedance of the culture relative to a sterile control when populations reach 10(6) to 10(7) per ml. The ratio of these changes was measured automatically, and the data were processed by computer. A linear relation was found between the log10 of the number of fecal coliforms in an inoculum and the time required for an electrical impedance ratio signal to be detected. Pure culture inocula consisting of 100 fecal coliforms in log phase or stationary phase were detected in 6.5 and 7.7 h, respectively. Standard curves of log10 fecal coliforms in wastewater inocula versus detection time, based on samples collected at a sewage treatment plant over a 4-month period, were found to vary from one another with time. Nevertheless, detection times were rapid and ranged from 5.8 to 7.9 h for 200 fecal coliforms to 8.7 to 11.4 h for 1 fecal coliform. Variations in detection times for a given number of fecal coliforms were also found among sewage treatment plants. A strategy is proposed which takes these variations into account and allows for rapid, automated enumeration of fecal coliforms in wastewater by the electrical impedance ratio technique.     

Automated electrical impedance technique for rapid enumeration of fecal coliforms in effluents from sewage treatment plants.

Fecal coliforms growing in a selective lactose-based broth medium at 44.5 degrees C generate a change in the electrical impedance of the culture relative to a sterile control when populations reach 10(6) to 10(7) per ml. The ratio of these changes was measured automatically, and the data were processed by computer. A linear relation was found between the log10 of the number of fecal coliforms in an inoculum and the time required for an electrical impedance ratio signal to be detected. Pure culture inocula consisting of 100 fecal coliforms in log phase or stationary phase were detected in 6.5 and 7.7 h, respectively. Standard curves of log10 fecal coliforms in wastewater inocula versus detection time, based on samples collected at a sewage treatment plant over a 4-month period, were found to vary from one another with time. Nevertheless, detection times were rapid and ranged from 5.8 to 7.9 h for 200 fecal coliforms to 8.7 to 11.4 h for 1 fecal coliform. Variations in detection times for a given number of fecal coliforms were also found among sewage treatment plants. A strategy is proposed which takes these variations into account and allows for rapid, automated enumeration of fecal coliforms in wastewater by the electrical impedance ratio technique.     

Survival of coliform bacteria in sewage sludge applied to a forest clearcut and potential movement into groundwater.

Anaerobically digested dewatered sludge (10 to 15 cm thick) was applied to a forest clearcut as a fertilizer source in northwest Washington on gravelly glacial outwash soil. This sludge is not microbiologically sterile and may contain pathogenic organisms. Fecal coliform bacterial counts in sludge applied in summer (July) fell from 1.08 X 10(5) to 358/g in 204 days and to 0/g in 267 days. Dieoff appeared more rapid in winter (January)-applied sludge, when colnts fell from 1.2 X 10(5) to 20/g in 162 days. Initial death rates were related to sludge temperature, moisture, pH, physical composition, and microbial competition. Aftergrowth of fecal coliforms occurred in warm summer and fall months, but counts were of similar magnitude to background levels in forest soils, where a maximum count of 54/g was recorded. Total coliform counts in fresh sludge ranged from 1.4 X 10(4) to 1.9 X 10(6)/g. Numbers stabilized at 10(3) to 10(4)/g in spring, fall, and summer, with lower numbers in winter. Both total and fecal bacteria moved from the sludge to the soil beneath, but few penetrated past the first 5 cm. The soil acts as an effective biological filter. Few fecal coliform bacteria were recorded in the groundwater, generally being less than 5/100 ml and mostly 0/100 ml. A maximum count of 52/100 ml was recorded. Groundwater contamination from vertical movement of potential pathogens appears unlikely, but hazards from surface runoff and direct handling in the first year may arise.     

Survival of coliform bacteria in sewage sludge applied to a forest clearcut and potential movement into groundwater.

