Antibiotic Resistance Bacteria and Antibiotic Markers In Recombinant Bacteria
Jim Bynum, VP                                                                                                                                                                                      January 1, 2010
Help For Sewage Victims

Scientists have known for a long time that genetic material can be exchanged between bacteria in the gut. Then it was
discovered antibiotics could cause antibiotic bacteria to emerge within the bacterial group being treated. Later in genetic
recombination scientists used genetic antibiotic genes as markers to prove genetic transfer of target genes in
genetically modified crops and genetically modified organisms. What no one wants to discuss is that scientists, including
EPA, have also known antibiotic bacteria were entering sewage and drinking water treatment plants where they were
amplified in treated sewage sludge, effluent, reclaimed water and drinking water. The rise of antibiotic resistant
infections are blamed on drugs, doctors and farmers.  No one considers that it is the policy of the United States
Agencies to spread these antibiotic resistant bacteria on food crops, grazing land, parks, forest, school grounds, home
lawns and gardens. Ricki Lewis explains some of the history, but ignores the treatment plants, federal policy and all of
the studies.

The Rise of Antibiotic-Resistant Infections
by Ricki Lewis, Ph.D. -- FDA
When penicillin became widely available during the second world war, it was a medical miracle, rapidly vanquishing the
biggest wartime killer--infected wounds. Discovered initially by a French medical student, Ernest Duchesne, in 1896, and
then rediscovered by Scottish physician Alexander Fleming in 1928, the product of the soil mold Penicillium crippled
many types of disease-causing bacteria. But just four years after drug companies began mass-producing penicillin in
1943, microbes began appearing that could resist it.
FDA Consumer magazine (September 1995)

Frederick Griffith (1879 - 1941) was a British medical officer. In 1928, in what is today known as
Griffith's experiment, he discovered the principle of DNA transference between bacteria.
Griffith was trying to make a vaccine to prevent pneumonia infections in the epidemics after World
War I by using two strains of the Streptococcus pneumoniae bacterium. The rough strain (R strain)
did not cause pneumonia when injected into mice and was not covered with a polysaccharide
capsule. The smooth strain (S strain) did have a polysaccharide capsule and was deadly when
injected, causing pneumonia and killing the mice in a day or two. When the S strain was heated to
inactivate it and then injected into mice, it produced no ill effects in the subjects. However, when
dead S coupled with live R was injected into the mouse, the mouse died. After isolating bacteria from
the blood of the mice, Griffith discovered that the normally nonpathogenic R bacteria had acquired
polysaccharide capsules. The bacteria isolated from the mice infected with the mixture of live R and
heat inactived S were all of the S strain, and maintained this phenotype over many generations.
Griffith hypothesized that some "transforming principle" from the heat inactivated S strain converted
the R strain to the virulent S strain. It wasn't until several years later that Griffith's "transforming
principle" was identified as DNA.

The medical importance of infective drug resistance, especially multiple drug resistance, is
apparently limited to Japan at present, but it could become a serious and world-wide problem
in the future. The fact that R factors can be transferred to every genus of
and to other genera, including V. comma, by nonpathogenic bacteria such as E. coli constitutes
a serious public health problem. In individuals who ingest E. coli which contain R factors, the
E. coli and other bacterial strains already present in the intestinal tract can be converted to drug
resistance. Such an individual will become a carrier of drug-resistant intestinal flora. If
infection with pathogenic bacteria such as salmonellae, shigellae, and V. comma occurs,
these pathogenic organisms could easily acquire R factors from the intestinal flora.

Incidence of Infectious Drug Resistance Among Lactose-Fermenting Bacteria Isolated from Raw and Treated Sewage
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
The conclusions to be drawn from this investigation are that multiply antibiotic-resistant
occur in significant numbers in both raw and treated sewage and that in at least 50% of these bacteria resistance is
determined by transmissible R factors. Assuming that most of the strains examined were of human origin, the R factors
identified by their patterns of resistance and the frequency of specific R factors may reflect the level of infectious drug
resistance existing in the intestinal flora of the general population at
any given time. Routine surveillance of sewage at periodic intervals for the detection and characterization
of prevailing R factors may serve as a means of detecting significant changes in the resistance patterns of prevailing R
factors and of detecting changes in the frequency of specific R factors to be found in the general population.

Non-Chromosomal Antibiotic Resistance in Bacteria, III :* Isolation of the Discrete Transfer Unit of the R-Factor RI
Stanley N. Cohen and Christine A. Miller
Abstract. A covalently-closed circular DNA species, banding at a buoyant density of p = 1.709 g/cm3 in CsCl, has been
identified in antibiotic-sensitive colonies of E. coli strain AB2463 (rec A-) after mating with a Proteus mirabilis
strain that carries the R-factor, Ri. This plasmid, which represents a stable segregant of Ri that has lost all of the drug
resistance determinants present on the parent R-factor but which has retained its ability to be transferred by
conjugation, fulfills the functional definition of the R-factor transfer unit (RTF).
Proceedings of the National Academy of Sciences Vol. 67, No. 2, pp. 510-516, October 1970

Genetic Mapping of Antibiotic Resistance in Markers Bacillus subtilis*
C. Goldthwaite, D. Dubnau, and I. Smith
Abstract. Several antibiotic resistance markers in Bacillus subtilis have been mapped by three-factor transduction
crosses using bacteriophage PBS1. These markers, which are thought to be located in genes coding for ribosomal
proteins, are within a segment comprising about 5 per cent of the B. subtilis genetic map, close to the major group of
rRNA cistrons. The order of genetic makers in this region was found to be cysA14 mic-1 t str-1 (ery-1, ole-2) spc-2 bry-2
lin-2. The positions of neo-2, nea-1, and kan-2 are uncertain, although they are located in this region to the right of
Proceedings of the National Academy of Sciences Vol. 65, No. 1, pp.96-103, January 1970

Incidence of Infectious Drug Resistance Among Fecal Coliforms Isolated from Raw Sewage
Abstract: 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.
APPLIED MICROBIOLOGY, Mar. 1971, p. 487-491, Vol. 21, No. 3

Transfer Among Erwinia spp. and Other Enterobacteria of Antibiotic Resistance Carried on R Factors
Department of Bacteriology, University of California, Davis, California 95616
Abstract: Antibiotic resistance carried on R factors was transferred by conjugation from Escherichia coli B/r and Shigella
flexneri la to Erwinia spp. Tetracycline resistance (TetR) carried on R factor R100 drd-56 was transferred from E. coli
B/r to strains of Erwinia amylovora, E. aroideae, E. atroseptica, E. chrysanthemi, E. cytolytica, E. dissolvens, E.
herbicola, E. nigrifluens, and E. nimipressuralis, but not to strains of Erwinia carotovora, E. carnegieana, E.
dieffenbachiae, E. oleraceae, and E. quercina. Multiple antibiotic resistance (chloramphenicol, streptomycin,
tetracycline; ChlR-StrR-TetR) carried on R factor SR1 was transferred from a clinical isolate of S. flexneri la to strains of
E. aroideae, E. chrysanthemi, E. herbicola, and E. nigrifluens, but not to strains of other Erwinia spp. The frequency of
this transfer was low with receptive cultures of Erwinia spp. and E. coli (F- strain). Antibiotic resistance in the
exconjugants showed varying degrees of stability in the presence or absence of acridine orange, depending on the
strain tested. The frequencies of segregation to drug susceptibility in the presence of acridine orange, though low,
suggest that the elements exist as plasmids in the majority of the Erwinia exconjugants. Multiple antibiotic resistance
(ChlR-StrR-TetR) was found to segregate into various resistance classes (ChlR-StrR,,StrR-TetR, TetR, StrR, and none)
in these exconjugants. The exconjugants of E. amylovora, E. herbicola, and E. nigrifluens, to which R100 drd-56 was
transferred from E. coli B/r, were sensitive to the male (F)-specific phage M13. There was a positive correlation between
the susceptibility of exconjugants to the F-specific phage M13 and their ability to transfer R100 drd-56 to the recipient
cultures of Escherichia coli, Erwinia herbicola, Salmonella typhimurium, and Shigella dysenteriae. Exceptions were,
however, noted with Erwinia dissolvens and E. nimipressuralis exconjugants harboring R100 drd-56; these
exconjugants, although not susceptible to M13, transferred R100 drd-56 to the recipient cultures. The frequency of
transfer of R100 drd-56 and the levels of resistance to tetracycline in Erwinia exconjugants were found to differ markedly
depending upon the strain employed. Transfer of multiple antibiotic resistance (ChlR-StrR-TetR) from Erwinia
exconjugants was not obtained in preliminary trials with an E. coli F- strain as the recipient culture.
JOURNAL OF BACTIOLOGY, Oct. 1972, p. 576-584, Vol. 112, No. 1

