Persistence and Distribution of Erysipelothrix rhusiopathiae and Bacterial Indicator Organisms on Land Used for Disposal of Piggery Effluent

Erysipelothrix rhusiopathiae was isolated from 15 of 40 effluents collected from commercial piggeries. The organism was isolated from soil and pasture of experimental disposal sites for up to two weeks after application of effluent naturally infected with Ery. rhusiopathiae. The organism was more commonly isolated from soil than pasture. Times for 90% reduction (T₉₀ values) of indicator organisms over a six week period following the effluent applications where Ery. rhusiopathiae was detected for 7 d or more, were 8–19 d for faecal coliforms in top soil and 5–12 d on pasture. T₉₀ for faecal streptococci was 10–14 d in soil and 8–11 d on pasture. Laboratory investigations indicated that the death rate of Ery. rhusiopathiae was up to six times greater than Escherichia coli in soil at field capacity. In dry soil the differences in die-out rate of the two organisms was less marked.     

Classification of antibiotic resistance patterns of indicator bacteria by discriminant analysis: use in predicting the source of fecal contamination in subtropical waters

The antibiotic resistance patterns of fecal streptococci and fecal coliforms isolated from domestic wastewater and animal feces were determined using a battery of antibiotics (amoxicillin, ampicillin, cephalothin, chlortetracycline, oxytetracycline, tetracycline, erythromycin, streptomycin, and vancomycin) at four concentrations each. The sources of animal feces included wild birds, cattle, chickens, dogs, pigs, and raccoons. Antibiotic resistance patterns of fecal streptococci and fecal coliforms from known sources were grouped into two separate databases, and discriminant analysis of these patterns was used to establish the relationship between the antibiotic resistance patterns and the bacterial source. The fecal streptococcus and fecal coliform databases classified isolates from known sources with similar accuracies. The average rate of correct classification for the fecal streptococcus database was 62.3%, and that for the fecal coliform database was 63.9%. The sources of fecal streptococci and fecal coliforms isolated from surface waters were identified by discriminant analysis of their antibiotic resistance patterns. Both databases identified the source of indicator bacteria isolated from surface waters directly impacted by septic tank discharges as human. At sample sites selected for relatively low anthropogenic impact, the dominant sources of indicator bacteria were identified as various animals. The antibiotic resistance analysis technique promises to be a useful tool in assessing sources of fecal contamination in subtropical waters, such as those in Florida.     

A Note on the Persistence of Salmonella havana and Faecal Coliforms on a Naturally Contaminated Piggery Effluent Disposal Site

Following application of effluent from apparently healthy pigs to land, Salmonella havana was isolated from kilogram samples, irregularly and at low frequency, for eight months from soil and for nearly two from pasture. Faecal coliform organisms on the effluent disposal site decreased rapidly in numbers during the first month following application of effluent at the start of the autumn. They were then maintained at about 100 organisms/g of topsoil until late in the following spring.     

Bacterial pollution indicators in the intestinal tract of freshwater fish

A study was made of the occurrence, distribution, and persistence of coliforms, fecal coliforms, and fecal streptococci in the intestinal tract of freshwater fish. A total of 132 fish representing 14 different species were used in various phases of these experiments. Examination of the intestinal contents of 78 fish from moderately polluted sections of the Little Miami River indicated that fecal coliform densities were lowest in bluegills (less than 20 per gram) and highest in catfish (1,090,000 per gram). Levels of fecal streptococci for these two species were 220 and 240,000 per gram, respectively. The occurrence of fecal coliforms in fish caught in this stream reflected the warm-blooded-animal-pollution level of the water. All fish used in this phase of the study were caught during July, August, and September when the water temperatures were between 13 and 18 C. The fate of fecal coliforms and Streptococcus faecalis in the fish intestine indicated that these organisms can probably survive and multiply when fish and water temperatures are 20 C or higher, but only when the organisms are retained in the gut for periods beyond 24 hr. Based on the biochemical reactions for 3,877 coliform strains isolated from 132 freshwater fish of 14 different species, 91.4% of all strains were composed of five IMViC types. In a similar study of the biochemical reactions of 850 streptococci isolated from the intestinal tract of 55 freshwater fish, the predominant strains included S. faecalis and various closely associated biotypes. No consistently recurring pattern for either coliforms or streptococci could be developed to identify species of fish investigated. The composition of the intestinal flora is, however, related in varying degree to the level of contamination of water and food in the environment.     

