A PERFECT STORM OF ANTIBIOTIC RESISTANCE
Chapter 5
Laboratories/Antibiotic Resistant Bacteria 9/09/2011
Back to Myth # 4
In 1928, Scottish physician Alexander Fleming is credited with discovering that fungus produces penicillin, even though
French medical student, Ernest Duchesne, is said to have actually discovered it in 1896. Drug companies began mass-
producing penicillin in 1943. Shortly thereafter researchers found penicillin resistant bacteria.
Also in 1928, British medical officer Fred Griffith made an equally profound discovery, the "transforming principle" that
live bacteria could absorb DNA from its environment. In this case, he used a heat killed pathogenic smooth strain of the
Streptococcus pneumoniae bacterium and a live non-pathogenic rough strain of Streptococcus pneumoniae. Neither
strain would cause disease alone. However, when both were injected into a mouse, Transformation occurred and it died.
http://www.experiment-resources.com/transforming-principle.html
In 1945, Selman A. Waksman, et al., New Jersey Agricultural Experiment Station, Rutgers University at New Brunswick,
reported on “STRAIN SPECIFICITY AND PRODUCTION OF ANTIBIOTIC SUBSTANCES. V. STRAIN RESISTANCE OF
BACTERIA TO ANTIBIOTIC SUBSTANCES, ESPECIALLY TO STREPTOMYCIN.” They said, “Different strains of the
same species of bacteria are found to vary greatly in their sensitivity to a given antibiotic substance. This phenomenon
has an important bearing upon-the utilization of the substance for chemotherapeutic purposes, where a knowledge of
the sensitivity of the particular strain of a given organism responsible for a certain disease becomes of paramount
importance. It has been definitely established that when a culture of an organism is grown in the presence of a certain
antibiotic substance, it becomes resistant to increasing concentrations of the substance; strains may thus be obtained
that have become adapted to the action of the antibiotic agent and that require far greater concentrations for growth
inhibition as compared with the parent or mother culture from which they were isolated.” http://www.ncbi.nlm.nih.
gov/pmc/articles/PMC1078787/pdf/pnas01675-0007.pdf
In a 1946 Letter to the Editor, E. L. Tatum and Joshua Lederberg, Yale University, discussed the
“Detection of Biochemical Mutants of Microorganisms.” They said, “Biochemical or nutritional mutants of microorganisms
have many uses in biochemistry and in chemical genetics, 1 but their application to many specific problems has been
limited by the effort that must be spent to obtain the specific mutants required in a given instance. The methods that
have been described previously* may be summarized briefly as follows: Mutations are induced in a culture of the
microorganism by any of a variety of agents, including x-radiation, ultraviolet light, and nitrogen mustard gas.3 Even with
the most efficient mutating agents, only a small fraction of the cells in the culture are mutants. -- The method has been
applied primarily to the detection of mutants in ultraviolet-treated Escherichia coli, but there is no apparent reason why it
should not be equally applicable to any organism which forms compact colonies when grown on minimal agar medium. --
It has, furthermore, been used to isolate a variety of biochemical mutants from x-ray and ultraviolet-treated E. coli,
including strains blocked at some point in the synthesis of proline, methionine, histidiie, isoleucine, cystine, thiamine, or
p-aminobenzoic acid.” http://www.jbc.org/content/165/1/381.full.pdf
In 1947, E. L. Tatum and Joshua Lederberg, Yale University, reported on “Gene Recombination in the Bacterium
Escherichia Coli.” They said, “The study of inheritance in bacteria has, for the most part, been confined to the
investigation. of mutational changes in the course of clonal reproduction. – The conception that bacteria have no sexual
mode of reproduction is widely entertained. This paper will be devoted to the presentation of evidence for the
occurrence in a bacterium of a process of gene recombination, from which the existence of a sexual stage may be
inferred. – Sherman and Wing (1937) have described experiments designed to detect recombinations of fermentative
characters in mixed cultures of various Escherichia coli and Aerobacter aerogenes strains. – A discussion of hereditary
processes in bacteria must take into account the extensive work on transformation of pneumococcal types, first
described by Griffith (1928) and culminating in the isolation of the transforming principle in chemically characterizable
form by Avery, MacLeod, and McCarty (1944). These studies have revealed that, under special experimental
conditions, a product isolated from a serologically specific, smooth, pneumococcus culture will convert cells of a
nonspecific rough culture to the smooth type characteristic of the source of the transforming principle.”
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC518375/pdf/jbacter00658-0015.pdf
Also in 1947, Joshua Lederberg, Yale University, reported on “GENE RECOMBINATION AND LINKED SEGREGATIONS
IN ESCHERICHIA COLI.” Lederberg said, “Mutations for resistance to specific bacteriophages or bacterial viruses have
proven to be exceedingly useful. They are readily obtained as spontaneous mutants by plating a large number of
sensitive bacteria with the particular virus in question; only resistant mutants escape lysis and may be recovered as
"secondary" colonies (fig. I). Resistant mutants are readily freed from residual virus by serial single colony isolation.
