A PERFECT STORM OF ANTIBIOTIC RESISTANCE
section 1
Basic Antibiotic Resistance History 9/09/2011
Back to Myth # 4
The difference between an antibiotic and a drug is that anti-biotics are self-defense toxins produced by soil bacteria and
fungi to kill antagonistic organisms, while a drug is a man made synthetic toxin used to kill toxin producing organisms
that invade the body. Bacteria and fungi also have DNA (genes) that make them resistance to those toxins (anti-biotic).
Our troubles really got to rolling down hill when the researchers learned to insert antibiotic resistant marker genes in
genetically modified organisms (GMOs).
The toxin resistance genes produced by nature may be passed on to other bacteria through natural Conjugation (direct
horizontal gene transfer between bacteria), Transduction (bacterial infection by a phage (virus) that has incorporated
the resistant gene) or Transformation (bacteria pick up the gene directly from the environmental remains of dead
bacteria). Transformation was first discovered in 1928 by British medical officer Fred Griffith. By 1948, researchers had
discovered methods for horizontal gene transfer as well as viral transfer of genetic material. In the 1970s, researchers
discovered methods to use Calcium chloride to directly insert genetic material, including an antibiotic resistant gene into
bacteria to confirm the transformation into a new Chimera that could not normally exist in nature, such as E. coli 0157:
H7, which contains a Central American Shigella gene. Herbert Boyer and Stanley Cohen reported in 1973 that they had
used Calcium chloride to successfully insert salmonella and staphylococcus genes into E. coli making it resistant to
streptomycin and several other antibiotics. The patent application for the process was filed in 1974, the first E. coli 0157:
H7 infection was documented in 1975, and the patent was issued to Boyer and Cohen in 1980, who assigned it to
Stanford University. This opened the field of genetic engineering and the construction of specialized bacteria that could
not exist in nature to create new chemicals and medicines such as insulin.
Resistant organisms have been with us since the first bacteria was exposed to the toxin (anti-biotic) of an adversarial
bacteria or a toxic metal that could damage its cells. The common denominator for all resistant studies are that
researchers have stressed bacteria with chlorine and other disinfectants, sulfur drugs, x-radiation, ultraviolet light,
nitrogen mustard, metals and antibiotics to isolate mutant strains that survive the experience. In the process more
dangerous resistant clones are created, and released to the environment through treated sewage effluent and sludge
because they were once thought to be harmless with a low probability of survival.
Herbert W. Boyer, Ph.D, explained the scientific thinking concerning the disposal of bacterial experiments in sewers in
1994 interviews for the oral history “Recombinant DNA Research at UCSF and Commercial Application at Genentech.”
Boyer said, “At that time, we were facing up to a general awareness in the scientific community about the "hazards" of
this type of research. People were concerned that maybe we would put the wrong kind of gene into a bacterium if we
tried using eukaryotic [ animals, plants and fungi] DNA. In particular, if we carried out recombination between a bacterial
plasmid and an animal virus that caused cancers in various laboratory animals, they worried that we might end up with a
sewer full of bacteria carrying genes that would cause cancer, and that sort of thing. – I think it was part of the rhetoric,
part of the concern. There were some microbiologists who probably were overly concerned about putting E. coli into the
sewer, which would be like peeing in the ocean, as some say. When I was a graduate student, postdoc, I used to grow
ten liters of bacteria and put the effluent down the drain. And you would sonicate bacteria in the laboratory without even
taking great precautions.”
http://content.cdlib.org/ark:/13030/kt5d5nb0zs/
We now know that peeing in the ocean is not as benign an act as Boyer thought. Ships are now required to have waste
treatment plants on board because the ocean environment can be destroyed if enough people pee in it. Congress
recognized the problem with the “Marine Protection, Research and Sanctuaries Act of 1972 (Public Law 92-532).” in
1988, Congress banned ocean-dumping of sewage sludge and industrial waste because pathogens and chemicals were
destroying the ocean environment. Authors S.M. Goyal, University of Minnesota at St. Paul and C. Gerba, University of
Arizona at Tucson, explained some of the reasons for the ban in the EPA document “Qualitative Pathogen Risk
Assessment for Ocean Disposal of Municipal Sludge (EPA/600/6-88/010 ... May 1986).” The last frontier with no laws to
prevent environmental damage from sludge dumping was agricultural land and your lawn.
