Where real-time measures are not essential, or for use in confirming their validity, it is logical to consider monitoring of indicator organisms as surrogates for pathogens themselves. For example, certain bacteria present in feces might be used to indicate the presence of fecal pollution and hence the likelihood that pathogens are present. Other organisms might be appropriate for other purposes, such as for evaluating swimming pools.
Because a major public health concern traditionally has been contamination of drinking, shellfish harvesting, and recreational waters with sewage, considerable effort has gone into developing methods for microbial indicators of fecal contamination in such waters. However, although several of these tests are used routinely and are generally reliable, it is important to remember the purpose for which they were developed. These standard indicators may not be appropriate for all applications, especially those for which they were not envisioned, such as microorganisms in aerosols.
Characteristics of an Ideal Indicator Organism The ideal microbial indicator of fecal contamination would have a number of characteristics. (Note that similar characteristics could be enumerated for other types of indicators as well.) First, it should be present in human feces in high numbers, making the test sensitive and thereby avoiding false negative results (in which a test says that contamination is absent even though it is really present). In fact, the ideal would be that the indicator to pathogen ratio would be high and stable. However, it is recognized that a constant ratio, at least, is not feasible, since the concentration of pathogens will vary based on the rate of infection in the population.
On the other hand, the indicator organism should be absent from uncontaminated materials (avoiding false positive results). In many cases, though, its presence in the feces of other warm-blooded animals would be desirable, since some diseases can be spread from animal to humans, but this would depend on the particular application.
The indicator organism should also die off in the environment at a slightly slower rate than the pathogens. Dying too quickly could lead to false negatives, while dying too slowly would lead to false positive results.
In terms of the actual testing, it would be very nice if rapid, easy, and inexpensive methods were available. Also, it would be preferable that the indicator organism itself is not pathogenic. After all, we do not want a large number of labs growing up vast quantities of potentially disease-causing organisms.
As might be expected, no ideal indicators are known. However, a number of methods have been developed that meet the criteria above for indicators of fecal contamination or other purposes to a useful extent. Several of these are described below. An important source for the detailed procedures is Standard Methods for the Examination of Water and Wastewater (Clesceri et al., 1998).
Total Coliforms Total coliforms are aerobic or facultatively anaerobic, gram-negative, nonsporeforming rods that ferment lactose (milk sugar) with acid and gas production (gas production not considered in an alternative procedure) within 48 hours (24 hours in the alternative test) at 35°C. This is an operationally defined group of microorganisms: that is, organisms that show up as positive in the test are defined as members of the group. The test is designed to include selected members of the bacterial family Enterobacteriaceae, especially many E. coli. This species is common (but not predominant) in the intestinal tract of humans and other warm-blooded animals. Also, it is closely related to Salmonella and Shigella, two of the bacteria historically of greatest concern for waterborne disease; thus its die-away in the environment is likely to be similar. However, not all E. coli are coliforms (e.g., some strains cannot ferment lactose), and not all coliforms are E. coli. Examples of other members of the family Enterobacteriaceae that may include coliforms are Enterobacter, Klebsiella pneumoniae, and Citrobacter. Additionally, some coliforms can grow and survive in soil or water. Also, while they serve as reasonably good indicators for Salmonella and Shigella, their survival of disinfection and various environmental factors may differ substantially from that of some other disease agents, including other bacteria, protozoa, and viruses.
Still, total coliforms are one of the most useful indicator groups for their intended purpose. About 106 mL-1 are commonly found in raw sewage. (In a practice dating from the early years of the field, microbial counts in water are often expressed "per 100 mL''; the same unit also is sometimes used for wastewater, so that this number of coliforms would be 108 per 100 mL.) They are used routinely to monitor the microbial safety of drinking water supplies. Total coliform counts also may be helpful in evaluating the fecal contamination of some foods and the disinfection efficiency for wastewaters. Both an MPN and an alternative membrane filtration procedure are available (Section 11.5.2).
Fecal Coliforms Since not all coliforms are of fecal origin, it would be useful to have a test that was more selective and included only fecal bacteria. The fecal coliform test attempts to achieve this mainly by incubation at 44.5°C, hoping by the higher temperature to eliminate bacteria that are better adapted to the lower soil and water temperatures of the ambient environment. The test is partially successful in meeting its goals. It is estimated that about 90% of the coliforms that grow at 44.5°C are of fecal origin (10% false positives); however, only about 90% of the coliforms of fecal origin can grow at the elevated temperature (10% false negatives). Additionally, precise control of the temperature is critical, as even 0.5°C higher can prevent the growth of many fecal coliforms, while slightly lower temperatures allow the growth of more nonfecal organisms.
Fecal coliforms are considered good indicators for outdoor swimming pools and recreational waters. (See also the fecal coliform/fecal strep ratio, below.) Again, both MPN and membrane filtration methods can be used.
Fecal Streptococci/Enterococci Although also operationally defined, the fecal streptococci consist mainly of Streptococcus faecalis, S. faecium, S. avium, S. bovis, S. equinus, and S. gallinarum; the enterococci include only S. faecalis, S. faecium, S. avium, and S. gallinarum. The first two species tend to be more common in humans and rats, whereas the others tend to be more common in chickens, cattle, horses, and domestic fowl (birds), respectively. However, they are not truly host-species specific.
The fecal streptococci are common inhabitants of the intestinal tracts of warm-blooded animals. In fact, humans are atypical in that they usually have a greater number of coli-forms than fecal streptococci; in most other species, the fecal streptococci are more numerous. Thus, for fresh contamination (the ratio changes with age), a fecal coliform/ fecal streptococci ratio of >4 may be considered indicative of human pollution, whereas a ratio <0.7 suggests an animal source.
The enterococci are now usually considered the best indicator for recreational surface waters, particularly ocean beaches. Once again, both MPN and membrane filtration methods are available. Incubation is at 35°C for 24 to 48 hours, depending on the test used.
Heterotrophic Plate Count The heterotrophic plate count (HPC), sometimes called the standard or total plate count, uses a nonselective medium in an attempt to include as many of the bacteria present as possible. Incubation is at 35°C for 48 hours or at 20 to 28°C for 5 to 7 days. One use of the HPC is that increases can indicate problems in water treatment processes or potable water distribution systems. High counts in indoor pools indicate poor disinfection and/or overuse or inadequate cleaning. Available tests include pour plate, spread plate, and membrane filtration methods.
Specific Organisms In some cases, tests for specific organisms may be desirable. For example, in indoor swimming pools and whirlpools or spas, tests for Pseudomonas aer-uginosa and Staphylococcus are recommended. These organisms serve not only as indicators of microbial loadings from skin and mucous membranes, but are themselves pathogens of potential concern. (HPC serves as a recommended indicator of disinfection efficiency for these systems.) Clostridium perfringens, which as an endosporeformer can persist for long periods in the environment, has been used to monitor the dispersal of wastewater treatment sludges disposed of in the ocean (now banned in the United States). Some coliphage (viruses that infect E. coli) have been used to monitor disinfection. Coliphage F2 has been found to be very heat resistant and was thus used as a conservative indicator (one that provides an extra margin of safety) during the development of criteria for pathogen destruction during composting. In monitoring microbial aerosols generated during composting operations, Aspergillus fumigatus has sometimes been used, since its spores will survive longer than coliforms, and since it is itself of concern.
These examples are meant to be illustrative rather than exhaustive. Remember, the available indicators were developed for certain purposes and should not be adopted arbitrarily for other situations.
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