A PERFECT STORM OF ANTIBIOTIC RESISTANCE
Section 10
Antibiotic Resistance Bacteria in Sewage Wastewater Treatment Plants. 9/09/2011
Back to Myth # 4
EPA's cheap coliform and fecal coliform tests are only for members of the gram-negative Enterobacteriacea family
under certain time and temperature circumstances. According to Kenneth Todar, University of Wisconsin-Madison
Department of Bacteriology, “The enterobacteriaceae include agents of food poisoning and gastroenteritis, hospital-
acquired infections, enteric fevers (e.g. typhoid fever) and plague. They also cause infections in domestic, farm and zoo
animals and include an important group of plant pathogens. Their host range includes animals ranging from insects to
humans, as well as fruits, vegetables, grains, flowering plants, and trees.”
Most experts claim that only members of the Enterobacteriacea family that show some minor growth activity when
incubated at 112.1°F (fecal coliform) are from human fecal material. However, according to EPA's top expert, Mark C.
Meckes, “most strains of Escherichia coli will ferment lactose under the elevated temperature test for fecal coliform and
therefore will meet the definition of “fecal coliform.” Similarly, some strains of Klebsiella will also ferment lactose under
these same test conditions and will meet the definition of “fecal coliform.” Yet, these experts claim these deadly
pathogens are only indicators of fecal pollution. Like E. coli, Klebsiella pneumoniae is second only to E. coli as a
urinary tract pathogen. Klebsiella pneumoniae is a common antibiotic resistant hospital-acquired infectious agent, an
enteroinvasive food-borne pathogen, and a cause of necrotizing fasciitis (i.e., flesh-eating bacteria syndrome).
Most experts also claim that members of the Enterobactericea family incubated at 95°F (coliform) are just indicators of
potential fecal pollution. According to Meckes, “The total coliform group consists of several genera of bacteria
belonging to the family Enterobacteriaceae. Fecal indicator bacteria are used to assess the microbiological quality of
water because although not typically disease causing, they may be found in relatively high densities following a pollution
event and they have been (but are not always) detected in concert with some waterborne disease-causing (pathogenic)
organisms.”
In reality, most experts know that antibiotic resistant Enterobacteriacea pathogens in sludge/biosolids and reclaimed
water are being spread on agricultural land used for grazing cattle and raising food crops as well as on school grounds,
parks, forests and sold for home lawns and gardens. As a result many plant and soil bacteria have picked up virulent
and antibiotic resistant genes to become human pathogens in community settings as our food and water is
contaminated.
In 1969, Alton B. Sturtevant, Jr., and Thomas W. Feary, The Medical Center, University of Alabama in Birmingham,
researched the “Incidence of Infectious Drug Resistance Among Lactose-Fermenting Bacteria Isolated from Raw and
Treated Sewage.” They said, “Since its discovery in Japan in 1959, infectious drug resistance, mediated by episomal
elements known as R factors, has been shown to be an important factor in the spread of multiple antibiotic resistance
among all members of the Enterobacteriaceae as well as to unrelated gram negative bacteria such as Pseudomonas
aeruginosa, Vibrio cholerae, and Pasteurella pestis . – Raw and treated sewage samples were examined for antibiotic-
resistant, lactose fermenting bacteria. Approximately 1% of the total lactose-fermenting bacteria were multiply resistant.
Of these organisms, 50% were capable of transferring all or part of their resistance to a drug-sensitive recipient. Only
43% of those isolated on media containing a single antibiotic were capable of resistance transfer, whereas 57% of those
recovered on multiple antibiotic plates transferred resistance. R factors conferring resistance to chloramphenicol,
streptomycin, and tetracycline; streptomycin and tetracycline; and ampicillin, streptomycin, and tetracycline accounted
for 22, 19, and 15%, respectively, of those identified. The data indicate a significant level of infectious drug resistance
among the intestinal bacteria of the urban population.” Antibiotic resistance was found in all members of the
Enterobacteriaceae family (coliform) as well as unrelated gram negative bacteria such as Pseudomonas aeruginosa,
Vibrio cholerae, and Pasteurella pestis. They said, “There was no significant difference in the incidence of drug-
resistant bacteria in raw or treated sewage.” Furthermore, they added, “Only 43% of the strains selected by a single
antibiotic were capable of transferring all or part of their resistance to a sensitive recipient, whereas 57% of those
selected by multiple antibiotics were capable of transfer.” Nineteen different R factors were found among the 30 multiple
resistant strains grown on antibiotics. http://thewatchers.us/EPA/2/1969-drug-resistance-sewage.pdf
In 1970, Alton B. Sturyevant, Gail H. Cassell, and Thomas W. Fearly published a similar study on heat resistant
Entrobacteriacea, “Incidence of Infectious Drug Resistance Among Fecal Coliforms Isolated from Raw Sewage.” They
said, “Although members of the coliform group are present in large numbers in the feces of warm-blooded animals, they
may also be found in association with soil or plants.” Therefore, to distinguish between fecal and nonfecal coliforms,
typical coliform colonies were incubated for 24 hours at 44.5°C (112.1°F). They fail to acknowledge that true fecal
coliforms from humans are released at less that 100°F anal temperature and less than 5% will show up in the test at
112.1°F. According to them, “Raw sewage was examined for the incidence of antibiotic-resistant coliforms present
among both total and fecal coliforms. In both groups, it was found that approximately 3% of the coliform bacteria were
resistant to two or more antibiotics. Of these organisms, 48% were capable of transferring all or part of their antibiotic
resistance to an antibiotic-sensitive, F-, derivative of Escherichia coli K-12. Among the R factors identified, those
conferring resistance to streptomycin-tetracycline, ampicillinstreptomycin-tetracycline, and ampicillin or ampicillin-
streptomycin accounted for 23, 20, and 15%, respectively, of the total R factors detected. The data indicate a significant
level of infectious drug resistance among the fecal coliforms of the urban population. The data indicate further that
because of the high incidence of coliform bacteria found to be doubly resistant to streptomycin and tetracyline, the
inclusion of these antibiotics in selective media used for routine total or fecal coliform counts may serve to identify
domestic sources of pollution.” They also said, “100 lactose-positive [antibiotic resistant coliform] colonies were
randomly picked for further study. Of these 100 isolates, 80 were shown to be typical E. coli and 20 were shown to be
either members of the Citrobacter group or the Klebsiella-Enterobacter group. -- Characterization of these [antibiotic
resistant fecal colifom] isolates revealed that 92 were typical E. coli strains, whereas 9 belonged to either the Citrobacter
or Klebsiella-Enterobacter group.” What is disturbing since the previous study was that incidence of an R factor
conferring single resistance to ampicillin had increased from 2% 1 year ago, to 15% in the present study.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC377208/pdf/applmicro00113-0102.pdf
In 1976, Marylyn D. Cooke, Cawthron Institute, Nelson, New Zealand, reported on “Antibiotic Resistance Among
Coliform and Fecal Coliform Bacteria Isolated from Sewage, Seawater, and Marine Shellfish.” She said, “Seawater and
shellfish samples collected in the vicinity of a marine sewage outfall were examined for the incidence of antibiotic
resistance among coliform and fecal coliform bacteria over a 2-year period. Seventy percent or more of these two
groups of bacteria from both sources were resistant to one or more antibiotics. Forty-five percent of the isolates
resistant to streptomycin or tetracycline were capable of transferring all or part of their resistance pattern to an antibiotic-
susceptible strain of Escherichia coli K-12.” Cooke states, “Antibiotic resistance, mediated by extrachromosomal
elements or R factors, is widespread among the Enterobacteriaceae. R factors may mediate resistance to as many as
eight antibiotics simultaneously and confer resistance to heavy metals such as nickel, mercury, and cobalt. They are
transmissible among gram-negative bacteria such as Escherichia coli, Salmonella, and Shigella and to other unrelated
bacteria such as Pseudomonas aeruginosa.” Cooke fails to indicate that Escherichia coli, Salmonella, and Shigella are
members of the Enterobacteriacea family and three of the coliform group when incubated at 95°F as well as the fecal
coliform group when incubated at 112.1°F. She states, “Coliform bacteria, generally regarded as nonpathogenic
indicators of pollution, are often used to study the bacteriological quality of water and foods.”
