A PERFECT STORM OF ANTIBIOTIC RESISTANCE
Section 11
Antibiotic Resistance in Water Revised 11/18/2011
Back to Myth # 4
Christiaan Eijkman became the primary expert in 1904 when he developed the fecal coliform glucose broth fermentation
test at 114.8°F for thermotolerant Bacillus Coli (Escherichia coli) in water to indicate fecal pollution. Somehow, Eijkman
was able to convince other experts, including government agencies, that E. coli found in water tests at 98.6°F or below
was not of human origin and therefore had no sanitary significance. The original coliform test was adopted by the Public
Health Service in 1914 to evaluate potential fecal contamination of drinking water by Bacillus coli (E. coli). The test was
later expanded to coli-like-forms of bacteria using a combination of four isolation methods, Indole, Methyl Red, Voges
Proskauer and Citrate utilization collectively known as IMViC test. Total coliforms (family Enterobacteriacea – including
E. coli) isolated by this method is the tests name for a small class of gram-negative bacteria that ferment lactose to
produce gas and acid when incubated at 35°C (95°F) for 24-48 hours. The coliform Enterobacteriacea test group
include animal, human, plant, soil and water bacteria that now cause antibiotic resistant infections in humans.
I believe it is worth repeating that this deadly family of Enterobacteriacea “coliform” include well known enteric
pathogens such as: E. coli; Klebsiella; Citrobacter; Enterobacter; Shigella; Salmonella; and Yersinia as well as less
known families such as, Averyella; Budvicia aquatica; Buttiauxella noackiae; Calymmatobacterium; Cedecea;
Edwardsiella; Ewingella; Hafnia alvei; Kluyvera; Koserella; Leclercia adecarboxylata; Leminorella; Moellerella
wisconsensis; Morganella; Pantoea; Photorhabdus; Proteus; Providencia; Rahnella aquatilis; Serratia; Tatumella;
Xenorhabdus; and Yokenella regensburgei.
The experts agree that 5% of the required monthly drinking water test may contain any or all of the above listed
pathogenic coliform bacteria. However, when coliforms were found in the tests, a fecal coliform test is required for
thermotolerant forms of the pathogenic bacteria, Should thermotolerant bacteria be found in the tests, a test for E. coli
is required. If the test is not positive for E. coli, the drinking water is considered to be pure no matter what the level of
bacteria, viruses, helmenths, fungi, yeast or protozoa in the water. Moreover, no consideration is given to antibiotic
resistant organisms.
According to EPA's Mark Meckes, “From late 1968 to early 1981, Central America was afflicted by an R+ S [Shigella]
dysenteriae pandemic. During the first year of the epidemic, in Guatemala alone, 12,500 deaths were recorded. The
causative organism was spread mainly by contaminated water and carried resistance to streptomycin, tetracycline,
chloramphenicol, and sulfadiazine.”
In 1969, W. O. K. Grabow, et al., reported on reclaimed/recycled water in “The bactericidal effect of lime
flocculation/flotation as a primary unit process in a multiple system for the advanced purification of sewage works
effluent.” They said, “An important advantage of using lime as flocculant in water purification processes is that bacteria
are not only removed by coagulation but also destroyed by the hydroxide alkalinity. This communication deals with the
bactericidal effect of lime in a flocculation/ flotation unit which is the primary and key process in a multiple system
experimental plant for the reclamation of potable water from secondary treated sewage effluent. Laboratory and field
tests revealed a marked difference in the survival of Gram-negative, Gram-positive and acid fast bacteria. Previous
studies on the bactericidal effect of lime in water treatment processes included only Gram-negative organisms. These
bacteria were considerably more sensitive than the other two groups and possible reasons for the differentiation in
resistance are discussed. Raising the pH value of humus tank effluent to 11·5 for a period of 1 hr destroyed all Gram-
negative bacteria and reduced the plate count by more than 99 per cent. Surviving organisms consisted mainly of spore-
formers. This treatment had almost no effect on mycobacteria.” (causes tuberculosis and leprosy)
http://www.sciencedirect.com/science/article/pii/0043135469900773
In 1972, the death rate in Guatemala and El Salvador was over 200 per 100,000 people. The unusual aspects were that
prior to the outbreak, Epidemic Shigella had virtually disappeared from the world for 30 years and all strains had been
antibiotic sensitive. Also, in 1972 CDC reported an epidemic typhoid fever outbreak in Mexico, on a scale
unprecedented in modern times. The Mexican outbreak was caused by Salmonella Typi.
http://www.usludgefree.org/pdf/hfw/hfw_meckes.pdf
http://jid.oxfordjournals.org/content/126/2/215.extract
In 1974, Leah K Koditscheka and Paul Guyre, Montclair State College, reported on “Resistance transfer fecal coliforms
isolated from the whippany river.” They said, “Three sites on the Whippany River, a tributary of the Passaic Watershed
in Morris County, N.J., were monitored for resistance transfer coliforms. A high percentage of fecal coliform isolates
showed multiple antibiotic resistance. A significant number of lac− and lac− isolates proved to be donors of transmissible
antibiotic resistance plasmids (R donors), by conjugation with a recipient strain of Salmonella gallinarum. Re-
examination of testing methods used for recreational and potable water sources is suggested by these findings.”