Nonchromosomal Antibiotic Resistance in Bacteria: Genetic Transformation of Escherichia coli by R-Factor DNA*
(CaCI2/extrachromosomal DNA/plasmid)
ABSTRACT Transformation of E. coli cells treated with CaCJ2 to multiple antibiotic resistance by purified R-factor DNA is
reported. Drug resistance is expressed in a small fraction of the recipient bacterial population almost immediately after
uptake of DNA, but full genetic expression of resistance requires subsequent incubation in drugfree medium before
antibiotic challenge. Transformed bacteria acquire a closed circular, transferable DNA species having the resistance,
fertility, and sedimentation characteristics of the parent R factor. Covalently-closed, catenated, and open (nicked)
circular forms of R-factor DNA are all effective in transformation, but denaturation and sonication abolish the
transforming ability of R factor DNA in this system.
Proc. Nat. Acad. Sci. USA Vol. 69, No. 8, pp. 2110-2114, August 1972

Abstract: TRANSFER of resistance (R) factors occurs readily between certain Gram negative organisms in the
laboratory, and there has been a tendency to assume that it will occur with equal facility in commensal flora, or in
lesions. The Gram-negative flora of the gut is particularly relevant in this connection, because it provides a large
population of organisms that might be involved in R-factor transfer and may cause exogenous or autogenous infection.
Since most R factors contain enough DNA to specify at least 50 gene products (Clowes, 1972), a closer examination
of the nature of an R factor is needed if it is to be “identified” with certainty. A study of the number and type of
resistance determinants-often only three or four of the total number of gene products specified-is inadequate;
and much of the earlier work has been questioned on these grounds
J. MED. MICROBIOL.-VOL. 6: 1973) 461

Construction of Biologically Functional Bacterial Plasmids In Vitro
(R factor/restriction enzyme/transformation/endonuclease/antibiotic resistance)
ABSTRACT: The construction of new plasmid DNA species by in vitro joining of restriction endonucleasegenerated
fragments of separate plasmids is described. Newly constructed plasmids that are inserted into Escherichia
coli by transformation are shown to be biologically functional replicons that possess genetic properties
and nucleotide base sequences from both of the parent DNA molecules. Functional plasmids can be obtained
by reassociation of endonuclease-generated fragments of larger replicons, as well as by joining of plasmid
DNA molecules of entirely different origins.
Proc. Nat. Acad. Sci. USA Vol. 70, No. 11, pp. 3240-3244, November 1973

Aminoglycoside Antibiotic-Inactivating Enzymes in Actinomycetes Similar to Those Present in Clinical Isolates of
Antibiotic-Resistant Bacteria (streptomyces/origin of R-factors/gentamicin-acetate)
These enzymes catalyze reactions identical to those catalyzed by enzymes found in gram-negative bacteria
containing R (antibiotic resistance)-factors. The discovery of these enzymes suggests the possibility of an
evolutionary relationship between the aminoglycosideinactivating enzymes (produced by resistance determinants) in
bacteria containing R-factors and similar enzymes found in the actinomycetes.
Streptomycin (SPT) R + E. coli, Staph. aureu,  P. aeruoinoea
Neomycin-kanamycin (NPT) I R + E. coli,  P. aeruginoea, Staph. aureu

Patent application for Recombinant Plasmid Chimera by Cohen and Boyer-- issued 1980
Patent to create E. coli 0157 using calcium chloride as an agent for genetic engineering -- the subject  invention
provides a technique, whereby a replicon and gene can coexist in a plasmid, which is capable of
being introduced into a unicellular organism, which could not exist in nature.  Conveniently, genes are
available, which provide for antibiotic or heavy metal resistance or polypeptide resistance, e.g.
colicin. Therefore, by growing the bacteria on a medium containing a bacteriostatic or bacteriocidal
substance, such as an antibiotic, only the transformants having the antibiotic resistance will survive.
Illustrative antibiotics include tetracycline, streptomycin, sulfa drugs, such as sulfonamide, kanamycin,
neomycin, penicillin, chloramphenicol, or the like.

Genome Construction Between Bacterial Species In Vitro: Replication and Expression of Staphylococcus Plasmid Genes
in Escherichia coli
(transformation/R plasmid/antibiotic resistance/restriction endonuclease/recombination)
ABSTRACT:  Genes carried by EcoRI endonucleasegenerated fragments of Staphylococcus plasmid DNA have
been covalently joined to the E. coli antibiotic-resistance plasmid pSClOl, and the resulting hybrid molecules have
been introduced into E. coli by transformation. The newly constructed plasmids replicate as biologically functional
units in E. coli, and express genetic information carried by both of the parent DNA molecules. In addition, electron
microscope heteroduplex analysis of the recombinant plasmids indicate that they contain DNA sequences derived
from E. coli and Staphylococcus aureus. Recombinant molecules can transform other E. coli cells for penicillin-
resistance markers originally carried by the staphylococcal plasmid, and can be transferred among E. coli strains by
conjugally proficient transfer plasmids.
Proc. Nat. Acad. Sci. USA Vol. 71, No. 4, pp. 1030-1034, April 1974

R Factor Transfer in Rhizobium leguminosarum
R factors of the compatibility class P were transferred between strains of Eschericha coli K12 and Rhizobium
leguminosarum. These R factors were stable in R. leguminosarum and conferred similar levels of antibiotic resistance to
those in the corresponding R+ E. coli K12 hosts, with the exception of carbenicillin resistance which was greatly
reduced. Transfer between R. leguminosarum strains was by conjugation and was stimulated by conditions favouring
spheroplast formation. R factor mediated recombination could not be demonstrated
Journal of General Microbiology 84 (1974), 188-198;

Antibiotic Resistance Plasmids of Staphylococcus aureus and Their Clinical Importance
Since the discovery of transferable antibiotic resistance in 1959, a fairly clear picture of the process of conjugation in
the Enterobacteriaceae has emerged, or at least of the process as it occurs in the laboratory. Although a huge amount
of circumstantial evidence seems to establish beyond doubt that transfer of plasmids among these organisms has
occurred in nature, it has often been impossible to calculate the frequency of transfer. This has resulted largely from the
difficulty of identifying specific strains over many years.
BACTEROLOGICAL REVIEWS, Mar. 1975, p. 1-32, Vol. 39, No. 1