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC429643/
In 1976, W.O.K. Grabow, et al., reported on the “Behaviour in conventional sewage purification processes of coliform
bacteria with transferable or non-transferable drug-resistance.” They said, “Transferable (R+) and non-transferable
(R−) resistance of coliform bacteria in settled sewage and after biofiltration, secondary sedimentation, chlorination and
sand filtration was investigated. The percentage of R− coliforms resistant to ampicillin (A), chloramphenicol (C) or
streptomycin (S), but not kanamycin (K) or tetracycline (T), was slightly reduced through the purification system. On the
average the percentage of R+ coliforms resistant to one or more of the same drugs was reduced by about 50%. This
reduction was mainly accomplished by biofiltration and sand filtration. The ratio of transferable to non-transferable
resistance for A, C, K or T increased during secondary sedimentation and chlorination. R factor transfer in water may
explain these findings. Rapid passage over stony surfaces in biological and sand filters is unfavourable for conjugation
and may damage sex pilli while R factor transfer can be expected under the relatively stagnant conditions in
sedimentation and chlorination tanks. The incidence of R factors conferring resistance to all five drugs simultaneously
increased through the purification system. These R factors are probably large and may carry markers which determine
resistance to sewage purification processes. Between 30 and 40% of all R factors mediated resistance to at least four of
the five drugs studied. The incidence of Escherichia coli I among R+ coliforms varied but did not exceed 50%. The
finding that conventional sewage purification has a limited effect on the incidence of drug resistance in bacteria
supports the view that sewage should be treated by more advanced methods prior to discharge into the environment.”
http://www.sciencedirect.com/science/article/pii/0043135476900105
In 1979, W. J. Kelch and J. S. Lee, Oregon State University at Corvallis, reported on “Antibiotic Resistance Patterns of
Gram-Negative Bacteria Isolated from Environmental Sources.” That said, “The presence in waterways of both
potentially pathogenic gram-negative bacteria and fecal coliforms containing transferable R-factors raises the question
of whether resistance transfer may actually occur in streams, rivers, bays, and other waterways.” They found, “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. – 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 other groups were, Aeromonas, Bacillus, Proteus, Arthrobacter,
Lactobacillus, Klebsiella, Plesiomonas, Pectobacterium, Chromobacterium, Serratia, Enterobacter, Staphylococcus, and
Micrococcus. According to them, “... except for the fecal coliforms, most of these bacteria are presumably of soil origin.”
They also pointed out that, “Grabow et al. (8) reviewed the public health implications of drug-resistant coliforms in water
supplies, and suggested that the prevalence of these drug-resistant bacteria requires reevaluation of water quality
standards as well as more advanced purification of sewage prior to discharge into the environment.” They said, “The
data suggest that antibiotic-resistant fecal coliforms may survive better than sensitive organisms in surface waters.”
Their conclusion was that, “These results strongly suggest that the antibiotic resistance patterns in these bacterial
groups are very similar, perhaps indicating similar mechanisms for the development of this resistance, and also that bay
isolates were contributed by the tributaries and that, likewise, tributary isolates were contributed by runoff from the
pastures.” http://thewatchers.us/EPA/5/1978-antibiotic-water.pdf
In 1980, Henry W. Talbot, Jr., et al., Oregon State University at Corvallis, reported on “Antibiotic Resistance and Its
Transfer Among Clinical and Nonclinical Klebsiella Strains in Botanical Environments.” They said, “A total of 183 isolates
of Klebsiella from drinking water, market vegetables, wood, sawdust, industrial effluents, and human and animal origin
were examined for susceptibility to 10 antibacterial agents. Incidence of resistance to two or more antibiotics tested was:
65% of the human clinical isolates, 59% among bovine mastitis, and 24% among the nonclinical isolates. The five
different multiple resistance patterns among nonclinically derived Klebsiella were also found among the human and
bovine mastitis isolates. Statistical analyses revealed that patterns of resistance among Klebsiella isolates from drinking
water, market vegetables, and industrial effluents were highly correlated with each other and with resistance patterns of
human clinical isolates. Antibiotic resistance was transferred between Klebsiella growing in two habitat-simulated
environments (growing radish plants and aqueous sawdust suspensions). Transconjugants were detected in 5 of 21
and 6 of 21 mating pairs, respectively. Average transconjugants/donor ranged from 10-3 to 10-6 in Penassay broth,
from 10-6 to 10'- on radish plants, and from 10-5 to 108 in sawdust suspensions. Although antibiotic resistance transfer
under simulated environmental conditions can occur, regrowth of clinical strains is probably the major cause for the
widespread occurrence of antibiotic-resistant Klebsiella in the nonclinical environment.” According to them, “... most
antibiotic-resistant Klebsiella isolated from environmental sources may first have been of human or animal origin, since it
is unlikely that they would encounter selective pressures to develop these same patterns of resistance. Further support
for this suggestion is the fact that the majority of the multiply antibiotic-resistant Klebsiella isolated from nonclinical
habitats in this study were fecal coliform positive. These fecal coliform-positive K. pneumoniae species constituted some
85% of the clinical isolates in a related investigation (4). It has also been shown that fecal coliforn-positive clinical
isolates of Klebsiella are capable of rapidly proliferating and colonizing a variety of botanical milieu (22). Therefore, it is
reasonable to expect that regrowth of antibiotic- resistant, fecal coliform-positive K. pneumoniae will occur in nature.”
They said, “In other studies, Shooter et al. found coliforms, including Klebsiella, in foods consumed raw in hospitals,
canteens, and schools (36). Hospital foods, and salads in particular, were found to occasionally contain sufficient levels
of coliforms to lead to their establishment in the bowel.”
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC291290/pdf/aem00231-0117.pdf
In the 1981 study, “R factors in coliform-fecal coliform sewage flora of the prairies and Northwest
Territories of Canada,” James B. Bell, et al., Environment Canada, Environmental Protection Service, Microbiological
Laboratory, said, “Coliform and fecal coliform populations found in the raw sewages and final sewage effluents of the
prairie provinces and the Northwest Territories were examined for antibiotic resistance and the possession of R factors.
