Regrowth -- Reactivation of Starved, Desiccated or Chemical/Heat
Injured Bacteria                                                          47+ studies
                 AKA   Viable But Nonculturable (VBNC)

Regrowth is the result of viable starved, desiccated  or injured bacteria being resuscitated by
nutrients, moisture or DNA repair as noted in the following studies. Remember that the EPA Standard
1681 is designed to test for <5% of E. coli and Klebsiella (
fecal coliform) that will grow at  44.5°C.

1971 --
Growth Rate of Escherichia coli at Elevated Temperatures: Limitation by Methionine
When Escherichia coli growing in minimal medium is shifted from 37 C to any temperature in the
range 40 to 45 C, the growth rate immediately assumes a new, lower value, characteristic of that
temperature. The decrease is shown to be due, in several strains, to decreased activity of the first
enzyme of the methionine pathway, homoserine trans-succinylase, which thus appears to be more
heat-sensitive than any other essential enzyme in the cell. This sensitivity does not involve
progressive denaturation of the enzyme; rather, the response to a shift of temperature, in either
direction, is immediate and reversible.
J Bacteriol. 1971 August; 107(2): 391-396
http://jb.asm.org/cgi/content/abstract/107/2/391

1971 -- Growth Rate of Escherichia coli at Elevated Temperatures: Reversible Inhibition of
Homoserine Trans-Succinylase
The preceding paper (10) showed that the growth of Escherichia coli is slowed, without killing, at 40
to 45 C, and that in the several strains tested the cause is a decrease in the activity of homoserine
trans-succinylase. These temperatures are now shown to inhibit the enzyme directly, in crude
extracts and after partial purification. The effect is rapid and is immediately reversible, unlike the
progressive and slowly reversible changes of conventional denaturation.
JOURNAL OF BACTERIOLOGY, Aug. 1971, p. 397-400
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC246938/pdf/jbacter00369-0023.pdf

1972 -- Synchronization of Cell Division in Escherichia coli by Elevated Temperatures: a
Reinterpretation
When an exponentially growing culture was transferred from 37 to 43.8 C, the growth rate was
reduced by about 80% (4); a shift back to 37 C after 16 min at the higher temperature resulted in a
synchronization of cell division
JOURNAL OF BACITROLOGY, Mar. 1972, p. 1316-1318, Vol. 109, No. 3
http://jb.asm.org/cgi/reprint/109/3/1316.pdf

1973 -- EPA and USDA acknowledged liming sludge does not kill salmonella. The original numbers
of Salmonella reappear in about 30 days.
http://thewatchers.us/science/properties-sludge-8.html

1973 -- Regrowth of coliforms and fecal coliforms in chlorinated wastewater effluent.
Observations made both in the field in chlorinated effluent, and in laboratory experiments show that
coliforms are capable of regrowth in chlorinated wastewater. Under field conditions regrowth of
coliforms in chlorinated effluent held in a storage reservoir for similar to 3 days appeared inversely
correlated to (1) The residual chlorine in the storage reservoir and (2) the number of coliforms
surviving chlorination. In the laboratory experiments regrowth occurred after initial doses as high as
11 ppm total chlorine even when there was no chemical inactivation of the chlorine. Fecal
coliforms were not detectable in 10-ml of samples after chlorination. Since coliforms and fecal
coliforms are capable of regrowth in chlorinated sewage effluent and admixtures of it, the sanitary
significance of the number of coliforms after storage or in receiving bodies of water is difficult to
interpret. Thus standards might be based on the number of coliforms, or fecal coliforms detected in
effluent s immediately after chlorination. However, this would not be justified if in addition to
coliforms, pathogenic bacteria can regrow in chlorinated effluents.
Water Research [Water Res.]. Vol. 7, no. 4, pp. 537-546. 1973.
http://md1.csa.com/partners/viewrecord.php?
requester=gs&collection=ENV&recid=5193474&q=fecal+coliform+effluent&uid=1020137&setcookie=yes

1979 --
Mechanism of growth delay induced in Escherichia coli by near ultraviolet radiation
ABSTRACT Continuously growing cultures of E. coli B/r were irradiated with a fluence of broad-band
near-ultraviolet radiation (315-405 nm) sufficient to cause extensive growth delay and complete
cessation of net RNA synthesis. Chloramphenicol treatment was found to stimulate resumption of
DNA synthesis, similar to that observed with chloramphenicol treatment after amino-acid starvation.
E. coli strains in which amino-acid starvation does not result in cessation of RNA synthesis ("relaxed"
or ret strains) show no cessation of growth and only a slight effect on the rate of growth or of RNA
synthesis. These findings show that such near-UV fluences do not inactivate the RNA synthetic
machinery but affect the regulation of RNA synthesis, in a manner similar to that produced by amino-
acid starvation. Such regulation is believed to be mediated through alterations in concentration of
guanosine tetraphosphate (ppGpp), and our estimations of ppGpp after near-UV irradiation are
consistent with such an interpretation. These data, combined with earlier published data, strongly
suggest that the mechanism of near-UV-induced growth delay in E. coil involves partial inactivation
of certain tRNA species, which is interpreted by the cell in a manner similar to that of amino-acid
starvation, causing a rise in ppGpp levels, a shut-off of net RNA synthesis, and the induction of a
growth delay.
Proc. Nat. Acad. Sd. Vol. 73, No. 1, pp. 59-63, January 1976
http://www.pnas.org/content/73/1/59.full.pdf

1979 -- FECAL COLIFORM AND E. COLI ESTIMATES, TIP OF THE ICEBERG
Pure cultures of E. coli, Klebsiella and Enterobacter, obtained from hospital patients and from
natural waters were tested for their growth patterns by spread plate and membrane filtration
procedures at the following temperatures; 35 °, 41.5 °, 43 °, 44.5 °, and 35°C for 4 h followed by 18
h at 44.5°C.
Results indicated that 44.5°C incubation produces the lowest population
estimate and that the application of the membrane filtration technique also reduced the
potential population.
Three water samples collected during June, August and November were
tested for fecal coliform and E. coli populations, with 11 different media (broth and agar) and
incubation temperatures of 35 °, 41.5 °, 43 °, 44.5 °, and 35°C for 4 h followed by 18 h at 44.5°C.
During the study, isolates were collected from all positive MPN tubes at each temperature and from
each MF medium-temperature regime, 24 to 50 isolates were collected. From the isolate data
corrected coliform (oxidase negative), fecal coliform and E. coli population estimates were made.
A sample of feces was diluted in lake water and maintained at 20°C for 56 days. Samples were
collected at various times and tested for fecal coliform densities using five media and the same
temperature regime as for the lake water samples. Data from these studies indicate that, depending
on the age of the population being measured, the temperature of the water sample, and the
temperature-media-procedure combination used, fecal coliform and E. coli population estimate
techniques measure from 5 to 100% of the potential population.
http://www.springerlink.com/content/vr0uq8q480078448/