Antibiotic Resistance Among Coliform and Fecal Coliform Bacteria Isolated from Sewage, Seawater, and Marine
Antibiotic resistance, mediated by extrachromosomal elements or R factors, is widespread among the
Enterobacteriaceae (22). R factors may mediate resistance to as many as eight antibiotics simultaneously and confer
resistance to heavy metals such as nickel, mercury, and cobalt (17). They are transmissible among gram-negative
bacteria such as Escherichia coli, Salmonella, and Shigella and to other unrelated bacteria such as Pseudomonas
aeruginosa (5).
The widespread use of antibiotics in agriculture and medicine is accepted as a major selective force in the increasing
reports of high incidence of antibiotic resistance among gram-negative bacteria (2, 15). Coliform bacteria, generally
regarded as nonpathogenic indicators of pollution (9), are often used to study the bacteriological quality of water and
foods. It has been demonstrated that antibiotic-resistant coliform bacteria from effluents and land runoff eventually may
enter marine receiving waters (7, 18-20).
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1976, p. 879-884, Vol. 9, No. 6

Replication and expression of plasmids from Staphylococcus aureus in Bacillus subtilis
(DNA/genetic transformation/molecular cloning/biohazards)
ABSTRACT One S. aureus plasmid coding for tetracycline resistance, pT127, and four plasmids (pC194, pC221,
pC223, and pUB112) coding for chloramphenic resistance have been introduced by transformation into B. subtifis. The
plasmids replicate in-and confer antibiotic resistance upon-their new host. These experiments show that the potential for
genetic exchange between diverse bacterial species is greater than has been commonly assumed.
Proc. Nat!. Acad. Sci. USA Vol. 74, No. 4, pp. 1680-1682, April 1977

Antibiotic Resistance Patterns of Gram-Negative Bacteria Isolated from Environmental Sources
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, --  were Aeromonas,
Bacillus, Proteus, Arthrobacter, Lactobacillus, Klebsiella, Plesiomonas, Pectobacterium, Chromobacterium, Serratia,
Enterobacter, Staphylococcus, and Micrococcus.-- 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.
Funded by: US Army Medical Research and Development Command , Fort Detrick, Frederick, Maryland 21701

Annual Report, September 1978
To be sure the majority of hospital acquired infections caused by gram negative organisms are still sensitive to most
antibiotics. Yet, the emergence of nosocomial pathogens which are multiply resistant is occurring throughout the
world (1,17). Moreover, organisms which were once considered to be minimally pathogenic, such as Serratia and
Proteus species, have now become common cause of infection (18). Undoubtedly, this situation is relitive to the
increasing prevalence of hospitalized patients with impaired host defenses as well as the multiple resistance of these
strains. Nor is the situation less serious in military hospitals than in civilian facilities.

Drug-Resistance and Enterotoxigenic Plasmids in Enteropathogenic Bacteria With Special Reference to Clinical
Isolates of San Lazaro Hospital*
Takeshi Yokota,** Ryushi Nozawa,** Reiko Sekiguchi** and Cesar V. Uylangco***
In 1948, Demerec in USA proposed the famous "Spontaneous Mutation and Selection" theory suggesting that the origin
of drug-resistant bacteria randomly occurred as spontaneous mutants during cell division and drugs play a role only as
the selective agents. This concept had been adopted until 1959 as the genetic background for drug-resistant bacteria
not only in vitro but also in vivo (Figure 2).
Although bacteria had been considered as an unisexual and bifissionally dividing primitive creature, various mechanisms
of gene transfer were discovered since 1946. There were chromosomal recombinations like sexual reproduction,
Transduction by bacteriophages, carrier of genes by virus-like parasite in. bacterial cells, and transformation by purified
DNA. With these discoveries, it became logical to assume that drug-resistance of bacteria can be transmitted to other
species by these phenomena. In 1959, Akiba et al and Ochitai et al in Japan discovered a phenomenon of transfer of
bacterial resistance from clinical isolates of Shigella flexneri and Escherichia coli to sensitive bacteria. It was the first
discovery of R factors.

Conjugative Transfer of Multiple Antibiotic Resistance Markers in Streptococcus pneumoniae
Abstract: Twenty antibiotic-resistant isolates of Streptococcus pneumoniae were investigated for conjugative transfer of
their drug resistance markers into streptococcal (groups B and D) and pneumococcal (encapsulated and non-
encapsulated) recipients. Of these, 7 wild-type donor pneumococci transferred all their resistance markers (except Pc
[penicillin], Su [sulfonamide], and Tp [trimethoprim]) into group D Streptococcus and non-encapsulated S. pneumoniae
recipients at a low frequency (10-5 to 10-6). The resistance markers transferred were Tc (tetracycline);
Tc and Cm (chloramphenicol); Tc and MLS (macrolides, lincosamides, and streptogramin B); Tc, MLS, Km (kanamycin),
and Cm. The transconjugants obtained retransferred their resistance markers into appropriate streptococcal or
pneumococcal recipients or both. The resistance markers of streptococcal transconjugants could not be cured by
chemical agents. All attempts to detect extrachromosomal deoxyribonucleic acid from pneumococcal or streptococcal
transconjugants were unsuccessful. The molecular weight of a streptococcal conjugative R plasmid (pIP501) was
investigated after transfer into the non-encapsulated S. pneumoniae recipient and was found to be similar to that of the
wild-type group B Streptococcus host (20 x 106).
JOURNAL OF BACTERIOLOGY, July 1980, p. 313-320, Vol. 143, No. 1

R-Plasmid Transfer Frequencies from Environmental Isolates of Escherichia coli to Laboratory and Fecal Strains
Outbreaks of food- and waterborne enteric disease caused by Shigella and Salmonella species which harbor pieces of
self-replicating deoxyribonucleic acid coding for multiple drug resistance (R plasmids) have been reported in many parts
of the world. That R+ coliforms may be the source of R plasmids for these pathogens has been known since 1959, when
researchers in Japan first isolated R+ strains of Shigella during a dysentery outbreak and recovered Escherichia coli
with the same resistance pattem from the same individuals. They also showed that R-plasmid exchange can occur
among E. coli, Shigella, and Salmonella species (29).
Outbreaks of waterborne enteric disease caused by Shigella, Salmonella, or enteropathogenic E. coli strains which
contain transferable R plasmids have rarely occurred in the United States, although the Shigella isolate obtained
during the only known outbreak of recreational waterbome dysentery possessed multiple drug resistance (22).
Nevertheless, there has been considerable concem that R plasmids in their coliform hosts are being disseminated into
environmental waters via the disposal of raw or marginally disinfected sewage and sludge.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1980, p. 756-764, Vol. 40, No. 4

Antibiotic-resistant bacteria in drinking water.
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.
Appl Environ Microbiol. 1981 August; 42(2): 277-283

Transposon-Mediated Multiple Antibiotic Resistance in Acinetobacter Strains
Abstract: Acinetobacter calcoaceticus subsp. anitratus, which is unusually resistant to multiple antibiotics, was the cause
of an epidemic of respiratory tract infections in patients in an intensive care unit. A representative isolate of the epidemic
strain was found to contain the aminoglycoside-modifying enzymes 3-N-acetyltransferase, 3'-phosphotransferase, and 3"
-adenylyltransferase, which confer resistance to gentamicin, kanamycin, and streptomycin, respectively. In addition, the
strain produced a cephalosporinase and was resistant to penicillins due to the production of a TEM-2 P-lactamase. The
bacterial isolate also exhibited resistance to chloramphenicol, tetracycline, and sulfonamides. The resistant phenotype
of this strain was similar to resistance patterns frequently observed in endemic hospital flora, suggesting that the
transfer of an R plasmid into Acinetobacter sp. may have occurred. However, antibiotic resistance could not be
transferred to any recipient by various mating procedures. After plasmid RP4 was transferred into an ampicillin- and
kanamycin-susceptible derivative of the epidemic strain, mobilization of resistance to chloramphenicol, gentamicin,
streptomycin, sulfonamides, and possibly tetracycline could be achieved. This mobilization was due to the
transposition of a 16-megadalton DNA sequence from the Acinetobacter chromosome into plasmid RP4. Insertion of the
transposable sequence occurred near the PstI and SmaI sites around position 22.5 on the physical map of plasmid RP4.
We suggest that a plasmid resistant to multiple antibiotics was transferred from the hospital flora into Acinetobacter sp.
but could not be maintained stably in this host. Instead, a multiply resistant DNA sequence was transposed and stably
integrated into the Acinetobacter chromosome. The occurrence of such multiply resistant transposons on conjugative
plasmids contributes greatly to the genetic variability of bacteria and may sometimes have serious epidemiological and
therapeutic consequences.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1982, p. 323-329, Vol. 22, No. 2