It was determined that 8.91% of the total coliform and 10.80% of the fecal coliform populations carried R factors. The
following numbers of combinations of R determinants were found: 39 in the Escherichia coli population, 6 in the
Citrobacter population, 20 in the Enterobacter populations, 10 in the Klebsiella populations, and 11 in the Aeromonas
populations. The maximum number of R determinants transferable simultaneously was seven; organisms with R factors
containing determinants for chloramphenicol usually contained determinants for ampicillin. Of the coliform and fecal
coliform populations, 2 to 4% were resistant to chloramphenicol in some provinces, and from 17 to 30% of the
populations were resistant to three or more antibiotics. It was calculated that coliforms containing R factors in the raw
sewage reached population levels of 1.5 X 10(7)/100 ml, and fecal coliforms containing R factors reached population
levels of 8.6 X 10(5) ml. Final effluent discharges to the receiving environment contained R factor-containing coliform
and fecal coliform populations of 3.1 X 10(4)/100 ml and 5.8 X 10 (2)/100 ml, respectively. The incidence of bacteria
containing R factors in sewage appears to be increasing with time, and their removal from sewage before discharge to
the receiving environment is desirable. Consideration of data on bacteria with R factors should be made in future water
quality deliberations and in discharge regulations.” Appl Environ Microbiol. 1981 August; 42(2): 204–210
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC243991/pdf/aem00189-0026.pdf
In 1982, Michael R. Altherr and Kenneth L. Kaswech, Florida Institute of Technology at Melbourne, reported on “In Situ
Studies with Membrane Diffusion Chambers of Antibiotic Resistance Transfer in Escherichia coli.” They said, “Since
multiply resistant strains with transferability were randomly encountered with almost equal frequency in sewage entering
the plant, sewage within the plant, and water downstream of the plant, we must conclude that there are multiple sources
of resistance traits. The observation of seven distinct antibiotic resistance patterns among the resistance transfer-
positive strains is indicative of multiple sources of R plasmids as well. – Periodically, high numbers of coliforms are found
in the effluent-receiving waters (Table 2). Although no R plasmid transfer was detected there, the occurrence of
transferable resistance is observed in coliforms isolated from these waters. This suggests that conditions may
transiently exist for R plasmid transfer in the downstream area. To our knowledge, this work is the only in situ study of
antibiotic resistance transfer with membrane diffusion chambers. The recombination frequencies recorded dispute the
conclusion of Linton et al. (21) that the conditions for R plasmid-mediated conjugation are not common to sewage.”
http://thewatchers.us/EPA/10/1982-antibiotic-resistance-sewage.pdf
In 1983, Maarit Niemi, National Board of Waters, Mervi Sibakov, National Public Health Institute, and Seppo Niemela,
Helsinki University, reported on “Antibiotic Resistance Among Different Species of Fecal Coliforms Isolated from Water
Samples” from sewage treatment plants, a farm, a river, a lake and the sea. Half of the resistant fecal coliform strains in
sewage were antibiotic-resistant Enterobacteriaceae species E. coli and the other half mainly Klebsiella and
Enterobacter strains. They said, “A total of 812 strains were identified from 14 water samples with different
concentrations of fecal coliforms” incubated at 44°C (111.2°F) for 24 hours – A total of 645 strains of the isolates (80%)
produced typical colonies on mFC agar, 76 strains (9%) were intermediate, and 90 strains (11%) were atypical.
Resistance to one or more of the four antibiotics or to sulfonamides was detected in 29% of the strains that produced
typical colonies, in 28% of the strains that produced intermediate colonies, and in 48% of the strains that produced
atypical colonies. The atypical strains would normally not be counted in the standard water analysis. Since, however,
most of them (71%) proved to be Enterobacteriaceae and their fractions in different samples were not significantly
different, we decided to include them in the analysis of antibiotic resistance. Furthermore, there is unpublished evidence
that lactose-positive coliforms may produce atypical colonies appearing lactose negative under the harsh conditions of
the mFC procedure. The high proportion of resistant strains (48%) in this group is chiefly due to Klebsiella pneumoniae
and Enterobacter cloacae strains with a high frequency of resistance (over 80%).”
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC242233/pdf/aem00170-0097.pdf
In 1983, James B. Bell, et al., Canada, reported on the “Influence of Sewage Treatment and Urbanization on Selection of
Multiple Resistance in Fecal Coliform Populations.” They said, “The fecal coliform populations found in the raw sewages
and final sewage effluents of mechanical treatment plants, a long-term retention lagoon, shorter term retention lagoons,
a remote northern Canada river, and a heavily urbanized prairie river were examined for antibiotic resistance and the
possession of R factors. It was determined that there was a decrease in the percentage of multiresistant fecal coliform
populations in the mechanical sewage treatment plants and shorter-term retention lagoons; however, there was an
increase in populations from the long-term retention lagoon. The percentage of the populations possessing
transmissible R factors was constant in the mechanical treatment and shorter-term retention facilities; however, the
ability to transmit was lost in 50%o of the infective population of the long-term retention facility. A striking contrast was
found between the populations of the remote northern Slave River and those of the urbanized Red River. Of the fecal
coliforms in the Slave River, 7.1% were multiresistant, and only 0.79% possessed transmissible R factors. The Red
River fecal coliform populations were 52.9% multiresistant, and 18.77% of the total population possessed transmissible
R factors. The influence of urbanization and the type of sewage treatment have been shown to affect the selection and
survival of multiresistant fecal coliforms and R+ fecal coliforms. Determination of other factors influencing the
development and the survival of these populations is needed for rational wastewater management and water quality
consideration.” http://aem.asm.org/cgi/reprint/46/1/227.pdf
In 1984, K. Kralikova, et al., reported on “Transferable resistance to gentamicin and other antibiotics in
Enterobacteriaceae isolates from municipal wastewater.” They said, “In two sets of Enterobacteriaceae and
Pseudomonas bacteria resistant to at least two antibiotics a distinctly upward trend was found in the incidence of strains
resistant to gentamicin. The strains examined were either routine isolates from three municipal wastewater treatment
facilities or from the Danube river samples collected near the outlet of municipal sewerage. The resistance to gentamicin
points to the representation of strains originating from hospitalized patients and its incidence among wastewater strains
is recordable since the summer of 1981. Gentamicin resistance transfer could be demonstrated in a sewage sludge
strain of Klebsiella pneumoniae resistant to seven antibiotics and in two multiresistant isolates from the river Danube.
Resistance transfers in the case of other antibiotics, especially those susceptible to beta-lactamase (ampicillin,
carbenicillin), were demonstrated in 10 out of the 24 di- and multiresistant strains tested. These findings show that both
municipal wastewater and water in streams may function as the reservoirs of strains bearing the determinants of
transferable resistance. Such strains may play an important role not only in the ecology and epidemiology of R
plasmids, but also in the accidental spread of the so-called DNA recombinants that might escape during gene
manipulations.” http://www.ncbi.nlm.nih.gov/pubmed/6470479
In 1984, Elena Alcaide and Esperanza Garay, Universidad de Valencia at Campus de Burjasot, found. “R-Plasmid
Transfer in Salmonella spp. Isolated from Wastewater and Sewage-Contaminated Surface Waters”. They said, “There
are many studies dealing with the antibiotic resistance of gram-negative bacilli, especially members of the
Enterobacteriaceae, from fecal specimens because of the increasing risk of gastroenteritis caused by strains carrying R
plasmids. The majority of the work has been done with coliforms; several papers have also been published on
environmental strains of members of the Enterobacteriaceae with R factors, and a few papers report on antibiotic
resistance in Salmonella strains isolated from aquatic environments. – water-borne outbreaks of enteric pathogens
carrying R factors have already led to large numbers of deaths due to diagnostic problems and the failure of an
adequate response to antibiotic treatment – Of the 52 serotypes isolated, 25 showed resistance to one or more
compounds. Salmonella infantis, Salmonella panama, Salmonella agona, Salmonella anatum, and Salmonella
typhimurium were most numerous and more frequently isolated from the two types of water analyzed. These five
serotypes accounted for 63.6% of the resistant strains. Of the 16 resistant S. typhimurium isolates, 9 were resistant to
tetracycline. The strains came from one of the discharging sites at Albufera Lake with high levels of fecal coliforms –
The sewage isolates, apart from showing a high transfer frequency, exhibited multiple resistance to five and six of the
antibiotics tested.” http://aem.asm.org/cgi/reprint/48/2/435.pdf
1985 Centers for Disease Control (CDC) – Enterobacteriaceae members
“44.5°C is the temperature setting of water baths used for the fecal coliform test.”