Anaerobically digested dewatered sludge (10 to 15 cm thick) was applied to a forest clearcut as a fertilizer source in northwest Washington on gravelly glacial outwash soil. This sludge is not microbiologically sterile and may contain pathogenic organisms. Fecal coliform bacterial counts in sludge applied in summer (July) fell from 1.08 X 10(5) to 358/g in 204 days and to 0/g in 267 days. Dieoff appeared more rapid in winter (January)-applied sludge, when colnts fell from 1.2 X 10(5) to 20/g in 162 days. Initial death rates were related to sludge temperature, moisture, pH, physical composition, and microbial competition. Aftergrowth of fecal coliforms occurred in warm summer and fall months, but counts were of similar magnitude to background levels in forest soils, where a maximum count of 54/g was recorded. Total coliform counts in fresh sludge ranged from 1.4 X 10(4) to 1.9 X 10(6)/g. Numbers stabilized at 10(3) to 10(4)/g in spring, fall, and summer, with lower numbers in winter. Both total and fecal bacteria moved from the sludge to the soil beneath, but few penetrated past the first 5 cm. The soil acts as an effective biological filter. Few fecal coliform bacteria were recorded in the groundwater, generally being less than 5/100 ml and mostly 0/100 ml. A maximum count of 52/100 ml was recorded. Groundwater contamination from vertical movement of potential pathogens appears unlikely, but hazards from surface runoff and direct handling in the first year may arise.     

Influence of seasonal environmental variables on the distribution of presumptive fecal Coliforms around an Antarctic research station

Factors affecting fecal microorganism survival and distribution in the Antarctic marine environment include solar radiation, water salinity, temperature, sea ice conditions, and fecal input by humans and local wildlife populations. This study assessed the influence of these factors on the distribution of presumptive fecal coliforms around Rothera Point, Adelaide Island, Antarctic Peninsula during the austral summer and winter of February 1999 to September 1999. Each factor had a different degree of influence depending on the time of year. In summer (February), although the station population was high, presumptive fecal coliform concentrations were low, probably due to the biologically damaging effects of solar radiation. However, summer algal blooms reduced penetration of solar radiation into the water column. By early winter (April), fecal coliform concentrations were high, due to increased fecal input by migrant wildlife, while solar radiation doses were low. By late winter (September), fecal coliform concentrations were high near the station sewage outfall, as sea ice formation limited solar radiation penetration into the sea and prevented wind-driven water circulation near the outfall. During this study, environmental factors masked the effect of station population numbers on sewage plume size. If sewage production increases throughout the Antarctic, environmental factors may become less significant and effective sewage waste management will become increasingly important. These findings highlight the need for year-round monitoring of fecal coliform distribution in Antarctic waters near research stations to produce realistic evaluations of sewage pollution persistence and dispersal.     

Influence of Seasonal Environmental Variables on the Distribution of Presumptive Fecal Coliforms around an Antarctic Research Station

Factors affecting fecal microorganism survival and distribution in the Antarctic marine environment include solar radiation, water salinity, temperature, sea ice conditions, and fecal input by humans and local wildlife populations. This study assessed the influence of these factors on the distribution of presumptive fecal coliforms around Rothera Point, Adelaide Island, Antarctic Peninsula during the austral summer and winter of February 1999 to September 1999. Each factor had a different degree of influence depending on the time of year. In summer (February), although the station population was high, presumptive fecal coliform concentrations were low, probably due to the biologically damaging effects of solar radiation. However, summer algal blooms reduced penetration of solar radiation into the water column. By early winter (April), fecal coliform concentrations were high, due to increased fecal input by migrant wildlife, while solar radiation doses were low. By late winter (September), fecal coliform concentrations were high near the station sewage outfall, as sea ice formation limited solar radiation penetration into the sea and prevented wind-driven water circulation near the outfall. During this study, environmental factors masked the effect of station population numbers on sewage plume size. If sewage production increases throughout the Antarctic, environmental factors may become less significant and effective sewage waste management will become increasingly important. These findings highlight the need for year-round monitoring of fecal coliform distribution in Antarctic waters near research stations to produce realistic evaluations of sewage pollution persistence and dispersal.     

Evaluation of m-T7 agar as a fecal coliform medium

m-T7 agar, designed to improve recoveries of injured total coliforms, was evaluated for its effectiveness as a fecal coliform medium. The time and temperature of preincubation were found to be crucial to the optimal recovery of fetal coliforms. Isolation rates for fecal coliforms on m-T7 agar from sewage effluents were the highest when plates were preincubated at 37 degrees C for 8 h before transfer to 44.5 degrees C for 12 h. The medium was found to produce consistently higher fecal coliform counts than all the other methods tested. Recoveries were 3.1 times greater than the standard m-FC method and 1.7 times greater than the two-layer enrichment, temperature acclimation procedure. Verification rates for fecal coliforms isolated on m-T7 agar averaged 89.0%, whereas verification rates for m-FC agar averaged only 82.8%. Both media isolated similar fecal coliform populations. The advantages of a single medium, highly effective for the isolation of both total and fecal coliforms, are discussed.     