Resistance to a given virus may be scored by streaking a loopful of bacteria on an EMB or nutrient agar plate at right
angles to a previous streak of the virus suspension. – The detection of recombinants is based upon the inability of
biochemical mutant bacteria to proliferate in the absence of their specific growth substances. – The recombination of
genetic factors and their segregation into prototroph recombinants of Escherichia coli have been studied. It was found
that genetic markers behaved as if they were part of a system of linked genes.” http://www.ncbi.nlm.nih.
gov/pmc/articles/PMC1209393/pdf/505.pdf
In 1948, M. Demerec, Department of Genetics, Carnegie Institution of Washington at Cold Spring Harbor, NY, offered
evidence in the study, “ORIGIN OF BACTERIAL RESISTANCE TO ANTIBIOTICS'” that resistance was a natural
phenomenon. Demerec said, “In this brief review of the problem of the genetic aspects of the origin of bacterial
resistance to antibiotics, I intend to discuss mainly work done in my laboratory. I shall (1) offer evidence that bacterial
resistance to penicillin and streptomycin is not induced by these compounds but originates spontaneously through
genetic changes comparable to gene mutations; (2) describe the resistance patterns observed in experiments with
penicillin and streptomycin; and (3) outline a possible mechanism responsible for resistance, and for the differences
between the resistance patterns observed with penicillin and those observed with streptomycin. – From the knowledge
gained concerning the mechanism of origin of resistance, it is concluded that in treatment with penicillin the
development of highly resistant strains can be avoided by application of the penicillin in doses sufficiently large to
prevent survival of first-step resistant mutants. In treatment with streptomycin, however, the development of highly
resistant strains cannot be prevented; effective treatment does not eliminate all bacteria, but it probably reduces their
number to a level at which the organism is able to eliminate them.” http://www.ncbi.nlm.nih.
gov/pmc/articles/PMC518544/pdf/jbacter00647-0081.pdf
In 1949, Bernard D. Davis, U. S. Public Health Service, Tuberculosis Research Laboratory, Cornell University, Medical
College, reported on “The Isolation Of Biochemically Deficient Mutants Of Bacteria By Means Of Penicillin.” Davis said,
“It is a simple matter to isolate bacterial mutants when the mutants can proliferate or survive in an environment which
suppresses or eliminates the parent strain. There is consequently no difficulty in obtaining mutants, even of low
frequency, which differ from the parent strain by being resistant to antibacterial chemicals or viruses, or by having
decreased nutritional requirements. Mutants with increased nutritional requirements, however, have been much less
convenient to isolate. Recently developed techniques permit a considerable improvement over the earlier practice of
random selection, but still permit selection from only a few hundred colonies per agar plate. This paper is concerned
with a method of obtaining biochemically deficient mutants from very much larger populations. The method is based on
the unusual mode of action of penicillin, which sterilizes only growing bacteria. Mutants which are unable to grow in
minimal medium therefore survive, while the predominant non-mutant population is sterilized. – The use of penicillin to
isolate biochemically deficient mutants was also developed independently by J. Lederberg and N. Zinder.
Communications by these investigators and by the author are being published in the December 1948 issue of the
Journal of the American Chemical Society.”
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1062948/pdf/pnas01538-0010.pdf
In 1950 scientists C. P. Miller and Marjorie Bohnhoff, reviewed “The Development of Bacterial Resistance to
Chemotherapeutic Agents.” The review discussed the spontaneous mutation of bacteria from their normal status of
streptomycin sensitivity to the resistance stage. The preferred method of recombination was to use nutrient deficient
bacteria which could not form colonies on minimal culture media. A second option was to use mutants based on specific
drug resistances.
http://www.annualreviews.org/doi/abs/10.1146/annurev.mi.04.100150.001221?journalCode=micro
In 1952, Norton D. Zinder and Joshua Lederberg, University of Wisconsin at Madison, reported on the “GENETIC
EXCHANGE IN SALMONELLA.” They said, “In doing recombination experiments with Streptomycin resistant mutants of
Salmonella typhimurium containing phages (viruses) in 1952, Norton D. Zinder and Joshua Lederberg used the term
transduction, rather than sexual reproduction, for what is now referred to as horizontal transfer. The gene
recombination (transductions) were from mutants to the wild type. The unusual aspect of the study was the reference to
33 strains of Salmonella and 2 E. coli strains absorbing FA (bacteria product/filtrable agent) particles prior to genetic
transfer of genes. Some 19 forms of Salmonella and 4 E. coli strains would not a absorb the FA particles. It was thought
the FA was connected to a bacteriophage that produces a single transduced clone. They found that a recombinant
strain could revert to the parent strain. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC169409/pdf/jbacter00011-0097.pdf
One of the most interesting experiments was reported by Rolf Freter, Stritch School of Medicine and Graduate School,
Loyola University, in his 1956 study “EXPERIMENTAL ENTERIC SHIGELLA AND VIBRIO INFECTIONS IN MICE AND
GUINEA PIGS”. He found that by feeding mice antibiotics it was possible rid the intestinal tract's normal E. coli flora and
thereby create long term asymptomatic infections by feeding the mice streptomycin-resistant strains of Shigella flexneri
or Vibrio cholerae. The most unusual finding was that upon introducing a streptomycin-resistant strain of E. coli into
the intestinal tract Shigella flexneri or Vibrio cholerae were quickly eliminated. http://www.ncbi.nlm.nih.