In Situ Studies with Membrane Diffusion Chambers of Antibiotic Resistance Transfer in Escherichia coli
Coliform bacteria were isolated from raw sewage and sewage effluent-receiving waters and tested for their antibiotic
susceptibility patterns and their ability to transfer antibiotic resistance to Escherichia coli K-12 C600. An environmental
isolate of E. coli (MA527) capable of transferring antibiotic resistance to C600 was mated, both in vitro and in situ, with
an antibiotic-sensitive E. coli environmental isolate (MA728). In situ matings were conducted in modified membrane
diffusion chambers, in the degritter tank at the Grant Street (Melbourne, Fla.) sewage treatment facility, and in the
sewage effluent-receiving waters in Melbourne, Fla. The transfer frequencies in situ were 3.2 x 10-5 to 1.0 X 10-6,
compared with 1.6 X 10-4 to 4.4 x 10-5 observed in vitro. Transfer was shown to occur in raw sewage but was not
detected in the effluent-receiving waters. The presence of a 60-megadalton plasmid species in both donor and
transconjugants, but not in the recipients, provided physical evidence for the transfer of antibiotic resistance in

R-Plasmid Transfer in a Wastewater Treatment Plant (1982)
Resistance transfer among gram-negative rods has posed a significant threat in human nosocomial infections (13, 24)
and in other animal infections where antimicrobial prophylaxis has provided the selective pressure necessary to
maintain R plasmids in the etiological agent(s)

Because of the high incidence of resistance among nosocomial bacteria, considerable research has been conducted on
in vivo R-plasmid transfer. These studies have demonstrated that transfer does occur, and at relatively high rates, in
wounds (21, 24) and in the gastrointestinal (3, 35), urinary, and respiratory tracts (13) of warm blooded animals,
including humans.

Generally, bacteria with the greatest levels of resistance have been isolated from environments with the
greatest potential for significant contamination by antimicrobial agents, e.g., hospitals and hospital sewage effluent (9,
12, 17), commercial fisheries (40), and abattoirs (15, 31). However, resistant bacteria have also been isolated from
apparently nonselective environments, including plants (38), estuaries (28), deep ocean water and sediment (19, 34),
and drinking water (4).

Grabow et al. (16) and Grabow and Prozesky (17) have shown a tendency for progressively larger numbers of
multiple-resistant organisms to occur as sewage treatment proceeds toward the plant outfall and the receiving water.
Kushner has observed that exposure to chlorine in sewage plants and elsewhere leads to an increased frequency of
antibiotic resistance in coliform bacteria (D. J. Kushner, personal communication).

Enteric bacteria have been examined for their ability to transfer antibiotic resistance in a wastewater treatment plant.
Resistant Salmonella enteritidis, Proteus mirabilis, and Escherichia coli were isolated from clinical specimens and
primary sewage effluent. Resistance to ampicillin, chloramphenicol, streptomycin, sulfadiazine, and tetracycline was
demonstrated by spread plate and tube dilution techniques. Plasmid mediation of resistance was shown by ethidium
bromide curing, agarose gel electrophoresis, and direct cell transfer. Each donor was mated with susceptible E. coli and
Shigella sonnei. Mating pairs (and recipient controls) were suspended in unchlorinated primary effluent that had been
filtered and autoclaved. Suspensions were added to membrane diffusion chambers which were then placed in the
primary and secondary setting tanks of the wastewater treatment plant. Resistant recombinants were detected by replica
plating nutrient agar master plates onto xylose lysine desoxycholate agar plates that contained per milliliter of medium
10 ,ug of ampicillin, 30 ,ug of chloramphenicol, 10 ,ug of streptomycin, 100 ,ug of sulfadiazine, or 30 ,ug of tetracycline.
Mean transfer frequencies for laboratory matings were 2.1 x 10-3. In situ matings for primary and secondary settling
resulted in frequencies of 4.9 x 10- 5 and 7.5 x 10-5, respectively. These values suggest that a significant level of
resistance transfer occurs in wastewater treatment plants in the absence of antibiotics as selective agents.

Effect of UV Light Disinfection on Antibiotic-Resistant Coliforms in Wastewater Effluents
It is evident from this work as well as from the work of others (10, 13-15, 29) that antibioticresistant
coliforms are entering the aquatic environment via treated municipal wastewater effluents. This work demonstrates that
UV light disinfection can effectively reduce the number of total coliforms both sensitive and resistant to antibiotics in an
activated sludge effluent. This work also points out that there is a significant increase in the percentage of the surviving
total coliform population resistant to tetracycline and chloramphenicol after UV irradiation.  -- Total coliforms and total
coliforms resistant to streptomycin, tetracycline, or chloramphenicol were isolated from filtered activated sludge effluents
before and after UV light irradiation. -- the percentage of the total surviving coliform population resistant to tetracycline
or chloramphenicol was significantly higher than the percentage of the total coliform population resistant to those
antibiotics before UV irradiation. -Multiple drug resistance patterns of 300 total coliform isolates revealed that 82% were
resistant to two or more antibiotics. Furthermore, 46% of these isolates were capable of transferring antibiotic
resistance to a sensitive strain of Escherichia coli.  -- From late 1968 to early 1981, Central America was afflicted by an
R+ S. dysenteriae pandemic (11). During the first year of the epidemic, in Guatemala alone, 12,500 deaths were
recorded. -- [The Central American Shigella dysenteriae gene appears to have translocated to E. coli in California to
create E. coli 0157:H7] --

Selection of antibiotic-resistant standard plate count bacteria during water treatment.
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.
Appl Environ Microbiol. 1982 August; 44(2): 308-316

Comparison of Epidemiological Markers Used in the Investigation of an Outbreak of Methicillin-Resistant
Staphylococcus aureus Infections
Abstract: An outbreak of nosocomial infections was caused by a single strain of methicillin-resistant (MR)
Staphylococcus aureus. This strain was followed as it was transmitted from the index case to 17 patients, 3 hospital
personnel, and 12 items in the hospital environment. The MR S. aureus strain was traced by using four specific
epidemiological markers: antibiogram, phage type, production of aminoglycoside-inactivating enzymes, and plasmid
pattern. These markers were assessed for their reliability in differentiating the epidemic S. aureus strain from
resident nonepidemic strains and for the ease and rapidity with which they determined differences. The epidemic strain
was resistant to beta-lactam antibiotics, gentamicin, erythromycin, clindamycin, and rifampin. Resistance to rifampin
was the only unique marker in the antibiogram which distinguished the epidemic strain from the indigenous strains, and
it was the easiest marker to use for screening isolates from culture surveys. Phage typing was poorly reproducible
and did not yield results rapidly enough to be useful for ongoing epidemiology. The epidemic strain produced a unique
aminoglycoside-inactivating enzyme (3'-phosphotransferase) which distinguished it from indigenous gentamicin-resistant
staphylococci, but this marker was not easily identified, nor was identification helpful during the course of the
investigation. Plasmid pattern analysis was rapidly performed (in less than 24 h), allowed many isolates to be examined
at a time, was stable and reproducible, and yielded a unique fingerprint which distinguished the epidemic strain from all
indigenous isolates. Plasmid pattern analysis is a promising epidemiological tool for MR S. aureus outbreaks in which
epidemic strains lack unique antibiotic resistance markers.
JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1983, P. 395-399, Vol. 18, No. 2