Boyer and Cohen's work led to programs such as University of Arizona's “BIOTECH Project” run by Dr. Nadja Anderson.
The BIOTECH Project supports Biotechnology/Molecular Biology activities in middle school and high school classrooms.
One of the projects is “Bacterial Transformation by Mystery DNA.” According to the Teacher's Guide, Bacterial
Transformation is now a very simple process in which students “'poke holes' in the bacteria using chemicals, allowing the
DNA to flow into bacteria.” As the Guide explains, DNA is shaped in a little circle, called a plasmid, into which several
genes can be inserted. In this case one of the plasmid genes codes for fluorescent and a second codes for ampicillin
resistance, which will allow the chimera bacteria to grow on the antibiotic. Ampicillin is unique in that it does not kill
bacteria, it just keeps them from producing cell walls. Calcium chloride is used to 'poke holes' in cooled bacteria as well
as cancel the negative charge in the plasmid. The experiment is then heat shocked at 42°C (107.6°F) for less than a
minute to seal the holes. E. coli is then incubated at 37°C (98.6°F) for 24 hours to reveal a lawn of antibiotic resistant
colonies with a green fluorescent glow under Ultraviolet light. http://biotech.biology.arizona.edu/labs/transformation(TG).
html
While unique forms of plasmids and viruses can be constructed in laboratories to create bacteria that could not exist in
nature by Conjugation, Transformation or Transduction, nature can do the same thing. In fact, “BioBricks,” with unusual
characteristics can be purchased from commercial laboratory supply houses to construct an even more dangerous
bacteria such as the Shigella Toxin E. coli (STEC) 0104:H4 found in the recent German foodborne outbreak. Prior to the
early genetic engineering experiments by researchers, E. coli and Klebsiella were fairly benign bacteria only associated
with hospital infections/deaths as were most of the coliform strains incorporated into STEC 0104:H4. There is no doubt
E. coli STEC 0104:H4 is a monstrosity that could not have existed in nature.
The German foodborne outbreak caused by E. coli strain STEC O104:H4, infected over 4,000 people, caused kidney
complications in 851 and killed 51 people. The supposition is that the outbreak was caused by Egyptian sprout seeds
which makes it a true international bacteria composed of genes from the following members of the gram negative
Enterobacteriacea (coliform) family. There were genes from about 112 different strains of E. coli in the make up. Major
gene contributors were E. coli laboratory strain K 12 (Stanford 1922), E. coli O26:H11 (strain 11368/EHEC--Japan
2001), E. coli (strain 55989 / EAEC--Central African Republic 2002), E. coli O44:H18 (strain 042/EAEC--Lima, Peru in
1983), E. coli O103:H2 (strain 12009 / EHEC--Japan 2001), E. coli O111:H- (strain 11128 / EHEC--Japan 2001), E. coli
O157:H7 (strain EC4115 / EHEC (?)), E. coli O157:H7 (strain TW14359 / EHEC--Michigan USA 2006).
Minor gene contributors to E. coli O104:H4 were the Enterobacteriacea (coliforms), Salmonella typhi, Serratia
marcescens , Shigella boydii , Shigella dysenteriae, Shigella flexneri, Yersinia pestis (Black Plague), Citrobacter koseri,
Klebsiella pneumoniae, Pantoea sp., Proteus mirabilis, and other non-coliform bacteria including Bacillus cereus,
Haemophilus ducreyi, Vibrio cholerae
Antibiotic drug resistance genes include: beta-lactamic, aminoglycoside, macrolide, polymyxin, tetracycline, fosfomycin
and deoxycholate, novobiocin, chloramphenicol, bicyclomycin, norfloxacin and enoxacin and 6-mercaptopurine. Metal
resistant genes included those for tellurium, mercury, nickel, copper, zinc and cobalt. http://blog.ohnosequences.
com/2011/06/275/
Now a new antibiotic from gram positive bacteria,“bisin”, on the horizon in the group known as “Lantibiotics” is causing a
media feeding frenzy. The intent is to market 'bisin' as a natural preservative rather than an antibiotic. One headline
states “Lucky accident slashes food poisonings.” Dennis Avery, senior fellow with the Hudson Institute in Washington,
Director for Global Food Issues cgfi.org and formerly a senior analyst for the Department of State, claims “A new natural
food additive, discovered in a laboratory accident, is now ready to slash by half the number of hospitalizations and
deaths from food-borne bacterial poisoning across the Western World.” http://canadafreepress.com/index.