Evaluation of m-T7 agar as a fecal coliform medium.

m-T7 agar, designed to improve recoveries of injured total coliforms, was evaluated for its effectiveness as a fecal coliform medium. The time and temperature of preincubation were found to be crucial to the optimal recovery of fetal coliforms. Isolation rates for fecal coliforms on m-T7 agar from sewage effluents were the highest when plates were preincubated at 37 degrees C for 8 h before transfer to 44.5 degrees C for 12 h. The medium was found to produce consistently higher fecal coliform counts than all the other methods tested. Recoveries were 3.1 times greater than the standard m-FC method and 1.7 times greater than the two-layer enrichment, temperature acclimation procedure. Verification rates for fecal coliforms isolated on m-T7 agar averaged 89.0%, whereas verification rates for m-FC agar averaged only 82.8%. Both media isolated similar fecal coliform populations. The advantages of a single medium, highly effective for the isolation of both total and fecal coliforms, are discussed.     

Coliforms in aerosols generated by a municipal solid waste recovery system.

Airborne total and fecal coliform concentrations averaged 2.1 X 10(3) and 9.9 X 10(2)/m3, respectively, inside an operating solid waste recovery system. Installation of dust control equipment reduced these levels by 50%. Frequency of recovery of coliforms also dropped by 15%.     

Monitoring of fecal pollution in coastal waters by use of rapid enzymatic techniques.

Enzyme assays for 4-methylumbelliferyl-beta-D-galactopyranosidase and 4-methylumbelliferyl-beta-D-glucuronidase activities were used for rapid detection (25 min) of fecal water pollution and to determine the impact of sewage discharge in coastal waters. Two coastal areas were investigated: (i) an estuary characterized by a high degree of contamination downstream of a discharge from a sewage treatment plant and a low degree of water renewal and (ii) a fjord with a low degree of pollution and a high degree of water renewal. Statistical analysis showed that a global correlation curve could be used to estimate concentrations of culturable fecal coliform bacteria in the two coastal areas, although environmental factors important for cell physiology (e.g., salinity) varied at different sampling locations. The sensitivity limit for detection of 4-methylumbelliferyl-beta-D-glucuronidase activity corresponded to bacterial concentrations on the order of 10 to 100 CFU/100 ml. The 4-methylumbelliferyl-beta-D-galactopyranosidase assay was less sensitive because of a higher rate of substrate autohydrolysis. The detection limit corresponded to bacterial concentrations on the order of 100 to 1,000 fecal coliforms per 100 ml.     

Coliforms as a measure of sewage contamination of the River Zambezi

The effect of releasing untreated sewage from Victoria Falls Town into the Zambezi river was determined by bacteriological examination of water samples collected upstream of Victoria Falls and for 22 km downstream. Most probable numbers of faecal coliforms and Escherichia coli were estimated. Water upstream of the falls, on the Zimbabwe side of the river, contained between seven and 130 E. coli per 100 ml. This section of the river was free from major sources of faecal pollution. Below the falls, but before the Victoria Falls Town sewage outfall, numbers of E. coli were between 1.8 X 10(2) and 1.4 X 10(4)/100 ml, indicating the existence of a sewage discharge other than that from Victoria Falls Town. The river was also highly polluted from the Victoria Falls Town sewage outfall to a point 18.6 km downstream. The highest E. coli count was 3.3 X 10(4)/100 ml and declined slowly to 1.4 X 10(3)/100 ml 18.6 km downstream of the outfall.     