A Cryptic Plasmid from Shigella sonnei
It is not uncommon to find multiple species of cryptic plasmids in bacteria isolated from natural environments (Stahly et
ai., 1978; Gonzhlez et al., 1980). We have reported previously a large number of plasmids in clinical isolates of Shigella
sonnei (Jamieson et al., 1979). Individual isolates have, in addition to self-transmissible and drug-resistance plasmids,
up to eight cryptic plasmids which can be distinguished on the basis of their mobilities in agarose gels. These cryptic
plasmids range in size from 1.5 to 7.2 kb but only one plasmid was found to be common to all strains studied. This 1.5
kb plasmid was shown to be mobilized efficiently by a co-resident self-transmissible plasmid in crosses with laboratory
strains of Escherichia coli
Journal of’ General Microbiology (1983), 129, 15 13 -1 525. Printed in Great Brituin.

Antibiotic Resistance Among Different Species of Fecal Coliforms Isolated from Water Samples
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. -- 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%). -- E. coli, 60%o; K. pneumoniae, 13%; E. cloacae, 11%; other Enterobacteriaceae, 6%; unidentified and
non-Enterobacteriaceae, 10%. -- If the incidence of resistance to antibiotics and synthetic antimicrobial drugs is to be
compared in different areas and at different times, it is necessary to identify the bacteria and to use the same set of
antimicrobial drugs in the tests. It may be very misleading to record only the resistance incidence of "fecal coliforms"
counted as typical colonies. The proportion of Klebsiella species is decisively important.

Association of metal tolerance with multiple antibiotic resistance of bacteria isolated from drinking water.
Bacterial isolates from the drinking water system of an Oregon coastal community were examined to assess the
association of metal tolerance with multiple antibiotic resistance. Positive correlations between tolerance to high levels
of Cu2+, Pb2+, and Zn2+ and multiple antibiotic resistance were noted among bacteria from distribution waters but not
among bacteria from raw waters. Tolerances to higher levels of Al3+ and Sn2+ were demonstrated more often by raw
water isolates which were not typically multiple antibiotic resistant. A similar incidence of tolerance to Cd2+ was
demonstrated by isolates of both water types and was not associated with multiple antibiotic resistance. These results
suggest that simultaneous selection phenomena occurred in distribution water for bacteria which exhibited unique
patterns of tolerance to Cu2+, Pb2+, and Zn2+ and antibiotic resistance.
Appl Environ Microbiol. 1984 June; 47(6): 1238-1242

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

Transferable resistance to gentamicin and other antibiotics in Enterobacteriaceae isolates from municipal wastewater.
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.

Large Plasmids Associated with Virulence in Shigella Species Have a Common Function Necessary for Epithelial Cell
Large plasmids (120 to 140 megadaltons) associated with virulence of Shigella sonnei, S. flexneri 2a and S. dysenteriae
1 were transferred from each strain into Escherichia coli K-12 and avirulent S. flexneri lb strains by ampicillin transposon
(Tnl)-mediated conduction. Strains with the virulence plasmid could penetrate tissue culture cells irrespective of the
original host of the plasmid.

Conjugative Transfer of Staphylococcal Antibiotic Resistance Markers in the Absence of Detectable Plasmid DNA
Abstract: Eleven Staphylococcus aureus clinical isolates were tested for transfer of resistance markers by transduction
and filter mating. The resistance markers of six of the strains could be transferred only by transduction; however, the
five remaining strains transferred their resistance both by transduction and filter mating. The resistance markers that
were cotransferred in filter matings (transfer of resistance to penicillin and streptogramin A was accompanied, in each
case, by the transfer of one or more markers, i.e., resistance to aminoglycosides, cadmium, or tetracycline, depending
on the donor) were not cotransduced. The filter mating transfers were recA independent and were observed with both
Staphylococcus aureus and Staphylococcus epidermidis recipients. Experiments to elucidate the mechanism of transfer
by filter mating suggested that conjugation requiring cell-to-cell contact may have been involved. These transfers
occurred in the absence of detectable plasmid DNA.

Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus
Charles J.Thompson"s4, N.Rao Moval, Richard Tizard2, Reto Crameri"15, Julian E.Davies"14, Marc Lauwereys3
and Johan Botterman3
Abstract: A gene which confers resistance to the herbicide bialaphos (bar) has been characterized. The bar gene was
odginally cloned from Streptomyces hygroscopicus, an organism which produces the tripeptide bialaphos as a
secondary metabolite. Bialaphos contains phosphinothricin, an analogue of glutamate which is an inhibitor of glutamine
synthetase. The bar gene product was purified and shown to be a modifying enzyme which acetylates phosphinothricin
or demethylphosphinothricin but not bialaphos or glutamate. The bar gene was subcloned and its nucleotide sequence
was determined. Interspecific transfer of this Streptomyces gene into Escherichia coli showed that it could be used as a
selectable marker in other bacteria. In the accompanying paper, bar has been used to engineer herbicide-resistant
The EMBO Journal vol.6 no.9 pp.2519-2523, 1987

The occurrence of antibiotic resistant coliforms in the waste water of a water treatment plant]
[Article in German]
Stelzer W, Ziegert E.
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.
Zentralbl Mikrobiol. 1988;143(6):415-23.

Review of Progress in the Biotechnology-Microbial Control Agent Risk Assessment
EPA ORD/ 600/9-90-029
--research results indicate for the first time that a GEM can induce non-transient changes on microbe-mediated
ecological processes in non-sterilized  soil.
one major concern was over viable but nonculturable recombinant microbes.
3,000 patents issued between 1980-84. Patent Office has backlog of several thousand more.
Six reports dealt with the survival of and potential adverse  human health effects caused by GEMs in several model and
test systems
Human health research needs
1. infectivity/pathogenicity questions
2. allergic reaction issues
3. toxicity, and
4. mogulation effects

Bacteria from commercial products are known to translocate from the gut to the spleen and liver in mouse models.
Pulmonary exposure caused death presumed to be from vegetable cells.
Viral biotechnology agents are toxic to human cells.

Evaluation of Exposure Markers
EPA/600/S4-90/034 Apr.1991
Assay to evaluate DNA damage from genotoxics

Behavior of drug resistant fecal coliforms and R plasmids in a wastewater treatment plant
[Article in Japanese]
Nakamura S, Shirota H.
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.
Nippon Koshu Eisei Zasshi. 1990 Feb;37(2):83-90.