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC271578/pdf/jcm00114-0072.pdf
In 1985, Micheal V. Walter and John W. Vennes, University of North Dakota School of Medicine at Grand Forks,
reported on the “Occurrence of Multiple-Antibiotic-Resistant Enteric Bacteria in Domestic Sewage and Oxidation
Lagoons.” They said, “The discovery of R+ coliforms and fecal coliforms in sewage and the possibility that these R
factors may be transferred to other microorganisms during conventional sewage treatment have led to calls for the
reevaluation of water quality standards to take into account the presence of these R+ coliforms – MAR coliforms were
isolated from every facet of the Grand Forks sewage system on every sampling date. The MAR coliform population
constituted 5% of the total coliform population in June 1983, as measured in the composite raw influent, and then
decreased to 0.35% of the total coliform population in January 1984. – There was however, some indication that MAR
coliforms had an ecological advantage over non-MAR coliforms in the oxidation lagoons (Table 1). Their numbers were
found to constitute 66% of the population in June but only 0.28% in October. – Five different genera of MAR coliforms
were isolated. – Studies of the effect of temperature on transfer efficiency revealed a sharp optimum of 35°C. Total
transfers declined above and below 35°C.” They only list wild-type E. coli, Enterobacter cloacae, Citrobacter freundii,
and Klebsiella oxytoca transferring the R factor to E. coli K 12. They added that, “All genera isolated had species unable
to transfer MAR to the E. coli K-12 receiver.”
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC291771/pdf/aem00230-0199.pdf
In 1986, A M Baya, et al., University of Maryland at College Park, reported on “Coincident Plasmids and Antimicrobial
Resistance in Marine Bacteria Isolated From Polluted and Unpolluted Atlantic Ocean Samples” from sewage effluent and
outfall confluence samples at the Barceloneta Regional Treatment Plant in Barceloneta, Puerto Rico, outfall confluence
samples at Ocean City, Md. and uncontaminated open ocean areas. Their study states that the use of antibiotics by sick
people was the cause of antibiotic resistance in chronically polluted waters rather that treated sewage released to rivers
and marine waters. They said, “Treated sewage was found to contain large numbers of bacteria simultaneously
possessing antibiotic resistance, chemical resistance, and multiple bands of plasmid DNA. Bacteria resistant to penicillin,
erythromycin, nalidixic acid, ampicillin, m-cresol, quinone, and bis(tributyltin) oxide were detected in nearly all samples,
but only sewage outfall confluence samples yielded bacterial isolates that were resistant to streptomycin. Bacteria
resistant to a combination of antibiotics, including kanamycin, chloramphenicol, gentamicin, and tetracycline, were
isolated only from sewage effluent samples. It is concluded that bacterial isolates derived from toxic chemical wastes
more frequently contain plasmid DNA and demonstrate antimicrobial resistance than do bacterial isolates from domestic
sewage-impacted waters or from uncontaminated open ocean sites.”
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC239059/pdf/aem00141-0141.pdf
In 1986, G. R. Fincha and D. W. Smith reported on “The survival of antibiotic‐resistant escherichia coli in an activated
sludge plant”. They said, “”Antibiotic‐resistant Escherichia coli were monitored in the raw influent wastewater and
effluent of a plug‐flow activated sludge wastewater treatment plant. A statistically significant increase in the proportion of
multiple antibiotic‐resistant E. coli was observed in the effluent of the plant. This suggests that conventional secondary
treatment selects for antibiotic‐resistant E. coli.” http://www.tandfonline.com/doi/abs/10.1080/09593338609384436
In 1986, Philip McPherson and Michael A. Gealt reported on the “Isolation of Indigenous Wastewater Bacterial Strains
Capable of Mobilizing Plasmid pBR325.” They said, “Members of the family Enterobacteriaceae have been isolated from
raw wastewater, identified, and characterized with respect to their plasmid content and antibiotic resistance. Several
strains possessing both antibiotic resistance and high-molecular-weight plasmid(s) transferred their resistance
characteristics to recipient cells during a 25 h coincubation. Eight were characterized (six Escherichia coli and two
Klebsiella pneumoniae); each produced 102 to 107 transconjugants per ml by the end of the incubation period. They
were also able to mobilize pBR325 from a laboratory E. coli strain into plasmid-free recipients to yield 102 to 107
transconjugants per ml. These transconjugants possessed phenotypic characteristics specified by pBR325, the R
plasmid, and the chromosome of the recipient. Many transconjugants exhibited recombinational rearrangements of the
acquired plasmid material.” http://www.ncbi.nlm.nih.gov/pmc/articles/PMC238985/pdf/aem00140-0040.pdf
In 1988, W. Stelzer and E. Ziegert, Forschungsinstitut für Hygiene und Mikrobiologie Bad Elster, reported on “[The
occurrence of antibiotic resistant coliforms in the waste water of a water treatment plant].” They said, “Colony counts
and total coliforms of the investigated biological treatment plant were decreased by more than 90% on average. In
activated sludge processes the colony counts increased, whereas total coliforms did not show significant differences
between raw sewage and activated sludge processes. With regard to the antibiotics tetracycline, chloramphenicol,
kanamycin and gentamicin raw sewage samples contained on average 10(3) antibiotic resistant coliforms/ml. From agar
plates supplemented with antibiotics a total of 896 strains were characterized. Single resistant E. coli strains (29.3%)
were isolated most frequently from agar plates supplemented with tetracycline. However, coliforms isolated from agar
plates which were supplemented with chloramphenicol, kanamycin and gentamicin showed a prevalent resistance to 5
and 6 antibiotics tested. The variety of resistance patterns of gentamicin-resistant coliforms was determined by few
plasmids encoding gentamicin resistance only.” http://www.ncbi.nlm.nih.gov/pubmed/3223110
In 1990, S. Nakamura and H. Shirota, Ube College, Reported on the “Behavior of drug resistant fecal coliforms and R
plasmids in a wastewater treatment plant”. They said, “Fecal coliforms were isolated from the inlet, the primary
sedimentation tank, the activated sludge digestion tank, the final settling tank, the outlet and the return activated sludge
drain at the municipal wastewater plant in Ube City, and examined for drug resistance and presence of R plasmids. Drug
concentrations employed to distinguish resistant isolates from sensitive isolates were 25 micrograms/ml for tetracycline,
kanamycin, chloramphenicol and streptomycin, 50 micrograms/ml for ampicillin, nalidixic acid and rifampicin, and 200
micrograms/ml for sulfisoxazole, respectively. Of a total of 900 isolates, 45.7% were drug resistant and 51.1% of them
carried R plasmids. The further along that wastewater had progressed through the treatment process the greater the
tendency was for appearance of the multiresistant isolates. These isolates also were shown to simultaneously carry
transferable R plasmids. Observed resistant patterns of R plasmids were mainly multiple and encoded to resistance to
tetracycline, chloramphenicol, streptomycin and sulfisoxazole. It became clear that multiplication of R plasmids took
place in the activated sludge digestion tank. This study show that drug resistance transfer mediated by these R
plasmids may occur in actual wastewater treatment plants.”
