Distribution and Characterization of Kepone-Resistant Bacteria in the Aquatic Environment

Effects of the chlorinated insecticide Kepone on the ecology of Chesapeake Bay and James River bacteria were studied. Kepone-resistant bacteria present in a given environment were found to reflect the degree of fecal and/or high organic pollution of the sampling sites, based on total numbers and generic composition of the populations of Kepone-resistant bacteria. The presence of Kepone-resistant bacteria was found to be correlated (α = 0.01) with total coliforms, fecal coliforms, and total aerobic viable heterotrophic bacteria, but not with Kepone concentration, since Kepone-resistant bacteria were present in locations where Kepone could not be detected by the analytical methods used in this study. Only gram-negative bacteria, predominantly Pseudomonas, Vibrio, and Aeromonas spp., were found to be resistant to ≥10 μg of Kepone per ml. Gram-positive bacteria, i.e., Bacillus and Corynebacterium spp., were generally sensitive to ≥0.1 μg of Kepone per ml. From results of cluster analysis of taxonomic data, we determined that characteristics of Kepone-resistant bacteria included: resistance to pesticides and heavy metals; degradation of oil; positive oxidase and catalase reactions; and nitrate reduction. From results of the ecological and taxonomic analyses, we conclude that Kepone resistance in estuarine bacteria is due to the physicochemical composition of the gram-negative cell wall and not prior exposure to Kepone. Therefore, the presence of Kepone-resistant bacteria cannot serve as an indicator of Kepone contamination in the aquatic environment where gram-negative bacteria are predominant.     

Antibiotic resistance patterns of gram-negative bacteria isolated from environmental sources.

A total of 2,445 gram-negative bacteria belonging to fecal coliform, Pseudomonas, Moraxella, Acinetobacter, and Flavobacterium-Cytophaga groups were isolated from the rivers and bay of Tillamook, Oregon, and their resistances to chloramphenicol (25 microgram/ml), streptomycin (10 microgram/ml), ampicillin (10 microgram/ml), tetracycline (25 microgram/ml), chlortetracycline (25 microgram/ml), oxytetracycline (25 microgram/ml), neomycin (50 microgram/ml), nitrofurazone (12.5 microgram/ml), nalidixic acid (25 microgram/ml), kanamycin (25 microgram/ml), and penicillin G (10 IU/ml) were determined. Among fecal coliforms the bay isolates showed greater resistance to antibiotics than those from tributaries or surface runoff. No such well-defined difference was found among other bacterial groups. The antibiotic resistance patterns of gram-negative bacteria from different sources correlated well, perhaps indicating their common origin. The antibiotic resistance patterns of gram-negative bacteria of different general also correlated well, perhaps indicating that bacteria which share a common environment also share a common mode for developing antibiotic resistance.     

Evaluation of broiler litter with reference to the microbial composition as assessed by using 16S rRNA and functional gene markers

Very little is known about the microbial composition of animal bedding wastes, including poultry litter, and what is known has been deduced from standard culture methods, by which some fastidious organisms that exist in the environment may not be detected. We evaluated the bacterial composition of poultry litter by using a combination of culture and molecular detection. Total aerobic bacteria in poultry litter were detected by culture at 10(9) CFU/g of material. Enteric bacteria such as Enterococcus spp. and coliforms composed 0.1 and 0.01%, respectively, of the total aerobic cultivatable bacteria in poultry litter; no Salmonella strains were detected by culture. In order to characterize the most abundant bacterial groups, we sequenced 16S ribosomal DNA (rDNA) genes amplified by PCR with microbial community DNA isolated from poultry litter as the template. From the 16S rDNA library, 31 genera were identified. Twelve families or groups were identified with lactobacilli and Salinococcus spp. forming the most abundant groups. In fact, 82% of the total sequences were identified as gram-positive bacteria with 62% of total belonging to low G+C gram-positive groups. In addition to detection of 16S rDNA sequences associated with the expected fecal bacteria present in manure, we detected many bacterial sequences for organisms, such as Globicatella sulfidofaciens, Corynebacterium ammoniagenes, Corynebacterium urealyticum, Clostridium aminovalericum, Arthrobacter sp., and Denitrobacter permanens, that may be involved in the degradation of wood and cycling of nitrogen and sulfur. Several sequences were identified in the library for bacteria associated with disease in humans and poultry such as clostridia, staphylococci, and Bordetella spp. However, specific PCR targeting other human and veterinary pathogens did not detect the presence of Salmonella, pathogenic Escherichia coli, Campylobacter spp., Yersinia spp., Listeria spp., or toxigenic staphylococci. PCR and DNA hybridization revealed the presence of class 1 integrons with gene cassettes that specify resistance to aminoglycosides and chloramphenicol. Only from understanding the microbial community of animal wastes such as poultry litter can we manage animal disease and limit the impact of animal waste on the environment and human and animal health.     