http://www.sciencedirect.com/science/article/pii/0043135474900190
1974, W. O. K. Grabow, et al., National Institute for Water Research, South African Council for Scientific and Industrial
Research, reported on “Drug resistant coliforms call for review of water quality standards.” They said, “The therapeutic
value of antimicrobial drugs is diminishing due to the rapid increase of resistant bacteria. A current prominent type of
resistance is mediated by R factors (extrachromosomal nucleic acid elements) which may cause high level resistance to
many drugs. These factors may also provide resistance to other antibacterial agents such as u.v. light, heavy metals,
bacteriocins and phages, and may enhance the virulence and infectivity of pathogens. Intestinal Gram-negative bacteria
like coliforms may act as reservoirs of R factors and transfer them to pathogens. Evidence is presented that sewage
polluted water may play an important role in the spread of coliform and other bacteria carrying R factors. Since coliforms
have joined forces with bacteria increasingly involved in disease, they can no longer be regarded as harmless indicators
of faecal pollution. This calls for a re-evaluation of water quality standards and for more advanced purification of
sewage prior to discharge into the environment.”
http://www.sciencedirect.com/science/article/pii/0043135474900025
In 1978, W. J. Kelch and J. S. Lee, Oregon State University at Corvallis, reported on the “Antibiotic Resistance Patterns
of Gram-Negative Bacteria Isolated from Environmental Sources.” They said, “A total of 2,445 gram-negative bacteria
belonging to fecal coliform, Pseudomonas, Moraxella, Acinetobacter, and Flavobacterium-Cytophaga groups were
isolated from the rivers and bay of Tillamook, Oregon, and their resistances to chloramphenicol (25 ug/ml), streptomycin
(10 ,ug/ml), ampicillin (10 Ag/ml), tetracycline (25 ,ug/ml), chlortetracycline (25 ,tg/ml), oxytetracycline (25 ,tg/ml),
neomycin (50 ,ug/ml), nitrofurazone (12.5 ,ug/ml), nalidixic acid (25 ,ug/ml), kanamycin (25 ,ug/ml), and penicillin G (10
IU/ml) were determined. Among fecal coliforms the bay isolates showed greater resistance to antibiotics than those from
tributaries or surface runoff. No such well-defined difference was found among other bacterial groups. The antibiotic
resistance patterns of gram-negative bacteria from different sources correlated well, perhaps indicating their common
origin. The antibiotic resistance patterns of gram-negative bacteria of different genera also correlated well, perhaps
indicating that bacteria which share a common environment also share a common mode for developing antibiotic
resistance.” http://aem.asm.org/cgi/reprint/36/3/450.pdf
In 1980, James B. Bell, Wendy R. Macrae, and Garth E. Elliott, Environment Canada, Environmental Protection Service,
reported on the “Incidence of R Factors in Coliform, Fecal Coliform, and Salmonella Populations of the Red River in
Canada.” They said, “Coliforns, fecal coliforms, and Salmonella were isolated from the Red River, Manitoba, Canada,
and identified. These organisms were then examined for resistance to 12 antibiotics. Some fecal coliforms were resistant
to all 12 antibiotics, and 18% of the Salmonella isolates were resistant to one or more antibiotics. A total of 52.9% of the
fecal coliforms resistant to three or more antibiotics were able to transfer single or multiple resistance (R) determinants
to the Salmonella recipient, and 40.7% could transfer R determinants to the Escherichia coli recipient. Of the resistant
Salmonella, 57% transferred one or two determinants to the Salmonella recipient, and 39% transferred one or two
determinants to the E. coli recipient. It was calculated that populations of fecal coliforms containing R factors were as
high as 1,400 per 100 ml and that an accidental intake of a few milliliters of water could lead to transient or permanent
colonization of the digestive tract. Consideration of data on bacteria with R factors should be made
in future water quality deliberations and in discharge regulations.”
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC291610/pdf/aem00239-0066.pdf
In 1980, David R. Shaw, EPA, and Victor J. Cabelli, University of Rhode Island, reported on “R-Plasmid Transfer
Frequencies from Environmental Isolates of Escherichia coli to Laboratory and Fecal Strains.” They said, “Multiple-drug-
resistant strains of Escherichia coli were isolated from the water at an estuarine site. They represented about 8.3% of
the total E. coli population. Fifty-five strains, representing each of the 32 resistance patterns identified, were mated with
an E. coli K-12 F- strain.” While they noted outbreaks of Shigella, Salmonella, or enteropathogenic E. coli strains which
contain transferable R plasmids were rare in the United States, there was a concern they were being disseminated into
water by the disposal of raw or disinfected sewage and sludge. They said, “The concern is that these plasmids are
being transferred to enteric, bacterial pathogens in sewage, environmental waters, or individuals who harbor R+ E. coli
previously ingested in the course of water-associated activities, notably swimming and shellfish consumption. – The
basis for concern is that multiple-drug resistant coliforms have been isolated from sewage, its receiving waters, and
sludge-dumping areas, where they comprised between 1 and 50% of the coliform population.” However, the conclusion
was that there was little probability of infections even for swimmers.” http://aem.asm.org/cgi/reprint/40/4/756.pdf
In the 1981 study, “Antibiotic-resistant bacteria in drinking water”, J. L. Armstrong, et al., said, “We analyzed drinking
water from seven communities for multiply antibiotic-resistant (MAR) bacteria (bacteria resistant to two or more
antibiotics) and screened the MAR bacterial isolates obtained against five antibiotics by replica plating. Overall, 33.9%
of 2,653 standard plate count bacteria from treated drinking waters were MAR. Two different raw water supplies for two
communities carried MAR standard plate count bacteria at frequencies of 20.4 and 18.6%, whereas 36.7 and 67.8% of
the standard plate count populations from sites within the respective distribution systems were MAR. Isolate
identification revealed that MAR gram-positive cocci (Staphylococcus) and MAR gram-negative, nonfermentative rods
(Pseudomonas, Alcaligenes, Moraxella-like group M, and Acinetobacter) were more common in drinking waters than in
untreated source waters. Site-to-site variations in generic types and differences in the incidences of MAR organisms
indicated that shedding of MAR bacteria living in pipelines may have contributed to the MAR populations in tap water.