http://www.ncbi.nlm.nih.gov/pubmed/2131972
In 1998, Luca Guardabassi, et al, The Royal Veterinary and Agricultural University, Denmark, reported on “Antibiotic
Resistance in Acinetobacter spp. Isolated from Sewers Receiving Waste Effluent from a Hospital and a Pharmaceutical
Plant”. They said, “The possible increase of antibiotic-resistant bacteria in sewage associated with the discharge of
wastewater from a hospital and a pharmaceutical plant was investigated by using Acinetobacter species as
environmental bacterial indicators. The level of susceptibility to six antimicrobial agents was determined in 385
Acinetobacter strains isolated from samples collected upstream and downstream from the discharge points of the
hospital and the pharmaceutical plant. Results indicated that while the hospital waste effluent affected only the
prevalence of oxytetracycline resistance, the discharge of wastewater from the pharmaceutical plant was associated with
an increase in the prevalence of both single- and multiple-antibiotic resistance among Acinetobacter species in the
sewers.” The found, “The relatively low incidence of antibiotic resistance observed in our study suggests that these
microorganisms are not inherently resistant to any particular class of drugs but more likely have a predisposition to
develop resistance under conditions of antibiotic selective pressure (e.g., hospital environments).” Their conclusion, “It
is generally agreed that the selection and dissemination of resistant bacteria in nature should be avoided in order to
ensure effective treatment against infectious disease in humans and maintain an ecological balance that favors the
predominance of a susceptible bacterial flora in nature. According to the results of this study, factors other than the
indiscriminate use of antibiotics in human medicine, animal husbandry, and agriculture may disrupt the microbial
balance in favor of resistant bacteria.” http://www.ncbi.nlm.nih.gov/pmc/articles/PMC106754/
In 2003, K. Kümmerer, Freiburg University Hospital, Freiburg, Germany, reported on the “Significance of antibiotics in
the environment.” He said, “During the past decade, concern has grown about the adverse effects the use and disposal
of pharmaceuticals might potentially have on human and ecological health. Research has shown that after passing
through wastewater treatment, pharmaceuticals, amongst other compounds, are released directly into the environment.
– In a report by the House of Lords, it is stated that: ‘resistance to antibiotics and other anti-infective agents constitutes
a major threat to public health and ought to be recognized as such more widely than it is at present’. Therefore, the
European Union (EU) recommends the prudent use of antimicrobial agents in human medicine. With respect to the
causes of resistance, the focus is on the use of antimicrobials in hospitals, by medical practitioners, i.e. in prescriptions
and in animal husbandry. ‘…Coordination between human, veterinary and environment sectors should be ensured and
the magnitude of the relationship between the occurrence of antimicrobial resistant pathogens in humans, animals and
the environment should be further clarified…’. However, very little is known about their contribution to the level of
bacterial resistance in the environment and its significance. Also, surprisingly, little is known about the extent of
environmental occurrence, transport, and ultimate fate and effects of pharmaceuticals in general, as well as of
antibiotics in particular. – The dilution of hospital effluents by municipal sewage will lower the concentration of antibiotics
only moderately, because municipal waste water also contains antibiotic substances and disinfectants from households,
veterinary sources and to a minor extent from livestock. – Cyclosporin A was shown to degrade only after some months
in samples of wet garden soil, despite the fact that several degrading strains have been isolated from soil. – Resistant
bacteria may be selected by antibiotic substances in hospital effluent, municipal sewage, aeration tanks, the anaerobic
digestion process of STPs or in soil. Furthermore, resistant bacteria are excreted and discharged into sewage or soil
and other environmental compartments. Resistant and even multi-resistant pathogenic bacteria have been detected in
wastewater and STPs, as well as in other environmental compartments. Furthermore, in arid regions, wastewater
containing resistant bacteria and antibiotics is used for irrigation, and sewage sludge serves as a fertilizer. This allows
resistant bacteria to enter the food chain directly. ” http://jac.oxfordjournals.org/content/52/1/5.full
In 2003, F.F. Reinthaler, et al., Institute of Hygiene, University of Graz, reported on “Antibiotic resistance of E. coli in
sewage and sludge.” They said, “The aim of the study is the evaluation of resistance patterns of E. coli in wastewater
treatment plants without an evaluation of basic antibiotic resistance mechanisms. Investigations have been done in
sewage, sludge and receiving waters from three different sewage treatment plants in southern Austria. A total of 767 E.
coli isolates were tested regarding their resistance to 24 different antibiotics. The highest resistance rates were found in
E. coli strains of a sewage treatment plant which treats not only municipal sewage but also sewage from a hospital.
Among the antimicrobial agents tested, the highest resistance rates in the penicillin group were found for Ampicillin (AM)
(up to 18%) and Piperacillin (PIP) (up to 12%); in the cephalosporin group for Cefalothin (CF) (up to 35%) and
Cefuroxime-Axetil (CXMAX) (up to 11%); in the group of quinolones for Nalidixic acid (NA) (up to 15%); and for
Trimethoprime/Sulfamethoxazole (SXT) (up to 13%) and for Tetracycline (TE) (57%). Median values for E. coli in the
inflow (crude sewage) of the plants were between 2.0 x 10(4) and 6.1 x 10(4)CFU/ml (Coli ID-agar, BioMerieux 42017)
but showed a 200-fold reduction in all three plants in the effluent. Nevertheless, more than 10(2)CFU E. coli/ml reached
the receiving water and thus sewage treatment processes contribute to the dissemination of resistant bacteria in the
environment.” http://www.ncbi.nlm.nih.gov/pubmed/12697213
In 2003, Audra Morse and Andrew Jackson,Texas Tech University, reported on the "Fate of a Representative
Pharmaceutical in the Environment" at the City of Lubbock’s Water Reclamation Plant (LWRP). They make a point that
thermotolerant E. coli, citrobacter and Klebsiella (i.e., Fecal coliform) are removed, but the system selects for non-
thermotolerant E. coli. They said, "Mezriou and Baleux (1994) investigated the antibiotic resistance of 879 E. coli strains
isolated from raw domestic sewage and the effluent from aerobic lagoons and activated sludge plants. Both aerobic
lagoons and activated sludge plants are used to reduce the BOD5 leaving the treatment facility. The results of this
study indicate that the aerobic lagoons were effective in removing fecal coliforms in the wastewater, but the
system selected for antibiotic resistant E. coli by selecting for E. coli. The number of antibiotic resistant strains of E. coli
in the effluent increased as compared to the influent. For both the inflow and outflow, the incidence of antibiotic
resistance increased as the number of antibiotics was reduced from seven to one. -- In the LWRP, bacteria were
resistant to multiple antibiotics. The greatest concern was that antibiotic resistant bacteria in the effluent of the LWRP
may spread the genetic information encoding antibiotic resistance to organisms in the environment of the LWRP outfall.
