A PERFECT STORM OF ANTIBIOTIC RESISTANCE
Section 7
Chlorine/Antibiotic Resistance in Bacteria 9/09/2011
Back to Myth # 4
Since chlorine is used as a primary disinfectant in drinking water treatment plants, sewage (wastewater) treatment
plants, swimming pools, hot tubs, etc., many people think it is benign. Few people consider that chlorine disinfectants
produce dangerous carcinogenic byproducts including bromate, chlorite, haloacetic acids (HAA5), and total
trihalomethanes (TTHMs), which cause bladder and rectal cancer, anemia in infants, nervous system dysfunction,
kidney and liver problems, as well as spontaneous abortion. However, the focus here is that these uses are creating not
only chlorine resistant bacteria but also antibiotic resistant bacteria.
In 1928, Fred 0. Tonney, et al., Bureau of Laboratories and Research, Department of Health at Chicago, could have
been talking about the industry's “sound science' when they reported on the “The Minimal "Chlorine Death Points" of
Bacteria, I. Vegetative Forms” They said, “It has not been determined definitely what organism may be considered a
safe index of effective chlorine, treatment or whether a given organism can be used as an index under different
conditions. B. coli [fecal coliform] has been almost universally accepted as the index of safety in the treatment of water
supplies. Its acceptance, however, has been based largely on theoretical considerations and on the analogy of its
greater resistance to heat and longer survival than the common pathogens which may be present in water.”
Basically, what the industry has ignored is that Bacillus coli (E. coli/fecal coliform) and other pathogens infect people at
normal body temperature of 37°C (98.6°F), the industry and regulators tell us that based on “sound science”, E. coli is
only evidence of fecal contamination if it continues to show some minor activity at 112.1°F. The scientific reality is that
before your fecal material reached 112.1°F, you would most likely be either dead or brain damaged at the minimum.
http://ajph.aphapublications.org/cgi/reprint/18/10/1259.pdf
Also, in 1978, Joseph L. Melnick, Charles P. Gerba, & Craig Wallis, Baylor College of Medicine, reported on “Viruses in
water.” They said, “Attention is drawn in this paper to the increasing problem of viral contamination of water and
shellfish, particularly since growing demands for available water resources by a rising world population and expanding
industry will make the recycling of wastewater almost inevitable in the future. The problem of eliminating viruses
pathogenic for man from water is considered in the light of present water treatment procedures, which are often
inadequate for that purpose. Man may be exposed to waterborne viruses through the consumption of contaminated
water, shellfish, or crops, as a result of recreational activities involving water, or from aerosols following the spraying of
crops with liquid wastes. – It is 30 years since studies on the presence of human enteric viruses in water began in
earnest, but the public health significance of these viruses in water has yet to be ascertained. This has been due in part
to the inapparent nature of most of the infections caused by these viruses and the lack of methods for their detection in
many places. Studies have shown that enteric viruses easily survive present sewage treatment methods and many can
persist for several months in natural waters.-- Russian investigators have reported the isolation of enteric viruses on
several occasions from drinking water in distribution systems; b the water treatment plant from which the water came was
found to be functioning normally (the process included chlorination) during the periods when viruses were recovered.
Viruses have also been isolated from drinking water in South Africa.c In a Romanian study, coxsackieviruses were
detected in 2 of 65 drinking water samples; the drinking water treatment consisted of flocculation with aluminium sulfate
and lime followed by sand filtration.4 More recently, poliovirus has been detected on several occasions in treated
drinking water containing free residual chlorine from a community in the United States.e – Because of the presence of
large amounts of organic matter in effluents from activated sludge plants, large reductions of virus are
not possible because the chlorine combines with the residual organics. – A recent report on the development of a
progressively more chlorine-resistant poliovirus strain, after a series of repeated sublethal exposures, has created
additional concern that chlorine-resistant enteric viruses could arise in nature. ”
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2395640/pdf/bullwho00441-0002.pdf
Yet, in the intervening years, Gerba has promoted contaminated sewage sludge use based on his failure to find
bacterial pathogens and ignoring viruses. Eight years later, Gerba became the ultimate EPA expert on pathogens after
he moved to the University of Arizona and authored “Development of a Qualitative Pathogen Risk Assessment
Methodology for Municipal Sludge Landfilling.” (EPA/600/6-88/006 – 1986). The report addresses potential risks
from pathogens present in municipal sludge disposed of in landfills. Since that time, Gerba has become EPA's premiere
expert on failing to find any problems, or risks, with the disposal of pathogen contamination of sludge on agricultural
land and food crops. The short version doesn't mention the author, but it does mention the lack of data – which was also
true for part 503. http://thewatchers.us/EPA/1/1988-pathogen-risk-landfill.pdf
The lower resistance of laboratory strains of virus was shown in a 1980 study by P. T. Shaffer, et al., who reported on
“Chlorine resistance of poliovirus isolants recovered from drinking water.” They said, “Poliovirus 1 isolants were
recovered from finished drinking water produced by a modern, well-operated water treatment plant. These waters
contained free chlorine residuals in excess of 1 mg/liter. The chlorine inactivation of purified high-titer preparations of
two such isolants was compared with the inactivation behavior of two stock strains of poliovirus 1, LSc and Mahoney.