Detection of Acinetobacter spp. in rural drinking water supplies.

A bacteriological survey was conducted of untreated, individual groundwater supplies in Preston County, W.Va. Nearly 60% of the water supplies contained total coliforms in excess of the U.S. Environmental Protection Agency maximum contaminant level of 1 CFU/100 ml. Approximately one-third of the water systems contained fecal coliforms and/or fecal streptococci. Acinetobacter spp. were detected in 38% of the groundwater supplies at an arithmetic mean density of 8 CFU/100 ml and were present in 16% of the water supplies in the absence of total coliforms, posing some concern about the usefulness of total coliforms as indicators of the presence of this opportunistic pathogen. Slime production, a virulence factor for A. calcoaceticus, was not significantly different between well water isolates and clinical strains, suggesting some degree of pathogenic potential for strains isolated from groundwater. In addition, several Acinetobacter isolates were able to interfere with sheen production by some coliform bacteria on M-Endo medium, adding further to the possible significance of Acinetobacter spp. in groundwater supplies.     

Detection of Acinetobacter spp. in rural drinking water supplies

A bacteriological survey was conducted of untreated, individual groundwater supplies in Preston County, W.Va. Nearly 60% of the water supplies contained total coliforms in excess of the U.S. Environmental Protection Agency maximum contaminant level of 1 CFU/100 ml. Approximately one-third of the water systems contained fecal coliforms and/or fecal streptococci. Acinetobacter spp. were detected in 38% of the groundwater supplies at an arithmetic mean density of 8 CFU/100 ml and were present in 16% of the water supplies in the absence of total coliforms, posing some concern about the usefulness of total coliforms as indicators of the presence of this opportunistic pathogen. Slime production, a virulence factor for A. calcoaceticus, was not significantly different between well water isolates and clinical strains, suggesting some degree of pathogenic potential for strains isolated from groundwater. In addition, several Acinetobacter isolates were able to interfere with sheen production by some coliform bacteria on M-Endo medium, adding further to the possible significance of Acinetobacter spp. in groundwater supplies.     

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.     

Relationship among fecal coliforms, Escherichia coli, and Salmonella spp. in shellfish.

The relationship of fecal coliforms, Escherichia coli, and Salmonella spp. was examined in freshly harvested and stored shellfish. In 16 of 40 freshly collected oyster samples, fecal coliform levels were above the recommended wholesale level suggested by the National Shellfish Sanitation Program (less than or equal to 230/100 g), and Salmonella spp. were present in three of these samples. Salmonella spp. were not, however, present in any sample containing less than 230 fecal coliforms per 100 g. Analysis of the data suggests that low fecal coliform levels in both fresh and stored oysters are good indicators of the absence of Salmonella spp., but that high levels of fecal coliforms are somewhat limited in predicting the presence of Salmonella spp. E. coli levels correlated very strongly with fecal coliform levels in both fresh and stored oysters and clams, suggesting that there is no advantage in replacing fecal coliforms with E. coli as an indicator of shellfish quality.     

Relationship among fecal coliforms, Escherichia coli, and Salmonella spp. in shellfish

The relationship of fecal coliforms, Escherichia coli, and Salmonella spp. was examined in freshly harvested and stored shellfish. In 16 of 40 freshly collected oyster samples, fecal coliform levels were above the recommended wholesale level suggested by the National Shellfish Sanitation Program (less than or equal to 230/100 g), and Salmonella spp. were present in three of these samples. Salmonella spp. were not, however, present in any sample containing less than 230 fecal coliforms per 100 g. Analysis of the data suggests that low fecal coliform levels in both fresh and stored oysters are good indicators of the absence of Salmonella spp., but that high levels of fecal coliforms are somewhat limited in predicting the presence of Salmonella spp. E. coli levels correlated very strongly with fecal coliform levels in both fresh and stored oysters and clams, suggesting that there is no advantage in replacing fecal coliforms with E. coli as an indicator of shellfish quality.     

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.     

Microbiological Evaluation of Incinerator Operations

Incineration is currently a widely used method for the disposal of municipal solid waste in major American cities. The efficacy of several incinerator types to destroy bacteria associated with solid waste was evaluated, with emphasis on fecal and food sources. Samples of solid waste and its residue after incineration, taken from four incinerators of different design, were homogenized in phosphate buffer at pH 7.5. Samples of these homogenates were quantitatively examined for (i) total bacterial cell number, (ii) total coliforms, (iii) fecal coliforms, and (iv) heat-resistant spore-formers. Survival of coliforms in the residue after incineration was considered an indication of inadequate incinerator design or operation, or both. Of the four incinerators tested, only one produced residue devoid of fecal coliforms; three others produced residue containing fecal coliform populations of 5 to 2,400 per g. An inverse relationship was noted between the efficacy of incinerators in destroying fecal coliforms and the heat resistance (80 C) of total bacterial populations surviving in their respective residues. This could be due to the selection of heat-resistant cells during incineration.     