We conclude that the treatment of raw water and its subsequent distribution select for standard plate count bacteria
exhibiting the MAR phenotype.” http://aem.asm.org/cgi/content/abstract/42/2/277
In 1982, J. L. Armstrong, et al., reported on the “Selection of antibiotic-resistant standard plate count bacteria during
water treatment.” They 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 1992, A. Gaur et al., Industrial Toxicology Research Centre at Lucknow, reported on the “Transferable antibiotic
resistance among thermotolerant coliforms from rural drinking water in India.” They found, “A total of 231 thermotolerant
[fecal] coliforms was isolated from rural drinking water from four states of India. Of these, 220 isolates were resistant to
ampicillin, chloramphemicol, streptomycin and tetracycline. Multiple (MAR), double and single antibiotic resistances were
observed in 31.4, 48.6 and 13.7% of the isolates, respectively. Out of 177 antibiotic-resistant isolates examined for
transmissibility, only 15.3% were able to transfer their resistances to Escherichia coli K-12 recipient. The resistances
were transferred by 32.5% of MAR, 21.9% of double resistant and 7.6% of single resistant isolates. Ampicillin resistance
was transferable in 14.69% strains while resistances for the rest of the antibiotics were transferable in less than 4%
strains. MAR strains of E. coli and Klebsiella sp. showed highest levels of R-plasmid transfer.”
http://www.ncbi.nlm.nih.gov/pubmed/1499665
In 1995, E. W. Rice, et al., EPA, reported on the “Occurrence of High-Level Aminoglycoside Resistance in
Environmental Isolates of Enterococci.” They said, “High-level resistance to aminoglycosides was observed in
environmental isolates of enterococci. Various aquatic habitats, including agricultural runoff, creeks, rivers, wastewater,
and wells, were analyzed. Strains of Enterococcus faecalis, E. faecium, E. gallinarum, and other Enterococcus spp.
demonstrated multiple antibiotic resistance patterns to aminoglycosides. – Enterococci are recognized as important
human pathogens in both community- and nosocomial-acquired infections. – Enterococci exhibit inherent low-level
aminoglycoside resistance; the MICs are between 2 and 16 mg/ml (8). In recent years, a number of enterococcal strains
have acquired high-level aminoglycoside resistance (HLAR); the MICs are $2,000 mg/ml (3, 8, 12). The occurrence of
HLAR in enterococci is an example of acquired resistance as opposed to intrinsic resistance. Enterococcus faecalis
infections are often treated with synergistic combinations of a cell wall-active agent and an aminoglycoside. The addition
of a cell wall-active agent, such as penicillin or vancomycin, typically results in enhanced killing of enterococci. However,
when E. faecalis isolates acquire HLAR, the synergism with a cell wall-active antibiotic is lost (8). In these instances,
medical treatment options are limited. – In this study, two sewage isolates of E. faecium were found to exhibit high-level
resistance to gentamicin. Furthermore, the occurrence of high-level gentamicin resistance was also seen for two
isolates of E. faecalis, one from sewage and one from an urban stream. The occurrence of community-acquired HLAR
enterococcal infections is a matter of continuing concern. Nachamkin et al. (9) noted that 7% of infections in their study
were community acquired. A careful review of patients to exclude those who had recently resided in a hospital or chronic
care facility prompted these investigators to conclude that a reservoir for gentamicin-resistant enterococci existed in the
community. Our findings suggest that environmental sources may contribute to the dissemination of HLAR enterococci.
The increasing prevalence of HLAR enterococci associated with nosocomial disease and the current finding of these
organisms in environmental samples should be a matter of concern for both clinicians and public health authorities.”
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC167293/pdf/610374.pdf
In 2003, Joseph O. Falkinham, III, Virginia Tech at Blacksburg, reported on “The changing pattern of nontuberculous
mycobacterial disease.” Falkinham said, “Nontuberculous mycobacteria are human opportunistic pathogens whose
source of infection is the environment. These include both slow-growing (eg, Mycobacterium kansasii and
Mycobacterium avium) and rapid-growing (eg, Mycobacterium abscessus and Mycobacterium fortuitum) species.
Transmission is through ingestion or inhalation of water, particulate matter or aerosols, or through trauma. The historic
presentation of pulmonary disease in older individuals with predisposing lung conditions and in children has been
changing. Pulmonary disease in elderly individuals who lack the classic predisposing lung conditions is increasing.