1981 -- Unique Temperature-Sensitive Nutritional Requirements of Bacteremic Escherichia coli
Isolates
Of 50 strains of Escherichia coli isolated from blood cultures of bacteremic patients, 14 (28%) were
unable to grow on minimal medium at 42°C, compared to only 2 of 50 nonbacteremic strains. In 7 of
the 14 bacteremic strains, growth at 42°C was restored by adding nicotinic acid. These unique
temperature-sensitive auxotrophic patterns warrant evaluation as a marker correlating with clinical
pathogenicity in E. coli.
JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1981, p. 606-608 Vol. 13, No. 3
http://jcm.asm.org/cgi/reprint/13/3/606.pdf

1981 -- Factors Affecting Salmonellae Repopulation in Composted Sludges
The repopulation potential and recovery of Salmonella sp. and their close relatives Arizona spp. and
Citrobacter spp. in sewage sludge which had been composted was examined. Salmonellae growth in
previously composted sludge was found to occur in the mesophilic temperature range (20 to 40
degC), require a moisture content of -20%, and require a carbon/nitrogen ratio in excess of 15:1.
These results also indicated that some enteric bacteria, upon desiccation, became
dormant and in this state were highly resistant to both heat and radiation.
Optimal recoveries in the low bacteria sample occurred at the 21% moisture level at 28 to 36 degC
after a 5-day incubation. The population increased more than four orders of magnitude under these
conditions.
The indigenous salmonellae initiating this growth had survived in a desiccated
state for over 1 year prior to providing the proper moisture-temperature combination for
the repopulation to occur
. --- as long as a demonstrated potential exists for repopulation of
salmonellae in a commercial soil amendment product produced from composted sludge, a potential
health hazard exists for the user.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1981, p. 597-602
http://thewatchers.us/EPA/2/1981-salmonella-regrowth-compost.pdf

1984 --
Effect of chlorination on antibiotic resistance profiles of sewage-related bacteria.
A total of 1,900 lactose-fermenting bacteria were isolated from raw sewage influent and chlorinated
sewage effluent from a sewage treatment plant, as well as from chlorinated and neutralized dilute
sewage, before and after a 24-h regrowth period in the laboratory. Of these isolates, 84% were
resistant to one or more antibiotics. Chlorination of influent resulted in an increase in the proportion
of bacteria resistant to ampicillin and cephalothin, the increase being most marked after regrowth
occurred following chlorination. Of the other nine antibiotics tested, chlorination resulted in an
increased proportion of bacteria resistant to some, but a decrease in the proportion resistant to the
remainder. Multiple resistance was found for up to nine antibiotics, especially in regrowth
populations. Identification of about 5% of the isolates showed that the highest proportion of
Escherichia coli fell in untreated sewage. Some rare and potentially pathogenic species were
isolated from chlorinated and regrowth samples, including Yersinia enterocolitica, Yersinia pestis,
Pasteurella multocida, and Hafnia alvei. Our results indicate that chlorination, while initially lowering
the total number of bacteria in sewage, may substantially increase the proportions of antibiotic-
resistant, potentially pathogenic organisms.
Appl Environ Microbiol. 1984 July; 48(1): 73–77.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC240314/pdf/aem00152-0081.pdf

1985 -- Viable but Non-Culturable Vibrio cholerae and Related Pathogens in the Environment:
Implications for Release of Genetically Engineered Microorganisms
In a series of microcosm and field studies, we have observed that over time V. cholerae and related
human pathogens enter a viable but non-culturable state. Direct viable counts by epifluorescent
microscopy are consistently higher than corresponding plate counts. Thus, the assumption that
microorganisms, including pathogens, "die-off" or "decay" in the marine environment must be re-
evaluated, since stressed or nutrient starved cells are unable to grow and be enumerated by
standard plate count methods. Furthermore, animal passage reveals pathogenicity persisting for
such "non-viable"cells. Indirect immunofluorescent microscopy offers a more sensitive detection
system for environmental sampling for human pathogens and therefore, a more valid estimation of
population size. One implication of these findings for the release of genetically engineered
organisms is that highly specific methods of detection and monitoring are required, with direct
detection by fluorescent antibody the most reliable at the present time.
Bio/Technology  3, 817 - 820 (1985)
http://www.nature.com/nbt/journal/v3/n9/abs/nbt0985-817.html

1986 -- Viable but nonculturable stage of Campylobacter jejuni and its role in survival in the natural
aquatic environment.
Conditions influencing the survival of Campylobacter jejuni in the natural aquatic environment have
been determined. Release of Campylobacter spp. into natural waters by animal hosts is postulated
to play a key role in the maintenance of viability and transmission of the organism in the
environment. Laboratory flask microcosms containing filter-sterilized stream water were used to test
C. jejuni for the ability to remain viable in simulated natural systems. The microcosms were
compared with the biphasic and shaking broth procedures used routinely for growth of
Campylobacter spp. in the research laboratory. The stream-water microcosms were analyzed to
determine effects of temperature and aeration on the survival of a well-characterized C. jejuni strain
isolated originally from a human campylobacteriosis patient. Morphological characteristics were
evaluated by phase-contrast microscopy and scanning or transmission electron microscopy. Survival
curves were quantified on the basis of plate counts, epifluorescent microscopy, optical density
measurements, and direct viable counts associated with protein synthesis in the absence of DNA
replication. A significant difference was observed between results of direct enumeration, i.e., direct
viable counts or acridine orange direct counts, and those from spread plate cultures. In all cases,
increasing temperature of cultivation resulted in decreased recoverability on laboratory media, due
possibly to an increased metabolic rate, as analyzed by CO2 evolution in the presence of
radiolabeled glutamate. Stream water held at low temperature (4 degrees C) sustained significant
numbers of campylobacters for greater than 4 months. Microcosms, aerated with shaking, exhibited
logarithmic decline in recoverable C. jejuni, while stationary systems underwent a more moderate
rate of decrease to the nonculturable state.(
Appl Environ Microbiol. 1986 September; 52(3): 531-538
http://aem.asm.org/cgi/content/abstract/52/3/531

1987 -- Survival Strategies of Bacteria in the Natural Environment
It is useful to review the observations reported during the past two decades that relate to the viable
but nonculturable state. Jannasch (74) differentiated between those bacteria that merely survive and
those that grow in marine waters. Nearly a decade later, Fliermans and Schmidt (46) combined
autoradiography and immunofluorescence methods to identify metabolic activity of individual
Nitrobacter cells that did not respond to laboratory culture. Dawes (33) interpreted endogenous
metabolism as a survival strategy of microorganisms that endure for significant periods of time in the
absence of nutrients. Hoppe (71) employed autoradiographic methods to demonstrate that free-
living bacteria that did not form colonies on routinely employed bacteriological media were
responsible, nevertheless, for the active metabolism of organic substrates
MICROBIOLOGICAL REVIEWS, Sept. 1987, p. 365-379 Vol. 51, No. 3
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC373117/pdf/microrev00050-0073.pdf

1987 -- Regrowth of Salmonellae in Composted Sewage Sludge
decrease may only be temporary because this pathogen can survive and grow without a human host.
[
the implication is that regrowth may be contributed to recontamination by birds]
http://thewatchers.us/EPA/1/1987-Salmonella-regrowth-compost.pdf