The surviving fraction of virus derived from the two natural isolants was shown to be orders of magnitude greater than
that of the standard strains. These results raise the question whether indirect drinking water standards based on free
chlorine residuals are adequate public health measures, or whether direct standards based on virus determinations
might be necessary.”
http://aem.asm.org/cgi/content/abstract/40/6/1115
In a 1982 study, “R-Plasmid Transfer in a Wastewater Treatment Plant”, Patrick A. Mach and D. Jay Grimes, University
of Wisconsin at La Crosse, said, “Enteric bacteria have been examined for their ability to transfer antibiotic resistance in
a wastewater treatment plant. Resistant Salmonella enteritidis, Proteus mirabilis, and Escherichia coli were isolated from
clinical specimens and primary sewage effluent. They found, “Mean transfer frequencies for laboratory matings were 2.1
x 10-3. In situ matings for primary and secondary settling resulted in frequencies of 4.9 x 10- and 7.5 x 1lo-, respectively.
These values suggest that a significant level of resistance transfer occurs in wastewater treatment plants in the absence
of antibiotics as selective agents.” Moreover, “Because of the high incidence of resistance among nosocomial [hospital]
bacteria, considerable research has been conducted on in vivo [within the living] R-plasmid transfer. These studies have
demonstrated that transfer does occur, and at relatively high rates, in wounds and in the gastrointestinal, urinary, and
respiratory tracts of warmblooded animals, including humans.” Furthermore, “Kushner has observed that exposure to
chlorine in sewage plants and elsewhere leads to an increased frequency of antibiotic resistance in coliform bacteria (D.
J. Kushner, personal communication). – In addition, the use of natural isolates, rather than highly mutated fragile
laboratory strains (e.g., E. coli K-12 strains) no doubt facilitated transfer. – Harada and Mitsuhashi, studying the transfer
kinetics of Escherichia-Shigella systems, showed temperature and pH to be the primary abiotic factors controlling in vitro
[laboratory] transfer. They found that temperature ranges of 25 to 45°C (37°C optimum) and pH ranges of 5.0 to 9.0
(7.5 optimum) supported plasmid transfer. – bacteria in a sewage treatment plant are certainly present in high enough
numbers and in close enough proximity to one another to effect significant transfer and retransfer of R plasmids and of
other transferable extrachromosomal genetic elements, including chimeric plasmids [Hybrid or genetically mixed
plasmid].” http://thewatchers.us/EPA/2/1982-r-transfer-treatment-plants.pdf
In a 1982 follow up study, “Selection of antibiotic-resistant standard plate count bacteria during water treatment,” J. L.
Armstrong, et al., Terrestrial Microbial Ecology/Biotechnology Program, U.S. Environmental Protection Agency, said,
“Standard plate count (SPC) bacteria were isolated from a drinking-water treatment facility and from the river supplying
the facility. All isolates were identified and tested for their resistance to six antibiotics to determine if drug-resistant
bacteria were selected for as a consequence of water treatment. Among the isolates surviving our test procedures,
there was a significant selection (P less than 0.05) of gram-negative SPC organisms resistant to two or more of the test
antibiotics. These bacteria were isolated from the flash mix tank, where chlorine, alum, and lime are added to the water.
