Tests for Bacteria in Sewage, Water and Food                                                                          July 2009

                                                                                                                                 updated August 28, 2009
In the past, EPA did not want you to know the definition of fecal coliform or coliform. The probability is that not many
people at EPA or the state regulators know exactly what they are or how it began. E. coli is the primary fecal coliform.
The current theory is you can have tons of shit (sludge/biosolids) spread all around you home, on your food crops,
on your children's play area, even school grounds and there is no problem. Just don't touch the shit in your

According to microbiologist Charles Gerba, Ph.D., there is roughly a tenth of a gram of fecal matter in worn undies;
that translates to about 100 million E. coli bacteria, which, in ferreting through your laundry, may get on your hands.
Then, if you rub your eyes or, God forbid, eat a sandwich, you could get sick. If you're a regular hamper digger, wash
your hands after rooting around in there.

Scientists have brought a newly-discovered bacterium back to life after more than 120,000 years in hibernation. It
raises hopes that dormant life might be revived on Mars.
Earlier bugs found in the Canadian Arctic were brought back to life after 30,000 years.

Modern Surgery,  By John Chalmers Da Costa 1919
The Bacillus coli communis, called also Bacterium coli commune, the colon bacillus, or the bacillus of Escherich
[E. coli] was discovered in feces by Emmerich in 1885. Under ordinary conditions this is a putrefactive bacillus
inhabiting the intestinal canal and feces invariable contains it. It is found in the mouth, nose, and vagina, on the skin,
and under the nails. The bacillus is normally found in water, even in water regarded by users as pure. It has already
been stated that this ordinarily harmless micro-organism may, under certain conditions, acquire pathogenic power
and enter the circulation. This bacterium grows best in air, but it can also grow when air is excluded.

This bacillus may be responsible for appendicitis, peritonitis, inflammation of the genito-urinary tract, pneumonia,
inflammation of the intestine, leptomeningitis, perineal abscess, cholangitis, cholecystitis, myelitis, puerperal fever,
wound infections and septicemia. It is the cause of many abscesses about the intestine and responsible for many
ischiorectal abscess. From the pus of an appendicitis abscess we may perhaps obtain culture of Escherich's bacillus,
but usually find also streptococci or staphylococci, and sometimes pneumococi.  Colon bacilli introduced into the
system by tainted food may be responsible for epidemic pneumonia . A few years ago there was such an epidemic in
Middlesbrough, England (Oliver, in "Brit. Med. Jour." April 30, 1910)

D. H. BERGEY, Laboratory of Hygiene, University of Pennsylvania
A group of facultative thermophilic bacteria has been discovered which develop at room temperature, about 20°C.,
and have their optimum temperature at about 50°C., and their maximum temperature at about 60°C. In this group
belong some of the well-known spore-forming soil organisms. Two distinct types of true thermophilic bacteria have
been found in the laboratory, viz., spore-forming and non-sporeforming rods, and thread forms belonging in the class


The author has felt for some time that before much light will be thrown on the true relative incidence of B. coli and B.
aerogenes in water and in feces, etc., it will be necessary first, so to modify our preliminary enrichment media, or other
conditions, as to enable the investigator to isolate or suppress eitier B. coli or B. aerogenes at will.

it has been observed that the maximum rate of multiplication of B. coli is at about 45° C.

The author observed that in peptone lactose media at 43° C. (in a water bath) all the cultures of B. coli (16.) grew
luxuriantly as evidenced by strong turbidity in 24 hours, but 69 percent showed no gas or only a bubble in 24 hours.
Of 20 cultures of B. aerogenes, on the other hand, 16 showed no growth, 2 slight, and 2 grew luxuriantly.
[Aerobacter, Enterobacter, Klebsiella,  are all aerogenes]

In 1904 Eijkman reported that Bacillus coli [E. coli - i.e. fecal coliform] from man and warmblooded animals has the
ability to ferment glucose broth, with acid and gas production, when incubated at 46°C. it is selective for Bacillus coli
[E. coli] and that other organisms in water are inhibited or destroyed. It is complete in twenty-four hours.