Pulmonary disease and hypersensitivity pneumonitis have been linked with occupational or home exposures to
nontuberculous mycobacteria. There has been a shift from Mycobacterium scrofulaceum to M avium in children with
cervical lymphadenitis. Further, individuals who are immunosuppressed due to therapy or HIV-infection are at a greatly
increased risk for nontuberculous mycobacterial infection. The changing pattern of nontuberculous mycobacterial
disease is due in part to the ability of these pathogens to survive and proliferate in habitats that they share with
humans, such as drinking water. The advent of an aging population and an increase in the proportion of
immunosuppressed individuals suggest that the prevalence of nontuberculous mycobacterial disease will increase.”
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2094944/
In 1997, Salina Pareen, et al., University of Florida, reported on the “Association of Multiple-Antibiotic-Resistance
Profiles with Point and Nonpoint Sources of Escherichia coli in Apalachicola Bay.” They said, “A total of 765 Escherichia
coli isolates from point and nonpoint sources were collected from the Apalachicola National Estuarine Research
Reserve, and their multiple-antibiotic-resistance (MAR) profiles were determined with 10 antibiotics. E. coli isolates from
point sources showed significantly greater resistance (P < 0.05) to antibiotics and higher MAR indices than isolates from
nonpoint sources. Specifically, 65 different resistance patterns were observed among point source isolates, compared
to 32 among nonpoint source isolates. Examples of this contrast in MAR profiles included percentages of isolates with
resistance to chlortetracycline-sulfathiazole of 33.7% and to chlortetracycline-penicillin G-sulfathiazole of 14.5% for point
source isolates versus 15.4 and 1.7%, respectively, for nonpoint source isolates. MAR profile homology, based on
coefficient similarity, showed that isolates from point sources were markedly more diverse than isolates from nonpoint
sources. Seven clusters were observed among point source isolates, with a coefficient value of approximately 1.8. In
contrast, only four clusters were observed among nonpoint source isolates. Covariance matrices of data displayed six
very distinct foci representing nonpoint source E. coli isolates. Importantly, E. coli isolates obtained directly from human
and animal feces also clustered among point and nonpoint sources, respectively. We conclude that E. coli MAR profiles
were associated with point and nonpoint sources of pollution within Apalachicola Bay and that this method may be useful
in facilitating management of other estuaries.” http://aem.asm.org/cgi/reprint/63/7/2607
In 2003, Matthew T. Roe, et al., Texas A&M University at College Station, reported on “Antimicrobial Resistance Markers
of Class 1 and Class 2 Integron-bearing Escherichia coli from Irrigation Water and Sediments.” They said, “Municipal
and agricultural pollution affects the Rio Grande, a river that separates the United States from Mexico. Three hundred
and twenty-two Escherichia coli isolates were examined for multiple antibiotic resistance phenotypes and the prevalence
of class 1 and class 2 integron sequences. Thirty-two (10%) of the isolates were resistant to multiple antibiotics. Four
(13%) of these isolates contained class 1–specific integron sequences; one isolate contained class 2 integron–specific
sequences. Sequencing showed that the class 1 integron–bearing strain contained two distinct gene cassettes, sat-1
and aadA. Although three of the four class 1 integron–bearing strains harbored the aadA sequence, none of the strains
was phenotypically resistant to streptomycin. These results suggest that integron-bearing E. coli strains can be present
in contaminated irrigation canals and that these isolates may not express these resistance markers. – Leaking septic
tanks and wastewater effluent discharges result in fecal contamination levels as high as 2,000 CFU/mL of fecal
coliforms. – Overall, these results suggest that the irrigation canals and sediments associated with the Rio Grande are
contaminated by bacteria of fecal origin that contain antimicrobial resistance genes. – This study did not investigate the
possibility that other integron-bearing nonfecal bacteria were present.”
http://www.cdc.gov/ncidod/EID/vol9no7/02-0529.htm
In 2004, Ronald J. Ash, et al., Washburn University at Topeka, reported on “Antibiotic Resistance of Gram-Negative
Bacteria in Rivers, United States.” They said, “Bacteria with intrinsic resistance to antibiotics are found in nature. Such
organisms may acquire additional resistance genes from bacteria introduced into soil or water, and the resident bacteria
may be the reservoir or source of widespread resistant organisms found in many environments. We isolated antibiotic-
resistant bacteria in freshwater samples from 16 U.S. rivers at 22 sites and measured the prevalence of organisms
resistant to β-lactam and non β-lactam antibiotics. Over 40% of the bacteria resistant to more than one antibiotic had at
least one plasmid. Ampicillin resistance genes, as well as other resistance traits, were identified in 70% of the plasmids.