As a consequence, changing the antibiotic resistance properties of the bacteria in an ecosystem may disrupt the
ecosystem, and the water may be a health hazard to end users. For example, say a person uses the LWRP water to
irrigate his/her farmland. During a visit at the farm, the farmer cuts their hand and the reclaimed wastewater comes into
contact with the wound. Bacteria present in the water may infect the cut and the
farmer may be hospitalized with a difficult infection to cure. This scenario is possible with the use of reclaimed
wastewater." http://water.usgs.gov/wrri/02grants/prog-compl-reports/2002TX60B.pdf
In 2004, Rafael Szczepanowski, et al., Universität Bielefeld, reported on “Antibiotic multiresistance plasmid pRSB101
isolated from a wastewater treatment plant is related to plasmids residing in phytopathogenic bacteria and carries eight
different resistance determinants including a multidrug transport system.“ They said, “Ten different antibiotic resistance
plasmids conferring high-level erythromycin resistance were isolated from an activated sludge bacterial community of a
wastewater treatment plant by applying a transformation-based approach. One of these plasmids, designated pRSB101,
mediates resistance to tetracycline, erythromycin, roxythromycin, sulfonamides, cephalosporins, spectinomycin,
streptomycin, trimethoprim, nalidixic acid and low concentrations of norfloxacin. Plasmid pRSB101 was completely
sequenced and annotated. Its size is 47 829 bp. Conserved synteny exists between the pRSB101 replication/partition
(rep/par) module and the pXAC33-replicon from the phytopathogen Xanthomonas axonopodis pv. citri. The second
pRSB101 backbone module encodes a three-Mob-protein type mobilization (mob) system with homology to that of IncQ-
like plasmids. Plasmid pRSB101 is mobilizable with the help of the IncP-1α plasmid RP4 providing transfer functions in
trans. A 20 kb resistance region on pRSB101 is located within an integron-containing Tn402-like transposon. The
variable region of the class 1 integron carries the genes dhfr1 for a dihydrofolate reductase, aadA2 for a
spectinomycin/streptomycin adenylyltransferase and blaTLA-2 for a so far unknown Ambler class A extended spectrum
β-lactamase. The integron-specific 3′-segment (qacEΔ1-sul1-orf5Δ) is connected to a macrolide resistance operon
consisting of the genes mph(A) (macrolide 2′-phosphotransferase I), mrx (hydrophobic protein of unknown function) and
mphR(A) (regulatory protein). Finally, a putative mobile element with the tetracycline resistance genes tetA (tetracycline
efflux pump) and tetR was identified upstream of the Tn402-specific transposase gene tniA. The second ‘genetic load’
region on pRSB101 harbours four distinct mobile genetic elements, another integron belonging to a new class and
footprints of two more transposable elements. A tripartite multidrug (MDR) transporter consisting of an ATP-binding-
cassette (ABC)-type ATPase and permease, and an efflux membrane fusion protein (MFP) of the RND-family is
encoded between the replication/partition and the mobilization module. Homologues of the macrolide resistance genes
mph(A), mrx and mphR(A) were detected on eight other erythromycin resistance-plasmids isolated from activated sludge
bacteria. Plasmid pRSB101-like repA amplicons were also obtained from plasmid-DNA preparations of the final effluents
of the wastewater treatment plant indicating that pRSB101-like plasmids are released with the final effluents into the
environment.” http://mic.sgmjournals.org/content/150/11/3613.full.pdf
In 2005, C. Gallert, et al., reported on “Antibiotic resistance of bacteria in raw and biologically treated sewage and in
groundwater below leaking sewers.” They said, “More than 750 isolates of faecal coliforms [thermotolerant
enterobacteriacea], (>200 strains), enterococci (>200 strains) and pseudomonads (>340 strains) from three wastewater
treatment plants (WTPs) and from four groundwater wells in the vicinity of leaking sewers were tested for resistance
against 14 antibiotics. Most, or at least some, strains of the three bacterial groups, isolated from raw or treated sewage
of the three WTPs, were resistant against penicillin G, ampicillin, vancomycin, erythromycin, triple sulfa and
trimethoprim/sulfamethoxazole (SXT). Only a few strains of pseudomonads or faecal coliforms were resistant against
some of the other tested antibiotics. The antibiotic resistances of pseudomonads, faecal coliforms and enterococci from
groundwater varied to a higher extent. In contrast to the faecal coliforms and enterococci, most pseudomonads from all
groundwater samples, including those from non-polluted groundwater, were additionally resistant against
chloramphenicol and SXT. Pseudomonads from sewage and groundwater had more multiple antibiotic resistances than
the faecal coliforms or the enterococci, and many pseudomonads from groundwater were resistant to more antibiotics
than those from sewage. The pseudomonads from non-polluted groundwater were the most resistant isolates of all. The
few surviving faecal coliforms in groundwater seemed to gain multiple antibiotic resistances, whereas the enterococci
lost antibiotic resistances. Pseudomonads, and presumably, other autochthonous soil or groundwater bacteria, such as
antibiotic-producing Actinomyces sp., seem to contribute significantly to the gene pool for acquisition of resistances
against antibiotics in these environments.”
http://www.springerlink.com/content/q6476vq232724483/
Also, in 2005, Leen De Gelder, et al., University of Idaho at Moscow, reported on “Plasmid Donor Affects Host Range of
Promiscuous IncP-1ß Plasmid pB10 in an Activated-Sludge Microbial Community.” They said, “Horizontal transfer of
multiresistance plasmids in the environment contributes to the growing problem of drug-resistant pathogens. Even
though the plasmid host cell is the primary environment in which the plasmid functions, possible effects of the plasmid
donor on the range of bacteria to which plasmids spread in microbial communities have not been investigated. In this
study we show that the host range of a broad-host-range plasmid within an activated-sludge microbial community was
influenced by the donor strain and that various mating conditions and isolation strategies increased the diversity of
transconjugants detected. To detect transconjugants, the plasmid pB10 was marked with lacp-rfp, while rfp expression
was repressed in the donors by chromosomal lacIq. The phylogeny of 306 transconjugants obtained was determined by
analysis of partial 16S rRNA gene sequences. The transconjugants belonged to 15 genera of the - and -Proteobacteria.
The phylogenetic diversity of transconjugants obtained in separate matings with donors Pseudomonas putida SM1443,
Ralstonia eutropha JMP228, and Sinorhizobium meliloti RM1021 was significantly different. For example, the
transconjugants obtained after matings in sludge with S. meliloti RM1021 included eight genera that were not
represented among the transconjugants obtained with the other two donors. Our results indicate that the spectrum of
hosts to which a promiscuous plasmid transfers in a microbial community can be strongly influenced by the donor from
which it transfers.”