A study of 147 samples of water from private wells, collected in rural districts adjacent to Chicago, showed a close
correlation between the separate count of Bact. coli, and the presence of probable sources of fecal pollution as
indicated by the sanitary survey. The separate Bact. aerogenes count and the colonaerogenes index by the standard
method (American Public Health Association, 1925) also were uniformly positive in this class of sanitary surveys.
The Bact. aerogenes counts in the absence of Bact. coli were grouped mainly with doubtful sanitary surveys, in which
potential rather than imminent danger of fecal pollution was indicated. The standard method indices were also
uniformly positive in this group of sanitary surveys.

A growing question for practical consideration in water control is the relative sanitary significance of Bact. coli and
Bact. aerogenes s criteria of fecal pollution. It is generally agreed that Bact. coli s strictly of fecal origin, and its
presence in water therefore is ccepted as evidence of dangerous pollution. Bact. aerogenes,
however, occupies a doubtful position as an organism of fecal origin. Some observers regard it as of the same fecal
significance as Bact. coli, while others consider it of little, if any, value as indicating contamination from fecal sources.
There appears to be growing evidence in support of the latter view (Chen and Rettger (1920); Koser (1927); Hinman
(1925); Tonney and Noble (1930)). Certainly, the great predominance of Bact. aerogenes over Bact. coli at large in
nature needs some explanation other than the assumption that both are of direct fecal origin, since in feces itself the
reverse relationship is true. In previous studies we have found the ratio of Bact. coli to Bact. aerogenes, by the
differential plate count, to be about 100 to 1 in feces of both human and animal origin, and on the other hand about 1
to 20 in soils and vegetation (Tonney and Noble (1930)).
1. That Bact. aerogenes survives longer and in greater numbers than Bact. coli outside of the animal body.
2. That Bact. aerogenes at large in nature multiplies to a greater extent than Bact. coli in the same environment.
3. That Bact. aerogenes is primarily of non-fecal origin and finds its way into the animal body with the food, hence
appearing irregularly

Routine Use of a Modified Eijkman Medium in the Examination of Oysters, Crabmeat, and Other Substances
Since the Eijkman test [fecal coliform] has been designed to eliminate members of the coli-aerogenes group [coliform]
not B. coli [ E. coli] it should be expected that many less confirmations for this group would be made from the Eijkman
broth tubes. Accordingly, 27.6 per cent of the lactose broth tubes were confirmed for members of the coli-aerogenes
group against 14.1 per cent of the Eijkman tubes. These figures include B. coli of course. The percentage of non-B.
coli members of the coli-aerogenes group are 21 .2 for lactose broth and 5. 2 per cent for Eijkman broth. -- With coli-
aerogenes organisms outnumbering B. coli 50 to 100 times (depending on the method), comparatively small
variations in the probable numbers of B. coli as determined by the two methods becomes of comparatively less
Am J Public Health Nations Health. 1935 June; 25(6): 720–724.

Erwinia, a genus of bacterial plant pathogens, has long been recognized as closely related to the Escherichia-
Aerobacter group. This view, although readily accepted, has been the object of little comparative study; the
relationship is usually taken for granted, the result being a relatively poor understanding of
the similarities of the two groups. On the basis of its disease-producing ability in plants, Bergey et al. (1939) gives the
group tribal ranking, Erwineae, in the family
Enterobacteriaceae. Other than the obvious facts that the species of
Erwinia are peritrichously flagellated, gram negative, do not form spores and ferment many carbohydrates, including
lactose, our knowledge of the relationship is meager. The possibility that the Erwinial might be coliform organisms has
been given comparatively little thought.
The manner in which species of Erwinia ferment lactose would tend to classify them as aberrant
coliforms. On the
basis of their IMViC patterns, the majority would seem to approximate Escherichia freundii. On the other hand,
according to their gelatin-liquefying ability, motility and production of acid in glycerol, the relationship seems to be
closer to Aerobacter cloacae.