Coliforms removal in two UASB + ASP based systems

Two full scale up-flow anaerobic sludge blanket (UASB) reactor with activated sludge process based STPs (43 Ml d⁻¹ and 100 Ml d⁻¹ capacities) purely for municipal wastewater having two different aeration systems (surface and diffused) were continuously evaluated for one year. Total coliforms (TC) and fecal coliforms (FC) were measured along with other performance monitoring parameters in raw sewage, UASB effluents, and in final effluents. The concentration of total and fecal coliforms ranged from 9.3 × 10⁶ to 1.2 × 10¹⁴ MPN/100 ml and 9.3 × 10⁶ to 2.4 × 10¹³ MPN/100 ml respectively in raw sewage. At both the plants, it was observed that finally treated effluent still contained significant number of total coliforms (5.71 × 10⁵ and 6.7 × 10⁵) and fecal coliforms (3.67 × 10⁵ and 2.2 × 10⁵) after overall removal of 99.9%, which is still greater than the permissible limit of 1000 MPN/100 ml and needs further treatment. Seasonal variation indicated the effective removal of coliforms in raw sewage was in summer. Fecal coliforms are highly correlated with phosphorous and total coliform concentrations.     

Isolation of Klebsielleae from within living wood.

Previous studies from this laboratory have documented the presence of coliform bacteria emanating from wooden reservoirs containing finished drinking water. Coliforms were identified as Klebsiella pneumoniae and Enterobacter spp. In the present report, evidence is presented which suggests that the origin of these coliforms is from the wood used to construct the reservoirs. In liquid expressed from freshly cut redwood, total bacterial counts in the range of 10(5) to 10(6)/ml were commonly observed. When present, coliform counts were over 10(3)/ml of expressed liquid. E. agglomerans was the most prevalent coliform present, but Klebsiella was isolated from freshly cut logs. Citrobacter freundii was also occasionally isolated. No fecal coliform-positive Klebsiella were obtained from any of the samples. Highest total bacteria and coliform counts were observed in sapwood specimens. Coliforms were present throughout sapwood as evidenced by contact plating serial sections of freshly cut wood. Scanning electron micrographs illustrate the presence of bacterial colonies within sapwood tracheids. Other wood species also contained coliform bacteria but in numbers lower than found in redwood.     

Isolation of Klebsielleae from within living wood.

Previous studies from this laboratory have documented the presence of coliform bacteria emanating from wooden reservoirs containing finished drinking water. Coliforms were identified as Klebsiella pneumoniae and Enterobacter spp. In the present report, evidence is presented which suggests that the origin of these coliforms is from the wood used to construct the reservoirs. In liquid expressed from freshly cut redwood, total bacterial counts in the range of 10(5) to 10(6)/ml were commonly observed. When present, coliform counts were over 10(3)/ml of expressed liquid. E. agglomerans was the most prevalent coliform present, but Klebsiella was isolated from freshly cut logs. Citrobacter freundii was also occasionally isolated. No fecal coliform-positive Klebsiella were obtained from any of the samples. Highest total bacteria and coliform counts were observed in sapwood specimens. Coliforms were present throughout sapwood as evidenced by contact plating serial sections of freshly cut wood. Scanning electron micrographs illustrate the presence of bacterial colonies within sapwood tracheids. Other wood species also contained coliform bacteria but in numbers lower than found in redwood.     

Dynamics of Aeromonas hydrophila, Aeromonas sobria, and Aeromonas caviae in a sewage treatment pond.

The spatiotemporal dynamics of Aeromonas spp. and fecal coliforms in the sewage treatment ponds of an urban wastewater center were studied after 20 months of sampling from five stations in these ponds. Isolation and identification of 247 Aeromonas strains were undertaken over four seasons at the inflow and outflow of this pond system. The hemolytic activity of these strains was determined. The Aeromonas spp. and the fecal coliform distributions showed seasonal cycles, the amplitude of which increased at distances further from the wastewater source, so that in the last pond there was an inversion of the Aeromonas spp. cycle in comparison with that of fecal coliforms. The main patterns in these cycles occurred simultaneously at all stations, indicating control of these bacterial populations by seasonal factors (temperature, solar radiation, phytoplankton), the effects of which were different on each bacterial group. The analysis of the Aeromonas spp. population structure showed that, regardless of the season, Aeromonas caviae was the dominant species at the pond system inflow. However at the outflow the Aeromonas spp. population was dominated by A. caviae in winter, whereas Aeromonas sobria was the dominant species in the treated effluent from spring to fall. Among the Aeromonas hydrophila and A. sobria strains, 100% produced hemolysin; whereas among the A. caviae strains, 96% were nonhemolytic.