php/article/39467
AOL's Huff Post Food section headline. “Bisin, New 'Natural' Preservative, Could Extend Shelf Life Of Meat, Dairy For
Years.” http://www.huffingtonpost.com/2011/08/15/bisin-new-preservative_n_927174.html
Xanthe Clay, food columnist for the Daily Telegraph, has a different take with her headline, “Bisin might make burgers
that never go off, but it doesn't make them fresh.” http://blogs.telegraph.co.uk/culture/xantheclay/100055442/bisin-might-
make-burgers-that-never-go-off-but-it-doesn't-make-them-fresh/
All the hype is over an August 2011 press release from the University of Minnesota which didn't promise much, and
was not completely factual, in the statement that it has “received a patent for a naturally occurring lantibiotic—a peptide
produced by a harmless bacteria—that could be added to food to kill harmful bacteria like Salmonella, E. coli, and
listeria. The lantibiotic is the first natural preservative found to kill gram-negative bacteria, typically the harmful kind. “It’s
aimed at protecting foods from a broad range of bugs that cause disease,” said Dan O’Sullivan, Professor of Food
Science and Nutrition, University of Minnesota. “Of the natural preservatives, it has a broader umbrella of bugs that it
can protect against.” – The lantibiotic could be used to prevent harmful bacteria in meats, processed cheeses, egg and
dairy products, canned foods, seafood, salad dressing, fermented beverages, and many other foods. In addition to food
safety benefits, lantibiotics are easy to digest, nontoxic, do not induce allergies, and are difficult for dangerous bacteria
to develop resistance against.” http://fscn.cfans.umn.
edu/newsandevents/2011NewsEvents/Researchers_discover_natural_preservative_that_kills_foodborne_bacteria/index.
htm
According to the Van der Donk Group at the University of Illinois-Urbana/Champaign, “The van der Donk group focuses
on the mode of action and mechanism of biosynthesis of two classes of antibiotics that have been underexplored but
have great potential for human therapeutic use, lantibiotics and phosphonate antibiotics (J. Org. Chem. 2006 71, 9561-
9571.). – Lantibiotics are ribosomally synthesized peptide antibacterial agents. After ribosomal synthesis the peptides
are modified to their bioactive forms by multi-enzyme complexes. – We are interested in the mechanism of lantibiotics for
a variety of reasons. First, the enzymes that produce lantibiotics in nature are amazingly impressive catalysts. One of
the enzymes studied in the laboratory breaks 14 chemical bonds and forms 10 new chemical bonds with defined stereo-,
regio-, and chemoselectivity! Despite this fantastic control over chemical reactivity, we have shown that the biosynthetic
enzymes are remarkably promiscuous with respect to their substrate specificity. This opens the intriguing perspective
that these enzymes can be used for re-engineering the structures of naturally occurring lantibiotics to improve their
properties with respect to human therapeutic use. Another reason for our interest in lantibiotics is the unusual
observation that the only commercially used lantibiotic, nisin, has been used for over 40 years in more than 80 countries
without widespread occurrence of bacterial resistance.”