In Vitro Studies of Semisynthetic α- (Substituted-Ureido) Penicillins

The activity of three alpha-(substituted-ureido) penicillins was evaluated in vitro against 599 clinical isolates of gram-negative bacilli, by use of the broth-dilution technique. At a concentration of 12.5 mug or less/ml, BL-P1597 inhibited 90% of isolates of Pseudomonas sp., 56% of Enterobacter sp., 67% of indole-positive Proteus spp., 72% of Escherichia coli, and 85% of Proteus mirabilis. BL-P1654 had similar activity, whereas BL-P1532 was much less active. At a concentration of 25 mug or less/ml, BL-P1597 also inhibited nearly 60% of isolates of Klebsiella sp. and nearly 40% of Serratia sp. BL-P1597 and BL-P1654 were as active as ampicillin and carbenicillin against E. coli and P. mirabilis. They were less active than carbenicillin against indole-positive Proteus spp. Both drugs were substantially more active than carbenicillin against Pseudomonas sp. A strain of Pseudomonas sp. which developed resistance to carbenicillin also developed resistance to the alpha-(substituted-ureido) penicillins simultaneously.     

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.     

Microbial impact of Canada geese (Branta canadensis) and whistling swans (Cygnus columbianus columbianus) on aquatic ecosystems.

Quantitative and qualitative analyses of the intestinal bacterial flora of Canada geese and whistling swans were carried out with the finding that wild birds harbor significantly more fecal coliforms than fecal streptococci. The reverse was typical of captive and fasting birds. Neither Salmonella spp. nor Shigella spp. were isolated from 44 migratory waterfowl that were wintering in the Chesapeake Bay region. Enteropathogenic Escherichia coli were detected in seven birds. Geese eliminated 10(7) and swans 10(9) fecal coliforms per day. Results of in situ studies showed that large flocks of waterfowl can cause elevated fecal coliform densities in the water column. From the data obtained in this study, it is possible to predict the microbial impact of migratory waterfowl upon aquatic roosting sites.     

Microbial impact of Canada geese (Branta canadensis) and whistling swans (Cygnus columbianus columbianus) on aquatic ecosystems.

Quantitative and qualitative analyses of the intestinal bacterial flora of Canada geese and whistling swans were carried out with the finding that wild birds harbor significantly more fecal coliforms than fecal streptococci. The reverse was typical of captive and fasting birds. Neither Salmonella spp. nor Shigella spp. were isolated from 44 migratory waterfowl that were wintering in the Chesapeake Bay region. Enteropathogenic Escherichia coli were detected in seven birds. Geese eliminated 10(7) and swans 10(9) fecal coliforms per day. Results of in situ studies showed that large flocks of waterfowl can cause elevated fecal coliform densities in the water column. From the data obtained in this study, it is possible to predict the microbial impact of migratory waterfowl upon aquatic roosting sites.     

Bacteria associated with a marine planktonic copepod in culture. I. Bacterial genera in seawater, body surface, intestines and fecal pellets and succession during fecal pellet degradation

In laboratory experiments, the bacterial flora of the zooplanktonmicrobial environments seawater, fecal pellets and associatedwith the external and internal surfaces of the copepod Acartiatonsa(Dana) were examined. The bacteria associated with fecal pelletswere dominated by Bacillus spp., Cytophaga/Flavobacterium spp.,Vibrio spp. and Pseudomonas spp. The same genera were foundin the seawater (0.22 7mu;m filtered) in which the pellets wereincubated. The bacteria showed a characteristic growth succession,and the abundance increased several orders of magnitude in theseawater during incubation of the pellets, indicating growthand proliferation based on the disintegrating/degrading fecalpellets. A carbon budget calculation revealed that organic matterfrom degrading fecal pellets could cover the carbon demand forthe growing bacterioplankton. The composition of the bacterialcommunity in the seawater and the fecal pellets also indicateda colonization of the pellets from bacterioplankton. The compositionof the bacteria associated with the copepods showed that bacterialgenera characterized as surface associated were preferentiallyassociated with fecal pellets, animal surfaces and intestines.This suggests a specific intestinal flora in the cultivatedcopepods composed of 10³ culturable bacteria per intestine (colony-formingunits, c.f.u.) or 10⁵ bacteria per intestine (acridine orangedirect counts, AODC), possibly colonizing the intestine passivelyduring filtration of algae. The activity of the bacterial communitieswas examined by the numencal ratio c.f.u.:AODC, where 1–19%of the bacteria were found active, with no significant differencebetween microbial environments.     