The most common resistant organisms belonged to the following genera: Acinetobacter, Alcaligenes, Citrobacter,
Enterobacter, Pseudomonas, and Serratia.” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2730334/
In 2004, Shivi Selvaratnam and J. D. Kunberger, reported on the “Increased frequency of drug-resistant bacteria and
fecal coliforms in an Indiana Creek adjacent to farmland amended with treated sludge.” They said, “Many studies
indicate the presence of human pathogens and drug-resistant bacteria in treated sewage sludge. Since one of the main
methods of treated sewage disposal is by application to agricultural land, the presence of these organisms is of concern
to human health. The goal of this study was to determine whether the frequency of drug-resistant and indicator bacteria
in Sugar Creek, which is used for recreational purposes, was influenced by proximity to a farmland routinely amended
with treated sludge (site E). Surface water from 3 sites along Sugar Creek (site E, 1 upstream site (site C) and 1
downstream site (site K)) were tested for the presence of ampicillin-resistant (AmpR) bacteria, fecal and total coliforms
over a period of 40 d. Site E consistently had higher frequencies of AmpR bacteria and fecal coliforms compared with
the other 2 sites. All of the tested AmpR isolates were resistant to at least 1 other antibiotic. However, no isolate was
resistant to more than 4 classes of antimicrobials. These results suggest that surface runoff from the farmland is
strongly correlated with higher incidence of AmpR and fecal coliforms at site E.” http://www.ingentaconnect.
com/content/nrc/cjm/2004/00000050/00000008/art00012?crawler=true
In 2006, Amy Pruden, et al., Colorado State University, reported on “Antibiotic Resistance Genes as Emerging
Contaminants: Studies in Northern Colorado.” They said, This study explores antibiotic resistance genes (ARGs) as
emerging environmental contaminants. The purpose of this study was to investigate the occurrence of ARGs in various
environmental compartments in northern Colorado, including Cache La Poudre (Poudre) River sediments, irrigation
ditches, dairy lagoons, and the effluents of wastewater recycling and drinking water treatment plants. Additionally, ARG
concentrations in the Poudre River sediments were analyzed at three time points at five sites with varying levels of
urban/agricultural impact and compared with two previously published time points. It was expected that ARG
concentrations would be significantly higher in environments directly impacted by urban/agricultural activity than in
pristine and lesser-impacted environments. Polymerase chain reaction (PCR) detection assays were applied to detect
the presence/absence of several tetracycline and sulfonamide ARGs. Quantitative real-time PCR was used to further
quantify two tetracycline ARGs (tet(W) and tet(O)) and two sulfonamide ARGs (sul(I) and sul(II)). The following trend was
observed with respect to ARG concentrations (normalized to eubacterial 16S rRNA genes): dairy lagoon water >
irrigation ditch water > urban/agriculturally impacted river sediments (p < 0.0001), except for sul(II), which was absent in
ditch water. It was noted that tet(W) and tet(O) were also present in treated drinking water and recycled wastewater,
suggesting that these are potential pathways for the spread of ARGs to and from humans. On the basis of this study,
there is a need for environmental scientists and engineers to help address the issue of the spread of ARGs in the
environment.”
http://pubs.acs.org/doi/abs/10.1021/es060413l
In 2006, P. W. Ramteke & Suman Tewari, Allahabad Agricultural Institute (Deemed University), reported on the
“Serogroups of Escherichia coli from Drinking Water.” They said, “Fifty seven isolates of thermotolerant E. coli [fecal
coliform] were recovered from 188 drinking water sources, 45 (78.9%) were typable of which 15 (26.3%) were
pathogenic serotypes. Pathogenic serogroup obtained were 04 (Uropathogenic E. coli, UPEC), 025 (Enterotoxigenic E.
coli, ETEC), 086 (Enteropathogenic E. coli, EPEC), 0103 (Shiga-toxin producing E. coli, STEC), 0157 (Shiga-toxin
producing E. coli, STEC), 08 (Enterotoxigenic E. coli, ETEC) and 0113 (Shigatoxin producing E. coli, STEC). All the
pathogenic serotypes showed resistance to bacitracin and multiple heavy metal ions. Resistance to streptomycin and
cotrimazole was detected in two strains whereas resistance to cephaloridine, polymixin-B and ampicillin was detected in
one strain each. Transfer of resistances to drugs and metallic ions was observed in 9 out of 12 strains studied.
Resistances to bacitracin were transferred in all nine strains. Among heavy metals resistance to As3+ followed by Cr6+
were transferred more frequently. – From 188 drinking water samples 57 isolates of E. coli were recovered and 45
(78.9%) of them were typeable. Half of the E. coli isolates belonged to nonpathogenic serotypes (50.0%) and only
26.3% E. coli were confirmed pathogenic serotypes. – In the present investigation STEC was the major group of
pathogens (5 nos.) representing three serotypes i.e., 0103, 0157 and 0113. In this study resistance to one or more
antibiotics was observed in 49.2% of the STEC strains, with some strains exhibiting multidrug resistance. – Shiga-like
toxin producing E. coli (STEC) cause a broad range of symptoms in human including hemorrhagic colitis and the often
deadly hemorrhagic uramic syndrome. – Recently E. coli 0157:H7 was found to cause infections of central nervous
system and brain damage. – The genome of E. coli is of high plasticity allowing it to gain or lose genes at a relatively
high frequency. Adding to the plasticity is the fact that many antibiotic resistance and virulence genes in E. coli are
located on mobile elements like plasmids. Since these factors are encoded on mobile genetic elements there is
possibility of emergence of new combinations of virulence genes in the future.” http://www.aseanenvironment.
info/Abstract/41015613.pdf
In 2008, Joseph W. Duris, et al., U.S. Geological Survey, reported on the “Gene and Antigen Markers of Shiga-toxin
Producing E. coli from Michigan and Indiana River Water: Occurrence and Relation to Recreational Water Quality
Criteria.” They said, “The relation of bacterial pathogen occurrence to fecal indicator bacteria (FIB) concentrations used
for recreational water quality criteria (RWQC) is poorly understood. This study determined the occurrence of Shiga-toxin
producing Escherichia coli (STEC) markers and their relation to FIB concentrations in Michigan and Indiana river water.