The Coliform Group - I. The Boric Acid Lactose Broth Reaction of Coliform IMViC Types
Several methods have been suggested for the accurate, rapid, and convenient detection and enumeration
of Escherichia coli in potable waters, stream samples, sewage, and in certain food products. The first of these
procedures was described by Eijkman who proposed a "fecal coli" test that he believed differentiated
between coliforms from the gut of warm-blooded animals and strains originating from cold-blooded ones. The method
was based on the assumption that only fecal types of coliforms from warm-blooded animals
could grow in glucose broth at 46 C with the production of gas. Twenty-five years later a close relationship
between the positive Eijkman reaction and indole production was observed by Leiter (1929) and he suggested that
the combination of the two positive tests was practically specific for E. coli.

Rapid Presumptive Identification of Enteropathogenic Escherichia coli in Faecal Smears by means of
Fluorescent Antibody.
This study for E. coli in fecal material points out some of the problems with testing. For medical testing
E. coli contaminated samples are incubated at 37 deg C rather than the higher 44.5 deg C when used as a fecal
coliform indicator in food, water and sludge. It is one of the first studies to point out the reduction in numbers from
fresh bacteria in a test to those of older bacteria in a test will be caused by dehydration and that rehydration of
bacteria will cause the number of positive cultures to increase.  It would be another 20 years before this viable but
nonculturable state was described in the 1982 paper from the laboratory of Rita Colwell (Xu et al., 1982), Survival and
viability of nonculturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment. Microb. Ecol.
8, 313-323.

THE SCIENCE of sanitary water bacteriology began in 1880 when von Fritsch described Klebsiella
pneumonia and K. rliinoscleromatis as organisms characteristic of human fecal contamination. A short time later
Escherich identified Bacillus coli [Escherich coli] as an indicator of fecal pollution. Both observers considered human
feces as dangerous pollution while the feces of other warm-blooded animals were not so regarded.
From this origin the current "coliform group" developed to include numerous micro-organisms
of diverse biochemical and serologic characteristics, natural sources and habitats, as well as controversial
sanitary interpretations.
1. The most acceptable temperature of incubation for separation of the fecal coliform group is 44.5° C. in a water
4. The test can be used only as a confirmatory procedure from coliform cultures growing on a nonselective medium.
Fecal coliforin organisms may be considered indicators of recent fecal pollution by warmblooded animals.

Comparison of the Recovery of Escherichia coli from Frozen Foods and Nutmeats by Confirmatory Incubation in EC
Medium at 44°5 and 45°5 C
In 85.3% of the cases, the parallel tubes of EC broth incubated for 24 hr at 44°5 and 45°5 C gave rise to identical E.
coli responses of positive, false positive, and negative. The remaining 14.7% of the reactions represent the
qualitative difference between the two temperatures. The EC test at 45.5 C was more specific for E. coli, since two- to
threefold fewer false positives were produced at this temperature than at 44.5 C. However, fecal E. coli recoveries
were slightly higher (4%Vc) at the lower temperature. Incubating the EC tubes from the interval of 24 to 48 hr gave
rise to an additional 4.3% of E. coli recovery, but this was accompanied by an excessive production of false positives
(75.9%), representing a 3.5-fold decrease in specificity.