Streptomycin resistance in particular was more frequent in this population as compared with bacteria in the untreated
river water (P less than 0.01). SPC bacteria from the clear well, which is a tank holding the finished drinking water at the
treatment facility, were also more frequently antibiotic resistant than were the respective river water populations. When
15.8 and 18.2% of the river water bacteria were multiply antibiotic resistant, 57.1 and 43.5%, respectively, of the SPC
bacteria in the clear well were multiply antibiotic resistant. Selection for bacteria exhibiting resistance to streptomycin
was achieved by chlorinating river water in the laboratory. We concluded that the selective factors operating in the
aquatic environment of a water treatment facility can act to increase the proportion of antibiotic-resistant members of the
SPC bacterial population in treated drinking water.” http://aem.asm.org/cgi/content/abstract/44/2/308
In a 1984 study, “Effect of chlorination on antibiotic resistance profiles of sewage-related bacteria”, G E Murray, et al.,
said, “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, [Black Plague] 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.” http://aem.asm.org/cgi/content/abstract/48/1/73
In 1985, J. M. Kuchta, et al., reported on the “Enhanced chlorine resistance of tap water-adapted Legionella
pneumophila as compared with agar medium-passaged strains.” They said, “Previous studies have shown that bacteria
maintained in a low-nutrient "natural" environment such as swimming pool water are much more resistant to disinfection
by various chemical agents than strains maintained on rich media. In the present study a comparison was made of the
chlorine (Cl2) susceptibility of hot-water tank isolates of Legionella pneumophila maintained in tap water and strains
passaged on either nonselective buffered charcoal-yeast extract or selective differential glycine-vancomycin-polymyxin
agar medium. Our earlier work has shown that environmental and clinical isolates of L. pneumophila maintained on agar
medium are much more resistant to Cl2 than coliforms are. Under the present experimental conditions (21 degrees C,
pH 7.6 to 8.0, and 0.25 mg of free residual Cl2 per liter, we found the tap water-maintained L. pneumophila strains to be
even more resistant than the agar-passaged isolates. Under these conditions, 99% kill of tap water-maintained strains of
L. pneumophila was usually achieved within 60 to 90 min compared with 10 min for agar-passaged strains. Samples
from plumbing fixtures in a hospital yielded legionellae which were "super"-chlorine resistant when assayed under
natural conditions. After one agar passage their resistance dropped to levels of comparable strains which had not been
previously exposed to additional chlorination. These studies more closely approximate natural conditions than our
previous work and show that tap water-maintained L. pneumophila is even more resistant to Cl2 than its already
resistant agar medium-passaged counterpart.” http://aem.asm.org/cgi/content/abstract/50/1/21
In 1985, M. S. Harakeh, et al., School of Medicine, Stanford University, Stanford and J. C. Hoff, EPA, reported on the
“Susceptibility of Chemostat-Grown Yersinia enterocolitica and Klebsiella pneumoniae to Chlorine Dioxide.” They said,
“The resistance of bacteria to antimicrobial agents could be influenced by growth environment. The susceptibility of two
enteric bacteria, Yersinia enterocolitica and Klebsiella pneumoniae, to chlorine dioxide was investigated. – Growth
conditions can profoundly influence the susceptibility of microorganisms to disinfectants. We have previously shown that
growth of Escherichia coli at submaximal rates due to nutrient limitation and at relatively low temperatures greatly
increases the resistance of this bacterium to chlorine dioxide and phenylphenol. Although coliforms are generally
regarded as a suitable indicator organism, there is reason to doubt this premise (13), and we therefore decided to
determine whether other organisms responded to nutrient limitation in the same manner as E. coli. – If antecedent
growth conditions do influence resistance of bacteria in general, then the current practice for environmental
management of microorganisms will need to be reexamined. This practice relies exclusively on batch culture-grown
bacteria for determining the efficacy of various disinfectants. However, it is very unlikely that the nutrient excess
conditions present in batch cultures are similar to those present either in the environment or in the human
body, since in most natural environments bacteria grow at submaximal rates owing to severe nutrient limitation. – The
results presented here demonstrate that slowly growing populations of Y. enterocolitica and K. pneumoniae grown under
nutrient limitation are considerably more resistant to disinfection by chlorine dioxide than populations grown at more
rapid rates. We have previously shown the same phenomenon for populations of E. coli and Legionella pneumophila
with respect to their susceptibility to chlorine dioxide and another disinfectant agent, phenylphenol. Thus, it may
generally be stated that slowly growing populations of bacteria tend to be more resistant to disinfectant agents than their
counterparts grown more rapidly. – In conclusion, populations grown under conditions that more closely approximate the
natural environment, e.g., low temperatures and growth at submaximal rates, exhibit enhanced resistance to
disinfectants..” http://www.ncbi.nlm.nih.gov/pmc/articles/PMC238346/pdf/aem00147-0085.pdf
In 1988, M. W. LeChevallier, et al., American Water Works Service Co., Inc., Belleville Laboratory, reported on the
“Factors promoting survival of bacteria in chlorinated water supplies.” They said, “Results of our experiments showed
that the attachment of bacteria to surfaces provided the greatest increase in disinfection resistance. Attachment of
unencapsulated Klebsiella pneumoniae grown in medium with high levels of nutrients to glass microscope slides
afforded the microorganisms as much as a 150-fold increase in disinfection resistance. Other mechanisms which
increased disinfection resistance included the age of the biofilm, bacterial encapsulation, and previous growth
conditions (e.g., growth medium and growth temperature). These factors increased resistance to chlorine from 2- to 10-
fold. The choice of disinfectant residual was shown to influence the type of resistance mechanism observed. Disinfection
by free chlorine was affected by surfaces, age of the biofilm, encapsulation, and nutrient effects. Disinfection by
monochloramine, however, was only affected by surfaces. Importantly, results showed that these resistance mechanisms
were multiplicative (i.e., the resistance provided by one mechanism could be multiplied by the resistance provided by a
second mechanism).”