Using 67 fecal coliform (FC) bacteria cultures from 41 river sites in multiple watersheds, we evaluated the occurrence of
five STEC markers: the Escherichia coli (EC) O157 antigen and gene, and the STEC virulence genes eaeA, stx1, and
stx2. Simple isolations from selected FC cultures yielded viable EC O157. By both antigen and gene assays, EC O157
was detected in a greater proportion of samples exceeding rather than meeting FC RWQC (P < 0.05), but was unrelated
to EC and enterococci RWQC. The occurrence of all other STEC markers was unrelated to any FIB RWQC. The eaeA,
stx2, and stx1 genes were found in 93.3, 13.3, and in 46.7% of samples meeting FC RWQC and in 91.7, 0.0, and 37.5%
of samples meeting the EC RWQC. Although not statistically significant, the percentage of samples positive for each
STEC marker except stx1 was lower in samples that met, as opposed to exceeded, FIB RWQC. Viable STEC were
common members of the FC communities in river water throughout southern Michigan and northern Indiana, regardless
of FIB RWQC. Our study indicates that further information on the occurrence of pathogens in recreational waters, and
research on alternative indicators of their occurrence, may help inform water-resource management and public health
decision-making.” https://www.soils.org/publications/jeq/articles/38/5/1878
In 2009, M.R. Shakibaie, et al., Kerman University of Medical Sciences, reported on “Horizontal transfer of antibiotic
resistance genes among gram negative bacteria in sewage and lake water and influence of some physico-chemical
parameters of water on conjugation process.” They said, “Transfer of antibiotic resistance genes among gram negative
bacteria in sewage and lake water and easy access of these bacteria to the community are major environmental and
public health concern. The aim of this study was to determine transfer of the antimicrobial resistance genes from
resistant to susceptible gram negative bacteria in the sewage and lake water by conjugation process and to determine
the influence of some physico-chemical parameters of sewage and lake water on the transfer of these resistance genes.
For this reason, we isolated 20 liter of each sewage and lake water from coconut area within university campus and
Lingambudi lake respectively in Mysore city, India, during monsoon season and studied different physical parameters of
the water samples like pH, temperature, conductivity, turbidity and color as well as chemical parameters like BOD, COD,
field DO and total chloride ion. The gram negative bacteria were isolated and identified from the above water samples
using microbiological and biochemical methods and their sensitivity to different antibiotics was determined by disc
diffusion break point assay. Conjugation between two multiple antibiotic resistant isolates Pseudomonas aeuginosa and
E.coli as donor and E.coli Rif r (sensitive to antibiotics) as recipient were carried out in 5ml sterile sewage and lake
water. All isolates were resistant to Am, moderately resistant to Te and E, while majority were sensitive to Cip, Gm and
CAZ antibiotics. Horizontal transfer of antibiotic resistance genes by conjugation process revealed transfer of Gm, Te
and E resistant genes from Ps. aeruginosa to E.coli Rif r recipient with mean frequency of ± 2.3 x 10 -4 in sewage and ±
2.6 x 10 -6 in lake water respectively. Frequency of conjugation in sewage was two fold more as compared to lake water
(p<0.05). Co- transfer study revealed simultaneous transfer of above resistant markers together to the recipient cells.
As the above results indicate, due to selective pressure in sewage (presence of antibiotics), the isolates from sewage
were more resistant to different antibiotics as compared to those from lake water. Furthermore, these resistance genes
can transfer to sensitive bacteria by conjugation. Physico-chemical parameters of water may play role in this process.”
http://www.jeb.co.in/journal_issues/200901_jan09_spl/paper_08.pdf
In 2009, C. Faria, et al., Universidade Católica Portuguesa, reported on “Antibiotic resistance in coagulase negative
staphylococci isolated from wastewater and drinking water.” They said, “This study reports the antibiotic resistance
patterns of coagulase negative staphylococci (CNS) isolated from a drinking water treatment plant (WTP), a drinking
water distribution network, responsible for supplying water to the consumers (WDN), and a wastewater treatment plant
(WWTP), responsible for receiving and treating domestic residual effluents. Genotyping and the 16S rRNA gene
sequence analysis demonstrated a higher diversity of species both in the WTP (6 species/19 isolates) and WWTP (12
species/47 isolates) than in the WDN (6 species/172 isolates). Staphylococcus pasteuri and Staphylococcus epidermidis
prevailed in the WTP and WDN and Staphylococcus saprophyticus in the WWTP. Staphylococci with reduced
susceptibility (resistance or intermediary phenotype) to beta-lactams, tetracycline, clindamycin and erythromycin were
observed in all types of water and belonged to the three major species groups. The highest resistance rate was found
against erythromycin, presumably due to the presence of the efflux pump encoded by the determinant msrA, detected in
the majority of the resistant isolates. This study demonstrates that antibiotic resistant CNS may colonize different types
of water, namely drinking water fulfilling all the quality standards.” http://repositorio.ucp.pt/bitstream/10400.