Isolation of Acinetobacter from Soil and Water    
[at this time it was not a pathogen]
An enrichment culture procedure for isolating members of the genus Acinetobacter from soil and water is described. It
involves the use of vigorously aerated mineral media at relative low pH, supplemented with acetate or other suitable
carbon source and nitrate as nitrogen source. With this method, virtually all samples of soil and water yielded
representatives of this genus. Semiquantitative comparisons of the numbers of Acinetobacter and of all bacteria
capable of aerobic growth in a complex medium revealed that Acinetobacter constituted no less than 0.001% of the
heterotrophic aerobic population in soil and water and was one of the predominant organisms in some water

Enumeration of Potentially Pathogenic Bacteria from Sewage Sludges
[list of bacteria found in sludge]
Hess and Breer (18) found Salmonella species in 90% of the sludges they examined and observed that the organisms
could survive for up to 72 weeks in sludges that had been applied to land. -- Such sludges could pose a significant
health risk through the contamination of vegetables, surface waters, and groundwaters. --
Mycobacterium tuberculosis was found by Jensen (21) in the sewage and digested sludge of towns with tuberculosis
sanitoria. The tubercle bacilli were found to survive for as long as 11 to 15 months in sludge on drying beds,  -- Greer
(15) observed densities of up to 107 Clostridium perfringens organisms per g in
sludge, but virtually none in the wastewater effluent.

The Evaluation of the Mutagenicity of Municipal Sewage Sludge
Mutagenic activity has been observed in sludges from Chicago and Sauget, Illinois. Sludges from primarily domestic
sewage do not apparently contain mutagins. The Tradescantia data clear indicate that whole or Chicago sludge
induces chromosome aberrations in meiotic cells. The aqueous phase is much less mutagenic than whole sludge and
it appears that a major portion of the mutagenic agents remain in the sludge solids. The in situ tests of sludge
amended soil using Chicago sludge and the maize wx locus assay demonstrates that mutagins are available to the
plant when sludge is used as a soil amendment. Toxic as well asmutagenic responses were observed. -- These
studies indicate that mutagens present in sludge-amended soil are transported into crop plant and can induce
genetic damage to germ sells......

Methods for the Detection of  Microorganisms in the Environment  
EPA 703-R-92-001 - 1992
Detection by Culturing
Not all microorganisms isolated from the environment will be culturable and thus detectable by these methods.
Markers conferring on antibiotic resistance can either be transferred to a host from another organism or obtained by
selection. One concern is the transfer of the antibiotic resistance determinants in situ to pathogenic microorganism or
the indigenous microbial population.
It has been pointed out that the use of microorganisms resistant to heavy metals in environmental studies
can contribute to spread of resistance in clinically important antibiotics as both are plasmid-borne.
Most Probable Number (MPN): This method is less precise than the plate count technique. Also, both viable count
procedure can be affected by microbial inhibition caused by other microbes present in the growth medium. Viable but
nonculturable microorganisms may not grow under conditions of MPN incubations. The lower level of reported
sensitivity is 10 cells per unit; however statistical variability can be relatively large, for example compared to the plate
count techniques, and may prevent detection of statistically significant differences.

A novel DNA-binding protein with regulatory and protective roles in starved Escherichia coli
The ability to maintain biological integrity during periods of starvation is essential for the survival of an organism.
Many microbes have evolved sophisticated developmental pathways that lead to the formation of highly resistant
differentiated cells in response to nutritional deprivation --  The starved Escherichia coli cell, in contrast to a dormant
spore, remains metabolically active and can quickly resume growth when the required nutrients are available (Matin
1991; Siegele and Kolter 1992). It is therefore not surprising that after the initial starvation induced switch in gene
expression, these cells maintain a low level of gene expression even after many days in stationary phase -- Growing
cells have the capacity to respond rapidly to changing environments by differentially expressing setsof genes. Thus, a
growing cell can cope with stresses that generate cellular damage by inducing the expression of a variety of repair
genes. When a microorganism finds itself in nutritionally limiting conditions, which in nature must be most of the time,
it must still be able to protect itself from environmental damage to assure its survival. A starved microorganism must
prepare in advance for the possibility that it may encounter environmental assaults, as its capacity for rapid response
is severely compromised. Many of the E. coli genes that are induced in response to starvation and whose expression
depends on cr s, play important roles in protecting the organism {Hengge-Aronis et al. 1991; Lange and Hengge-
Aronis 1991b; McCann et al. 1991).

