A PERFECT STORM OF ANTIBIOTIC RESISTANCE
Section 9
Ultraviolet (UV) Radiation/Antibiotic Resistant Bacteria 9/09/2011
Back to Myth # 4
There are many “experts” who promote ultraviolet light and other radiation as the ultimate methods to “kill” disease
causing organisms in sewage, water and food in spite of all the studies available proving these methods just create
more resistant bacteria. Yet, they are sold to the public as safe methods of disinfection.
In 1944, C. H. Gray and E. L. Tatum, Stanford University, reported on the “X-Ray Induced Growth Factor Requirements
in Bacteria.” They said, “The specific growth factor requirements of bacteria result from the inability of these organisms
to synthesize particular essential substances from simpler constituents.1' 2 In some instances such deficiencies have
been shown to result from an inability to carry out definite chemical reactions in a biosynthesis.3 These defects in
synthesis are characteristic of each cell of a given strain of bacteria, and are in addition transmitted to each descendant
cell. Similar defects in certain biosyntheses have been induced in the ascomycete Neurospora by irradiation and have
been shown to result from mutations in specific genes. – Two species of bacteria have been used in this investigation:
Escherichia coli (K-12) and Acetobacter melanogenum (M.A. 6.1). – The bacteria were treated with x-rays produced in a
Westinghouse "Duocondex" unit containing an industrial type oil-cooled tube operated at 160 kv. and 25 ma. Twenty-
four or 48-hour-old liquid cultures of bacteria were exposed to a total irradiation of 180,000 r units given at a rate of
4000 per minute. Preliminary tests had shown that at this dosage over 99.99 per cent of the cells were killed. – All of the
available evidence is consistent with the view that the new strains of E. coli and A. mekrnogenum were produced as a
result of the x-ray treatment. No variant strains of either organism were obtained following treatment with ultraviolet light,
although as many isolations were made and tested as after x-ray treatment. – X-rays are known to increase the rate of
gene mutations in other organisms and the mutation or inactivation of single genes seems the most reasonable
explanation for the origin of the variant strains obtained since each differs from the original strain in having lost the
ability to synthesize specific growth factors. – These results prove that the capacity of bacteria for carrying out specific
biochemical reactions can be modified by x-ray treatment, and suggest that biosyntheses in bacteria are controlled by-
specific genes.” http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1078737/pdf/pnas01669-0028.pdf
In 1945, E. L. Tatum, Stanford University, reported on the “X-ray Induced Mutant Strains of Escherichia Coli.” Tatum
said, “In a previous paper the production by means of x-ray treatment of a number of growth-factors requiring mutant
strains of Escherichia coli and of Acetobacter melanogenum was described. These results were interpreted as indicating
an analogy with the induction by irradiation of true gene mutations in Neurospora with similar biochemical changes.
Comparable changes have been induced in other strains of E. coli. The present work was undertaken with the aims of
producing more "biochemical" mutant strains of E. coli for biochemical study, of providing convincing evidence of
derivation of these strains and of comparing the mutation rates of irradiated and control cultures. A considerable
number of new mutant strains have been obtained after further irradiation of two biochemical mutant strains previously
described. These new strains require growth-factors in addition to those required by the parent strains. The double
requirements of the new strains thus provide reasonable certainty of their derivation from their parent strains. The
irradiation significantly raised the mutation rate from one in 2000 isolations to 16 in the same number of isolations.
Furthermore, the biochemical specificities of those mutant strains which have been analyzed are consistent with the view
that each differs from the original stock in only one biochemical reaction. The evidence therefore supports the view that
treatment with x-rays produces heritable defects in synthetic reactions in bacteria, biologically and biochemically
analogous to those resulting from single gene mutations in Neurospora. This is presumptive evidence for the existence
of genes in bacteria, perhaps contained in the nuclear structures which have been observed in a number of bacteria.”