14/3292/1/Antibiotic%20resistance%20in%20coagulase.PDF
The European Commission summarized the C. Faria, et al., study in a 2009 news release. It said, “‘Coagulase-negative
staphylococci’ (CNS) are common and usually harmless. However, some CNS can cause skin infections when introduced
medically or if present in wounds. The urban water cycle is an important means of transport of microorganisms like CNS
and recently the risk of infection by CNS has increased. Although the European Drinking Water Directive sets quality
standards for drinking water, it does not recommend screening for staphylococci. – [because] – Antibiotic resistant CNS
in water for human consumption is a risk for which little or no assessment is available. In addition, the behaviour of CNS
may change depending on other environmental stress factors. For example, the over-use of antibiotics might encourage
an increase in CNS.”
http://www.ncbi.nlm.nih.gov/pubmed/19324394
http://ec.europa.eu/environment/integration/research/newsalert/pdf/155na4.pdf
In 2009, A.H. Shar, et al., Shah Abdul Latif University, reported on the “Antibiotic susceptibility of thermo-tolerant
Escherichia coli 2 isolated from drinking water of Khairpur City, Sindh, Pakistan.” They said, “A total 72 drinking water
sample were collected and analyzed by membrane filtration method during 1 year study from various points in Khairpur
City. Out of these 58 (80.55%) samples were found to be contaminated with thermo-tolerant Escherichia coli 2 [fecal
coliform]. The susceptibility of these isolates to 35 antibiotics was studied by disc diffusion method and the organism
was highly sensitive to levoflaxin, cefipime, enoxobid, noroxin, tarivid, ciproxin, avelox, amikacin, kanamycin, rocifin,
pipenedic acid and slightly sensitive to cravit, naladixic acid, neomycin, cefizox, fortum cefotaxime, cefizox, fortum,
tobramycin and cefoperoxone. The resistance against 16 antibiotics such as meropenem, linkomycin, fusidic acid,
orbenin, penicillin, streptomycin, bacitracin, minocin, zinacef, amoxil, ceclor, claracid, cephalexin, augmentin, cephradin
and dalacin was shown by these isolates. We report the presence of multi-drug resistance in thermo-tolerant
Escherichia coli isolated in municipal water with different levels of prevalence in Khairpur City. In this study a higher
number of positive results were obtained in all sampling points indicating the more fecally polluted municipal water.”
http://docsdrive.com/pdfs/ansinet/pjbs/2009/648-652.pdf
In 2009, L. M. Fincher, et al., Indiana Univ. at Kokomo, reported on the “Occurrence and antibiotic resistance of
Escherichia coli O157:H7 in a watershed in north-central Indiana.” They said, “Rivers and streams can become
contaminated with microorganisms from various sources: human sewage and leaking septic tanks, discharge of treated
wastewater into source water, animal waste (domestic and wildlife), and storm water. These various sources of biological
pollution are reservoirs for many types of microorganisms such as bacteria, viruses, and protozoa – Common bacterial
pathogens in water include Salmonella sp, Shigella sp., E. coli O157:H7, Pseudomonas aeruginosa, Aeromonas
hydrophila, mycobacteria, Helicobacter pylori, and various others. – The Wildcat Creek in north-central Indiana is an
impaired stream with historically high fecal coliform counts. This study evaluated the presence of both fecal coliforms
and Escherichia coli O157:H7 at five sites in rural and urban areas in the eastern part of the Wildcat Creek watershed.
Escherichia coli O157:H7 was isolated by immunomagnetic separation. Shiga-like toxin genes (stx1 and stx2) were
detected in selected isolates by polymerase chain reaction (PCR) amplification. Isolates of E. coli O157:H7 were also
tested by the Kirby-Bauer method for their resistance to eight different antibiotics. Fecal coliform counts were high at
two sites located downstream from the city of Kokomo. Escherichia coli O157:H7 was found to be a common occurrence
in both the urban and rural parts of the Wildcat Creek watershed, being detected at least twice from each site. This
bacterium was also found at various times of the year. Additionally, isolates of antibiotic resistant E. coli O157:H7 were
detected from various sites along the stream, especially in sites located in the city and downstream from the urban area,
suggesting that human activities might be associated with the dissemination of these bacteria.”
http://www.ncbi.nlm.nih.gov/pubmed/19329688
In 2009, D.S. Pontes, et al., Universidade Federal de Minas Gerais, reported on “Multiple antimicrobial resistance of
gram-negative bacteria from natural oligotrophic lakes under distinct anthropogenic influence in a tropical region.” They
said, “The aim of this study was to evaluate the resistance to ten antimicrobial agents and the presence of bla ( TEM1 )
gene of Gram-negative bacteria isolated from three natural oligotrophic lakes with varying degrees of anthropogenic
influence. A total of 272 indigenous bacteria were recovered on eosin methylene blue medium; they were characterized
for antimicrobial resistance and identified taxonomically by homology search and phylogenetic comparisons. Based on
16S ribosomal RNA sequences analysis, 97% of the isolates were found to be Gram-negative bacteria; they belonged to
11 different genera. Members of the genera Acinetobacter, Enterobacter, and Pseudomonas predominated. Most of the
bacteria were resistant to at least one antimicrobial. The incidence of resistance to beta-lactams, chloramphenicol, and
mercury was high, whereas resistance to tetracycline, aminoglycosides, and nalidixic acid was low. There was a great
frequency of multiple resistances among the isolates from the three lakes, although no significant differences were
found among the disturbed and reference lakes. The ampicillin resistance mechanism of 71% of the isolates was due to
the gene bla ( TEM1 ). Our study suggests that multiresistant Gram-negative bacteria and the bla ( TEM1 ) gene are
common in freshwater oligotrophic lakes, which are subject to different levels of anthropogenic inputs.”