1988 --
Occurrence of Pathogens in Distribution and Marketing Municipal Sludges
[Class A Sludge -- aka Biosolids]
"Although the use of sludge as a soil amendment is attractive, it is not without potential health risks.
Toxic chemicals, including heavy metals and industrial organics, may enter the food chain and
present long-term health risks." The plague causing bacteria Yersinia (pestis?) was consistently
found in static pile compost. CDC authorities state, "Outbreaks in people still occur in rural
communities or in cities." significant increases in bacterial populations, including salmonellae,
occurred during subsequent production of commercial soil amendment products.
http://deadlydeceit.com/D_M_sludge.html

1991 -- Viable but nonculturable bacteria in drinking water.
Klebsiella pneumoniae, Enterobacter aerogenes, Agrobacterium tumefaciens, Streptococcus
faecalis, Micrococcus flavus, Bacillus subtilis, and Pseudomonas strains L2 and 719 were tested for
the ability to grow and maintain viability in drinking water. Microcosms were employed in the study to
monitor growth and survival by plate counts, acridine orange direct counts (AODC), and direct viable
counts (DVC). Plate counts dropped below the detection limit within 7 days for all strains except
those of Bacillus and Pseudomonas. In all cases, the AODC did not change. The DVC also did not
change except that the DVC, on average, were ca. 10-fold lower than the AODC.
Appl Environ Microbiol. 1991 March; 57(3): 875-878
http://aem.asm.org/cgi/content/abstract/57/3/875


1991 -- Survival of pathogenic micro-organisms and parasite in excreta, manure and sewage sludge
most pathogenic agents can survive the treatment process" and the sewage treatment process
causes some of the pathogenic disease organisms to be absorbed or enclosed in faecal particles
during the treatment process. "Therefore," according to Strauch, "sewage sludge is rightly described
as a concentration of pathogens." the study " reported that two groups of researchers had found
that pathogenic disease organisms will be taken up inside the food crops. -- Salmonella has survived
in forest stands between 424 and 820 days -- "In any case, the agricultural utilization of hygienically
dubious sewage sludge poses a risk for the whole national economy"
http://www.ncbi.nlm.nih.gov/pubmed/1782431

1994 -- Luminescence-based detection of activity of starved and viable but nonculturable bacteria.
ABSTRACT: A naturally luminescent bacterium, Vibrio harveyi, and two bacteria, Escherichia coli
and Pseudomonas fluorescens, which had been genetically marked with luminescence were starved
in liquid medium at 4 and 30 degrees C for 54 days. Total cell concentrations and concentrations of
culturable and viable cells were determined by acridine orange staining, dilution plate counting, and
direct viable counting, respectively, and population activity was measured by luminometry. V. harveyi
became nonculturable but maintained viability during starvation at 4 degrees C and maintained both
culturability and viability at 30 degrees C. In contrast, E. coli became viable but nonculturable during
starvation at 30 degrees C but not at 4 degrees C. Luminescence of nonculturable cells of both
strains, and culturable cells of V. harveyi, decreased to background levels during starvation.
Luminescence of starved culturable cells of E. coli also fell below background levels but occasionally
increased to detectable values. Viable, nonculturable forms of P. fluorescens were not detected at
either temperature, and cells starved at 4 degrees C showed no decrease in luminescence
measured during incubation of samples at 25 degrees C. Following incubation of late-log-phase cells
with yeast extract and nalidixic acid, changes in light output directly paralleled changes in cell length,
as observed during direct viable counting. Quantification of changes in luminescence following
incubation of starved cells with yeast extract enabled measurement of the activity of both culturable
and viable but nonculturable cells. Measurement of luminescence was significantly more sensitive,
rapid, and convenient in quantifying activity following nutrient amendment than measurement of
changes in cell length
Appl Environ Microbiol. 1994 April; 60(4): 1308-1316
http://aem.asm.org/cgi/content/abstract/60/4/1308


1994 -- PHYSIOLOGY AND MOLECULAR GENETICS OF VIABLE BUT NON-CULTURABLE
MICROORGANISMS
Gram-negative bacteria are known to enter a viable but non-culturable (VBNC) state, in which they
no longer grow on conventional media, but remain intact and retain viability. This phenomenon has
been regarded, in many ways, to be analogous to sporulation in Gram-positive bacteria. We have
investigated the presence of spoO-like genes in Vibrio cholerae and Escherichia coli by polymerase
chain reaction using primers based on conserved regions of Bacillus subtilis spoOA and spoOF
genes. We did not identify regions of V. cholerae or E. coli that exhibited complete homology to
spoO genes from B. subtilis and, therefore, concentrated on an alternate approach of transposon
mutagenesis. Over 2,500 transposon mutants of V. cholerae were screened under low nutrient
conditions in artificial sea water for an altered VBNC response, compared to the wild type. Mutant
JR09H1 entered the VBNC state more rapidly than the wild type at both 25oC and 4oC.
http://www.isb.vt.edu/brarg/brasym94/chowd.htm

1996 -- "Pathogen Destruction and Biosolids Composting" in Biocycle of June of 1996,
"There is some evidence that coliforms and Salmonella sp. can survive prolonged exposure to
temperatures of 55 C." They cite a study done by Droffner and Brinton (1995) using DNA gene
probes, where they detected E. coli and Salmonella sp. in samples collected from an in-vessel
composting facility after the first 15 days of active composting at
a temperature above 55 C. In Table 5-4 Processes to Further Reduce Pathogens in A Plain English
Guide to the EPA Part 503 Biosolids Rule, composting time and temperature requirements for within-
vessel composting method was 55 C or higher for three days!
Droffner and Brinton found that it
took 56 days and 90 days for the densities of Salmonella sp. and E. Coli, respectively, to
decline below the detection limit
...These investigators also "cite evidence of mutant strains of E.
coli and Salmonella sp. resistant to thermal environments in composting." (p. 68)

The Study
MICROBIAL APPROACHES TO CHARACTERIZATION OF COMPOSTING PROCESSES
"this data also indicates that under certain conditions, thermo-tolerance spreads rapidly to
organisms not believed to be heat resistant. The dissemination of thermal tolerance may take place
through similar, complicated mechanisms as are responsible for the increase among bacteria of
antibiotic resistance."

"We have demonstrated, however, that the genera Escherichia, Salmonella and Pseudomonas all
have the capacity to produce mutants able to grow at elevated temperatures."
Compost Science & Utilization, Summer, 1994
http://woodsend.org/pdf-files/microbia.pdf

1997 -- Regrowth of faecal coliforms and salmonellae in stored biosolids and soil amended with
biosolids
Stabilised wastewater sludge (biosolids) has beneficial re-use properties but these are limited by the
presence of human pathogens. In two storage trials biosolids were stored in piles 1m high and
monitored for <60 weeks. Included in the monitoring programme were tests to determine the
concentrations of faecal coliforms, faecal streptococci and salmonellae. In both the soil amendment
trials and biosolids storage trials,
concentrations of indicator organisms and salmonellae
decreased through an extended hot, dry summer period
. Although these organisms were not
detected in the majority of samples taken during the summer,
repopulation of faecal coliforms
and salmonellae occurred in the trials following rainfall at the beginning of the winte
r. In
the case of one of the storage trials repopulation occurred following a period of 50 weeks when
salmonellae and faecal coliforms were not detected. When repopulation occurred, faecal coliform
concentrations increased to higher than those at the beginning of
the trials.
These results suggest that faecal coliforms and salmonellae were at
undetectable concentrations through the summer period but were able to grow when
provided with favourable conditions.
From this limited trial it was concluded that soil amended
with biosolids could not be considered free from pathogens for at least one year following
amendment.
Water Science and Technology Vol 35 No 11-12 pp 269–275 © IWA Publishing 1997
http://www.iwaponline.com/wst/03511/wst035110269.htm