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1078806/pdf/pnas01677-0003.pdf
In a 1946 letter to the editor, Joshua Lederberg and E. L Tatum, Yale University, reported on “Gene Recombination in
Escherichia Coli.” They said, “This paper will be devoted to the presentation of evidence for the occurrence in a
bacterium of a process of gene recombination,” Their primary research was to detect mutant bacteria. They noted that
biochemical and nutritional mutants had many uses but difficult to identify. They acknowledged that many agents could
be used to find resistant bacteria in culture. That included using x-radiation, ultraviolet light and nitrogen mustard gas to
detect mutants. However, only occasional strains could actually be determined to be resistant mutants. On the other
hand, they did find biochemical mutants from x-ray and ultraviolet-treated E. coli. “Gene Recombination in Escherichia
Coli.” was actually the name of Lederberg's 1947 Dissertation
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC518375/pdf/jbacter00658-0015.pdf.
In 1964, David Pettijohn and Philip Hanawalt, Stanford University, reported on the “Evidence for repair-replication of
ultraviolet damaged DNA in bacteria.” They said, “Our findings are consistent with the view that in ultraviolet-resistant
organisms a mechanism for repair replication exists in which damaged single-strand regions of the chromosome can be
excised and replaced, using the undamaged DNA strand as template.” http://www.sciencedirect.
com/science/article/pii/S0022283664802163
In 1969, Edward B. Marsh, Jr., Harvard Medical School, and David H. Smith, Children's Hospital Medical Center at
Boston, found “R Factors Improving Survival of Escherichia coli K-12 After Ultraviolet Irradiation.” They said, “Two R
factors which have the capacity to improve survival of some strains of Escherichia coli K-12 by approximately 60% after
ultraviolet light have been identified and characterized. – The ability to enhance survival can be separated from all other
identified R-factor functions. Improved survival does not result from improved excisional capacity, but does require an
intact host capacity for genetic recombination. – The effects of UV light on bacteria are principally the result of damage
to DNA – That substrains show differences in survival implies the presence of repair systems or the ability to bypass the
damage produced.” While they were unable to relate the research to wild type bacteria, they said, “A more probable
effect of the R factor would be to influence recombination. Such an effect would manifest itself as more successful repair
and, therefore, a higher survival.” http://jb.asm.org/cgi/reprint/100/1/128.pdf
In 1970, Daniel Billen and Laura Bruns, University of Florida at Gainesville, reported on the “Relationship Between
Radiation Response and the Deoxyribonucleic Acid Replication Cycle in Bacteria: Dependence on the Excision-Repair
System.” They said, “Prestarvation of Escherichia coli for required amino acids results in a marked enhancement in both
ultraviolet light (UV) or X-ray resistance for selective strains. Preventing protein synthesis by starvation for required
amino acids results in completion of the cycle of chromosomal replication then underway. We have investigated
the relationship between starvation-induced resistance enhancement (SIRE) and the excision-repair (Hcr) system in
several E. coli strains including E. coli B/r hcr+ and its isogenic mutant E. coli B/r hcr-. The following observations were
made. (i) The Hcr system is the major component of SIRE in UV-irradiated strain B/r. By using the Hcr+ strain, SIRE
increases the 10% survival dose from -400 ergs to /41,200 ergs/mm2. With the Hcr cells, the increase is from -45 ergs
to 60 ergs/mm2. (ii) Although prestarvation leads to a moderate enhancement of resistance to X irradiation, this effect is
not dependent on the Hcr system. (iii) The double mutant, E. coli B8.1 (hcr- exr-) is completely unable to express SIRE
whether studied with UV or X irradiation. It is concluded that the Hcr system is the major system responsible for SIRE in
UV-treated cells, whereas Exr (resistance to X rays) may be involved to a minor extent. The Exr character appears to be
required for SIRE expression in X-ray exposed cells.” http://www.ncbi.nlm.nih.