gov/pmc/articles/PMC2136576/pdf/411.pdf
By 1957, S. E. Luria and Jeanne W. Burrous, University of Illinois, report on ““HYBRIDIZATION BETWEEN ESCHERICHIA
COLI AND SHIGELL. They said, “Our experiments have proved that Shigella can mate with E. coli and shares with it a
common system of mating polarities. By hybridization we can create hybrids that would be considered as monstrosities
from the standpoint of traditional bacterial classification,” Not only that, but, “all our Lac+ hybrids, although they are
predominantly "Shigellas" (and probably still pathogenic) would never be detected as potential pathogens by the routine
diagnostic procedures. They would be discarded as "coliforms". http://www.ncbi.nlm.nih.
gov/pmc/articles/PMC289941/pdf/jbacter00516-0063.pdf
L. S. Baron, W. F. Carey, and W. M. Spilman, Walter Reed Army Institute of Research, followed up in 1959 with the
study, “GENETIC RECOMBINATION BETWEEN ESCHERICHIA COLI AND SALMONELLA TYPHIMURIUM.” They found
that recombination could be done much quicker with a highly fertile mutant of the K-12 strain. They referred to over 400
serotypes of Salmonella with each serotype containing large numbers of clones. Streptomycin-resistant strains were
used in the experiments. They were able to produce hybrids attributed to mating. They said, “As a result, it would not be
unreasonable to expect successful crosses of Salmonella X Escherichia, Salmonella X Salmonella, or Salmonella X
Shigella. The hybrids of such crosses would surely offer material of an exceptionally interesting nature for studies in
bacterial virulence.” http://www.pnas.org/content/45/7/976.full.pdf
In 1961, H. Schneider, Samuel B. Formal, and L. S. Baron, Catholic University of America and Walter Reed Army
Institute, presented evidence in “EXPERIMENTAL GENETIC RECOMBINATION IN VIVO BETWEEN ESCHERICHIA COLI
AND SALMONELLA TYPHIMURIUM” The study shows that genetic recombination can occur in the lumen of the intestine
and that hybrids can be detected and enumerated with the use of selective techniques. These results support the
premise that genetic recombination may have played a role in the evolution of the family Enterobacteriaceae. The data
indicate also that hybrids recovered from in vivo [living organism] matings are similar to those reported from in vitro [test
tube or petri dish] crosses of Research.” The supposition was based on feeding mice streptomycin resistant E. coli and
streptomycin-resistant F- S. typhimurium culture. Within 24 hours they were able to recover hybrid strains of bacteria
demonstrating has genetic recombination had occurred. http://jem.rupress.org/content/114/1/141.full.pdf
Also in 1961, Stanley Falkow, J. Marmur, W. F. Carey, W. M. Spilman and L. S. Baron, reported on “EPISOMIC
TRANSFER BETWEEN SALMONELLA TYPHOSA AND SERRATIA MARCESCENS.” They found that Salmonella
typhosa, strain ST-2 was rather unusual in that it utilized lactose. This strain was able to transfer this lactose utilization
factor to many strains of lactose negative members of the Escherichia, Salmonella and Shigella as well as Serratia with
ease in mixed cultures. Lactose use is the hallmark of the Enterobacteriacea family. They found that some Serratia
mutant hybrid strains could pass on the lactose utilization factor while others were very poor at passing on this genetic
material. This was significant due to the differences in the DNA base of both bacteria. http://www.ncbi.nlm.nih.
gov/pmc/articles/PMC1210232/pdf/703.pdf
Moreover, in 1961, Tsutomu Watanabe, and Toshio Fukasawa, Keio University School of Medicine, reported on
“EPISOME-MEDIATED TRANSFER OF DRUG RESISTANCE IN ENTEROBACTERIACEAE III. Transduction of Resistance
Factors.” They found that resistance to streptomycin, chloramphenicol, tetracycline, and sulfonamide, could be
transferred to Salmonella typhimurium strain LT-2 with phage virus P-22 and Escherichia coli strain K-12 with phage
virus P1kc. http://jb.asm.org/cgi/content/abstract/82/2/202
In 1962, Yoshinobu Sugino and Yukinori Hirota, Osaka University, reported on the “CONJUGAL FERTILITY
ASSOCIATED WITH RESISTANCE FACTOR R IN ESCHERICHIA COLI.” They explained the new resistance terminology
for genetic manipulation. They said, “R or R factor is a general term for the infectious drug-resistance factors
(Mitsuhashi, 1960; at the Meeting of Microbial Genetics at Mishima, Japan, it was agreed by investigators in this field to
use the term "R" for the multiple drug-resistance factor). R-infection is the contagious transmission of an R factor by cell
contact. R-mating and F-mating denote chromosome transfer from the donor cell during a mating process mediated by
an R or an F factor, respectively.” Furthermore, they said, “recombinants are formed by mating, or transfer of
comparatively large segments of chromosome by the conjugation process [cell-to-cell horizontal transfer], not by
transduction [transfer by virus] or transformation [DNA direct uptake]. The latter mechanisms of recombination transfer
only a limited size of chromosomal segment in a single transfer.” http://www.ncbi.nlm.nih.