1997 -- Resuscitation of Vibrio vulnificus from the Viable but Nonculturable State
Like many other gram-negative bacteria, the human pathogen Vibrio vulnificus is induced into a
viable but nonculturable (VBNC) state by incubation at low temperatures. The ability of any
bacterium to resuscitate from this dormant state would appear to be essential if the VBNC state is
truly a survival strategy. The question as to whether the culturable cells which appear following
removal of the inducing stress are a result of true resuscitation or of regrowth of a few residual
culturable cells has long been debated. V. vulnificus was examined for its ability to resuscitate from
this state following a temperature upshift. Several lines of investigation, including dilution studies,
determination of the time necessary for appearance of a culturable population, and the effects of
nutrient on recovery, all indicated that, at least for V. vulnificus, true resuscitation does occur. Our
studies further suggest that nutrient is in some way inhibitory to the resuscitation of cells in the VBNC
state and that studies which add nutrient in an attempt to detect resuscitation are able to detect only
residual culturable cells which might be present and which were not inhibited by the added nutrient.
Appl. Environ. Microbiol., Mar 1997, 1002-1005, Vol 63, No. 3
http://aem.asm.org/cgi/content/abstract/63/3/1002

1998 -- Enterococcus faecalis Gene Transfer under Natural Conditions in Municipal Sewage Water
Treatment Plants†
The ability of Enterococcus faecalis to transfer various genetic elements under natural conditions
was tested in two municipal sewage water treatment plants. Experiments in activated sludge basins
of the plants were performed in a microcosm which allowed us to work under sterile conditions;
experiments in anoxic sludge digestors were performed in dialysis bags. We used the following
naturally occurring genetic elements: pAD1 and pIP1017 (two so-called sex pheromone plasmids
with restricted host ranges, which are transferred at high rates under laboratory conditions); pIP501
(a resistance plasmid possessing a broad host range for grampositive bacteria, which is transferred
at low rates under laboratory conditions); and Tn916 (a conjugative transposon which is transferred
under laboratory conditions at low rates to gram-positive bacteria and at very low rates to gram-
negative bacteria). The transfer rate between different strains of E. faecalis under natural
conditions was, compared to that under laboratory conditions, at least 105-fold lower for the sex
pheromone plasmids, at least 100-fold lower for pIP501, and at least 10-fold lower for Tn916. In no
case was transfer from E. faecalis to another bacterial species detected. By determining the
dependence of transfer rates for pIP1017 on bacterial concentration and extrapolating to actual
concentrations in the sewage water treatment plant, we calculated that the maximum number of
transfer events for the sex pheromone plasmids between different strains of E. faecalis in the
municipal sewage water treatment plant of the city of Regensburg ranged from 105 to 108 events
per 4 h, indicating that gene transfer should take place under natural conditions.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1998, p. 626–632 Vol. 64, No. 2
http://aem.asm.org/cgi/reprint/64/2/626.pdf

1998 --  Starvation recovery of Staphylococcus aureus 8325-4
Nutrient limitation of Staphylococcus aureus induces a starvation-survival state which enables it to
survive until sufficient nutrients become available to support growth. The response of starved S.
aureus cells to nutritional upshift was analysed to characterize the recovery mechanism which results
in the resumption of rapid growth. S. aureus 83254 starved for 7 d in a chemically defined medium
limited for glucose was able to resume growth upon the addition of complex medium (brain heart
infusion broth) or a mixture of amino acids and glucose. The addition of either glucose or amino
acids alone did not lead to recovery of cells. Prior to the first cell division event, a lag period of about
120-150 min was observed, the duration of which was independent of the length of starvation
survival. During this lag period, RNA synthesis increased immediately upon the addition of nutrients
whilst protein synthesis was delayed by approximately 5 min. Cells rapidly enlarged within 30 min of
recovery, and initiation of chromosome replication could be detected after 90 min. Changes in the
profile of proteins expressed during the recovery period revealed that several starvation-specif ic
proteins were downregulated within 30 min, whilst other proteins were common to both starvation
and recovery. Two proteins were identified which were only transiently expressed during the first 60
min of recovery. Protein synthesis could be detected during recovery even if the cells had been
treated with the RNA synthesis inhibitor rifampicin for 30 min prior to the addition of recovery
nutrients, demonstrating that several proteins are translated from long-lived
mRNA transcripts present in starved cells.
Microbiology (1 998), 144, 1755-1 763
http://mic.sgmjournals.org/cgi/reprint/144/7/1755.pdf

1998 -- Effects of Starvation on Physiological Activity and Chlorine Disinfection Resistance in
Escherichia coli O157:H7
Escherichia coli O157:H7 can persist for days to weeks in microcosms simulating natural conditions.
In this study, we used a suite of fluorescent, in situ stains and probes to assess the influence of
starvation on physiological activity based on membrane potential (rhodamine 123 assay), membrane
integrity (LIVE/DEAD BacLight kit), respiratory activity (5-cyano-2,3-di-4-tolyl-tetrazolium chloride
assay), intracellular esterase activity (ScanRDI assay), and 16S rRNA content. Growth-dependent
assays were also used to assess substrate responsiveness (direct viable count [DVC] assay), ATP
activity (MicroStar assay), and culturability (R2A agar assay). In addition, resistance to chlorine
disinfection was assessed. After 14 days of starvation, the DVC values decreased, while the values
in all other assays remained relatively constant and equivalent to each other. Chlorine resistance
progressively increased through the starvation period. After 29 days of starvation, there was no
significant difference in chlorine resistance between control cultures that had not been exposed to
the disinfectant and cultures that had been exposed. This study demonstrates that E. coli O157:H7
adapts to starvation conditions by developing a chlorine resistance phenotype.
Appl Environ Microbiol. 1998 December; 64(12): 4658–4662.
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=90905

1998 -- Characterization of the Starvation-Survival Response of Staphylococcus aureus
The starvation-survival response of Staphylococcus aureus as a result of glucose, amino acid,
phosphate, or multiple-nutrient limitation was investigated. Glucose and multiple-nutrient limitation
resulted in the loss of viability of about 99 to 99.9% of the population within 2 days. The remaining
surviving cells developed increased survival potential, remaining viable for months. Amino acid or
phosphate limitation did not lead to the development of a stable starvation-survival state, and cells
became nonculturable within 7 days. For multiple-nutrient limitation, the development of the
starvation-survival state was cell density dependent. Starvation survival was associated with a
decrease in cell size and increase in resistance to acid shock and oxidative stress. There was no
evidence for the formation of a viable but nonculturable state during starvation as demonstrated by
flow cytometry. Long-term survival of cells was dependent on cell wall and protein biosynthesis.
Analysis of [35S]methionine incorporation and labelled proteins demonstrated that differential
protein synthesis occurred deep into starvation.
J Bacteriol, April 1998, p. 1750-1758, Vol. 180, No. 7
http://jb.asm.org/cgi/content/abstract/180/7/1750