gov/pmc/articles/PMC248094/pdf/jbacter00379-0144.pdf
In 1976, Evelyn M. Witkin, Rutgers University at New Brunswick, reported on "Ultraviolet Mutagenesis and Inducible DNA
Repair in Escherichia coli." Witkin said, "This article summarizes current evidence for the inducibility of mutagenic DNA
repair and explores some of its implications. Other aspects of UV mutagenesis in microorganisms have recently been
reviewed. – Enzymatic repair mechanisms operating in bacteria which minimize the damaging effects of UV radiation are
described in detail elsewhere The brief summary given here is intended only to provide sufficient background for
discussion of UV mutagenesis. – All or nearly all detectable UV-induced mutations occur before the second post
irradiation DNA replication. In wild-type strains, some UV-induced mutations occur before DNA replication via SOS repair
of gaps resulting from excision of UV photoproducts; others occur during or after the first post irradiation DNA replication
via SOS repair of damage (probably gaps in the daughter strands) caused by unexcised UV photoproducts passing
through a replication fork. There is some evidence that mutagens and carcinogens induce DNA repair activities in
mammalian cells. It is not known whether these activities are error-prone or whether they are induced in coordination
with other SOS-like functions in response to DNA damage. If so, at least two such functions (induction of latent virus and
of error-prone DNA repair activity) could contribute to carcinogenesis." http://www.ncbi.nlm.nih.
gov/pmc/articles/PMC413988/pdf/bactrev00054-0083.pdf
In EPA's 1982 study, “Effect of UV light disinfection on antibiotic-resistant coliforms in wastewater effluents”, Mark
Meckes said, “Total coliforms and total coliforms resistant to streptomycin, tetracycline, or chloramphenicol were isolated
from filtered activated sludge effluents before and after UV light irradiation. – the percentage of the total surviving
coliform population resistant to tetracycline or chloramphenicol was significantly higher than the percentage of the total
coliform population resistant to those antibiotics before UV irradiation. – Multiple drug resistance patterns of 300 total
coliform isolates revealed that 82% were resistant to two or more antibiotics. Furthermore, 46% of these isolates were
capable of transferring antibiotic resistance to a sensitive strain of Escherichia coli – research has demonstrated that
bacteria carrying transmissible R-factors are responsible for the spread of multiple antibiotic resistance among members
of the Enterobacteriaceae (such as E. coli, Salmonella typhi, and Shigella dysenteriae), Aeromonas and Yersinia
species, Pseudomonas aeruginosa, and Vibrio cholerae. – Several researchers have pointed out that wastewater,
treated or untreated, is a primary contributor of bacteria to the aquatic ecosystem. Studies have been conducted which
demonstrate that significant numbers of multiple drug-resistant coliforms occur in rivers, bays, bathing beaches, and
coastal canals. – Available information shows that conventional wastewater purification methods without disinfection are
not adequate for removal of antibiotic-resistant bacteria. – It is evident from this work as well as from the work of others
that antibiotic resistant coliforms are entering the aquatic environment via treated municipal wastewater effluents. – This
work also points out that there is a significant increase in the percentage of the surviving total coliform population
resistant to tetracycline and chloramphenicol after UV irradiation.”
http://www.usludgefree.org/pdf/hfw/hfw_meckes.pdf
In 1986, Nevine El Solh, et al., Institut Pasteur, reported on the “Conjugative Transfer of Staphylococcal Antibiotic
Resistance Markers in the Absence of Detectable Plasmid DNA.” They said, “Most of the drug resistance genes of
multiply resistant Staphylococcus aureus strains isolated in Europe are reported to be located on the chromosome. –
Although the [horizontal] transfer by conjugation of the antibiotic resistance genes in the absence of detectable plasmids
occurs in various bacterial genera reports of such conjugative transfer in staphylococci have so far been limited to
plasmid-borne determinants. We report in this study the transfer by conjugation of apparently chromosome-borne
resistance markers from wild-type S. aureus strains into staphylococcal recipients belonging to different species. – The
technique used for transduction was described previously. Transductions were performed either with the S. aureus
transducing phage [virus] 4i11, 1503, 1305, or 80a, propagated on the donor strains, or with the phage(s) harbored by
the donors and induced by UV irradiation.” http://thewatchers.us/EPA/10/1986-antibio-bacteria.pdf
In 1992, Michael P. Carty, et al., University of Cincinnati, Cincinnati, reported on the “Replication and Mutagenesis of
UV-Damaged DNA Templates in Human and Monkey Cell Extracts.” They said, “We have used in vitro DNA replication