http://aem.asm.org/cgi/content/abstract/54/3/649
In 1988, C. H. King, et al., College of Veterinary Medicine at Athens, reported on the “Survival of coliforms and bacterial
pathogens within protozoa during chlorination.” They said, “The susceptibility of coliform bacteria and bacterial
pathogens to free chlorine residuals was determined before and after incubation with amoebae and ciliate protozoa.
Viability of bacteria was quantified to determine their resistance to free chlorine residuals when ingested by laboratory
strains of Acanthamoeba castellanii and Tetrahymena pyriformis. Cocultures of bacteria and protozoa were incubated to
facilitate ingestion of the bacteria and then were chlorinated, neutralized, and sonicated to release intracellular bacteria.
Qualitative susceptibility of protozoan strains to free chlorine was also assessed. Protozoa were shown to survive and
grow after exposure to levels of free chlorine residuals that killed free-living bacteria. Ingested coliforms Escherichia coli,
Citrobacter freundii, Enterobacter agglomerans, Enterobacter cloacae, Klebsiella pneumoniae, and Klebsiella oxytoca
and bacterial pathogens Salmonella typhimurium, Yersinia enterocolitica, Shigella sonnei, Legionella gormanii, and
Campylobacter jejuni had increased resistance to free chlorine residuals. Bacteria could be cultured from within treated
protozoans well after the time required for 99% inactivation of free-living cells. All bacterial pathogens were greater than
50-fold more resistant to free chlorine when ingested by T. pyriformis. Escherichia coli ingested by a Cyclidium sp., a
ciliate isolated from a drinking water reservoir, were also shown to be more resistant to free chlorine. The mechanism
that increased resistance appeared to be survival within protozoan cells. This study indicates that bacteria can survive
ingestion by protozoa. This bacterium-protozoan association provides bacteria with increased resistance to free chlorine
residuals which can lead to persistence of bacteria in chlorine-treated water. We propose that resistance to digestion by
predatory protozoa was an evolutionary precursor of pathogenicity in bacteria and that today it is a mechanism for
survival of fastidious bacteria in dilute and inhospitable aquatic environments.”
http://aem.asm.org/cgi/content/abstract/54/12/3023
Some researchers do not take into account the ability of bacteria to slowly repair cell damage.
In the 1989 study, “[Development of antibiotic resistance in purified sewage effluents subjected to chlorination]”, G.
Morozzi reported, “Antibiotic-resistance is widely spread phenomenon in the environment because of uncontrolled
discharge of urban and animal wastewaters. Sewage treatment can significantly reduce the number of both sensitive
and resistant bacteria. A reduction of about 1.5 logarithmic units in faecal coliforms was observed during biological
treatment (3, 7), but a simultaneous increase in the percentage of resistant strains occurred because of not well
understood selection phenomena. The above reported bacterial reduction is not always sufficient to meet the quality
standards of Italian legislation required to discharge the treated effluents into surface waters, and so, chlorination
become a compulsory additional treatment whose impact on both sensitive and resistant microflora must be evaluated.