1999 -- Distibution of viable but non-culturable bacteria in wastewater treatment systems.
Abstract;Application of molecular methods to natural ecosystems has revealed the occurrence of
many unexpected phylogenetic groups of prokarytoes which have not yet been isolated as cultivable
strains. This is true for biological waste water treatment systems. Plate bacterial counts accounted
for 1 to 18% of total (DAPI) counts in municipal sewage activated sludge. Microscopic, molecular
genetic, and chemical biomarker approaches have suggested that the failure to detect many
bacteria as viable counts results from the occurrence of viable but non-culturable bacteria and from
the existence and diversity of bacteria undescribed so far.
Microbes Environ VOL.14;NO.2;PAGE.91-99(1999)
http://sciencelinks.jp/j-east/article/199919/000019991999A0591504.php

2002 -- A comparison of solid and liquid media for resuscitation of starvation- and low-temperature-
induced nonculturable cells of Aeromonas hydrophila
Like many other gram-negative bacteria, starved cells of Aeromonas hydrophila can be induced into
a viable but nonculturable (VBNC) state by incubation at low temperature, as shown here by using
various bacterial enumeration methods. Starved A. hydrophila strain HR7 cells at 4 °C reached the
nonculturable stage in about 45 days. The cells were resuscitated by either a solid medium
resuscitation method, using solid agar amended with H2O2-degrading agents, catalase or sodium
pyruvate, or a liquid medium resuscitation method, by incubating nonculturable cells in liquid media
containing these compounds before spreading onto plates. The liquid medium resuscitation method
using catalase resulted in nearly complete recovery of nonculturable cells.
Journal Archives of Microbiology Volume 173, Number 4 / March, 2000
http://www.springerlink.com/content/mhgpgpg2wmbewvf5/

2003 -- US Patent 6589771 - Methods for arousing dormant bacteria
http://www.patentstorm.us/patents/6589771/claims.html

2003  -- The effect of free chlorine on Burkholderia pseudomallei in potable water
Chlorine is widely used in public water supplies to provide a disinfection barrier. The effect of
chlorine disinfection on the water-borne pathogen Burkholderia pseudomallei was assessed using
multiple techniques. After exposure to chlorine viable bacteria were undetectable by conventional
plate count techniques; however, persistence of B. pseudomallei was verified by flow cytometry and
bacteria were recoverable following a simple one-step broth procedure. The minimum residual
chlorine concentration and contact time as prescribed by potable water providers in Australia was
insufficient to reduce a B. pseudomallei population by more than 2 log10. Chlorine had a
bacteriostatic effect only on B. pseudomallei; viable bacteria were recovered from water containing
up to 1000 ppm free chlorine. This finding has practical implications for water treatment in regions
where B. pseudomallei is endemic. Future work to assess the effect of alternative water disinfection
processes either singly or in sequence is necessary.
Water Research, Volume 37, Issue 18, November 2003, Pages 4425-4432
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V73-49G5NT1-1&_user=10&_coverDate=11%2F30%
2F2003&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1180034791&_rerunOrigin=google&_a
cct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=f241a1251956513fe462e2467a0ac818

2004 -- Confirmation of the presence of viable but non-cultureable bacteria in the stratosphere
The presence of viable, but non-cultureable, bacteria on membranes through which stratospheric air
samples were passed has been confirmed using viable fluorescent staining.
International Journal of Astrobiology 00 (00): 1–3 (2004)
http://eprints.whiterose.ac.uk/1555/1/wainrightm2.pdf

2004 --

2005 --
The Viable but Nonculturable State in Bacteria
It had long been assumed that a bacterial cell was dead when it was no longer able to grow on
routine culture media. We now know that this assumption is simplistic, and that there are many
situations where a cell loses culturability but remains viable and potentially able to regrow. This mini-
review defines what the "viable but nonculturable" (VBNC) state is, and illustrates the methods that
can be used to show that a bacterial cell is in this physiological state. The diverse environmental
factors which induce this state, and the variety of bacteria which have been shown to enter into the
VBNC state, are listed. In recent years, a great amount of research has revealed what occurs in cells
as they enter and exist in this state, and these studies are also detailed. The ability of cells to
resuscitate from the VBNC state and return to an actively metabolizing and culturable form is
described, as well as the ability of these cells to retain virulence. Finally, the question of why cells
become nonculturable is addressed. It is hoped that this mini-review will encourage researchers to
consider this survival state in their studies as an alternative to the conclusion that a lack of
culturability indicates the cells they are examining are dead.
The Journal of Microbiology, February 2005, p.93-100 Vol. 43, special issue (No. S)
http://www.ncbi.nlm.nih.gov/pubmed/15765062

2005 -- Viable but non-culturable bacteria: their impact on public health
The viability of a bacterial cell was traditionally determined by its ability to grow and produce
colonies. However, in recent years, many studies have revealed the ability of both Gram-positive and
Gram-negative bacteria to go into a viable but non-culturable (VBNC) state. In this state, bacteria
are still viable and show metabolic activity
and respiration, but cannot be shown as colony forming units by the conventional plate counts and
hence remain hidden1.
CURRENT SCIENCE, VOL. 89, NO. 10, 25 NOVEMBER 2005 p. 1650
http://www.ias.ac.in/currsci/nov252005/1650.pdf

2005 -- Potential Regrowth and Recolonization of Salmonellae and Indicators in Biosolids and
Biosolid-Amended Soil
This study evaluated the potential for conversion of Class B to Class A biosolids with respect to
salmonellae and fecal coliforms during solar drying in concrete lined drying beds. Anaerobically (8%
solids) and aerobically (2% solids) digested Class B biosolids were pumped into field-scale drying
beds, and microbial populations and environmental conditions were monitored.
Numbers of fecal
coliforms and salmonellae decreased as temperature and rate of desiccation increased
.
After 3 to 4 weeks, Class A requirements were achieved in both biosolids for the pathogens and the
indicators.
However, following rainfall events, significant increase in numbers was
observed for both fecal coliforms and salmonellae.
In laboratory studies, regrowth of fecal
coliforms was observed in both biosolids and biosolid-amended soil, but the regrowth of salmonellae
observed in the concrete-lined drying beds did not occur. These laboratory studies demonstrated
that pathogens decreased in numbers when soil was amended with biosolids. Based on serotyping,
the increased numbers of salmonellae seen in the concrete lined drying beds following rainfall
events was most likely due to recolonization due to contamination from fecal matter introduced by
animals and not from regrowth of salmonellae indigenous to biosolids. Overall,
we conclude that
the use of concrete-lined beds created a situation in which moisture added as rainfall
accumulated in the beds, promoting the growth of fecal coliforms and salmonellae
added
from external sources.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2005, p. 3701–3708 Vol. 71, No. 7
http://aem.asm.org/cgi/reprint/71/7/3701.pdf