Association of Multiple-Antibiotic-Resistance Profiles with Point and Nonpoint Sources of Escherichia coli in Apalachicola
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.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1997, p. 2607–2612 Vol. 63, No. 7

The Challenge of Antibiotic Resistance  (1998)
Certain bacterial infections now defy all antibiotics. The resistance problem may be reversible, but only if society begins
to consider how the drugs affect “good” bacteria as well as “bad”

Last year an event doctors had been fearing finally occurred. In three geographically separate patients, an often deadly
bacterium, Staphylococcus aureus, responded poorly to a once reliable antidote—the antibiotic
vancomycin. Fortunately, in those patients, the staph microbe remained susceptible to other drugs and was eradicated.
But the appearance of S. aureus not readily cleared by vancomycin foreshadows trouble.
What is more, strains of at least three bacterial species capable of causing life threatening illnesses (Enterococcus
faecalis, Mycobacterium tuberculosis and Pseudomonas aeruginosa) already evade
every antibiotic in the clinician’s armamentarium, a stockpile of more than 100 drugs.
Scientific American March 1998

Antibiotic Resistance in Acinetobacter spp. Isolated from Sewers Receiving Waste Effluent from a Hospital and a
Pharmaceutical Plant
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 Use of Antibiotic Resistance Markers to Develop Biotech Crops  (Public Relations Piece Claiming no harm)
Scientists use “antibiotic resistance markers” as a tool for recognizing when they have successfully introduced
a new gene into a plant cell. Some people have questioned whether cells containing these markers could be
transferred to bacteria in people or animals, making antibiotic medicines less effective. Extensive research has
concluded that the use of antibiotic resistance markers is safe.

Mechanisms of antibiotic resistance in bacterial biofilms
Bacteria that attach to a surface and grow as a biofilm are protected from killing by antibiotics. Reduced antibiotic
susceptibility contributes to the persistence of biofilm infections such as those associated with implanted devices. The
protective mechanisms at work in biofilms appear to be distinct from those that are responsible for conventional
antibiotic resistance. In biofilms, poor antibiotic penetration, nutrient limitation and slow growth, adaptive stress
responses, and formation of persister cells are hypothesized to constitute a multi-layered defense. The genetic and
biochemical details of these biofilm defenses are only now beginning to emerge. Each gene and gene product
contributing to this resistance may be a target for the development of new chemotherapeutic agents. Disabling biofilm
resistance may enhance the ability of existing antibiotics to clear infections involving biofilms that are refractory to
current treatments.
International Journal of Medical Microbiology, Volume 292, Issue 2, 2002, Pages 107-113

Long-Term Persistence of Resistant Enterococcus Species after Antibiotics To Eradicate Helicobacter pylori
Maria Sjo¨ lund, MSc; Karin Wreiber, MSc; Dan I. Andersson, PhD; Martin J. Blaser, MD; and Lars Engstrand, MD
Background: Antibiotic treatment selects for resistance not only in the pathogen to which it is directed but also in the
indigenous microflora.
Objective: To determine whether a widely used regimen (clarithromycin, metronidazole, and omeprazole) for
Helicobacter pylori eradication affects resistance development in enterococci.
Design: Cohort study.
Setting: Endoscopy units at 3 community hospitals in Sweden. Patients: 5 consecutive dyspeptic patients who were
colonized with H. pylori, had endoscopy-confirmed duodenal ulcer, and received antibiotic treatment, and 5 consecutive
controls with dyspepsia but no ulcer who did not receive treatment. Measurements: Fecal samples were obtained from
patients and controls before, immediately after, 1 year after, and 3 years after treatment. From each patient and
sample, enterococci were isolated and analyzed for DNA fingerprint, clarithromycin susceptibility, and presence of the
erm(B) gene.
Results: In treated patients, all enterococci isolated immediately after treatment showed high-level clarithromycin
resistance due to erm(B). In 3 patients, resistant enterococci persisted for 1 to 3 years after treatment. No resistance
developed among controls.
Conclusion: A common H. pylori treatment selects for highly resistant enterococci that can persist for at least 3 years
without further selection.
Ann Intern Med. 2003;139:483-487.

Human intestinal bacteria as reservoirs for antibiotic resistance genes
Abigail A. Salyers, Anamika Gupta and Yanping Wang
Until recently, bacterial pathogens were the primary focus of studies of antibiotic resistance genes and their spread.
Now, scientists are starting to wonder whether this focus is too narrow. Could the microflora of the human colon,
normally considered innocuous or beneficial, be playing a more sinister role in human health as reservoirs for
antibiotic resistance genes? The reservoir hypothesis is depicted in Figure 1. According to this view, human
intestinal bacteria not only share resistance genes among themselves but can also acquire from or donate resistance
genes to bacteria that are just passing through the intestine [1–3]. The possibility that resistance gene spread
in the human colon might be a serious threat to human health was first raised in connection with post-surgical
infections, which are usually caused by the normal microflora of the patient or the patient’s caretakers [4,5].
Recently, concern about resistance gene transfers in the human colon has expanded to include agriculture [6,7]
TRENDS in Microbiology Vol.12 No.9 September 2004

Environmental Contamination with Vancomycin-Resistant Enterococci from Hospital Sewage in Portugal
Vancomycin-resistant enterococci (VRE) were detected in samples of sewage obtained downstream of hospitals
of the Porto area in Portugal, and in samples from the Douro Estuary. Clonal analysis, Tn1546 typing,
and presence of putative virulence traits indicate the clinical origin of these isolates. This observation
highlights the importance of hospital sewage in the VRE contamination of the environment.

Persistence of resistant Staphylococcus epidermidis after single course of clarithromycin.
Sjölund M, Tano E, Blaser MJ, Andersson DI, Engstrand L.
We examined how a common therapy that includes clarithromycin affects normally colonizing Staphylococcus
epidermidis. Samples from the nostrils of 5 patients receiving therapy were collected before, immediately after, 1 year
after, and 4 years after treatment. From each patient and sample, S. epidermidis strains were isolated and analyzed for
clarithromycin susceptibility and presence of the erm(C) gene. We show that macrolide-resistant strains of S.
epidermidis were selected during therapy and that the same resistant strain may persist for 4 years, in the absence of
further antimicrobial treatment.
Emerg Infect Dis. 2005 Sep;11(9):1389-93.

Municipal Wastewater Treatment: A Novel Opportunity to Slow the Proliferation of Antibiotic-Resistant Bacteria?  
University of Minnesota researchers published data showing that extremely high numbers of
multi-drug resistant bacteria  (173) in effluent (treated water) at high levels are being released into the
environment from highly efficient, award winning, sewage wastewater treatment plants. Researchers were
very concerned when they found extremely fast transfer of the drug resistant gene between bacteria in
the treatment plant which confirmed EPA studies from the 80s. They appeared to be somewhat confused
because the bacteria taken out of the treated water were not detectable in sludge.

Frequency of transferable multiple antibiotic resistance amongst coliform bacteria isolated from a treated sewage
effluent in Antofagasta, Chile
The wastewater treatment plant constitutes an important reservoir of enteric bacteria which carry potentially transferable
resistance genes. This study has demonstrated that sewage treatment may result in the increase the proportion of
antibiotic resistant bacteria in effluent water. The use of the treated sewage may contribute to spread antibiotic
resistance in the environment, specially when regions poor in water resources re-use it in watering agricultural crops,
such as in northern part of Chile. This represents a dangerous public health risk, which needs future evaluation and

Antibiotic Resistance Genes as Emerging Contaminants: Studies in Northern Colorado†
On the basis of this study it is clear that ARGs are present in various environmental compartments, including river
sediments, irrigation ditch water, dairy lagoon water, DWTPs, and a WRP. Furthermore, quantitative techniques
incorporating Q-PCR provide a means to compare the concentrations of ARGs associated with the known urban and
agricultural impacts, which provides a more direct measure than previous culture  based methods. On the basis of this
occurrence survey, it is argued that ARGs are emerging contaminants that need to be further studied in the paradigm of
environmental science and engineering. The concept of ARGs as “pollutants” has also been suggested by Rysz and
Alvarez (46). It should be noted that besides the tetracycline and sulfonamide ARGs that were the focus of this study,
there are numerous other ARGs that have been described in the literature and likely even more that have not yet been
discovered, each potentially with its own unique properties.
Environ. Sci. Technol. 2006, 40, 7445-7450  (file 5)