The results obtained in the present research have demonstrated that chlorine concentrations in the range of 0.5-2 ppm
are able to reduce significantly the faecal coliforms concentrations and, in particular, treatment with 1 ppm of chlorine for
1 hour reduces the concentration of the above reported bacteria to the extent of 2 logarithmic units, so that their final
concentration are of the about 10(2)/100 ml. The surviving chlorine tolerant bacteria seem to be antibiotic resistant in
higher percentage than the chlorine sensitive ones and so, as a consequence, a significant increase in the antibiotic
resistance and multiresistance was observed in the chlorinated effluents. In this context it is interesting to underline the
larger variety of resistance patterns observed in the chlorine-resistant bacteria in comparison with the uniformity in the
resistance patterns observed in isolated from unchlorinated effluents. The selected chlorine-tolerant strains seem to be
less able to transfer their resistances under laboratory conditions, not because of curing effect of chlorine on the
plasmids but, probably, because of the damage to cellular cell envelopes.” http://www.ncbi.nlm.nih.gov/pubmed/2483077
In 1991, James Debartolomeis and Victor J. Cabelli, University of Rhode Island at Kingston, reported on the “Evaluation
of an Escherichia coli Host Strain for Enumeration of F Male-Specific Bacteriophages”. Bacteriophages are viruses that
live in bacteria. Somatic coliphages are DNA viruses that infect host cells via the outer cell membrane. They said,
somatic “coliphages, which initiate their infectious process by adsorbing to receptors on the bacterial envelope and
whose host cells are Escherichia coli strains such as B, C, and K-12 F-, are found consistently in sewage at levels
considerably greater than those of the enteroviruses. Nevertheless, these coliphages have not found acceptance as
water quality indicators, even by those individuals who recommended enterovirus standards. The objections to their use
have been their inconsistent presence in human feces, the possibility that they could encounter an appropriate host and
be replicated in environmental waters, and the greater sensitivity of some of these somatic coliphages to chlorination
than some of the enteroviruses.” Because of these concerns, they recommend the F male-specific bacteriophages.
They said, “These are single-stranded RNA viruses. Along with a group of filamentous, single-stranded DNA, fd-like
viruses, they compose the F male-specific bacteriophages. – The F male-specific phages, especially the fd-like
component, are extremely resistant to the viricidal effect of combined chlorine. However, the most compelling support for
the use of F male-specific phages as models for the environmental behavior of the important viral pathogens, at least
with regard to chlorination, was reported by Keswick et al.” They found that, of all the viruses examined, only the f2
phage was as resistant as the Norwalk virus to the cidal effects of chlorination.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC182946/pdf/aem00058-0035.pdf
In 1992, M. H .Stewart, and B. H. Olson, Metropolitan Water District of Southern California, reported on the
“Physiological studies of chloramine resistance developed by Klebsiella pneumoniae under low-nutrient growth
conditions.” They said, “This study investigated the physiological mechanisms of resistance to chloramines developed
by Klebsiella pneumoniae grown in a nutrient-limited environment. Growth under these conditions resulted in cells that
were smaller than cells grown under high-nutrient conditions and extensively aggregated. – Cell wall and cell membrane
lipids also varied with growth conditions. The ratio of saturated to unsaturated fatty acids in cells grown under low-
nutrient conditions was approximately five times greater than that in cells grown under high-nutrient conditions,
suggesting possible differences in membrane permeability. An analysis of sulfhydryl (-SH) groups revealed no
quantitative difference with respect to growth conditions. However, upon exposure to chloramines, only 33% of the -SH
groups of cells grown under low-nutrient conditions were oxidized, compared with 80% oxidization of -SH groups in cells
grown under high-nutrient conditions. The reduced effectiveness of chloramine oxidization of -SH groups in cells grown
under low-nutrient conditions may be due to restricted penetration of chloramines into the cells, conformational changes
of enzymes, or a combination of both factors. The results of this study suggest that chloramine resistance developed
under low-nutrient growth conditions may be a function of multiple physiological factors, including cellular aggregation
and protection of sulfhydryl groups within the cell.”
http://aem.asm.org/cgi/content/abstract/58/9/2918
Methylobacteria is one of the emerging pathogens that have been found in hospital tap water, water from dental units as
well as blood bank purification units. In 1995, A. Hiraishi, et al., Laboratory of Environmental Biotechnology, reported on
the “Phenotypic and genetic diversity of chlorine-resistant Methylobacterium strains isolated from various environments.”
They said, “Strains of pink-pigmented facultative methylotrophs which were isolated previously from various
environments and assigned tentatively to the genus Methylobacterium were characterized in comparison with authentic
strains of previously known species of this genus. Most of the isolates derived from chlorinated water supplies exhibited
resistance to chlorine, whereas 29 to 40% of the isolates from air, natural aquatic environments, and clinical materials
were chlorine resistant. None of the tested authentic strains of Methylobacterium species obtained from culture
collections exhibited chlorine resistance. Numerical analysis of phenotypic profiles showed that the test organisms tested
were separated from each other except M. organophilum and M. rhodesianum. The chlorine-resistant isolates were
randomly distributed among all clusters. The 16S ribosomal DNA (rDNA) sequence-based phylogenetic analyses
showed that representatives of the isolates together with known Methylobacterium species formed a line of descent
distinct from that of members of related genera in the alpha-2 subclass of the Proteobacteria and were divided into
three subclusters within the Methylobacterium group. These results demonstrate that there is phenotypic and genetic
diversity among chlorine-resistant Methylobacterium strains within the genus.” http://aem.asm.