2006
EPA acknowledges that Biosolids Compost may be hazardous to your Health in the 2006

Biosolids Technology Fact Sheet: Use of Composting for Biosolids Management
Potential environmental impacts may result from both composting operations and use
of the compost product
Composting is not a sterilization process and a properly composted product maintains an active
population of beneficial microorganisms that compete against the pathogenic members. Under some
conditions, explosive regrowth of pathogenic microorganisms is possible.
In addition to odors, other bioaerosols, such as pathogens, endotoxins, and various
volatile organic compounds, must also be controlled
Survival and presence of primary pathogens in the product.
Dispersion of secondary pathogens such as Aspergillus fumigatus, particulate matter, other airborne
allergens
The spores of A. fumigatus counts at composting facilities are high, and-- persons handling
composted biosolids being exposed to these spores is also high (Epstein, 1998).
While healthy individuals may not be affected, immunocompromised individuals may be at risk.
These organisms can potentially invade a normal, healthy human being and produce illness or
debilitation
http://deadlydeceit.com/EPA_Compost.html

2006 -- WEF: Study Finds Fecal Coliforms Appear to Reactivate in Centrifuge Dewatered Solids At
Four of Seven Facilities Tested
Based on the data collected in this study, researchers determined that more coliforms may have
actually been present after anaerobic digestion but were not picked up by the standard culturing
method. Researchers suspect that certain configurations of anaerobic digestion processes lead to
conditions which inhibit the ability of the coliforms to grow and be measured by SCMs. In other
words, indicator organisms may "hibernate" and become "non-culturable." This phenomenon is
referred to as "viable but non-culturable". The issue of viable but non-culturable (VBNC) bacteria
was advanced in the 1980s, and gained significant interest in medicine, the food industry, and many
other fields.
http://deadlydeceit.com/WERF-VBNC.html http://deadlydeceit.com/viable-nonculturable/03-CTS-
13Tweb.pdf

2006 -- COPPER INDUCES A VIABLE BUT NONCULTURABLE (VBNC) STATE IN ERWINIA
AMYLOVORA
Copper compounds, used to control Erwinia amylovora, have a strong effect on the culturability of
plant-associated bacteria. Recent studies have shown that some phytopathogenic bacteria enter
into a viable but nonculturable (VBNC) state in the presence of copper. This state, in which cells
progressively lose their culturability on non-selective solid media, is considered to be a survival
strategy under adverse environmental conditions. To determine whether copper kills or induces a
VBNC state in E. amylovora cells, mineral medium without copper or supplemented with 0.005, 0.01
and 0.05 mM Cu2+ was inoculated with 107 cfu/ml of the pathogen. Aliquots were taken regularly for
four months, and the total and viable cell counts were determined using the Live/Dead staining kit
and culturable cell counts were determined on King’s B medium. E. amylovora entered into a VBNC
state in the presence of the three copper concentrations assayed. It entered faster the VBNC state
with increased copper concentration: it entered at days 36, 1 and 0 (immediately after being
introduced in the medium) with 0.005, 0.01 and 0.05 mM Cu2+ respectively. Afterwards, the
restoration of culturability or resuscitation of copper-induced VBNC E. amylovora cells was achieved
by the addition of different copper complexing agents that could remove the free-copper ions.
Dilution studies were also performed to demonstrate that the resuscitated cells came from a true
resuscitation and not from the regrowth of any undetectable culturable cell. Finally, the pathogenicity
of both VBNC and resuscitated cells was evaluated by inoculation on immature pear fruits. Copper-
induced VBNC cells were virulent only for five days while resuscitated cells held their pathogenicity
for more than four months. Understanding the effect of copper against E. amylovora cells could help
to optimize fireblight control strategies.
ISHS Acta Horticulturae 704  X International Workshop on Fireblight
http://www.actahort.org/books/704/704_27.htm

2006 -- LONG-TERM STARVATION-SURVIVAL OF ERWINIA AMYLOVORA IN STERILE IRRIGATION
WATER
Abstract:
The role of irrigation water in disseminating Erwinia amylovora is not fully recognized, and the
survival of the bacterium in natural water has not been carefully investigated. This risk has been
underestimated, since it is generally considered that E. amylovora survives only for a short period in
water and its isolation from natural water samples has not been reported. The main goal of this study
has been to clarify whether E. amylovora could survive under nutrient starvation conditions usually
found in aquatic environments and if it is nonculturable on solid media when recovered from sterile
natural water. Infectivity of E. amylovora cells in water was evaluated by using immature pear fruits.
Total and viable cell counts were monitored by the Live/Dead viability kit, and culturability by plate
counts on King’s B medium. E. amylovora was able to survive in water from different sources
showing a long persistence in irrigation water and maintaining its infectivity for green pears.
However, a progressive loss of culturability on solid media by 2 to 3 logarithmic units during storage
time was observed, the rate at which cells became nonculturable dependent on the type of water
used. A significant difference in the time to nonculturability between cells maintained in irrigation
water and those kept in deionized water was observed, associated with the content of dissolved
compounds, which differ for these two types of water. Since bacterial cells maintained their viability, it
seems that the oligotrophic conditions found in natural aquatic environments could allow the survival
of the fireblight pathogen. Further, the maintenance of the pathogenicity of E. amylovora supports
the possible role of water as a reservoir and vehicle for transmission of this pathogen.
ISHS Acta Horticulturae 704  X International Workshop on Fireblight
http://www.actahort.org/books/704/704_12.htm

2007 -- Reactivation and growth of non-culturable indicator bacteria in anaerobically digested
biosolids after centrifuge dewatering
Abstract
Recent literature has reported that high concentrations of indicator bacteria such as fecal coliforms
(FCs) were measured in anaerobically digested sludges immediately after dewatering even though
low concentrations were measured prior to dewatering. This research hypothesized that the indicator
bacteria can enter a non-culturable state during digestion, and are reactivated during centrifuge
dewatering. Reactivation is defined as restoration of culturability. To examine this hypothesis, a
quantitative polymerase chain reaction (qPCR) method was developed to enumerate Escherichia
coli, a member of the FC group, during different phases of digestion and dewatering.
For
thermophilic digestion, the density of E. coli measured by qPCR could be five orders of
magnitude greater than the density measured by standard culturing methods (SCMs),
which is indicative of non-culturable bacteria
. For mesophilic digestion, qPCR enumerated up
to about one order of magnitude more E. coli than the SCMs. After centrifuge dewatering, the non-
culturable organisms could be reactivated such that they are enumerated by SCMs, and the
conditions in the cake allowed rapid growth of FCs and E. coli during cake storage.
WAT E R RE S E A R C H 41 (2007) 665 – 673
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V73-4MBJXYJ-1&_user=10&_coverDate=02%2F28%
2F2007&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1179901053&_rerunOrigin=scholar.
google&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=ec0f3599c4daa60b7dbfa72a039f434d

2008 -- Effect of total solids on fecal coliform regrowth in anaerobically digested biosolids
Abstract
Fecal coliform (FC) concentrations in anaerobically digested biosolids can increase during
centrifugal dewatering and afterwards in storage of dewatered cake.
The immediate increase
after centrifugation (reactivation) has been demonstrated to be the revitalization of fecal
coliforms that had become non-culturable
. The increase during storage (regrowth) has been
regarded as a subsequence of reactivated bacteria growing in a favorable environment. In this
paper, however, regrowth is demonstrated without preceding reactivation, using intensive laboratory
centrifugation to duplicate the levels of regrowth seen in full-scale centrifugation. Higher total solids
(TS) levels of the dewatered biosolids lead to greater magnitudes of FC increase. The final TS level
appears much more important than the level of shear imposed during centrifugation, based on
comparison of different centrifugation/dilution procedures used to obtain similar TS levels. The
greater TS levels also reduce methane production, suggesting that methanogens compete with, or
inhibit, the fecal coliforms. The addition of bromoethanesulfonate as a methanogen-specific inhibitor
decreased the production of methane gas, and also increased the number of fecal coliforms.
water r e s e arch 4 2 ( 2 0 0 8 ) 3 8 1 7 – 3 8 2 5
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V73-4STB0C1-1&_user=10&_coverDate=08%2F31%
2F2008&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1179906867&_rerunOrigin=scholar.
google&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=ccf9e9edb032ea7bfafc7f1f23c3d0e7

