There is no place on the earth surface where potentially dangerous drug resistant bacteria have not reached. They have infiltrated even into wilderness of virgin and barren islands including the Arctic region. After discovery of antimicrobial agents in the first half of 20th century, clinicians felt relieved as these wonder drugs substantially reduced the threat of infectious diseases. Over the years, antimicrobials have saved lives, eased the suffering of millions of people. And have contributed to the major gains in life expectancy (WHO 2000, 2005). However, these wonder drugs have started to loose ground rapidly. With each application of antibiotic to kill bacteria a new micro environment is created where the sensitive microbes get killed but the resistant organisms start to flourish. New selection pressure each time leads to rapid evolution in bacteria. As a result, now almost all important infection causing bacteria are armoured to survive in antibiotic loaded environment with much deadlier infective power.

1. Antimicrobial drug use and its
implications
The term antibiotic, meaning “substance against life," thus
“Antibiotic resistance means life”.
Antibiotic therapy, if indiscriminately used, may turn out to be a
medicinal flood that temporarily cleans and heals, but ultimately
destroys life itself (Felix Marti-Ibanez, 1955)
Bhoj R Singh
Section of Epidemiology, CADRAD, IVRI, Izatnagar

2. Bacteria
 Most numerous denizens of agriculture, with
populations ranging from 100 million to 3 billion in a
gram of soil.
 One bacterium is capable of producing 16 million
more in just 24 hours.
 Bacteria perform chemical transformations, including
degradation, disease suppression, disease, and
nutrient transformations.
 Cloud formation in Troposphere (NASA’s
Genesis and Rapid Intensification Processes
experiment).
 Generation of electricity: Using microbial fuel cell Bacillus
stratosphericus generates twice as much electricity as other
bacteria.

3. Bacteria
 I always thought the most significant thing that we ever found on the whole
goddamn Moon was that little bacteria who came back and lived and nobody
ever said shit about it. — Pete Conrad
 On April 20, 1967, unmanned lunar lander Surveyor 3, left a camera on
Moon. Two-and-a-half years later, on November 20, 1969, Apollo 12
astronauts Pete Conrad and Alan L. Bean recovered the camera. NASA
scientists examined it back and find still alive Streptococcus mitis, survived
for 31 months in the vacuum of the moon's atmosphere.
 Zero gravity: “Generally speaking, bacteria tend to grow better in
space,” Klaus (2004) said. “The first and most fundamental b
 Bacteria found in rocks taken from the cliffs at Beer have survived a
grueling year-and-a-half exposure to space conditions on the exterior
of the ISS and returned home alive, becoming the longest-lived
photosynthesizing microbes to survive in space.acterial response to
spaceflight is a shortened lag phase.” The bacteria sent was: OU-20
resembling the cyanobacteria genus Gloeocapsa

4. Bacteria in our body
 We are 90% bacteria and only 10% human(approximately 1014
versus 1013
). Sears,
2005.
 500 to 1000 species of bacteria live in the human gut and a roughly similar number on
the skin. Science, 2008; Grice et al., 2009.
 The human gut alone contains on average: 40,000 bacterial species 9 million unique
bacterial genes and 100 trillion microbial cells and only 1% of them are characterized
(Yang et al., 2009).
 332,000 genetically distinct bacteria belonging to 4,742 different species were
detected on hands of students using molecular techniques. Fierer et al., 2008.
 The mass of microorganisms are estimated to account for 1-3% total body mass. 1.0
to 2.26 kilograms of live bacteria inside our bodies (Berg, 1996; Raymond, 2012).
 Bacterial makeup of our body change little over time (National Human Genome
Research Institute in Bethesda, Maryland).
 Infections increase from overuse of antibiotics (Human Microbiome Project; HMP).
 They help in development of our immune system and how we fight off pathogens, they
influence our metabolism, our odor and even our behavior. Eisen, 2012.

5. How we get them?
 Any two hands – even belonging to the same person – had only 13%
of their bacterial species in common (Fierer et. al., 2008). It is due to
the influence of sex, handedness, and washing habits but relatively
unrelated with type of food intake.
 We get them from:
 From mothers while come to this world.
 From Air, Food, Water, Environment and friends and family.
 How we can get healthy bacteria?
 From healthy donors
 From excrements of the healthy persons
 Poop transplant (the best way to fight enteritis necroticans, Cl.
difficile)

6. Where bacteria are absent?
 Where there is no life?
 NASA’s Clean room: 100 types of bacteria, about 45 percent of
which were previously unknown to scientists.
 Two miles below the surface of a Greenland glacier – “ultrasmall
bacteria” in a glacial core – a habitat which is low-temperature, high-pressure,
reduced-oxygen, and nutrient-poor. The core was estimated to be 120,000 ye
 Deepest layer of the earth's crust –the Earth's oceanic crust has revealed a
new ecosystem living over a kilometer beneath our feet.
 In tissue sites once deemed sterile – The Relman Lab at Stanford used
real time PCR to target conserved regions of the bacterial 16S ribosomal
DNA (rDNA). They concluded that there is a substantial and “normal”
population of bacterial DNA sequences in the blood of even healthy
individuals.