org/cgi/content/abstract/61/6/2099
In 1998, H. Fitnat, et al., Stanford University Medical School, reported on “Vibrio cholerae O1 El Tor: Identification of a
gene cluster required for the rugose colony type, exopolysaccharide production, chlorine resistance, and biofilm
formation.” They said, “The rugose colony variant of Vibrio cholerae O1, biotype El Tor, is shown to produce an
exopolysaccharide, EPSETr, that confers chlorine resistance and biofilm-forming capacity. EPSETr production requires
a chromosomal locus, vps, that contains sequences homologous to carbohydrate biosynthesis genes of other bacterial
species. Mutations within this locus yield chlorine-sensitive, smooth colony variants that are biofilm deficient. The biofilm-
forming properties of EPSETr may enable the survival of V. cholerae O1 within environmental aquatic habitats between
outbreaks of human disease.” http://www.pnas.org/content/96/7/4028.short
In 1998, John T. Lisle, et al., Montana State University at Bozeman, reported on the “Effects of Starvation on
Physiological Activity and Chlorine Disinfection Resistance in Escherichia coli O157:H7.” They said, “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.” http://aem.asm.
org/cgi/content/abstract/64/12/4658
In 2000, Robert H. Taylor, et al., Virginia Polytechnic Institute and State University at Blacksburg, reported on “Chlorine,
Chloramine, Chlorine Dioxide, and Ozone Susceptibility of Mycobacterium avium.” They said, “Environmental and patient
isolates of Mycobacterium avium were resistant to chlorine, monochloramine, chlorine dioxide, and ozone. For chlorine,
the product of the disinfectant concentration (in parts per million) and the time (in minutes) to 99.9% inactivation for five
M. avium strains ranged from 51 to 204. Chlorine susceptibility of cells was the same in washed cultures containing
aggregates and in reduced aggregate fractions lacking aggregates. Cells of the more slowly growing strains were more
resistant to chlorine than were cells of the more rapidly growing strains. Water-grown cells were 10-fold more resistant
than medium-grown cells. Disinfectant resistance may be one factor promoting the persistence of M. avium in drinking
water.” http://aem.asm.org/cgi/content/full/66/4/1702
In 2004, Richa Shrivastava, et al., Biomembrane Division, Industrial Toxicology Research Centre, reported the
“Suboptimal chlorine treatment of drinking water leads to selection of multidrug-resistant Pseudomonas aeruginosa.”
They said,”The present study was undertaken to investigate the spectrum of bacteria present in the River Gomti water
before and after chlorination for drinking purposes. We observed that the strains of Pseudomonas aeruginosa that
survived chlorination on three out of seven occasions were resistant to almost all the antibiotics tested. The chlorine-
resistant bacteria had mucoid colonies and grew better at 24°C. All attempts to isolate the plasmid responsible for
chlorine resistance were unsuccessful. Laboratory experiments using different strains of the P. aeruginosa in distilled
water showed that only the resistant strain survived chlorine treatment at a dose of 500 μg/L. Similar results were
obtained when water collected from seven different sites on the River Gomti was treated with graded doses of chlorine.
At the higher dose of chlorine, all the bacteria died in 30 min, whereas with lower doses all the bacteria survived. The
present study underscores the importance of measuring water chlorine concentrations to assure they are sufficiently
high to remove pathogenic bacteria from drinking water. To our knowledge, this is the first report in the literature of the
selection of multidrug-resistant bacteria by suboptimal chlorine treatment of water.” http://www.sciencedirect.
com/science/article/pii/S0147651303001076
The following study shows that excessive amounts of chlorine does little to destroy bacteria in mixed organic matter.
Consider also that at 4 mg/L, chlorine is a contaminate in drinking water. In 2006, John J Macauley, et al., University of
Missouri at Rolla, reported on the “Disinfection of swine wastewater using chlorine, ultraviolet light and ozone.” They
said, “Veterinary antibiotics are widely used at concentrated animal feeding operations (CAFOs) to prevent disease and
promote growth of livestock. However, the majority of antibiotics are excreted from animals in urine, feces, and manure.