Multiple Antimicrobial Resistance in Plague:
An Emerging Public Health Risk Antimicrobial resistance in Yersinia pestis is rare, yet constitutes a significant
international public health and biodefense threat. In 1995, the first multidrug resistant (MDR) isolate of Y. pestis (strain
IP275) was identified, and was shown to contain a self-transmissible plasmid (pIP1202) that conferred resistance to
many of the antimicrobials recommended for plague treatment and prophylaxis. Comparative analysis of the DNA
sequence of Y. pestis plasmid pIP1202 revealed a near identical IncA/C plasmid backbone that is shared by MDR
plasmids isolated from Salmonella enterica serotype Newport SL254 and the fish pathogen Yersinia ruckeri YR71. The
high degree of sequence identity and gene synteny between the plasmid backbones suggests recent acquisition of
these plasmids from a common ancestor. In addition, the Y. pestis pIP1202-like plasmid backbone was detected in
numerous MDR enterobacterial pathogens isolated from retail meat samples collected between 2002 and 2005 in the
United States. Plasmid-positive strains were isolated from beef, chicken, turkey and pork, and were found in samples
from the following states: California, Colorado, Connecticut, Georgia, Maryland, Minnesota, New Mexico, New York and
Our studies reveal that this common plasmid backbone is broadly disseminated among MDR zoonotic pathogens
associated with agriculture. This reservoir of mobile resistance determinants has the potential to disseminate to Y. pestis
and other human and zoonotic bacterial pathogens and therefore represents a significant public health concern. PLoS
ONE 2(3): e309.  

Toxicogenomic Response to Chlorination Includes Induction of Major Virulence Genes in Staphylococcus aureus
M A T T H E W WOOK C H A N G , † F R E S H T E H T O G H R O L , * , ‡ A N D W I L L I A M E . B E N T L E Y §
School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637459, Center for
Biosystems Research, University of Maryland Biotechnology Institute, College Park, Maryland 20742, and Microarray
Research Laboratory, Biological and Economic Analysis Division, Office of Pesticide Programs, U. S. Environmental
Protection Agency, Fort Meade, Maryland 20755

Our results suggest that hypochlorous acid repressed transcription of genes involved in cell wall
synthesis, membrane transport, protein synthesis, and primary metabolism, while amino acid synthesis genes were
induced. Furthermore, hypochlorous acid induced transcription of genes encoding major virulence factors of S. aureus,
such as exotoxins, hemolysins, leukocidins, coagulases, and surface adhesion proteins, which all play essential roles in
staphylococcal virulence. This work implies that chlorination may stimulate production of virulence factors, which
provides new insight into host-pathogen interactions and effects of chlorine application for microbial control.
Environ. Sci. Technol. 2007, 41, 7570-7575

Antimicrobial Drug–Resistant Escherichia coli from Humans and Poultry Products, Minnesota and
Wisconsin, 2002–2004,
James R. Johnson,*† Mark R. Sannes,*†1 Cynthia Croy,*† Brian Johnston,*† Connie
Clabots,*† Michael A. Kuskowski,*† Jeff Bender,‡ Kirk E. Smith,§ Patricia L. Winokur,¶# and Edward A. Belongia**
Emerging Infectious Diseases • • Vol. 13, No. 6, June 2007
The food supply, including poultry products, may transmit antimicrobial drug–resistant Escherichia coli to humans.

Extended-Spectrum -Lactamases (ESBLs), Food, and Caphalosporin Use in Food Animals.
Peter Collignon and Frank Aarestrup,  These drug-resistant bacteria then spread to people via food and other
routes (e.g.,groundwater

Elimination of Indicators (TC, FC, FS) and Enterobacteriaceae Family Bacteria During the Sewage Treatment Process
The occurrence and removal of sanitary indicator bacteria (TC, FC, FS) and bacteria of Enterobacteriaceae family
during the standard sewage treatment process were investigated. In addition, we determined the antibiotic-resistance of
Enterobacteriaceae bacteria in raw and treated sewage. The result of the conducted study demonstrated that bacteria
of Enterobacteriaceae family were most numerous among the surveyed microorganisms, and fecal streptococci was the
least numerous bacteria group. The survey of antibiotic resistance of Enterobacteriaceae family demonstrated that
these bacteria are the least sensitive to streptomycin and erythromycin. The increase in the proportion of bacterial
strains resistance to tested antibiotics in the subsequent months of the study was correlated with the upward seasonal
trend in human population morbidity, and in turn the usage antibiotic therapy.
Polish Journal of Natural Science, Volume 22, Number 2 / June 2007

A seasonal study of the mecA gene and Staphylococcus aureus including methicillin-resistant S. aureus in a municipal
wastewater treatment plant
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. Taken together our results show that the mecA gene, S. aureus and MRSA occur
over the year at all sites investigated.
water research 4 3 ( 2 0 0 9 ) 9 2 5 – 9 3 2

Will Agricultural Biotechnology Lead to the Spread of Antibiotic Resistance? (Public Relations Piece claiming no
Bruce M. Chassy, Ph.D. Introducing a new trait to plants, by inserting DNA into target plant cells, can be a ―long
shot.‖ Sometimes only one in a thousand — or even one in a million — plant cells will take up the inserted DNA and
incorporate the new gene. So biotechnologists need a ―marker‖ to show up when they have hit the target. One such
marker is antibiotic resistance. If genes for antibiotic resistance are linked to genes for the desirable trait, researchers
can single out plant cells that have been transformed successfully by exposing all cells to an antibiotic. Plants cells that
successfully incorporated the combined genes (for the desirable plant trait and for antibiotic resistance) will survive the
antibiotic test and will grow into little mounds of transformed plant cells called a callus. Then the researchers can isolate
cells from the callus and regenerate whole plants with the newly introduced, desired trait. This means, however, that
some new varieties of plants with desirable traits, such as insect resistance or herbicide tolerance, may also carry a new
gene for antibiotic resistance.  

2009 Article Date: 30 May 2009 - 0:00 PDT
Antibiotic Resistant Bacteria Found In Fertilizer
Vancomycin resistant enterococci (VRE) have been found in sewage sludge, a by-product of waste-water treatment
frequently used as a fertilizer. Researchers writing in the open access journal Acta Veterinaria Scandinavica point out
the danger of antibiotic resistance genes passing into the human food chain.

Leena Sahlström, from the Finnish Food safety Authority, worked with a team of researchers from the Swedish National
Veterinary Institute to study sewage sludge from a waste-water treatment plant in Uppsala, Sweden. She 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 one link in this chain".