2008 -- 2008 -- Resuscitation of eleven-year VBNC Citrobacter.
Citrobacter freundii strain WA1 was stressed by incubation in seawater microcosms for eleven years.
After two years of starvation, no culturable strain was observed. Incubation of samples in nutrient-
rich broth medium not supplemented with growth factors, however, allowed resuscitation of VBNC
cells so that subsequent plating yielded observable colonies for significantly extended periods of
time.
Recovery of VBNC Citrobacter freundii was obtained by incubation in nutrient broth even after
eleven years of starvation
. To see whether the samples contained the same strain of Citrobacter
freundii inoculated 11 years ago. The complete 16S rRNA gene was PCR amplified and sequenced
from initial, stressed and revived strains of Citrobacter freundii strain WA1.The 16S rRNA gene
sequences from eleven-year stressed strains were homologous with a high degree of similarity to the
GenBank reference strain and were identical to each other. Copyright IWA Publishing 2008.
J Water Health. 2008 ;6 (4):565-568 18401122
http://www.ncbi.nlm.nih.gov/pubmed/18401122


2008 -- Long-Term Survival of Legionella pneumophila in the Viable But Nonculturable State After
Monochloramine Treatment
Abstract   Legionella pneumophila, a facultative intracellular human pathogen, can persist for long
periods in natural and artificial aquatic environments. Eradication of this bacterium from plumbing
systems is often difficult. We tested L. pneumophila survival after monochloramine treatment.
Survival was monitored using the BacLight Bacterial Viability Kit (Molecular Probes), ChemChrome
V6 Kit (Chemunex), quantitative polymerase chain reaction and culturability on buffered charcoal–
yeast extract agar. In nonculturable samples, regain of culturability was obtained after addition of the
amoeba Acanthamoeba castellanii, and esterase activity and membrane integrity were observed
after >4 months after treatment. These results demonstrate for the first time that L. pneumophila
could persist for long periods in biofilms into the viable but nonculturable (VBNC) state. Monitoring L.
pneumophila in water networks is generally done by enumeration on standard solid medium. This
method does not take into account VBNC bacteria. VBNC L. pneumophila could persist for long
periods and should be resuscitated by amoeba. These cells constitute potential sources of
contamination and should be taken into account in monitoring water networks.
Current Microbiology, Volume 57, Number 5 / November, 2008
http://www.springerlink.com/content/027652366r032245/

2008 -- Identification of culturable and originally non-culturable endophytic bacteria isolated from
shoot tip cultures of banana cv. Grand Naine
 
In this article we describe the identification of endophytic bacteria belonging to three groups isolated
from shoot tip cultures of banana cv. Grand Naine in a recent study (Thomas et al. 2008) based on
partial 16S rRNA gene sequence homology analysis. The first group included banana stocks that
displayed obvious colony growth on MS based tissue culture medium during the first in vitro
passage. The second group constituted stocks that were tissue index-negative for cultivable bacteria
initially but turned index-positive after a few to several (4-8) in vitro passages
while the third group
formed one sub-stock that turned index-positive after about 18 passages.
The organisms belonged
to about 20 different genera comprising of a, β, γ-proteobacteria, Gram-positive firmicutes and
actinobacteria. Visibly expressing easily cultured organisms during the first in vitro passage included
Enterobacter, Klebsiella, Ochrobactrum, Pantoea, Staphylococcus and Bacillus spp.
Organisms of
second group that were not detected or non-culturable originally constituted
Brevundimonas, Methylobacterium, Alcaligenes, Ralstonia, Pseudomonas,
Corynebacterium, Microbacterium, Staphylococcus, Oceanobacillus and Bacillus spp
. while
the third group that turned cultivable after extended in vitro culturing included mostly non-
filamentous actinobacteria (Brachybacterium, Brevibacterium, Kocuria and Tetrasphaera spp.). The
identification results suggested that the endophytes of second and third groups were not strictly
obligate or fastidious microbes but those surviving in viable but-non-culturable (VBNC) state and
displaying gradual activation to cultivable form during continuous tissue culturing. Several of the
organisms isolated are known as beneficial ones in agriculture while some organisms have possible
implications in human health. The use of tissue cultures for isolating uncommon endophytes is
discussed.
Plant Cell, Tissue and Organ Culture Volume 93, Number 1 / April, 2008
http://cat.inist.fr/?aModele=afficheN&cpsidt=20178481

2008 -- Using Fourier Transform Infrared (FT-IR) Absorbance Spectroscopy and Multivariate
Analysis To Study the Effect of Chlorine-Induced Bacterial Injury in Water
The effect of chlorine-induced bacterial injury on spectral features using Fourier transform infrared
(FT-IR) absorbance spectroscopy was studied using a mixed bacterial culture of (1:1) ca. 500
CFU/mL each Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 15442 in 0.9%
saline. Bacterial cells were treated with 0, 0.3, or 1.0 ppm of initial free chlorine (21 degrees C, 1 h of
contact time). Chlorine-injured and dead bacterial cells retained the ATR spectral properties of
uninjured or live cells in the region of C-O-C stretching vibrations of polysaccharides, indicative of
the cell wall peptidoglycan layer and lipopolysaccharide outer leaflet. This confirms the observations
of others that extensive bacterial membrane damage is not a key factor in the inactivation of bacteria
by chlorine. The bactericidal effect of chlorine caused changes in the spectral features of bacterial
ester functional groups of lipids, structural proteins, and nucleic acids, with apparent denaturation
reflected between 1800 and 1300 cm (-1) for injured bacterial cells. Three-dimensional principal
component analysis (PCA) showed distinct segregation and clustering of chlorine-treated and
untreated cells. Cells exposed to chlorine at 0.3 or 1.0 ppm could be distinguished from the
untreated control 73 and 80% of the time, respectively, using soft independent modeling of class
analogy (SIMCA) analysis. This study suggests that FT-IR spectroscopy may be applicable for
detecting the presence of injured and viable but not culturable (VBNC) waterborne pathogens that
are underestimated or not discernible using conventional microbial techniques.
http://pubs.acs.org/doi/abs/10.1021/jf801604p

2008 -- Detection of Viable but Nonculturable Escherichia coli O157:H7 Bacteria in Drinking Water
and River Water
A sensitive method for specific detection of viable Escherichia coli O157:H7 cells, including viable but
nonculturable (VBNC) cells, in water samples was developed. This method involved capture of the
bacterial cells on a low-protein-binding membrane and direct extraction and purification of RNA
followed by reverse transcription-PCR and electronic microarray detection of the rfbE and fliC genes
of E. coli O157:H7. It detected as few as 1 CFU of E. coli O157:H7 in diluted cultures, 3 to 4 CFU/liter
in tap water, 7 CFU/liter in river water, and 50 VBNC cells in 1 liter of river water, demonstrating the
best limit of detection reported to date for VBNC cells in environmental water samples.
Applied and Environmental Microbiology, March 2008, p. 1502-1507, Vol. 74, No. 5
http://aem.asm.org/cgi/content/full/74/5/1502?
maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fulltext=cfu&searchid=1&FIRSTINDEX=180&resourcetype=HWFIG