Consequently, the lagoons used to store these wastes can act as reservoirs of antibiotics and antibiotic-resistant
bacteria. There is currently no regulation or control of these systems to prevent the spread of these bacteria and their
genes for antibiotic resistance into other environments. This study was conducted to determine the disinfection potential
of chlorine, ultraviolet light and ozone against swine lagoon bacteria. Results indicate that a chlorine dose of 30 mg/L
could achieve a 2.2–3.4 log bacteria reduction in lagoon samples. However, increasing the dose of chlorine did not
significantly enhance the disinfection activity due to the presence of chlorine-resistant bacteria. The chlorine resistant
bacteria were identified to be closely related to Bacillus subtilis and Bacillus licheniformis. A significant percentage of
lagoon bacteria were not susceptible to the four selected antibiotics: chlortetracycline, lincomycin, sulfamethazine and
tetracycline (TET). However, the presence of both chlorine and TET could inactivate all bacteria in one lagoon sample.
The disinfection potential of UV irradiation and ozone was also examined. Ultraviolet light was an effective bacterial
disinfectant, but was unlikely to be economically viable due to its high energy requirements. At an ozone dose of 100
mg/L, the bacteria inactivation efficiency could reach 3.3−3.9 log” http://www.sciencedirect.
com/science/article/pii/S0043135406001928
In 2007, Matthew Wook Chang, et al., Nanyang Technological University at Singapore, reported on the “Toxicogenomic
Response to Chlorination Includes Induction of Major Virulence Genes in Staphylococcus aureus.” They said, “In this
work, genome-wide transcriptional analysis was performed to elucidate cellular response of S. aureus to hypochlorous
acid, an active antimicrobial product of chlorination in aqueous solution. Our results suggest that hypochlorous acid
repressed transcription of genes involved in cell wall synthesis, membrane transport, protein synthesis, and primary
metabolism, while amino acid synthesis genes were induced. Furthermore, hypochlorous acid induced
transcription of genes encoding major virulence factors of S. aureus, such as exotoxins, hemolysins, leukocidins,
coagulases, and surface adhesion proteins, which all play essential roles in staphylococcal virulence. This work implies
that chlorination may stimulate production of virulence factors, which provides new insight into host-pathogen
interactions and effects of chlorine application for microbial control. – Consequently, we propose that staphylococcal
pathogenesis can be increased during phagocyte-driven chlorine stress and during chlorine application for microbial
control.” http://www.bren.ucsb.edu/academics/courses/595JJ/Readings/Virulence_chlor.pdf
In 2009, Leah M. Feazela, et al., University of Colorado at Boulder, reported on “Opportunistic pathogens enriched in
showerhead biofilms.” They said, “In one case [showerhead BSK, a Denver Metro 1 showerhead sampled on three
occasions (Fig. 2)], attempted cleaning with bleach solution resulted in a 3-fold increase in the load of M. gordonae,
from approximately 25% of the assemblage sequences initially (BSK1Q) to 72% and 74% subsequently (BSK2Q and
BSK3Q). Although anecdotal, this observation is interesting in light of the general resistance of mycobacteria to
chlorine, which also may be one reason for the mycobacterial enrichment in municipal systems compared to well-water
fed systems (discussed below). – Furthermore, many species of biofilm-forming mycobacteria are chlorine-resistant, and
thus potentially can be enriched by chlorine disinfection protocols used by many municipalities. Consistent with this, we
only observed mycobacterial rRNA gene sequences in municipal water systems, not in untreated well water systems.”
http://www.pnas.org/content/106/38/16393.long
In 2009, S. Wang, et al., Illinois Institute of Technology at Chicago, reported on the “Transcriptomic response of
Escherichia coli O157:H7 to oxidative stress.” They said, “Chlorinated water is commonly used in industrial operations to
wash and sanitize fresh-cut, minimally processed produce. Here we compared 42 human outbreak strains that
represented nine distinct Escherichia coli O157:H7 genetic lineages (or clades) for their relative resistance to chlorine
treatment. A quantitative measurement of resistance was made by comparing the extension of the lag phase during
growth of each strain under exposure to sublethal concentrations of sodium hypochlorite in Luria-Bertani or brain heart
infusion broth. Strains in clade 8 showed significantly (P < 0.05) higher resistance to chlorine than strains from other
clades of E. coli O157:H7. To further explore how E. coli O157:H7 responds to oxidative stress at transcriptional levels,
we analyzed the global gene expression profiles of two strains, TW14359 (clade 8; associated with the 2006 spinach
outbreak) and Sakai (clade 1; associated with the 1996 radish sprout outbreak), under sodium hypochlorite or hydrogen
peroxide treatment. We found over 380 genes were differentially expressed (more than twofold; P < 0.05) after exposure
to low levels of chlorine or hydrogen peroxide. Significantly upregulated genes included several regulatory genes
responsive to oxidative stress, genes encoding putative oxidoreductases, and genes associated with cysteine
biosynthesis, iron-sulfur cluster assembly, and antibiotic resistance. Identification of E. coli O157:H7 strains with
enhanced resistance to chlorine decontamination and analysis of their transcriptomic response to oxidative stress may
improve our basic understanding of the survival strategy of this human enteric pathogen on fresh produce during
minimal processing.” http://iit.academia.
