Use of bacterial spores in monitoring water quality and treatment.

Because Clostridium perfringens spores are both specific to sewage contamination and environmentally stable, they are considered as possible conservative indicators of human fecal contamination and possible surrogates for environmentally stable pathogens. This review discusses the reasons and summarizes methods for monitoring spores in water. Cultural methods are still preferred over qPCR for routine water quality monitoring because of their low costs. Membrane filter (MF) methods are preferred over the more laborious and less accurate most probable number methods. The most commonly used MF media are TSC medium and mCP medium. TSC normally allows higher recoveries than mCP. TSC produces fewer false-positive results than mCP; however, it does produce more false-negatives. Two newer methods have substantial potential, CP Chromo Select agar, which allows better recoveries and greater specificity than mCP, and the Fung double tube method, which creates anaerobic conditions and allows enumeration of colonies in tubes in 5-6 hours. Aerobic spores are not associated with fecal contamination but they can be surrogates for environmentally stable pathogens in monitoring water for treatment efficacy; Bacillus cereus spores are normally measured on nutrient agar by the MF method.

Research considerations for more effective groundwater monitoring.

Since numerous pathogens occur in feces, water is monitored for fecal contamination using indicator organisms rather than individual pathogens. Although this approach is supported by health effects data in recreational waters, it is questionable when used for drinking water. Most outbreaks in groundwater occur in systems that have not violated the US EPA's maximum contaminant limit (MCL) for total coliforms within 12 months before the outbreak. Additionally, environmentally stable viruses and parasites are often detected in drinking water samples with no detectable indicators. Recent detections of Escherichia coli O157:H7 and Campylobacter jejuni in groundwaters in the apparent absence of indicators also cast some doubt on the worth of indicators for fecal bacterial pathogens. Individual pathogen monitoring is now technically achievable but currently unreasonable due to the number of possible pathogens and the costs involved. Several alternatives to pathogen monitoring could significantly reduce the frequency at which pathogens occur in waters testing negative for indicators: (i) increasing sample volumes for indicators, (ii) increasing monitoring frequency, (iii) using a suite of indicators, (iv) using a more conservative polymerase chain reaction (PCR) method, (v) sampling when fecal contamination is most likely present or (vi) any combination of these options.

Molecular comparison of Mycobacterium avium isolates from clinical and environmental sources.

We collected Mycobacterium avium isolates from clinical and drinking-water sources and compared isolates among themselves and to each other using molecular methods. Four clinical isolates were related to water isolates. Groups of indistinguishable clinical isolates were identified. The groups of identical clinical isolates suggest a common source of exposure.

Rare occurrence of heterotrophic bacteria with pathogenic potential in potable water.

Since the discovery of Legionella pneumophila, an opportunistic pathogen that is indigenous to water, microbiologists have speculated that there may be other opportunistic pathogens among the numerous heterotrophic bacteria found in potable water. The US Environmental Protection Agency (USEPA) developed a series of rapid in vitro assays to assess the virulence potential of large numbers of bacteria from potable water to possibly identify currently unknown pathogens. Results of surveys of potable water from several distribution systems using these tests showed that only 50 of the approximately 10,000 bacterial colonies expressed one or more virulence characteristics. In another study, 45 potable water isolates that expressed multiple virulence factors were tested for pathogenicity in immunocompromised mice. None of the isolates infected mice that were compromised either by treatment with carrageenan (CG), to induce susceptibility to facultative intracellular pathogens, or by cyclophosphamide (CY), to induce susceptibility to extracellular pathogens. These results indicate that there are very few potential pathogens in potable water and that the currently developed in vitro virulence screening tests give an overestimation of the numbers of heterotrophic bacteria that may be pathogens. Current efforts are focused on using the animal models to screen concentrated samples of waters known to contain large numbers of heterotrophic bacteria and newly discovered Legionella-like organisms that parasitize amoebae.

Persistence of nontuberculous mycobacteria in a drinking water system after addition of filtration treatment.