http://aem.asm.org/cgi/content/abstract/71/9/5309
In 2006, Juan Silva, et al., Universidad de Antofagasta, reported on the “Frequency of transferable multiple antibiotic
resistance amongst coliform bacteria isolated from a treated sewage effluent in Antofagasta, Chile.” They said, “The
results of this study suggested that wastewater treatment could reduce the total number of enteric bacteria in sewage,
but may increase the proportion of antibiotic resistant coliforms in effluent water at the Antofagasta plant. The high
values of resistance found to ampicillin was in general agreement with that reported by others, and is very common in
coliforms isolated from the human and animal intestines. Climatic conditions in summer months probably influenced
increases the proportion of resistant coliforms as demonstrated in our study, as shown by the higher percentages of
resistant isolates observed in the December samples. – Eight different species were identified by the biochemical tests,
with E. coli the most frequent species isolated from both raw and treated sewage. The resistant coliform species were
eliminated at different rates by the treatment, Escherichia coli decreased, whereas the proportion of Klebsiella sp
increased. – The percentage of multiple-resistant isolates increased at the end of the treatment process, exhibiting
resistance to as many as 8 antibiotics. – The bacterial conjugation experiments showed that a high percentage of multi-
resistant coliforms tested (89%) were able to partially or completely transfer their resistance patterns to E. coli K-12. A
high proportion of the multi-resistance was mediated by conjugative plasmids, which were easily transferred to E. coli K-
12. – The increase of multi-resistance in coliforms isolated from treated wastewater is explainable by bacterial
conjugation or other genetic mechanisms of change from antibiotic resistant to antibiotic sensitive bacteria by horizontal
gene transfer within the treatment plant. – The use of the treated sewage may contribute to spread antibiotic resistance
in the environment, – This represents a dangerous public health risk,”
http://ejb.ucv.cl/content/vol9/issue5/full/7/bip/index.html
In 2006, Timothy M. LaPara, et al., University of Minnesota, reported on “Municipal Wastewater Treatment: A Novel
Opportunity to Slow the Proliferation of Antibiotic-Resistant Bacteria?” They said, “Our research has demonstrated that
extremely high numbers of antibiotic resistant bacteria are released from municipal wastewater treatment plants, even
when disinfection is performed. – we learned that although a 99% inactivation looks encouraging, 1% of a very large
number (1015, or 1 quadrillion) still represents a very large number (1013, or 10 trillion) of antibiotic-resistant bacteria
that are released from the Metropolitan Wastewater Treatment Facility each day. The bacteria that we studied were all
pathogens or related to pathogens and all were resistant to multiple antibiotics. A substantial fraction of these bacteria
(greater than 50%) harbored genes encoding for tetracycline resistance. These bacteria frequently harbored integrons
(genes that allow bacteria to accumulate multiple genes for antibiotic resistance) and some of them were capable of
transferring their resistance to other bacteria. The frequency of lateral gene transfer of ciprofloxacin resistance, which
occurred in more than 40% of the strains we studied, is particularly worrisome because this trait is typically very rare
(less than 1%) among clinical strains of ciprofloxacin-resistant E. coli. Simply put, the bacteria that we detected in
municipal wastewater are some of the most resistant bacteria ever studied. – This ongoing research is particularly
pertinent because of a recent shift in policy that emphasizes the application of treated wastewater solids to land rather
than putting these residues into landfills—that is, the “environmental friendly” practice of applying wastewater solids to
land may have unexpected and undesirable consequences in terms of the proliferation of antibiotic-resistant bacteria.”
http://www.cura.umn.edu/reporter/06-Fall/LaPara_et_al.pdf
In 2007, A. J. Watkinson, et al., National Research Centre for Environmental Toxicology, University of Queensland, et
al., reported on “Antibiotic-Resistant Escherichia coli in Wastewaters, Surface Waters, and Oysters from an Urban
Riverine System.” They said, “The aim of this study was to investigate the rates of antibiotic resistance among
Escherichia coli isolates from a variety of sources, including wastewater treatment plants (WWTPs), surface waters
(including those directly influenced by WWTP discharges), and oysters affected by WWTP discharges. – A total of 462
isolates were tested for sensitivity to six antibiotics by using the CLSI disk susceptibility testing method. – The highest
incidence of bacterial resistance recorded was that for tetracycline (51%), followed by those for cephalothin (41%) and
sulfafurazole (32%). – Rates of AR and multiple-AR among isolates from surface water sites adjacent to wastewater
treatment plant (WWTP) discharge sites were significantly higher (P < 0.05) than those among other isolates, whereas
the rate of AR among isolates from oysters exposed to WWTP discharges was low (<10%).” http://www.ncbi.nlm.nih.
gov/pmc/articles/PMC2042091/
In 2007, A. J. Watkinson, et al., National Research Centre for Environmental Toxicology at Brisbane, reported on
“Antibiotic Resistance in Escherichia coli Isolates from Environmental Waters by Use of a Modified Chromogenic Agar.”
They said, “Bacterial contamination of surface waters, particularly contamination with fecally derived bacteria, has long
been a water quality issue due to the potential for disease transmission. Because of this and the potential for antibiotic
resistance, there is a new level of risk associated with these bacteria. Recent studies have also identified antibiotics
themselves in surface waters, and the role of these antibiotics in the development, transfer, and maintenance of
resistance is largely unknown. – All wastewater isolates growing on MI-R plates were confirmed to be resistant using the
CLSI disk susceptibility test. Bacterial resistance to ampicillin (38% ± 4% overall), sulfamethoxazole, tetracycline (21% ±
3% overall), and ciprofloxacin (6% ± 1%) were found in wastewater effluent. A successful trial was also conducted with
water collected from the Brisbane River, Australia. The levels of antibiotic resistance in E. coli ranged from 0 to 47% for
ampicillin, from 0 to 24% for tetracycline, from 0 to 63% for sulfamethoxazole, and from 0 to 1% for ciprofloxacin, with the
highest incidence of resistance associated with wastewater treatment plant discharges.”
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1855640/
In 2009, Stefan Borjessona, et al., Linkoping University, reported on “A seasonal study of the mecA gene and
Staphylococcus aureus including methicillin-resistant S. aureus in a municipal wastewater treatment plant.” They said,
“The spread of methicillin-resistant Staphylococcus aureus (MRSA), in which the mecA gene mediates resistance,
threatens the treatment of staphylococcal diseases. The aims were to determine the effect of wastewater treatment
processes on mecA gene concentrations, and the prevalence of S. aureus and MRSA over time. To achieve this a
municipal wastewater treatment plant was investigated for the mecA gene, S. aureus and MRSA, using real-time
PCR assays. Water samples were collected monthly for one year, at eight sites in the plant, reflecting different aspects
of the treatment process. The mecA gene and S. aureus could be detected throughout the year at all sampling sites.
MRSA could also be detected, but mainly in the early treatment steps. The presence of MRSA was verified through
cultivation from inlet water. The concentration of the mecA gene varied between months and sampling sites, but no
obvious seasonal variation could be determined. The wastewater treatment process reduced the mecA gene
concentration in most months. – Although the treatment process reduces mecA concentration, the gene is not removed
in the effluent water. Other studies have shown that the mecA genes could not be detected in soil, surface water and
drinking water biofilms (Borjesson et al., in press; Schwartz et al., 2003). Compared to these environments the effluent
water has high concentration of mecA, indicating that WWTP may be a potential source for spread of genes encoding
antibiotic resistance to other environments. – Because both mecA genes and S. aureus simultaneously occur in the
same environment there is potential for horizontal gene transfer giving rise to MRSA. The potential of S. aureus to
transfer genes encoding antibiotic resistance in wastewater has been shown. The occurrence and survival of S. aureus
in wastewater environments have not been well studied, but the indication from both cultivation and PCR is that S.
aureus occurs at low levels in municipal wastewaters and surface waters. In the present study S. aureus was detected
to almost the same extent in OUT as well as IN, and other groups have been able to isolate S. aureus from microbial
aerosols in WWTPs.” http://www.loudounnats.org/pdf/09WRAseasonalstudyofmecASaureusandMRSAinafull-
scaleWWTP.pdf
In 2009, Yongli Zhang, et al., University of Michigan, reported that “Wastewater treatment contributes to selective
increase of antibiotic resistance among Acinetobacter spp.” They said, “The occurrence and spread of multi-drug
resistant bacteria is a pressing public health problem. The emergence of bacterial resistance to antibiotics is common in
areas where antibiotics are heavily used, and antibiotic-resistant bacteria also increasingly occur in aquatic
environments. The purpose of the present study was to evaluate the impact of the wastewater treatment process on the
prevalence of antibiotic resistance in Acinetobacter spp. in the wastewater and its receiving water. During two different
events (high temperature, high-flow, 31 °C; and low-temperature, low-flow, 8 °C), 366 strains of Acinetobacter spp. were
isolated from five different sites, three in a wastewater treatment plant (raw influent, second effluent, and final effluent)
and two in the receiving body (upstream and downstream of the treated wastewater discharge point). – The prevalence
of antibiotic resistance in Acinetobacter isolates to AMC, CHL, RA, and multi-drug (three antibiotics or more) significantly
increased (pb0.01) from the raw influent samples (AMC, 8.7%; CHL, 25.2%; RA, 63.1%; multi-drug, 33.0%) to the final
effluent samples (AMC, 37.9%; CHL, 69.0%; RA, 84.5%; multi-drug, 72.4%), and was significantly higher (pb0.05) in the
downstream samples (AMC, 25.8%; CHL, 48.4%; RA, 85.5%; multi-drug, 56.5%) than in the upstream samples (AMC,
9.5%; CHL, 27.0%; RA, 65.1%; multi-drug, 28.6%). These results suggest that wastewater treatment process
contributes to the selective increase of antibiotic resistant bacteria and the occurrence of multi-drug resistant bacteria in
aquatic environments.”