systems from human HeLa cells and monkey CV-1 cells to replicate a UV-damaged simian virus 40-based shuttle vector
plasmid, pZ189. We found that replication of the plasmid was inhibited in a UV fluence-dependent manner, but even at
UV fluences which caused damage to essentially all of the plasmid molecules some molecules became completely
replicated. This replication was accompanied by an increase (up to 15-fold) in the frequency of mutations detected in
the supF gene of the plasmid. These mutations were predominantly G:C--A:T transitions similar to those observed in
vivo. Treatment of the UV-irradiated plasmid DNA with Escherichwa coli photolyase to reverse pyrimidine cyclobutane
dimers (the predominant UV-induced photoproduct) before replication prevented the UV-induced inhibition of replication
and reduced the frequency of mutations in supF to background levels. Therefore, the presence of pyrimidine
cyclobutane dimers in the plasmid template appears to be responsible for both inhibition of replication and mutation
induction. Further analysis of the replication of the UV-damaged plasmid revealed that closed circular replication
products were sensitive to T4 endonuclease V (a pyrimidine cyclobutane dimer-specific endonuclease) and that this
sensitivity was abolished by treatment of the replicated DNAwith E. coli photolyase after replication but before T4
endonuclease treatment. These results demonstrate that these closed circular replication products contain pyrimidine
cyclobutane dimers. Density labeling experiments revealed that the majority of plasmid DNA synthesized in vitro in the
presence of bromodeoxyuridine triphosphate was hybrid density whether or not the plasmid was treated with UV
radiation before replication; therefore, replication of UV-damaged templates appears to occur by the normal
semiconservative mechanism. All of these data suggest that replication of UV-damaged templates occurs in vitro as it
does in vivo and that this replication results in mutation fixation.” http://www.ncbi.nlm.nih.
gov/pmc/articles/PMC358932/pdf/molcellb00013-0559.pdf
In 2005, Satyendra Gautam, et al., reported on the “Gamma Irradiation of Municipal Sludge for Safe Disposal and
Agricultural Use.” They said, “For academic interest, the change in the cell count of residual fecal coliform was also
determined in the irradiated sludge when it was held in a liquid state overnight (approximately 24 hours) after the
radiation treatment. The coliform count was found to significantly increase when the irradiated sludge, withdrawn from
the closed circulation-loop system, was held overnight (Table 5). The increase was sometimes found to be more than
their numbers in nonirradiated sludge (Table 5). However, the counts were found to decline during a prolonged storage
beyond 10 days (data not shown). It was observed that, in closed-circulation loop, the coliforms could recover on
holding and multiply. The reduced microbial load provided competitive advantage to the survivors. To check the
possibility of repair and recovery, sludge samples were plated at different time intervals after the radiation treatment.
Interestingly, the coliform numbers remained low until approximately 9 hours of radiation treatment and then started to
increase(Table 6).”
http://www.redorbit.
com/news/science/268638/gamma_irradiation_of_municipal_sludge_for_safe_disposal_and_agricultural/
In 2009, Sungpyo Kim, et al., Columbia University at New York, reported on the “Propensity of activated sludge to
amplify or attenuate tetracycline resistance genes and tetracycline resistant bacteria: A mathematical modeling
approach.” They said, “The overall goal of this study was to quantify the propensity of the activated sludge (AS) process
at three wastewater treatment plants (WWTP) to amplify or attenuate tetracycline resistant bacteria (TRB) and
tetracycline resistance genes (TRG). Accordingly, the abundance and fraction of TRB and seven TRG in different unit
operations of these WWTP were analytically measured and modeled using a mass balance approach widely used for AS
design. Based on the model, the AS process of the different WWTP neither amplified nor attenuated the TRB and TRG
fractions. Of the TRG tested, the ribosomal protection genes, tet(O) and tet(W) were the most abundant, along the
treatment train of the WWTP, on all sampling dates and sampling locations. Significant amounts of TRB and TRG were
discharged in the effluent streams. Notably, in selected samples, the fraction of TRB increased in response to ultraviolet
disinfection of treated wastewater compared to chlorination. This study therefore implicates wastewater treatment
processes as significant point sources of tetracycline resistance determinants to the environment, and provides a
mathematical basis to compute the production capacity of these determinants in the AS process.”
http://www.sciencedirect.com/science/article/pii/S0045653509015252
Next Antibiotics Resistance in Sewage Wastewater Treatment Plants
Back to Myth # 4