Comparative Survival of Indicator Bacteria and Enteric Pathogens in Well Water

The comparative survival of various fecal indicator bacteria and enteric pathogens was studied in a stable well water supply by using membrane chambers. There was more variation in the 29 coliform cultures and they died more rapidly, as a group, than the 20 enterococcus cultures that were examined. The comparative survival of the organisms tested follows: Aeromonas sp. > the shigellae (Shigella flexneri, S. sonnei, and S. dysenteriae) > fecal streptococci > coliforms = some salmonellae (Salmonella enteritidis ser. paratyphi A and D, S. enteritidis ser. typhimurium) > Streptococcus equinus > Vibrio cholerae > Salmonella typhi > Streptococcus bovis > Salmonella enteritidis ser. paratyphi B. S. bovis had a more rapid die-off than did S. equinus, but both had significantly shorter half-lives than the other streptococci. The natural populations of indicator bacteria from human and elk fecal material declined similarly to the pure cultures tested, whereas the die-off of fecal streptococci exceeded the coliforms from bovine fecal material.     

Evaluation of Standard and Modified M-FC, MacConkey, and Teepol Media for Membrane Filtration Counting of Fecal Coliforms in Water

MacConkey agar, standard M-FC agar, M-FC agar without rosolic acid, M-FC agar with a resuscitation top layer, Teepol agar, and pads saturated with Teepol broth, were evaluated as growth media for membrane filtration counting of fecal coliform bacteria in water. In comparative tests on 312 samples of water from a wide variety of sources, including chlorinated effluents, M-FC agar without rosolic acid proved the medium of choice because it generally yielded the highest counts, was readily obtainable, easy to prepare and handle, and yielded clearly recognizable fecal coliform colonies. Identification of 1,139 fecal coliform isolates showed that fecal coliform tests cannot be used to enumerate Escherichia coli because the incidence of E. coli among fecal coliforms varied from an average of 51% for river water to 93% for an activated sludge effluent after chlorination. The incidence of Klebsiella pneumoniae among fecal coliforms varied from an average of 4% for the activated sludge effluent after chlorination to 32% for the river water. The advantages of a standard membrane filtration procedure for routine counting of fecal coliforms in water using M-FC agar without rosolic acid as growth medium, in the absence of preincubation or resuscitation steps, are outlined.     

Virus and bacteria removal from wastewater by land treatment.

Secondary sewage effluent and renovated water from four wells at the Flushing Meadows Wastewater Renovation Project near Phoenix, Arizona, in operation since 1967, were assayed approximately every 2 months in 1974 for viruses and enteric bacteria during flooding periods. No viruses of Salmonella sp. were detected in any renovated well water samples, and the numbers of fecal coliforms, fecal streptococci, and total bacteria were decreased by about 99.9% in the renovated well waters after the wastewater was filtered through about 9 m of soil.     

Occurrence of Coliforms, Fecal Coliforms, and Streptococci on Vegetation and Insects

This study considers the sanitary significance of coliforms, fecal coliforms, and streptococci isolated from 152 species of plants and 40 samples of insects. These specimens were collected from various ecological environments and grouped into several categories. Results indicate that typical coliforms of the warm-blooded animal gut contribute a relatively small percentage of the organisms associated with vegetation (14.1%) and insects, (14.9%). A total of 1,203 coliform strains from vegetation and 1,084 coliform strains from insects were classified as to IMViC type and fecal coliform. No type was predominant in either the vegetation or insect groupings. The biochemical results for 646 streptococci from vegetation and 226 cultures from insects were reported. The predominant group, Streptococcus fecalis, as defined by Sherman criteria, constituted a majority of all strains from vegetation and insects. The “Completed Coliform Test” is recommended for the examination of plant and insect specimens to eliminate the many anaerobic and aerobic sporeforming bacteria that frequently produce false positive reactions by the “Confirmed Test” procedure. These findings support the current interpretation of the significance of the fecal coliform test for stream investigations or for surface water quality evaluations.     

Effect of sunlight on survival of indicator bacteria in seawater.

The stability of the natural populations of fecal coliforms and fecal streptococci in raw sewage diluted 1:1,000 in seawater or phosphate-buffered water at 24 +/- 2 degrees C was markedly affected by the absence or presence of sunlight. In the absence of sunlight, these bacteria survived for days, whereas in the presence of sunlight 90% of the fecal coliforms and fecal streptococci were inactivated within 30 to 90 min and 60 to 180 min, respectively. The bactericidal effect of sunlight was shown to penetrate glass, translucent polyethylene, and at least 3.3 m of clear seawater, suggesting that the visible rather than the ultraviolet light spectrum of sunlight was primarily responsible for the observed bactericidal effect. However, these same sewage-borne bacteria were relatively resistant to the bactericidal effect of sunlight when diluted in fresh mountain stream waters. These results indicate that the presence of sunlight is a major factor controlling the survival of fecal coliforms and fecal streptococci in seawater.     

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.     

Evaluation of millipore HA and HC membrane filters for the enumeration of indicator bacteria.

Millipore type HA and HC membrane filters were compared for recoveries of total coliforms, fecal coliforms and fecal streptococci from natural waters and wastewater effluents. Bacterial and fecal coliform recovery was better on HC than on HA filters.     