http://www.graham.umich.edu/pdf/zhang-article.pdf
In 2009, T. Zhang, et al., The University of Hong Kong, reported on “Tetracycline resistance genes and tetracycline
resistant lactose-fermenting Enterobacteriaceae in activated sludge of sewage treatment plants.” They said, “Activated
sludges were sampled from five sewage treatment plants (STPs) distributed in three geographically isolated areas, i.e.,
Hong Kong (Shatin, Stanley), Shanghai (Minhang) in China, and the bay area in California (Palo Alto and San Jose) of
the United States. Among the tested 14 tetracycline resistance (tet) genes, nine genes encompassing efflux pumps
(tetA, tetC, tetE, and tetG), ribosomal protection proteins (tetM, tetO, tetQ, and tetS), and enzymatic modification (tetX)
were commonly detected in the STP sludge samples, whereas five genes encompassing efflux pumps [tetB, tetD, tetL,
tetK, and tetA(P)] were not detected in any sludge sample. Additionally, 109 lactose-fermenting Enterobacteriaceae
(LFE) strains were isolated from the activated sludge of the Shatin STP. Tetracycline-resistant (TR) LFE accounted for
32% of the total 109 LFE strains. The occurrence frequencies of tet genes among all TR-LEF strains varied from 0 to
91%, i.e., tetC (91%), tetA (46%), tetE (9%), tetG (6%), and tetD (6%). Finally, quantitative real-time polymerase chain
reaction was used to quantify the change of tetC and tetA genes as the indicator of TR-LEF in the Shatin and Stanley
STPs. The results showed that the concentrations of tetC and tetA genes in STP effluent ranged from 10(4) to 10(5)
copies/mL, significantly lower than those in the influent by 3 orders of magnitude.”
http://www.ncbi.nlm.nih.gov/pubmed/19544839
In 2009, L. Sahlström, et al., Finnish Food Safety Authority, reported on “Vancomycin resistant enterococci (VRE) in
Swedish sewage sludge.” They said, “Antimicrobial resistance is a serious threat in veterinary medicine and human
healthcare. Resistance genes can spread from animals, through the food-chain, and back to humans. Sewage sludge
may act as the link back from humans to animals. The main aims of this study were to investigate the occurrence of
vancomycin resistant enterococci (VRE) in treated sewage sludge, in a Swedish waste water treatment plant (WWTP),
and to compare VRE isolates from sewage sludge with isolates from humans and chickens. – Biochemical typing
(PhenePlate-FS) and pulsed field gel electrophoresis (PFGE) revealed prevalence of specific VRE strains in sewage
sludge for up to 16 weeks. No connection was found between the VRE strains isolated from sludge, chickens and
humans, indicating that human VRE did not originate from Swedish chicken. – This study demonstrated widespread
occurrence of VRE in sewage sludge in the studied WWTP. This implies a risk of antimicrobial resistance being spread
to new farms and to the society via the environment if the sewage sludge is used on arable land.”
http://www.ncbi.nlm.nih.gov/pubmed/19480649
http://www.actavetscand.com/content/pdf/1751-0147-51-24.pdf
In 2010, S. Börjesson, et al., Linkoping University, followed up their 2009 study with “Methicillin-resistant Staphylococcus
aureus (MRSA) in municipal wastewater: an uncharted threat?” They said, “The aim was: (i) To cultivate methicillin-
resistant Staphylococcus aureus (MRSA) from a full-scale wastewater treatment plant (WWTP), (ii) To characterize the
indigenous MRSA-flora, (iii) To investigate how the treatment process affects clonal distribution and (iv) To examine the
genetic relation between MRSA from wastewater and clinical MRSA. – Wastewater samples were collected during 2
months at four key sites in the WWTP. MRSA isolates were characterized using spa typing, antibiograms, SSCmec
typing and detection of Panton–Valentine leukocidin (PVL). – MRSA could be isolated on all sampling occasions, but
only from inlet and activated sludge. The number of isolates and diversity of MRSA were reduced by the treatment
process, but there are indications that the process was selected for strains with more extensive antibiotic resistance and
PVL+ strains. The wastewater MRSA-flora had a close genetic relationship to clinical isolates, most likely reflecting
carriage in the community. – This study shows that MRSA survives in wastewater and that the WWTP may be a potential
reservoir for MRSA.”
http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2009.04515.x/full
In 2010, Mariya Munir, et al., Michigan State University at East Lansing, reported on the “Release of antibiotic resistant
bacteria and genes in the effluent and biosolids of five wastewater utilities in Michigan.” They said, “The purpose of this
study was to quantify the occurrence and release of antibiotic resistant genes (ARGs) and antibiotic resistant bacteria
(ARB) into the environment through the effluent and biosolids of different wastewater treatment utilities including an
MBR (Membrane Biological Reactor) utility, conventional utilities (Activated Sludge, Oxidative Ditch and Rotatory
Biological Contactors-RBCs) and multiple sludge treatment processes (Dewatering, Gravity Thickening, Anaerobic
Digestion and Lime Stabilization). Samples of raw wastewater, pre- and post-disinfected effluents, and biosolids were
monitored for tetracycline resistant genes (tetW and tetO) and sulfonamide resistant gene (Sul-I) and tetracycline and
sulfonamide resistant bacteria. ARGs and ARB concentrations in the final effluent were found to be in the range of ND
(non-detectable)-2.33 x 10[6] copies/100 mL and 5.00 x 10[2] x 6.10 x 10[5] CFU/100 mL respectively. Concentrations
of ARGs (tetW and tetO) and 16s rRNA gene in the MBR effluent were observed to be 1- 3 log less, compared to
conventional treatment utilities. Significantly higher removals of ARGs and ARB were observed in the MBR facility (range
of removal: 2.57 to 7.06 logs) compared to that in conventional treatment plants (range of removal: 2.37 - 4.56 logs) ( p
< 0.05). Disinfection (Chlorination and UV) processes did not contribute in significant reduction of ARGs and ARB ( p >
0.05). In biosolids, ARGs and ARB concentrations were found to be in the range of 5.61 x 10[6] -4.32 x 10[9] copies/g
and 3.17 x 10[4] -1.85 x 10[9] CFU/g, respectively. Significant differences ( p < 0.05) were observed in concentrations of
ARGs (except tetW ) and ARB between the advanced biosolid treatment methods (i.e., anaerobic digestion and lime
stabilization) and the conventional dewatering and gravity thickening methods.”
http://www.egr.msu.edu/~xagorara/documents/Poster%20Mariya%20ARG%202011.pdf
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