There is evidence that drinking water may be a source of infections with pathogenic nontuberculous mycobacteria (NTM) in humans. One method by which NTM are believed to enter drinking water distribution systems is by their intracellular colonization of protozoa. Our goal was to determine whether we could detect a reduction in the prevalence of NTM recovered from an unfiltered surface drinking water system after the addition of ozonation and filtration treatment and to characterize NTM isolates by using molecular methods. We sampled water from two initially unfiltered surface drinking water treatment plants over a 29-month period. One plant received the addition of filtration and ozonation after 6 months of sampling. Sample sites included those at treatment plant effluents, distributed water, and cold water taps (point-of-use [POU] sites) in public or commercial buildings located within each distribution system. NTM were recovered from 27% of the sites. POU sites yielded the majority of NTM, with >50% recovery despite the addition of ozonation and filtration. Closely related electrophoretic groups of Mycobacterium avium were found to persist at POU sites for up to 26 months. Water collected from POU cold water outlets was persistently colonized with NTM despite the addition of ozonation and filtration to a drinking water system. This suggests that cold water POU outlets need to be considered as a potential source of chronic human exposure to NTM.

Nonpoint Pollution From Animal Sources and Shellfish Sanitation.

Many of the microorganisms pathogenic to both animals and man are transmitted via the fecal-oral route. Most of these pathogens could conceivably be transmitted through a shellfish vector. Bacteria potentially transmitted from animal to man via shellfish include most of the salmonellae, Yersinia enterocolitica , Yersinia pseudotuberculosis , Escherichia coli O157:H7, Campylobacter jejuni , and Listeria monocytogenes . The protozoa most likely to be transmitted this way are Giardia lamblia and Cryptosporidium spp. Because the enteric viruses are highly species-specific, they are not likely to be transmitted from animals to humans. There are environmental data showing that bacterial pathogens shed by both domestic and wild animals have been isolated from shellfish. However, there is little epidemiological evidence that illness outbreaks have been caused by shellfish harvested from waters polluted by animals. Unfortunately, epidemiological observations are of limited value because most illnesses are probably not recorded. In addition, more than half of the recorded outbreaks are of unknown etiology, and more than half of the shellfish implicated in illness outbreaks cannot be traced to their points of origin. More lenient bacteriological standards should not be established for waters affected only by animal pollution until health effects studies have been performed, and an indicator that differentiates between human and nonhuman fecal pollution is available. Most of the pollution that originates from domestic animals could be eliminated by simple and inexpensive measures.

Oral Infectivity of Vibrio vulnificus in Suckling Mice.

Lethal doses of 11 clinical and environmental isolates of Vibrio vulnificus were determined in suckling mice after oral challenge. With one exception, isolates that were virulent to iron-overloaded adult mice after intraperitoneal inoculation were highly lethal to the infant mice (>50% lethality at 105 CFU/mouse). The virulent isolate that failed to kill infant mice at 105 CFU had lost its invasiveness. Conditionally virulent isolates that were virulent only to simultaneously iron-overloaded and immunosuppressed adult mice required > 109 CFU to kill the infant mice. Avirulent isolates failed to kill at >109 CFU/mouse. There were no significant differences in the lethalities of clinical and environmental isolates. These findings demonstrated a close correlation between virulence in the iron-overloaded adult mouse and infectivity by the oral route.

Recovery of Aeromonas hydrophila from Oysters Implicated in an Outbreak of Foodborne Illness.

Potentially pathogenic Aeromonas hydrophila organism were isolated from oysters frozen at -72°C for 1-1/2 years. The oysters which had been associated with 472 cases of gastroenteritis in Louisiana in November 1982, were examined and found negative for Salmonella , pathogenic Vibrio parahaemolyticus , and diarrhetic shellfish poison. In 1983, oysters from the same shellfish growing area in Louisiana were implicated in seven cases of gastroenteritis caused by A. hydrophila . The oysters collected in 1982 were reexamined and found to contain A. hydrophila (MPN 9.3/100 g). Twenty-three of 28 strains identified by the MICRO-IS and API-20E systems were positive for at least one of the tests for virulence which included the suckling mouse test, the adrenal Y-1 mouse cell test, and hemolysin assays. Of five strains tested, all showed activity in the rabbit ileal loop. Although these results do not prove that A. hydrophila caused the outbreak in 1982, they suggest that in cases of foodborne illness involving oysters, A. hydrophila should be included in the screening tests.