gov/pmc/articles/PMC277988/pdf/jbacter00463-0048.pdf
Also, in 1962, Susumu Mitsuhashi et al, reported on the “COMBINATION OF TWO TYPES OF TRANSMISSIBLE DRUG-
RESISTANCE FACTORS IN A HOST BACTERIUM.” They pointed out that, “multiply drug-resistant Shigella strains
suddenly appeared in Japan in 1956, and that shigellae, E. coli, and E. freundii were resistant to the four drugs from the
very beginning Scientists are constantly looking for new organisms that produce antibiotic drugs.” Their research
showed that when two bacterium, S. flexneri and E. coli 0-26G, with the R factors for streptomycin, chloramphenicol,
tetracycline, and sulfonamide or chloramphenicol, tetracycline, cell to cell superinfected a third Shigella bacterium, the
R factor could then be transferred to a fourth E. coli K 12 bacterium through a phage virus. They noted that by two
separate R factors creating a superinfection in one bacterium, a third R factor was created. They were not sure if it
was a true recombination or caused by some unknown mechanism. http://www.ncbi.nlm.nih.
gov/pmc/articles/PMC277758/pdf/jbacter00459-0029.
pdf
Tsutomu Watanabe, Keio University School of Medicine, reviewed the drug resistance research in his 1963 study
“INFECTIVE HEREDITY OF MULTIPLE DRUG RESISTANCE IN BACTERIA.” He said, “The first isolation of shigellae with
multiple drug resistance was reported in a dysentery patient who had just returned from Hong Kong in 1955.” He noted
that in 1959 Japanese researchers had found that “multiple drug resistance can be easily transferred between shigellae
and Escherichia coli by mixed cultivation.” This was illustrated by the rise in multi-antibiotic resistance. In 1953, only
seven strains of bacteria, out of 4,900 strains tested, showed resistance. Five were resistant to streptomycin and two
were resistant to tetracycline. During 1960, 3,396 strains were tested with 29 strains resistant to streptomycin, 36
strains resistant to tetracycline, none were resistant to chloramphenicol alone, however, 61 strains were resistant to
chloramphenicol and streptomycin, 9 strains were resistant to chloramphenicol and streptomycin, 7 were resistant to
chloramphenicol and tetracycline, 308 strains were resistant to chloramphenicol, streptomycin and tetracycline. The
most interesting suggestion was that multiple drug resistant E. coli could transfer the multi-drug resistance to Shigella in
the intestinal tract. This would explain the phenomenon that when chloramphenicol was given for a sensitivity strain of
Shigella, multi-drug resistant were excreted.
http://thewatchers.us/EPA/2/1963-infective-drug-resistance.pdf
In 1966, Anthony B. Gonzalez, Department of Pathology, Tampa General Hospital, reported a new unique strain of
“Lactose-Fermenting Salmonella.” Gonzalez said, “Salmonella species are generally considered to be unable to
ferment lactose and sucrose according to Bergey's Manual. Recently, however, J. M. Bulmash, McD. Fulton, and J. Jiron
(J. Bacteriol. 89:259, 1965) and L. J. Kunz and W. H. Ewing (J. Bacteriol. 89:1629, 1965) have reported two separate
species of Salmonella capable of fermenting lactose and sucrose. The organism reported here again illustrates that
certain strains of Salmonella are capable of fermenting lactose and sucrose rapidly and can resemble very closely
lactose-fermenting members of the Enterobacteriaceae. The organism isolated was the etiological agent of a rapidly
fatal septicemia resulting from an infection of the uterus in a 21-year-old female during the first trimester of pregnancy,
who aborted spontaneously. The organism was isolated from the blood and placental tissue during the course of illness
and from postmortem blood and uterine.” Like the chimeric E. coli 0157:H7, this chimeric Salmonella would be blamed
on antibiotic use in agriculture in 2000.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC316096/pdf/jbacter00421-0307.pdf
In 1968, David Berkowitz, et al., Laboratory of Molecular Biology, National Institute of Arthritis and Metabolic Diseases at
Bethesda, reported on a “Procedure for Identifying Nonsense Mutations” in Salmonella. Using viruses, Berkowitz said,
“The mutant F'lac episomes were transferred from E. coli to S. typhimurium strains carrying suppressors which could
suppress the mutation in the introduced F'lac episome, thus enabling the Salmonella recipient to grow on lactose.
Donors and recipients were mixed on lactose agar, and lactose-positive colonies were selected. – A method has been
devised for the rapid identification of nonsense mutations (UAG, UAA, UGA codons) in Salmonella. The mutations to be
tested are reverted, and the revertants are replica-printed onto lactose plates spread with lawns of tester strains. These
tester strains contain F' lac episomes with nonsense mutations in the episomal Z gene. The revertants are infected with
the episome from the tester strain lawn. Because S. typhimurium is unable to ferment lactose, only those revertants
which have nonsense suppressors are able to grow on lactose. If colonies appear on the lactose plate, it may be
concluded that the original strain carries a nonsense mutation, since nonsense suppressors suppress the mutant
phenotype.” The mutants are classified as a lactose fermenting coliform.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC252275/pdf/jbacter00394-0251.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1983307/pdf/brmedj02032-0031.pdf
By 1970, Stanley N. Cohen and Christine A. Miller, Stanford University School of Medicine, reported on “Non-
Chromosomal Antibiotic Resistance in Bacteria, III : Isolation of the Discrete Transfer Unit of the R-Factor R1.” They
established that a plasmid (circle of DNA that is self-replicating) carrying the R factor had the ability to be transferred
horizontally cell-to-cell between bacteria. They found that E. coli with the plasmid that carries R1 factor appears to
express both resistance and transfer functions in that host. They also found that plasmids may carry either the transfer
factor or resistance factor as well and be transferred either individually or at the same time. http://thewatchers.