Survival of fecal microorganisms in marine and freshwater sediments.

The survival of culturable fecal coliforms, fecal streptococci, and Clostridium perfringens spores in freshwater and marine sediments from sites near sewage outfalls was studied. In laboratory studies, the inhibition of protozoan predators with cycloheximide allowed the fecal coliforms to grow in the sediment whereas the presence of predators resulted in a net die-off. C. perfringens spores did not appear either to be affected by predators or to die off throughout the duration of the experiments (28 days). Studies using in situ membrane diffusion chambers showed that, with the exception of C. perfringens, die-off of the test organisms to 10% of their initial numbers occurred in both marine and freshwater sediments within 85 days. The usual exponential decay model could not be applied to the sediment survival data, with the exception of the data for fecal streptococci. It was concluded that application of the usual decay model to the fecal coliform data was confounded by the complex relationship between growth and predation. The survival of seeded Escherichia coli in marine sediment was studied by using an enumeration method which detected viable but nonculturable bacteria. Throughout the duration of the experiment (68 days), the same proportion of E. coli organisms remained culturable, suggesting that sediment provides a favorable, nonstarvation environment for the bacteria.     

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.     

Characterization of the coliform and enteric bacilli in the environment of calves with colibacillosis.

In the first part of the present study the coliform and enteric bacilli in the environment of calves with colibacillosis were examined. The occurrence, number, and pathogenic properties of Escherichia coli in barnyard soils were obtained from six cattle ranches. The O and K serogroups of E. coli isolates obtained from the feces of calves with colibacillosis born at these cattle ranches were determined, and their serotypes were compared with the E. coli O and K serotypes found in soils. The results showed a reservoir of potentially pathogenic E. coli in barnyard soils contaminated with bovine feces. For the second part of this study, 6 healthy calves and 51 calves with colibacillosis were studied. The numbers of total aerobic heterotrophic bacteria, total streptococci, fecal streptococci, total coliforms, and fecal coliforms in the feces of calves were determined. In addition, coliform and enteric bacilli from the feces of both healthy and diseased calves were identified, and their indole, methyl red, Voges-Proskauer, citrate (IMViC) types were described. In parallel, the IMViC types of coliform and enteric bacilli isolated from barnyard soils previously contaminated with bovine feces were compared with those isolated from uncontaminated soils. All fecal specimens were also examined for the presence of rotavirus. No significant effect on the numbers of the bacterial types was found. The results suggest that the predominant IMViC types found in the feces of calves with colibacillosis originate from the soil. From this study it is apparent that the occurrence, number, and survival of E. coli in barnyard soils is related to ranch husbandry and sanitary practices.     

Characterization of the coliform and enteric bacilli in the environment of calves with colibacillosis

In the first part of the present study the coliform and enteric bacilli in the environment of calves with colibacillosis were examined. The occurrence, number, and pathogenic properties of Escherichia coli in barnyard soils were obtained from six cattle ranches. The O and K serogroups of E. coli isolates obtained from the feces of calves with colibacillosis born at these cattle ranches were determined, and their serotypes were compared with the E. coli O and K serotypes found in soils. The results showed a reservoir of potentially pathogenic E. coli in barnyard soils contaminated with bovine feces. For the second part of this study, 6 healthy calves and 51 calves with colibacillosis were studied. The numbers of total aerobic heterotrophic bacteria, total streptococci, fecal streptococci, total coliforms, and fecal coliforms in the feces of calves were determined. In addition, coliform and enteric bacilli from the feces of both healthy and diseased calves were identified, and their indole, methyl red, Voges-Proskauer, citrate (IMViC) types were described. In parallel, the IMViC types of coliform and enteric bacilli isolated from barnyard soils previously contaminated with bovine feces were compared with those isolated from uncontaminated soils. All fecal specimens were also examined for the presence of rotavirus. No significant effect on the numbers of the bacterial types was found. The results suggest that the predominant IMViC types found in the feces of calves with colibacillosis originate from the soil. From this study it is apparent that the occurrence, number, and survival of E. coli in barnyard soils is related to ranch husbandry and sanitary practices.     

R factors in coliform-fecal coliform sewage flora of the prairies and Northwest Territories of Canada.