Production of Enterotoxin by Vibrio vulnificus Isolates.

Weakly virulent isolates of Vibrio vulnificus that were lethal only to simultaneously iron-overloaded and immunosuppressed mice were tested for ability to cause fluid accumulation in the permanently ligated rabbit ileal loop. Unlike the highly virulent isolates, which caused septicemia and death in rabbits, these isolates caused significant fluid accumulation in the rabbit loops. Fluid accumulation was also observed when culture filtrates were tested, indicating the existence of an enterotoxin. Enterotoxin activity did not correlate with the hemolysin or protease activities. Only one of three enterotoxigenic isolates caused diarrhea when administered to temporarily ligated rabbit ileal loops, suggesting involvement of some other pathogenic determinant(s) such as colonization.

Radioimmunoassay for Clostridium perfringens Enterotoxin and Its Use in Screening Isolates Implicated in Food-Poisoning Outbreaks.

Fourteen isolates of Clostridium perfringens obtained from food-poisoning outbreaks were screened for enterotoxigenicity using a radioimmunoassay (RIA) that detects 1.0 ng of enterotoxin/ml. Only four of the isolates produced enterotoxin in concentrations too low to be detected by counterimmunoelectrophoresis when grown in Duncan-Strong sporulation (D-S) medium. Substitution of raffinose for soluble starch or addition of theobromine to the medium stimulated enterotoxin production by three of the four enterotoxin-positive isolates. Raffinose and theobromine did not stimulate enterotoxin production by isolates that were enterotoxin-negative in D-S medium. Enterotoxin production by the RIA-positive strains correlated with the numbers of heat-resistant spores they produced. The RIA-negative isolates produced approximately the same numbers of spores/ml as the high enterotoxin producers, and more spores/ml than strain H8 produced under optimum conditions. Therefore, inability to sporulate is not the cause for failure of these isolates to produce enterotoxin. Rabbit ileal loop assays showed that the two isolates that were lowest enterotoxin producers in vitro were highly active in vivo.

Evidence That Clostridium perfringens Produces Only One Enterotoxin.

Thirteen Clostridium perfringens isolates classified as nonenterotoxigenic by radioimmunoassay (RIA) were tested for biological activity in rabbit ileal loops to determine whether these organisms produced enterotoxins serologically unrelated to the classical C. perfringens enterotoxin. None of these strains was active in the ileal loop assays. The large number of RIA-negative isolates obtained from food-poisoning outbreaks is more likely due to the failure to isolate causative strains rather than to the existence of novel enterotoxins.

Examination of Bottled Water for Nontuberculous Mycobacteria.

The objective of this study was to examine bottled water for the presence of nontuberculous mycobacteria as a potential source of infection in AIDS patients. Twenty brands of bottled water commonly used in the Los Angeles area were tested for the presence of nontuberculous mycobacteria. The three brands most commonly used in the Los Angeles area were tested most frequently. Sixty-nine samples were filtered and the filters were treated using cetylpyridinium chloride, sodium hydroxide, or oxalic acid (or a combination of these) as decontaminants to remove background flora. An aliquot of each sample was untreated. The filters were placed on selective Middlebrook 7H10 agar plates containing 500 µg of cycloheximide per ml. Plates were examined at 3 and 8 weeks. No acid-fast organisms were found. Although no nontuberculous mycobacteria were observed in any samples tested, before recommending the use of bottled water as an alternative to tap water by high-risk patients, the possible presence of other contaminants must be considered.