Bacterial Contamination of Drinking Water Supplies in a Modern Rural Neighborhood

On six occasions during a 15-month period, the private well and spring water supplies in a modern rural neighborhood of 78 households were examined for total coliforms, fecal coliforms, Staphylococcus aureus, and standard plate count bacteria. More than one-third of the water supplies were unsatisfactory on at least one occasion as judged by standard plate counts over 10³/ml and the presence of coliforms, fecal coliforms, and/or S. aureus. Citrobacter freundii, Klebsiella pneumoniae, and Escherichia coli were the most frequently isolated total coliforms. At least 12 other genera of bacteria were identified from standard plate count agar. Coliform contamination was found to be higher after periods of rainfall, and high standard plate counts were more prevalent during warmer weather. These observations probably reflect leakage of surface water into improperly sealed wells or aquifer contamination during winter and the lack of chlorination to control microbial regrowth during the warm season. An inverse correlation was found between the presence of high standard plate counts and incidence of coliforms. Consumer education and at least a twice yearly monitoring of private water supplies (winter and summer) are suggested methods to signal that treatment may be necessary to reduce the risk of waterborne disease.     

Effects of kepone on growth and respiration of several estuarine bacteria

The toxicity of Kepone to mixed populations of estuarine microorganisms was determined by standard plate assays on Zobell marine medium containing 0.02, 0.20, and 2.0 mg of Kepone per liter. Under aerobic conditions, Kepone reduced the number of colony-forming units at all concentrations tested, but had no effect on the number of anaerobic microorganisms. Gram-positive organisms were more sensitive to Kepone than were gram-negative organisms. Growth of gram-negative isolates was not inhibited in nutrient broth, but was significantly inhibited in a minimal salts broth. Oxygen uptake by most isolates was reduced 25 to 100% by 20 ppm (20 mg/ml) of Kepone. Oxygen evolution was observed when several gram-positive isolates were exposed to Kepone concentrations of 20 ppm. Pentachlorophenol at concentrations above 28 ppm produced effects similar to those produced by Kepone. Inhibition of electron transport by Kepone was demonstrated by a significant reduction in the specific activities of NADH oxidases and succinooxidase.     

Effects of kepone on growth and respiration of several estuarine bacteria.

The toxicity of Kepone to mixed populations of estuarine microorganisms was determined by standard plate assays on Zobell marine medium containing 0.02, 0.20, and 2.0 mg of Kepone per liter. Under aerobic conditions, Kepone reduced the number of colony-forming units at all concentrations tested, but had no effect on the number of anaerobic microorganisms. Gram-positive organisms were more sensitive to Kepone than were gram-negative organisms. Growth of gram-negative isolates was not inhibited in nutrient broth, but was significantly inhibited in a minimal salts broth. Oxygen uptake by most isolates was reduced 25 to 100% by 20 ppm (20 mg/ml) of Kepone. Oxygen evolution was observed when several gram-positive isolates were exposed to Kepone concentrations of 20 ppm. Pentachlorophenol at concentrations above 28 ppm produced effects similar to those produced by Kepone. Inhibition of electron transport by Kepone was demonstrated by a significant reduction in the specific activities of NADH oxidases and succinooxidase.     

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.     

Effects of orally administered tetracycline on the intestinal community structure of chickens and on tet determinant carriage by commensal bacteria and Campylobacter jejuni

There is a growing concern that antibiotic usage in animal production has selected for resistant food-borne bacteria. Since tetracyclines are common therapeutic antibiotics used in poultry production, we sought to evaluate the effects of oral administration on the resistance of poultry commensal bacteria and the intestinal bacterial community structure. The diversity indices calculated from terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA amplicons did not indicate significant changes in the cecal bacterial community in response to oxytetracycline. To evaluate its effects on cultivable commensals, Enterococcus spp., Escherichia coli, and Campylobacter spp. were isolated from the cecal droppings of broiler chickens. Enterococcus spp. and E. coli expressed tetracycline MICs of >8 microg/ml and harbored a variety of tet resistance determinants regardless of the tetracycline exposure history of the birds. The enterococcal isolates possessed tetM (61%), tetL (25.4%), and tetK (1.3%), as well as tetO (52.5%), the determinant known to confer a tetracycline resistance phenotype in Campylobacter jejuni. E. coli isolates harbored tetA (32.2%) or tetB (30.5%). Tetracycline MICs remained at <2 microg/ml for Campylobacter isolates before and after tetracycline treatment of the chickens, even though isolates expressing MICs of >16 mug/ml were commonly cultured from flocks that did not receive oxytetracycline. The results imply that complex ecological and genetic factors contribute to the prevalence of antibiotic resistance arising from resistance gene transfer in the production environment.