Metabolic Activity of Pathogenic Bacteria during Semicontinuous Anaerobic Digestion
In natural environments such as anaerobic digesters, bacteria are frequently subjected to the stress of
nutrient fluxes because of the continual changes in the flow of nutrients, and to survive, they must be capable of
adapting readily to nutrient changes. In this study, the metabolic activities of Escherichia coli, Salmonella typhimurium,
Yersinia enterocolitica, Listeria monocytogenes, and Campylobacterjejuni were studied within culture bags  -- The
results show that the enteric bacteria investigated probably exist in transient states between different stages of growth
because of fluctuating nutrient levels during anaerobic digestion. -- Enteric bacteria such as Escherichia coli,
Salmonella enteritidis, and Campylobacter jejuni have a continual supply of nutrients within the mammalian gut.
However, it has been reported that these bacteria have a viable but nonculturable state under stressed conditions
which enables them to survive in low-nutrient environments such as soil and water

Application, Performance, and Costs of Biotreatment Technologies for Contaminated Soils
EPA/600/R-03/037, Sept. 2002
Microbial populations: Enumerating microorganisms from soils is by no means an exact science because of the
difficulty in removing the cells from the soil particles coupled with the fact that many soil microorganisms are simply
not culturable in the laboratory.

Statistical Analyses: Possible Reasons for Unreliability of Source Tracking Efforts
Analyses for the presence of indicator organisms provide information on the microbiological quality of water. Indicator
organisms recommended by the United States Environmental Protection Agency for monitoring the microbiological
quality of water include Escherichia coli, a thermotolerant coliform found in the feces of warm-blooded animals. These
bacteria can also be isolated from environmental sources such as the recreational and pristine waters of tropical rain
forests in the absence of fecal contamination. In the present study, E. coli isolates were compared to E. coli K12
(ATCC 29425) by restriction fragment length polymorphism using pulsed-field gel electrophoresis. Theoretically,
genomic DNA patterns generated by PFGE are highly specific for the different isolates of an organism and can be
used to identify variability between environmental and fecal isolates. Our results indicate a different band pattern for
almost every one of the E. coli isolates analyzed. Cluster analysis did not show any relations between isolates and
their source of origin. Only the discriminant function analysis grouped the samples with the source of origin. The
discrepancy observed between the cluster analysis and discriminant function analysis relies on their mathematical
basis. Our validation analyses indicate the presence of an artifact (i.e., grouping of environmental versus fecal
samples as a product of the statistical analyses used and not as a result of separation in terms of source of origin) in
the classification results; therefore, the large genetic heterogeneity observed in these E. coli populations makes the
grouping of isolates by source rather difficult, if not impossible.
Applied and Environmental Microbiology, August 2005, p. 4690-4695, Vol. 71, No. 8

Staphylococcus aureus ClpC Is Required for Stress Resistance, Aconitase Activity, Growth Recovery, and Death
The ability of Staphylococcus aureus to adapt to various conditions of stress is the result of a complex
regulatory response. Previously, it has been demonstrated that Clp homologues are important for a variety of
stress conditions, and our laboratory has shown that a clpC homologue was highly expressed in the S. aureus strain
DSM20231 during biofilm formation relative to expression in planktonic cells. Persistence and longterm survival are a
hallmark of biofilm-associated staphylococcal infections, as cure frequently fails even in the presence of bactericidal
antimicrobials.  --- Staphylococcus aureus is a major human pathogen (33) that is unabated as a significant cause of
morbidity and mortality in community-acquired and nosocomial infections. One reason contributing to its endemic
importance is thought to be the organism’s marked ability to survive under adverse environmental conditions --  
Additionally, many of the commonly utilized laboratory strains fail to form a biofilm (9), an important mechanism for
causing disease. --

- All are heterotrophic pathogenic Etiologic Agents
-- Also see Etiologic Agents  Viruses - Helminths - Protozoa - Fungus

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