us/EPA/10/1970-Cohen-antibio-bact.pdf
In 1972, Arun K. Chatterjee and Mortimer P. Starr, University of California, Davis, reported on “Transfer Among Erwinia
spp. and Other Enterobacteria of Antibiotic Resistance Carried on R Factors.” They took on the task of transferring
antibiotic resistant from Escherichia coli B/r and Shigella flexneri la to Erwinia spp. They said, “The species of Erwinia,
besides causing diseases in plants, are known to occur as saprophytes in soil, on plant surfaces as epiphytes, and as
pathogens of fish and of insects. In recent years, members of the genus Erwinia have been implicated also in causing
pathological conditions in man and other animals. They said, “The transmission of antibiotic resistance from E. coli and
S. flexneri to Erwinia spp. and the ability of the antibiotic-resistant erwinias to transmit resistance determinants to Erwinia
herbicola, Escherichia coli, Shigella dysenteriae, and Salmonella typhimurium suggest a very grave potential threat to
the public health. E. herbicola, strains of which are known to be widely distributed in nature, including hospitals, and
which are clinically significant, appears unique in that it accepts genetic elements readily from other enterobacteria and
is capable of transmitting these elements not only to other Erwinia spp. but also to other enterobacteria.” http://www.ncbi.
nlm.nih.gov/pmc/articles/PMC251447/pdf/jbacter00354-0596.pdf
The genetic world was turned upside down in 1972 when Stanley N. Cohen, Annie C. Y. Chang, and Leslie Hsu,
Stanford University School of Medicine, reported on the “Nonchromosomal Antibiotic Resistance in Bacteria: Genetic
Transformation of Escherichia coli by R-Factor DNA* (CaCI2/extrachromosomal DNA/plasmid).” They used purified R-
factor DNA and Calcium chloride (salt of calcium and chlorine) to multiply antibiotic resistance in E. coli by
transformation (i.e., direct uptake of R carrying plasmids). They said, “we find that the introduced R-factor DNA can
persist in such cells as an independently replicating plasmid, and can express both the fertility and antibiotic resistance
functions of the parent R factor. – The number of recipient bacteria exhibiting antibiotic resistance increases about
1000-fold during subsequent incubation in antibiotic-free medium and reaches a maximum in 1 hr.” http://www.pnas.
org/content/69/8/2110.full.pdf
In 1973, Jorge Olarte, and Emma Galindo, Hospital Infantil De Mexico. Reported on “Salmonella typhi resistant to
Chloramphenicol, Ampicillin, and Other Antimicrobial Agents: Strains Isolated During an Extensive Typhoid Fever
Epidemic in Mexico.” They said, “During 1972 a large epidemic, in excess of 10,000 cases, of typhoid fever occurred in
Mexico City, Pachuca, and other communities of Mexico. The main characteristic of the epidemic, in addition to the large
number of persons affected, was the prevalence of a strain of Salmonella typhi which was highly resistant to
chloramphenicol both in vivo and in vitro, and which belonged to a single phage type, Vi degraded approaching type A.
Of 493 strains of S. typhi studied during the outbreak, 452 (91.7%) were resistant to chloramphenicol (CM), tetracycline
(TC), streptomycin (SM), and sulfonamides (SU). The epidemic strain owes its resistance to an R factor which is easily
transferable to Escherichia coli K-12 and which appears to be stable. In the third month of the outbreak, a strain of S.
typhi resistant to CM, TC, SM, SU, ampicillin (AM), and kanamycin (KM) was isolated from a patient with severe typhoid
fever. During the following 9 months, six additional strains of S. typhi resistant to AM, CM, TC, SM, and SU were also
isolated. Transfer experiments to E. coli K-12 indicate that these strains are infected with two different R factors, one
causing CM, TC, SM, and SU resistance and the other causing AM or AM and KM resistance. The frequency of transfer
of the resistance in overnight crosses was in the order of 10–4 for CM, TC, SM, and SU and 10–6 for AM or AM, and
KM.” http://www.ncbi.nlm.nih.gov/pmc/articles/PMC444603/pdf/aac00354-0009.pdf
In 1973, Raoul Benveniste and Julian Davies, University of Wisconsin-Madison, reported on “Aminoglycoside Antibiotic-
Inactivating Enzymes in Actinomycetes Similar to Those Present in Clinical Isolates of Antibiotic-Resistant Bacteria
(streptomyces/origin of R-factors/gentamicin-acetate).” They found that several species of Actinomycetes produced
antibiotic inactivating enzymes similar to those found in antibiotic resistant pathogens. The supposition was that if
Actinomycetes were the source of antibiotics, they might also be the source of the R factor necessary to deactivate the
antibiotics. They found that some species of Streptomyces produce antibiotics while other species of Streptomyces
produce R factor inactivating enzymes. Their conclusion was, “The discovery of aminoglycoside-modifying enzymes in
actinomycetes catalyzing the same reactions as those found in clinical isolates of other bacteria suggests an origin for
resistance determinants. – The actinomycetes may excrete antibiotics into the soil in order to complete effectively with
other soil microorganisms for nutrients, and it could be that some gram negative or gram-positive bacteria have
acquired the inactivating enzymes in order to protect themselves against these antibiotics. Hill has shown that penicillin
is produced in soils inoculated with a wild-type Penicillium chrysogenum, and that Bacillus cereus strains constitutive for
B-lactamase production have a clear survival advantage in this soil.”