The researchers collected sludge from the plant every week for four months, for a total of 77 samples. Of these, 79%
tested positive for the drug resistant superbugs. Although VRE themselves are not generally considered to be highly
pathogenic, the danger is that they may pass on their resistance genes to other bacteria. Sahlström concludes, "Our
results demonstrate a need for more efficient hygienic treatment of sewage sludge, in order to avoid possible spread of
antimicrobial resistance through use of sewage sludge on arable land".
Vancomycin resistant enterococci (VRE) in Swedish sewage sludge
Leena Sahlström, Verena Rehbinder, Ann Albihn, Anna Aspan and Björn Bengtsson
Acta Veterinaria Scandinavica (in press)

Vancomycin resistant enterococci (VRE) in Swedish sewage sludge 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. -- Biochemical typing (PhenePlate-
FS) and pulsed field gel electrophoresis (PFGE) revealed prevalence of specific VRE strains in sewage sludge for up to
16 weeks. -- 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. Acta Veterinaria Scandinavica 2009, 51:24   (file 5)

Tetracycline Resistance Genes and Tetracycline Resistant Lactose-Fermenting Enterobacteriaceae in Activated Sludge
of Sewage Treatment Plants
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 104 to 105 copies/mL, significantly lower than those in the influent by 3 orders of magnitude.
Environ. Sci. Technol., 2009, 43 (10), pp 3455–3460

Screening for antibiotic-resistant bacteria in drinking water
This study assessed the diversity of CNS and their resistance to antibiotics in water from three different sources:
A drinking water treatment plant where raw water is treated
A water distribution network that receives treated water
A wastewater treatment plant where effluent is treated
Staphylococci were isolated from a total of 242 water samples. Although the majority (175) were from the water
distribution network, the water from the wastewater treatment plant showed the greatest diversity of species with 12
different species of CNS present. The large number of CNS in the water distribution network may represent an
undesired colonisation. Pinpointing the origins of this colonisation is not within the scope of this study, but the authors
have two suggestions. It may be that the CNS may not be completely eliminated during water treatment or there may be
undetectable micro-fissures (tiny cracks) in the network that allow CNS to intrude.

A seasonal study of the mecA gene and Staphylococcus aureus including methicillin-resistant S. aureus in a municipal  wastewater treatment plant 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. Taken together our results show that the mecA gene, S. aureus and MRSA occur over the year at all sites  investigated. Water Research
Volume 43, Issue 4, March 2009, Pages 925-932
23999569995% 23942072%23FLA%

Staphylococcus aureus, MRSA, methicillin, β-lactam, SCCmec, spa typing, Panton Valentine leukocidin, PVL, antibiotic  
resistance, antibiogram :
Methicillin-resistant S. aureus (MRSA) was recently detected in municipal wastewater, why there
is a need for further  studies to elucidate if MRSA in wastewater constitutes a health risk, and to determine how
wastewater treatment  processes affects MRSA. We cultivated MRSA from a full-scale wastewater treatment plant to
characterise the  indigenous MRSA-flora and to investigate how the wastewater treatment process affects the clonal
distribution. MRSA  isolates were characterised using spa typing, antibiograms, SSCmec typing and detection of Panton
Valentine  leukocidin (PVL) genes. We found that the wastewater MRSA-flora has a close genetic relationship to clinical
isolates,  but we also isolated novel spa types, primarily from the activated sludge treatment step. The number of
isolates and the  diversity of MRSA are reduced by the treatment process, but the process also selects for more
extensive antibiotic  resistant strains as well as for PVL positive strains.

Wastewater treatment contributes to selective increase of antibiotic resistance among Acinetobacter spp. 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 degrees C; and low-temperature,  low-flow,
8 degrees 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 antibiotic susceptibility phenotypes were determined by the  disc-diffusion
method for 8 antibiotics, amoxicillin/clavulanic acid (AMC), chloramphenicol (CHL), ciprofloxacin (CIP),  colistin (CL),
gentamicin (GM), rifampin (RA), sulfisoxazole (SU), and trimethoprim (TMP). The prevalence of antibiotic  resistance in
Acinetobacter isolates to AMC, CHL, RA, and multi-drug (three antibiotics or more) significantly increased  (p<0.01) 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 (p<0.05) 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. Sci
Total Environ. 2009 Jun 1;407(12):3702-6. Epub 2009 Mar 24.  

Mechanisms of Infectious Disease • Fall 2009, Lecture 2
Jonathan Dworkin, PhD
Genetic Basis of Variation in Bacteria
Transmission of genetic variation: R-plasmid conjugation
• R factors - Drug-resistance plasmids first isolated in late 1950's in Shigella during an outbreak of dysentery. The first
plasmid isolated carried resistant determinants to four different antibiotics: chloramphenicol, tetracycline, streptomycin,
and sulfonamides. Later the same plasmid was found in E. coli.
• In patients given oral tetracycline, the predominant fecal E. coli isolates carry tetracycline-resistance R
plasmids within one week.

I must include a PR piece which has no meaning in the real world or as a scientific study. This so called
study puts public health at risk by discussing
 heat inhibited thermotolerant antibiotic resistant E. coli
known in the wastewater industry as fecal coliform. Heat inhibited E. coli (fecal coliform) are incubated at
112.1 deg F for 24 hours before the colonies are counted to determine the most probable number of single
bacteria at the beginning of the test one day earlier. Sludge/biosolids is considered Class A if there are less
than 1,000 colonies at the end of the test. The high heat of the fecal coliform test inactivates many of the
bacteria since the optimum growth rate for E. coli (coliform) is 98.6 deg F when incubated for 24 hours. At
the optimum temperature E. coli will double every 20 minutes. As an example, if there was only one (1) E.
coli bacteria at the start of the test doubling every 20 minutes, in 12 hours the colony would be composed
of 68 billion, 719 million, 476 thousand, 736 hundred (68,719,4 76, 736) bacteria. That would be counted as
one (1) most probable number (MPN) colony forming unit (CFU) for the test result and the test still has 12
hours left -- but it would still be counted as one MPN.

Antibiotic resistant bacteria in wastewater processed by the Metropolitan Water Reclamation District of Greater Chicago  
 Rijal GK, Zmuda JT, Gore R, Abedin Z, Granato T, Kollias L, Lanyon R.
The Metropolitan Water Reclamation District of Greater Chicago (District) initiated a research study to determine the  
total numbers and percentages of antibiotic resistant fecal coliform (FC) bacteria in raw sewage (RS) entering and final  
effluents (FE) discharged from its seven Water Reclamation Plants (WRPs). The density of FC was determined on m-
FC agar containing ampicillin (ampR-16 microg/ml), gentamycin (genR-8 microg/ml), tetracycline (tetR-8 microg/ml), or  
all three antibiotics. The study was primarily undertaken to determine whether secondary sewage treatment at the  
District WRPs adequately reduces the numbers and percentages of FC(ampR), FC(tetR), FC(genR), FC(amp/tet/genR)  
in the FE. The numbers of ampR, tetR, genR, and amp/tet/genR FC observed in RS ranged from 2.0 x 10(5) to 1.1 x 10
(7), 9.5 x 10(4) to 2.2 x 10(6), 95 to 1.5 x 10(4) and 90 to 9.5 x 10(3) per 100 mL, respectively. Secondary sewage  
treatment without disinfection was shown to reduce the number of antibiotic resistant FC by two-three orders of  
magnitude. The numbers of FC(ampR), FC(tetR), FC(genR), and FC(amp/tet/genR) observed in non-disinfected FE  
ranged from 2.0 x 10(2) to 6.4 x 10(3), 2.2 x 10(2) to 4.1 x 10(3), 9 to <20 and 9 to <20 per 100 mL, respectively. The  
relative percentages of antibiotic resistant FC observed in FE followed the same trend observed in RS: FC(ampR) > FC
(tetR) > FC(genR) > FC(amp/tet/genR). Only one FC(amp/tet/genR) bacteria was found in this study indicating that  
multiple-antibiotic resistant FC was virtually eliminated by secondary sewage treatment. The results of multivariate  
regression analysis showed that the percentages of antibiotic resistant FC in the FE from all seven District WRPs were  
lower than the percentages of these organisms in RS (p<0.01). These results support the conclusion that secondary  
sewage treatment in the District effectively reduces the number of antibiotic resistant FC and that the environments of  
the District's seven WRPs are not conducive to the propagation or survival of antibiotic resistant fecal coliform bacteria.
Water Sci Technol. 2009;59(12):2297-304.

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