https://www.ncbi.nlm.nih.gov/pubmed/19504148
In 2009, Chuanwu Xi, et al., University of Michigan at Ann Arbor, reported on the “Prevalence of Antibiotic Resistance in
Drinking Water Treatment and Distribution Systems.” They said, “The occurrence and spread of antibiotic-resistant
bacteria (ARB) are pressing public health problems worldwide, and aquatic ecosystems are a recognized reservoir for
ARB. We used culture-dependent methods and quantitative molecular techniques to detect and quantify ARB and
antibiotic resistance genes (ARGs) in source waters, drinking water treatment plants, and tap water from several cities in
Michigan and Ohio. We found ARGs and heterotrophic ARB in all finished water and tap water tested, although the
amounts were small. The quantities of most ARGs were greater in tap water than in finished water and source water. In
general, the levels of bacteria were higher in source water than in tap water, and the levels of ARB were higher in tap
water than in finished water, indicating that there was regrowth of bacteria in drinking water distribution systems.
Elevated resistance to some antibiotics was observed during water treatment and in tap water. Water treatment might
increase the antibiotic resistance of surviving bacteria, and water distribution systems may serve as an important
reservoir for the spread of antibiotic resistance to opportunistic pathogens.”
http://aem.asm.org/cgi/content/abstract/75/17/5714
In 2011, Ivone Vaz-Moreira, Universidade Católica Portuguesa, reported on the “Diversity and antibiotic resistance
patterns of Sphingomonadaceae isolated from drinking water.” They said, “Sphingomonadaceae (n=86) were isolated
from a drinking water treatment plant (n=6), tap water (n=55), cup filler of dental chairs (n=21) and a water
demineralization filter (n=4). The bacterial isolates were identified based on the analysis of the 16S rRNA gene
sequence and intra-species variation was assessed on basis of atpD gene sequence analysis. The isolates were
identified as members of the genera Sphingomonas (n=27), Sphingobium (n=28), Novosphingobium (n=12),
Sphingopyxis (n=7) and Blastomonas (n=12). The susceptibility patterns to five classes of antibiotics were analysed and
compared for the different sites of isolation and taxonomic groups. Colistin resistance was observed to be intrinsic (92
%). The highest antibiotic resistance prevalence values were observed in members of the genera Sphingomonas and
Sphingobium and for beta-lactams, ciprofloxacin and cotrimoxazol. In tap water and in water from dental chairs, antibiotic
resistance was more prevalent than in the other samples, mainly due to the predominance of isolates of the genera
Sphingomonas and Sphingobium. These two genera presented distinct patterns of association of antibiotic resistance,
hinting different paths of resistance development. Antibiotic resistance patterns were often species- rather than site- or
strain-related, suggesting the importance of vertical resistance transmission in these bacteria. This is the first study
demonstrating the members of the family Sphingomonadaceae as potential reservoirs of antibiotic resistance in drinking
water.” http://aem.asm.org/cgi/content/abstract/AEM.00579-11v1
In the 2011 study, “Tertiary-Treated Municipal Wastewater is a Significant Point Source of Antibiotic Resistance Genes
into Duluth-Superior Harbor”, T.M. Lapara, et al., stated, “In this study, the impact of tertiary-treated municipal
wastewater on the quantity of several antibiotic resistance determinants in Duluth-Superior Harbor was investigated by
collecting surface water and sediment samples from 13 locations in Duluth-Superior Harbor, the St. Louis River, and
Lake Superior. Quantitative PCR (qPCR) was used to target three different genes encoding resistance to tetracycline
(tet(A), tet(X), and tet(W)), the gene encoding the integrase of class 1 integrons (intI1), and total bacterial abundance
(16S rRNA genes) as well as total and human fecal contamination levels (16S rRNA genes specific to the genus
Bacteroides ). The quantities of tet(A), tet(X), tet(W), intI1, total Bacteroides , and human-specific Bacteroides were
typically 20-fold higher in the tertiary-treated wastewater than in nearby surface water samples. In contrast, the
quantities of these genes in the St. Louis River and Lake Superior were typically below detection. Analysis of sequences
of tet(W) gene fragments from four different samples collected throughout the study site supported the conclusion that
tertiary-treated municipal wastewater is a point source of resistance genes into Duluth-Superior Harbor. This study
demonstrates that the discharge of exceptionally treated municipal wastewater can have a statistically significant effect
on the quantities of antibiotic resistance genes in otherwise pristine surface waters.” http://www.ncbi.nlm.nih.
gov/pubmed/21981654
Few experts stop to think our state and federal regulators are promoting spreading antibiotic resistance into our food
supply by the use of treated antibiotic resistance contaminated sewage effluent (reclaimed water) to irrigate food crops
we eat raw as well as recharge aquifers and to use sewage sludge (biosolids) as a fertilizer. Changing the name of
sewage effluent to reclaimed water and sludge to biosolids offers no protection and does little to convince the public of
its safety. To add to the problem, we now have to deal with the internalization of antibiotic resistant bacteria and DNA
into plants, both intentionally and accidentally.
Next Antibiotic Resistance in “Biotech” plants
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