http://thewatchers.us/EPA/5/1973-antibiotic-r.pdf
In 1973, Stanley N. Cohen, Annie C. Y. Chang, Herbert W. Boyer and Robert B. Helling, Stanford University School of
Medicine and University of California at San Francisco, reported on the “Construction of Biologically Functional Bacterial
Plasmids In Vitro (R factor/restriction enzyme/transformation/endonuclease/antibiotic resistance).” They said, “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.” According to the authors, “Controlled shearing of antibiotic
resistance (R) factor DNA leads to formation of plasmid DNA segments that can be taken up by appropriately treated
Escherichia coli cells and that recircularize to form new, autonomously replicating plasmids.” In fact, they were able to
construct a single plasmid from two different DNA species as well as a Plasmid from DNA fragments.”
http://www.pnas.org/content/70/11/3240.full.pdf
J. E. Beringer, Department of Genetics, John Innes Institute, reported on “R Factor Transfer in Rhizobiurn
Zegurninosarum.” Beringer studied the horizontal transfer of the R factor between E. coli and the symbiont pea bacteria
Rhizobium leguminosarum in 1974. He said, “These results show that R. leguminosarum, like many other Gram-negative
bacteria can act as a recipient or donor of P group R factors in mixed culture with other bacteria. The insensitivity of
this transfer to deoxyribonuclease, the wide range of R. leguminosarum strains that are able to act as recipients of R
factors, and the observation that transfer can occur both to and from E. coli, make conjugation the most likely mode of
transfer. Therefore, as was originally anticipated, R factor transfer experiments have provided a method for
demonstrating conjugation in R. leguminosarurn.”
http://thewatchers.us/EPA/2/1974-r-factor-transfer.pdf
In 1974, Stanley Cohen, Stanford University School of Medicine, and Herbert Boyer, University of California at San
Francisco, filed a patent application “Process for producing biologically functional molecular chimeras.” The patent
outline a way to create bacteria such as E. coli 0157 using calcium chloride as an agent for genetic engineering. -- The
application claims “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. The ability of genes
derived from totally different biological classes to replicate and be expressed in a particular microorganism permits the
attainment of interspecies genetic recombination. 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. They said, “The method provides a convenient and
efficient way to introduce genetic capability into microorganisms for the production of nucleic acids and proteins, such
as medically or commercially useful enzymes, which may have direct usefulness, or may find expression in the
production of drugs, such as hormones, antibiotics, or the like, fixation of nitrogen, fermentation, utilization of specific
feedstocks, or the like.” http://deadlydeceit.com/patent_E_coli.html
Also in 1974, Annie C. Y. Chang and Stanley Cohen reported on “Genome construction between bacterial species in
vitro: replication and expression of Staphylococcus plasmid genes in Escherichia coli.” They applied the patent process
to creating a hybrid plasmid by joining Staphylococcus plasmid DNA to an E. coli antibiotic-resistance plasmid then
inserting it into E. coli by direct uptake, thereby creating a clone bacteria that could not exist in nature. They said,
"Genes carried by EcoRI endonuclease-generated fragments of Staphylococcus plasmid DNA have been covalently
joined to the E. coli antibiotic-resistance plasmid pSC101, 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.”
http://www.ncbi.nlm.nih.gov/pubmed/4598290?dopt=Abstract&otool=stanford
In 1979, Yoshikatsu Murooka and Tokuya Harada, Osaka University, Suita-shi, reported on the “Expansion of the Host
Range of Coliphage P1 and Gene Transfer from Enteric Bacteria to Other Gram-Negative Bacteria.” They said,
“Recently, Goldberg et al. developed a method using PlclrlOOKM, which confers kanamycin resistance, for isolating
mutants sensitive to coliphage P1 from some strains of enteric bacteria. Using the same P1 phage and F' episomes, we
created intergeneric hybrid strains of enteric bacteria by transfer of the leucine and tyramine oxidase genes between
Klebsiella aerogenes, Escherichia coli, and Salmonella typhimurium. – The bacterial host range of coliphage P1 was
extended by using the heat inducible phage PlclrlOOKM. A gene for kanamycin resistance was transferred from
Escherichia coli to members of the family Enterobacteriaceae and some other genera of gram-negative bacteria. –
Most of the bacterial strains used here are important in agriculture or in microbial industry: K. aerogenes W70 produces
special enzymes, arylsulfatase (18), histidase (16), pentitol-degradating enzymes (17), or pullulanase (2, 11); K.
pneumoniae ATCC 8329 has nitrogen fixation genes (Y. Murooka, unpublished data); a strain of S. marcescens is used
to produce amino acids (10); A. faecalis var. myxogenes. 10C3K produces the gel-forming /B-1,3-glucan, curdlan (6); A.