http://vanderdonk.scs.uiuc.edu/van_der_Donk/lantibiotics.html
However, bacterial resistance was noted in the 1999 paper “Lantibiotics: biosynthesis, mode of action and applications”,
by Cindy van Kraaij, et al., NIZO Food Research, The Netherlands. They said, “Just before the discovery of penicillin by
Fleming, reports appeared in the literature that described potent antimicrobial substances produced by lactic acid
bacteria. In those days the nature of the inhibitory compound was not yet elucidated, but in retrospect it seems likely
that the inhibitory activity was caused by the production of the special class of peptides that forms the subject of this
review. It was found that many Gram-positive bacteria secrete compounds [e.g., nisin and subtilin] which are specifically
active against a wide range of other Gram-positive bacteria. This characteristic made these compounds attractive
candidates for application in either food preservation, e.g. by preventing spoilage or by inhibiting pathogens, or for
pharmaceutical use, e.g. to prevent or fight infections in humans or animals. – the first applications of nisin were
ventured in clinical and veterinary therapies. However, the intravenous use of nisin has not been further developed
since nisin shows a low stability at physiological pH. However, several protein-engineered derivatives of nisin Z have
been generated in recent years that show improved stability and these or others may extend the medical application of
nisin. Various alternative pharmaceutical applications of nisin have been considered. One that builds on the high activity
at low pH relates to the use of nisin to antagonize Helicobacter pylori which is the causative agent of gastric ulcers.138
Since this is a high value market, it is feasible that the development of specific nisin species that have been improved by
protein engineering could be rewarding. The same holds for the use of nisin to inhibit growth of multidrug resistant
pathogens. In a recent survey, the capacity of nisin to prevent growth of resistant Staphylococcus aureus or
Streptococcus pneumoniae was found to be promising, although resistance development was observed. Another
application is the use of nisin as a sanitizer against mastitis pathogens in cows that also include Streptococcus and
Staphylococcus spp. Finally, the use of nisin in personal-care products such as mouth wash or deodorants has also
been suggested.” http://gbb.eldoc.ub.rug.nl/FILES/root/1999/NatProdRepvKraaij/1999NatProdRepvKraaij.pdf
In 2010, Lee, Hyungjae and Hae-Yeong Kim, Kyung Hee University at Yongin, reported on “Lantibiotics, Class I
Bacteriocins from the Genus Bacillus.” They said, “Antimicrobial peptides exhibit high levels of antimicrobial activity
against a broad range of spoilage and pathogenic microorganisms. Compared with bacteriocins produced by lactic acid
bacteria, antimicrobial peptides from the genus Bacillus have been relatively less recognized despite their broad
antimicrobial spectra. These peptides can be classified into two different groups based on whether they are ribosomally
(bacteriocins) or nonribosomally (polymyxins and iturins) synthesized. Because of their broad spectra
and high activity, antimicrobial peptides from Bacillus spp. may have great potential for applications in the food,
agricultural, and pharmaceutical industries to prevent or control spoilage and pathogenic microorganisms. In this review,
we introduce ribosomally synthesized antimicrobial peptides, the lantibiotic bacteriocins produced by members of
Bacillus. In addition, the biosynthesis, genetic organization, mode of action, and regulation of subtilin, a well-investigated
lantibiotic from Bacillus subtilis, are discussed.” http://210.101.116.28/W_kiss2/05211540_pv.pdf
You might wonder if the politicians believe the scientific and media hype. After 30 years of government funding public
relations programs, research by engineers and soil scientists promoting the spreading of antibiotic resistant organisms
in sewage sludge on agricultural grazing land, food crops, parks, school grounds, home lawns and gardens on June 17,
2011, Senator Diane Feinstein (D-CA) introduced the Preservation of Antibiotics for Medical Treatment Act (PAMTA), S.
1211. The Bill's aim is to phase out the non-therapeutic use of medically important antibiotics in the livestock industry.
This will do little to protect agricultural products, water, public health or stop antibiotic resistance and drug resistance.
Feinstein's proposed resolution is an evolutionary political solution to a larger municipal and industry sewage disposal
liability problem that has led to drug and antibiotic resistance in the community. In the beginning, sewage was dumped in
the streets causing pathogen contaminated public drinking water. The politic solution was to create sewage treatment
farms which raised liability concerns for municipalities. This was followed by municipalities and industry releasing
pathogen and toxic contaminate raw sewage into surface waters which resulted in burning rivers such as the Cuyahoga
River in Ohio. The political solution was to create sewage treatment plants using bacteria and fungi to decompose
organic waste, which still released some pathogens and toxic chemicals to surface waters and created an even bigger
problem with tons of concentrated pathogens and toxic chemicals. The political solution for large Coastal municipalities
was to dump the sludge into the Ocean where toxic chemicals and pathogens destroyed the Ocean environment. The
political solution to that problem was to move sludge dumping from the Ocean to private agricultural grazing land which
resulted in the spread of antibiotic resistant pathogens among farm animals. Then fruits and vegetables from cropland
were included where there are no laws to prevent pathogen contaminated runoff from polluting surface water or food
products. The political solution was then expanded to sell or give away unlabeled pathogen contaminated sludge and
reclaimed water to schools and home owners which resulted in the spread of antibiotic resistant pathogens into the
community. Now, rather than attempt to solve the major problem of releasing antibiotic resistant pathogens into the
community, the political solution is to restrict the use of antibiotics by doctors and farmers. Researchers and politicians
have known the problem was developing, yet refused to address it.
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