Coliform and fecal coliform populations found in the raw sewages and final sewage effluents of the prairie provinces and the Northwest Territories were examined for antibiotic resistance and the possession of R factors. It was determined that 8.91% of the total coliform and 10.80% of the fecal coliform populations carried R factors. The following numbers of combinations of R determinants were found: 39 in the Escherichia coli population, 6 in the Citrobacter population, 20 in the Enterobacter populations, 10 in the Klebsiella populations, and 11 in the Aeromonas populations. The maximum number of R determinants transferable simultaneously was seven; organisms with R factors containing determinants for chloramphenicol usually contained determinants for ampicillin. Of the coliform and fecal coliform populations, 2 to 4% were resistant to chloramphenicol in some provinces, and from 17 to 30% of the populations were resistant to three or more antibiotics. It was calculated that coliforms containing R factors in the raw sewage reached population levels of 1.5 X 10(7)/100 ml, and fecal coliforms containing R factors reached population levels of 8.6 X 10(5) ml. Final effluent discharges to the receiving environment contained R factor-containing coliform and fecal coliform populations of 3.1 X 10(4)/100 ml and 5.8 X 10(2)/100 ml, respectively. The incidence of bacteria containing R factors in sewage appears to be increasing with time, and their removal from sewage before discharge to the receiving environment is desirable. Consideration of data on bacteria with R factors should be made in future water quality deliberations and in discharge regulations.     

Two-temperature membrane filter method for enumerating fecal coliform bacteria from chlorinated effluents.

Reports indicate that the standard membrane filter (MF) technique for recovery of fecal coliform bacteria from chlorinated sewage effluents is less effective than the multiple-tube (or most-probable-number [MPN]) procedure. A modified MF method was developed that requires a preincubation period of 5 h at 35 degrees C followed by 18+/-1 h at 44.5 degrees C. This procedure was evaluated by using both laboratory- and plant-chlorinated primary and secondary effluents. Results obtained by the modified MF method compared favorably with those of the MPN technique for the enumeration of fecal coliforms from chlorinated effluent. Agreement between these two methods was greatest with samples from secondary treatment plants. The average recovery of fecal coliforms by the standard MF procedure was only 14% that of the MPN method, whereas with the modified technique recovery was increased to 68% of the MPN counts. Enhanced recovery resulting from a simple modification in the incubation schedule makes the MF method a valuable adjunct for enumerating fecal coliforms from chlorinated effluents.     

Bacteriology of Dehydrated Space Foods

The initial bacteriological requirement established in 1964 for space foods by the U.S. Army Natick Laboratories are: a total aerobic plate count ( 300,000), chocolate ice cream cubes (20,000), and each of four samples of chocolate candy (12,000 to 61,000); (ii) coliforms: two out of three vanilla milk drinks (16 and 127) and one beef hash bar (14); (iii) fecal coliforms: one sample of chicken soup and gravy base positive; (iv) fecal streptococci: two samples of peanut cubes (40 and 108), coconut cubes (75), chicken soup and gravy base (2,650), beef soup and gravy base (33), and five out of six flavored milk drinks (23 to 300); (v) salmonellae: one each of chicken and beef soup and gravy base were positive.     

Two-temperature membrane filter method for enumerating fecal coliform bacteria from chlorinated effluents.

Reports indicate that the standard membrane filter (MF) technique for recovery of fecal coliform bacteria from chlorinated sewage effluents is less effective than the multiple-tube (or most-probable-number [MPN]) procedure. A modified MF method was developed that requires a preincubation period of 5 h at 35 degrees C followed by 18+/-1 h at 44.5 degrees C. This procedure was evaluated by using both laboratory- and plant-chlorinated primary and secondary effluents. Results obtained by the modified MF method compared favorably with those of the MPN technique for the enumeration of fecal coliforms from chlorinated effluent. Agreement between these two methods was greatest with samples from secondary treatment plants. The average recovery of fecal coliforms by the standard MF procedure was only 14% that of the MPN method, whereas with the modified technique recovery was increased to 68% of the MPN counts. Enhanced recovery resulting from a simple modification in the incubation schedule makes the MF method a valuable adjunct for enumerating fecal coliforms from chlorinated effluents.     

Seasonal Variations in Survival of Indicator Bacteria in Soil and Their Contribution to Storm-water Pollution

Survival of a fecal coliform (Escherichia coli) and a fecal streptococcus (Streptococcus faecalis var. liquifaciens) was studied through several years at shaded and exposed outdoor soil plots. Death rates for both organisms were calculated for the different seasons at both sites. The 90% reduction times for the fecal coliform ranged from 3.3 days in summer to 13.4 days in autumn. For the fecal streptococcus, 90% reduction times were from 2.7 days in summer to 20.1 days in winter. During summer, the fecal coliform survived slightly longer than the fecal streptococcus; during autumn, survival was the same; and in spring and winter the fecal streptococcus survived much longer than the fecal coliform. Both organisms were isolated from storm-water runoff collected below a sampling site when counts were sufficiently high in soil. Isolation was more frequent during prolonged rains, lasting up to 10 days, than during short rain storms. There was evidence of aftergrowth of nonfecal coliforms in the soil as a result of temperature and rainfall variations. Such aftergrowth may contribute to variations in bacterial count of storm-water runoff which have no relation to the sanitary history of the drainage area.