tumefaciens induces crown gall tumors in dicotyledonous plants (3); Flavobacterium sp. M64 forms succinoglucan
depolymerase (1); and P. amyloderamosa SB15 produces isoamylase, which in combination with ,B-amylase is used to
make maltose (8)”. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC243574/pdf/aem00203-0198.pdf
Also in 1979, Joe D. Cohen, et al., Albert Einstein College of Medicine, reported the “Functional expression in yeast of
the Escherichia coli plasmid gene coding for chloramphenicol acetyltransferase (chloramphenicol
resistance/Saccharomyces cerevisiae transformation/heterologous gene expression).” They said, “The Escherichia coli
R factor-derived chloramphenicol resistance (camr) gene is functionally expressed in the yeast Saccharomyces
cerevisiae. The gene was introduced by transformation into yeast cells as part of a chimeric plasmid, pYTII-LEU2,
constructed in vitro.” They said, “Recombinant Plasmids Used in This Work. Plasmid pYTI 1-LEU2 is a composite vector
consisting of the bacterial plasmid pBR325 (13), the yeast 2-nm DNA plasmid (14), and a fragment of DNA containing
the yeast LEU2 structural gene derived from plasmid pYeleulO.” Moreover, “By selecting for growth on high levels of
chloramphenicol, a number of mutants have already been isolated in which acetyltransferase [resistance] activity is
increased 20- to 40 fold over the levels observed in the primary "wild type" transformants.” http://www.pnas.
org/content/77/2/1078.full.pdf
In 1985, Haruo Watanabe, Institute of Public Health, and Akiko Nakamura, National Institute of Health, Tokyo, reported
“Large Plasmids Associated with Virulence in Shigella Species Have a Common Function Necessary for Epithelial Cell
Penetration.” They said, “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. – Plasmids pSS120, pSF140, and pSD140 were sufficient to
allow E. coli K-12 to invade culture cells in vitro but not to provoke keratoconjunctivitis in guinea pigs. – Our results
clearly demonstrated that plasmid pSS120 of S. sonnei encodes determinants necessary for invasion of culture cells
and that they function in E. coli, S. sonnei, and S. flexneri.”
http://thewatchers.us/EPA/11/1985-shigella-e-coli.pdf
In 1987, Charles J.Thompson, and International associates, reported on the “Characterization of the herbicide-
resistance gene bar from Streptomyces hygroscopicus.” They said, “A gene which confers resistance to the herbicide
bialaphos (bar) has been characterized. – Bialaphos is now being used in agriculture as a non-selective herbicide. –
The bar gene was originally cloned from Streptomyces hygroscopicus, an organism which produces the tripeptide
bialaphos as a secondary metabolite. – Interspecific transfer of this Streptomyces gene into Escherichia coli showed
that it could be used as a selectable marker in other bacteria.. – Streptomyces which produce antibotics have evolved
mechanisms to avoid the toxicity of their own products. Such strains often contain modifying enzymes which can
inactivate the antibiotics they produce. – Since Streptomyces spp. produce hundreds of different antibiotics their
resistance genes have been a rich source of selectable markers for the construction of both bacterial and animal cell
vectors. – In the accompanying paper, bar has been used to engineer herbicide-resistant plants.”
http://thewatchers.us/EPA/10/1987-antibio-herbicide.pdf
In 1994, Patrice Courvalin, University of Califomia at San Diego, reported on the “Transfer of Antibiotic Resistance
Genes between Gram-Positive and Gram-Negative Bacteria.” Courvalin said, “Four major mechanisms account for the
evolution of bacterial resistance to antibiotics that correlates with the use of the drugs: (i) emergence of "new"
opportunistic pathogenic soil microorganisms that are often multiresistant to antibiotics (e.g., Acinetobacter spp.); (ii)
emergence of "new" acquired resistance mechanisms (e.g., glycopeptide resistance in enterococci); (iii) occurrence of
mutations in genes located in the host chromosome (e.g., encoding DNA gyrase) or plasmid borne (e.g., for extended-
spectrum ,B-lactamases); and (iv) spread of "old" (i.e., already known) resistance genes into "new" bacterial hosts (i.e.,
genera or species that were previously uniformly susceptible). The last mechanism has been known since the early
finding that antibiotic resistance genes are often part of self-transferable plasmids or of transposable elements.