2008 -- Improving the Assessment of Risk from Pathogens in Biosolids: Fecal Coliform Regrowth,
Survival, Enumeration, and Assessment
Abstract: Reactivation or regrowth of fecal coliform bacteria in biosolids has recently become a
concern due to knowledge that Class B materials may fail to meet this criterion after storage or even
after land application. In this paper, data show the two types of fecal coliform increases that have
been characterized: immediate reappearance of large concentrations directly after dewatering; and
the rapid, but less immediate, increases that follow dewatering with some biosolids after dewatering.
The latter phenomenon is shown to extend over a time period of days prior to gradual decrease in
fecal coliform numbers. Modeling shows that anaerobic or fermentative growth cannot simulate
the observed growth, but that a straightforward biokinetic model can duplicate the observed
conditions if a doubling time of one hour is assumed, which is supported by literature. Thus regrowth
cannot be ruled out as the underlying phenomenon.
http://www.cepis.ops-oms.org/bvsaar/cdlodos/pdf/assessmentofrisk635.pdf

2009 -- Survey of Wastewater Indicators and Human Pathogen Genomes in Biosolids Produced by
Class A and Class B Stabilization Treatments
This study also reports the first detection (by culture or PCR) of Staphylococcus aureus and
Clostridium difficile in biosolids. Special attention should be given to the detection of S. aureus
genomes here as a link has been suggested between this organism and infection in residents living
near agricultural class B land application sites.
Applied and Environmental Microbiology, January 2009, p. 164-174, Vol. 75, No. 1
http://aem.asm.org/cgi/content/abstract/75/1/164


2009 -- Proteome analysis of the Escherichia coli heat shock response under steady-state conditions
E. coli is able to grow over a range of approximately 40°C. The normal temperature growth range is
located from 21°C to 37°C. The maximum temperature at which balanced growth can occur is
approximately 49°C. The growth rate of several strains of E. coli, including K-12 strains, is markedly
influenced in the high temperature range (40-45°C) by the availability of exogenous methionine [18].
In the absence of methionine, growth stops at 45°C. Between 40°C and 45°C, the growth rate is
limited by the absence of methionine. At these temperatures the activity of the first enzyme of the
methionine biosynthetic pathway, the homoserine transsuccinylase, is inhibited [19]. For these
reasons, the control of the methionine synthesis regulates many aspects of growth, since it appears
to be the most temperature-sensitive synthesis pathway in E. coli [18].
Proteome Science 2009, 7:36
http://www.proteomesci.com/content/7/1/36


Impact of Silver Nanoparticle Effects at Threshold Levels
Silver nanoparticles (AgNPs) are a frequently used nanomaterial with a wide range of industrial and
consumer applications, including fiber coating, detergents, and hydrogels and plastics to prevent
bacterial and fungal growth. Nanoparticles released from various nanotechnology-enhanced
consumer products will inevitably enter our sewers and wastewater treatment plants (WWTPs). What
effects, if any, they have on treatment processes is the subject of an ongoing WERF research
project, Impact of Silver Nanoparticles on Wastewater Treatment (U3R07).

Researchers from the University of Missouri are evaluating how silver nanoparticles will affect
bacterial growth during wastewater treatment. The team, led by Dr. Zhiqiang Hu, assistant professor
of civil and environmental engineering, has set up several lab-scale wastewater treatment modular
units using activated sludge processes designed to remove organic matter and nutrients in
wastewater. After a prolonged period of operation (>300 days), the researchers took the activated
sludge samples for nanotoxicity testing.

Results to date demonstrate that nitrifying bacteria are especially susceptible to inhibition by silver
nanoparticles. Researchers found that at a concentration of 0.4 mg/L total Ag, a mixture of positively
charged silver ions and AgNPs inhibited the growth of nitrifying bacteria by 11.5 percent. In an
experiment on shock loading of 100% AgNPs (lasting for 12 hours), researchers detected a peak
concentration of 0.75 mg/L total Ag in the activated sludge basin, and about 50% nitrifying bacterial
growth inhibition (or nitrification inhibition) accompanied with a slight accumulation of nitrite
concentration in wastewater effluent was observed.

The results suggest the accumulation of silver could have a detrimental effect on wastewater
treatment, if the concentration reaches threshold levels. Preliminary results from parallel studies of
anaerobic digestion, a commonly used solid stabilization process in wastewater treatment plants,
indicate that AgNPs at concentrations of 19 mg/L (19,000 ppb) or above in biomass started to inhibit
anaerobic microbial activities. Because most of the silver particles are in the activated sludge or
biosolids, the researchers will continue to examine the impact of these nanoparticles on anaerobic
digestion.

The U.S. Environmental Protection Agency has instituted a secondary drinking water standard for
silver of 100 ppb. Though typical silver effluent concentrations from sewage plants are low (at ppb
levels or below), increased use of nanosilver products poses new threats to sensitive water bodies.
U.S. EPA has set water quality criteria values for silver in salt and fresh water at 1.9 and 3.2 ppb,
respectively. The research team will provide a summary that identifies threshold levels of silver
nanoparticles in various treatment processes. Awareness of these threshold levels will directly
benefit wastewater treatment operators and state regulatory officials in making informed decisions
with regard to silver nanoparticles in wastewater treatment plants.

Look for the final published report Impact of Silver Nanoparticles on Wastewater Treatment (U3R07)
in May 2010.
February 4, 2010
Water Environment Federation
http://www.werf.org/AM/Template.cfm?
Section=Home&CONTENTID=13156&TEMPLATE=/CM/HTMLDisplay.cfm


Back to List
1-26-2010
Jim Bynum, VP
Help for Sewage Victims

Coliform exhibit optimum
growth in  the 20-37° C range
(68 - 98.6°F).  Total Coliform
are gram negative members of
the Family Enterobacteriaceae
that ferment lactose at 98.6°F
to produce gas and/or acid
within 48 hours. Other members
of the Family
Enterobacteriaceae
take a longer time to ferment
lactose

Fecal coliform exhibit minimal
growth at an elevated
temperature. They are a few
thermotolerant members of the
Family Enterobacteriaceae
that ferment lactose at
112.1°F  to produce gas
and/or acid within 48 hours.

E. coli and Klebsiella are the
primary fecal coliform that
exhibit less than 5% growth rate
at the elevated temperature.
They act as opportunistic
pathogens when they are
introduced into body
locations where they are not
normally found, especially if
the host is debilitated or
immunosuppressed.

While there may be some
non-pathogenic strains, the
International Escherichia and
Klebsiella Centre (WHO) has a
collection of approximately 60,000
E. coli strains, most of which are
clinical isolates. Clinical isolates
means they are pathogens
associated with disease.  About
half of the hospital acquired
infections are caused by the
coliform bacteria