edu/SiyunWang/Papers/162800/Transcriptomic_response_of_Escherichia_coli_O157_H7_to_oxidative_stress
In 2010, M. S. Gião, et al., Universidade do Minho, Campus de Gualtar at Braga, reported on the “Effect of Chlorine on
Incorporation of Helicobacter pylori into Drinking Water Biofilms.” They said, “The use of a specific peptide nucleic acid
(PNA) probe demonstrated that Helicobacter pylori persisted inside biofilms exposed to low concentrations of chlorine
(0.2 and 1.2 mg liter−1) for at least 26 days, although no culturable cells were recovered. Coupled with data obtained
using viability stains in pure culture, this result suggests that H. pylori can survive chlorination but remain undetectable
by culture methods, which can be effectively replaced by PNA hybridization. – In a recent study, we demonstrated that H.
pylori can be incorporated into drinking water biofilms and remain viable in the lower layers of these structures (11). It is
therefore important to understand the ability of this pathogen to be incorporated and survive in heterotrophic biofilms
formed in chlorinated waters. If this pathogen can remain viable under these conditions, it might therefore represent a
risk to public health when released into the bulk fluid. – As H. pylori is a fastidious microorganism that grows very slowly,
other species present may easily overgrow it, making it impossible to obtain culturable data which could give important
information.” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2832397/
One of the most unusual aspects of the following article is that one of the authors, Charles P. Gerba, University of
Arizona at Tucson, UA Water Quality Center and UA EPA Homeland Security Center, is one of the primary scientific
promoters of sludge disposal on agricultural land where runoff will contaminate surface and drinking water. Yet, in 2011,
Mark R Riley, et al., University of Arizona at Tucson, reported on the “Biological approaches for addressing the grand
challenge of providing access to clean drinking water.” They said, “In 1993 the largest waterborne outbreak of disease
ever documented occurred in Milwaukee, Wisconsin. Over 400,000 individuals developed gastroenteritis and perhaps
100 individuals died in response to consuming drinking water contaminated with the protozoan parasite Cryptosporidium
parvum [25]. The organism appeared in high concentrations in the city's water source after a period of heavy rains,
allowing some of the oocysts of the organism to penetrate the filtration barrier. Infections ranged from acute conditions,
such as diarrhea, to chronic conditions with symptoms that remained present even 10 years after initial infection.
Chlorine disinfection had little effect on the viability of the oocysts, and in fact, this organism was later detected in 60%
of the treated drinking water supplies in the United States. – Cryptosporidium is highly chlorine resistant and thus
presents a challenge for its elimination from water supplies. – Commonly used methods for water disinfection in pipes
rely on chlorination as the predominant method in part due to chlorine's wide range of activity and to its role as a
lingering disinfectant, which unfortunately also leads to harmful residuals. Ultraviolet (UV) light has been shown in some
cases to be equivalent to chlorine, but generates no disinfection residuals; the merit of such residuals is still in debate.
– Increased use of chlorine and chloramines has resulted in biofilm populations being supplemented with
Mycobacterium. The increased usage of UV light has resulted in increased discharge of adenovirus in surface waters;
the super-surviving fraction has caused the disinfection microbe inactivation curves not to be linear. Rotavirus has no
significant inactivation with UV light from 100-350 mJ cm2. – Naegleria is one of the most resistant water-based
pathogens to chlorine disinfectants and UV light; with 98% human mortality if infection occurs. – Bacillus sp. and
Pseudomonas sp. are among the most prevalent bacteria that form biofilms on RO and nanofiltration (NF) membranes
used for water treatment. Chlorination cannot be applied to reverse osmosis membranes because oxidants degrade the
membrane structure. Inorganic scaling can also arise due to phosphate and other inorganic ions present in wastewater
effluent.” http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3080283/
These disinfectants created antibiotic resistant bacteria are released to the environment in treated sewage effluent,
reclaimed water and sludge. However, the soil experts only research the potential damage certain metals will cause to
crops. Other experts research the potential use of metals resistant bacteria for bio-remediation of metal polluted sites.
Both types of experts neglect to consider the potential damage metals/antibiotic resistant organisms will do as they pass
through soil, water, plants, animals and into humans.
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