However, and until recently, it was thought that this type of genetic transfer only occurred between closely related
bacteria (1, 24). This review will focus on two recent notions: (i) the transfer of antibiotic resistance genes in natural
environments can occur between phylogenetically distant bacterial genera, in particular between gram-positive and
gram-negative bacteria; and (ii) conjugation is a mechanism of transfer of genetic information with a very broad host
range.” http://www.ncbi.nlm.nih.gov/pmc/articles/PMC284573/pdf/aac00371-0003.pdf
In 1994, Hilde Kruse and Henning Sorum, Norwegian College of Veterinary Medicine at Oslo, reported on the “Transfer
of Multiple Drug Resistance Plasmids between Bacteria of Diverse Origins in Natural Microenvironments”. They found,
“Plasmids harboring multiple antimicrobial-resistance determinants (R plasmids) were transferred in simulated natural
microenvironments from various bacterial pathogens of human, animal, or fish origin to susceptible strains isolated from
a different ecological niche. R plasmids in a strain of the human pathogen Vrbrio cholerae 01 El Tor and a bovine
Escherichia coli strain were conjugated to a susceptible strain of the fish pathogenic bacterium Aeromonas salmonicida
subsp. salmonicida in marine water. Conjugations of R plasmids between a resistant bovine pathogenic E. coli strain
and a susceptible E. coli strain of human origin were performed on a hand towel contaminated with milk from a cow with
mastitis. A similar conjugation event between a resistant porcine pathogenic E. coli strain and a susceptible E. coli strain
of human origin was studied in minced meat on a cutting board. Conjugation of R plasmids between a resistant strain of
the fish pathogenic bacterium A. salmonicida subsp. salmonicida and a susceptible E. coli strain of human origin was
performed in raw salmon on a cutting board. R plasmids in a strain of A. salmonicida subsp. salmonicida and a human
pathogenic E. coli strain were conjugated to a susceptible porcine E. coli strain in porcine feces. Transfer of the
different R plasmids was confirmed by plasmid profile analyses and determination of the resistance pattern of the
transconjugants. The different R plasmids were transferred equally well under simulated natural conditions and under
controlled laboratory conditions, with median conjugation frequencies ranging from 3 x 10-6 to 8 x 10-3. The present
study demonstrates that conjugation and transfer of R plasmids is a phenomenon that belongs to the environment and
can occur between bacterial strains of human, animal, and fish origins that are unrelated either evolutionarily or
ecologically, even in the absence of antibiotics. Consequently, the contamination of the environment with bacterial
pathogens resistant to antimicrobial agents is a real threat not only as a source of disease but also as a source from
which R plasmids can easily spread to other pathogens of diverse origins.” Antibiotic resistant organisms are released
to and from sewage treatment plants
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC201930/pdf/aem00028-0125.pdf
In 1994, M. Steinmetz and R. Richter, Laboratoire de Génétique des Microorganismes, Centre National de la
Recherche Scientifique at Thiverval-Grignon, said they have “Plasmids designed to alter the antibiotic resistance
expressed by insertion mutations in Bacillus subtilis, through in vivo recombination.” As an example, “Numerous
insertion mutations conferring resistance to antibiotics are available in Bacillus subtilis. However, many of them have
been constructed in vitro by inserting genes from the Staphylococcus aureus plasmids, pC194 or pE194, conferring
resistance to chloramphenicol (Cm) or erythromycin (Er). Others are insertions of the Enterococcus faecalis Tn917
transposon conferring resistance to Er. This paucity of resistance markers has been limiting the possibilities of
constructing and studying mutants carrying two or more of these mutations in the past. We constructed plasmids which
can be used to change the antibiotic resistance expressed by preexisting chromosomal insertions, through
transformation and homologous recombination. These vectors replace the pre-existing resistance to Cm or Er with new
resistances to neomycin (Nm), phleomycin (Pm), spectinomycin (Sp) or tetracycline (Tc).” http://www.ncbi.nlm.nih.
gov/pubmed/8181761
In 2001, Steve A. Carlson, et al., USDA, reported on “Antibiotic Resistance in Salmonella enterica Serovar Typhimurium
Exposed to Microcin-Producing Escherichia coli.” They said, “Microcin 24 is an antimicrobial peptide secreted by
uropathogenic Escherichia coli. Secretion of microcin 24 provides an antibacterial defense mechanism for E. coli. In a
plasmid-based system using transformed Salmonella enterica, we found that resistance to microcin 24 could be seen in
concert with a multiple-antibiotic resistance phenotype. This multidrug-resistant phenotype appeared when Salmonella
was exposed to an E. coli strain expressing microcin 24. Therefore, it appears that multidrug-resistant Salmonella can
arise as a result of an insult from other pathogenic bacteria. – A previous study demonstrated that Mcc24 has activity
against Salmonella enterica and most E. coli strains but not against Campylobacter or Listeria strains (18). We also
found that Mcc24 does not have activity against multiple-antibiotic-resistant Klebsiella pneumoniae (ATCC MCV37)
(data not shown).” http://www.ncbi.nlm.nih.gov/pmc/articles/PMC93088/
In 2010, Alison E. Barnhill, et al., Iowa State University, Ames, reported on the “Identification of
Multiresistant Salmonella Isolates Capable of Subsisting on Antibiotics.” They said, “A recent study found
that a diverse group of bacteria, including intrinsically resistant microbes (e.g., Pseudomonadales
and Burkholderiales), could subsist on and presumably catabolize antibiotics as a sole carbon source. This
group includes bacteria related to the pathogens Burkholderia cepacia and Serratia marcesens (5). This
phenotype has also previously been reported for catabolism of chloramphenicol by Streptomyces
(1) and beta-lactams by Leptospira (8) and Pseudomonas (7). – This study assessed the ability of Salmonella
(572 isolates) to subsist on 12 different antibiotics. The majority (11/12) of the antibiotics enabled
subsistence for at least 1 of 140 isolates. Furthermore, 40 isolates were able to subsist on more than one
antibiotic. Antibiotic resistance and antibiotic subsistence do not appear to be equivalent.”
http://aem.asm.org/cgi/reprint/76/8/2678
Antibiotic resistant bacteria don't stay in the soil. They move out of the soil in stormwater runoff, on dust,
on plants, on/in animals, birds, pests and humans where they meet and mate with pathogenic strains.
Eventually, they make their way into hospitals in food and water or on plants in pathogen contaminated
potting soil and pick up more resistant genes. Then the cycle starts all over with more and stronger
bacteria making their way back to the agricultural soil or your lawn and garden.
Next Antibiotic Resistance in Hospitals
Back to Myth # 4