7. Why bacteria persist so diversely?
 Need very few genes to persist –
Encephalitozoon cuniculi (a pathogen of
rabbits) has 2.9Mbp, encoding approximately
2,000 densely packed genes. Genome
Encephalitozoon intestinalis, at 2.3Mbp, a
severely compacted genome.
 Can take and give genes rapidly.
 Can survive anywhere at any temperature
(even in boiling springs), eat anything, breath
any air.

8. AMDR is Natural?
 Call of the wild: antibiotic resistance genes in natural
environments (Allen et al., 2010).
 Antibiotic resistance genes are detected in ‘antibiotic-
naïve’ strains isolated in pre-antibiotic era (before
1930).
 24% strains of Murray collection (1917-1952) are
resistant to ampicillin and tetracycline.
 Bacteria that may grow on antibiotics as the sole
carbon and nitrogen sources include: Pseudomonas
fluorescens (on streptomycin and penicillin); Burkholderia cepacia (on
Penicillin); Flavobacterium spp., Streptomyces venezuelae (on
chloramphenicol), actinomycetes cultured from forest (on telithromycin)

9. Use of anti-microbials in Nature
 Antibiotics have been produced for over 500 million years, dating
back to the Cambrian period and the emergence of vertebrate fish.
Antibiotic-like molecules, are likely to be even older than this.
 The non-protein amino acids that are found as components of
peptide antibiotics have been detected in meteorites and other
primordial sources (Davies, 2009).
 The fungus-growing ant system, in which ants carry an antibiotic-
producing actinomycete (a Pseudonocardia sp.) on their cuticle
and use this bacterium specifically for biocontrol of the fungal
garden parasite, Escovopsis sp. (Currie et al., 1999; Cafaro and
Currie, 2005)
 Biocontrol of the causative agent of potato scab, Streptomyces
scabies str. RB4, by the antibiotic-producing suppressive strain,
Streptomyces diastatochromogenes str. (Neeno-Eckwall et al.,
2001)
 Some autoinducers used in quorum sensing have antibiotic
activity (Kravchenko et al., 2008).
 Bacteria as natural pesticides in the biological pest control
(Bacillus thuringiensis) (Chattopadhyay et al., 2004)

10. Antimicrobials used by human
 We hardly know about 1% of antibacterial molecules in nature. Most of
these molecules cannot be detected, and the few that have been identified
some of them are the most used antibiotics (Baltz, 2008).
 Of the known antibiotics, < 1% of antimicrobial agents have medical
value (Marino, 2007).
 Antibiosis was first described in 1877 in bacteria when Louis Pasteur and
Robert Koch observed that an airborne bacillus could inhibit the growth
of Bacillus anthracis.
 Salvarsan Discovered by Paul Ehrlich was produced in 1932 at the Bayer
Laboratories and was used for treatment of syphilis.
 Alexander Fleming: Discovered penicillin in 1928, could not purify.
 In 1939, gramicidin from B. brevis. was one of the first commercially
manufactured antibiotics (Sykes, 2001).
 Ernst Chain and Howard Florey successfully purified penicillin, and in
1941 tested on human subjects.
 The term antibiotic was coined by Selman Waksman in 1942.

11. Use of Antimicrobials for Animals
 Antibiotics are used in food-producing animals for three major
reasons (CDC, 2009).
 A. in high doses for short periods to treat sick animals.
 B. in high doses for short periods to prevent diseases (after
weaning, or during transport). This use “usually involves
treating a whole herd or flock, which increases the likelihood
of selecting for organisms that are resistant to the antibiotic.”
 C. antibiotics are commonly given in the feed at low doses
for long periods to promote the growth of cattle, poultry, and
swine (How? No one knows exactly, 1-15% Improvement in
production, poorer the hygiene better the effect).
 A=17%, B+C=83% (About ten million Kg alone in USA, i.e., 70%
of total antibiotic production): AHI, 2009, Union of Concerned
Scientists, 2009.
 A+B+C=87%, 40% of it is added in to feed (APHA, 2009)

12. Use of Antimicrobials----Conti--
 By the mid-1990s the EU had authorised 9 antibiotics, plus the
antibacterials carbadox and olaquindox, for use in animal feed as
‘growth promoters’ and preventive antibiotic use had become a
routine aspect of intensive farming.
 FDA authorized the use of 18 antibiotics for ‘growth promotion’, of
which 8 were identical or chemically similar to drugs used in
human medicine
 By 1995 around 90% of poultry production units used feed
containing antibiotics.
 1999 in the US, 90% of the diets of recently weaned piglets, 70% of
the diets of growing pigs and 50% of the diets of ‘finishing’ pigs
contained some form of antibiotic.
 DANMAP (Denmark) estimated that in 1997, 80% of total
antibiotics produced were used in animals.
 In 2001, the Union of Concerned Scientists in the US estimated
that around 70% of all US antibiotic usage were in animals.
 November 2011 in US, 8 times more antimicrobials are used for
non-therapeutic purposes in the three major livestock sectors [i.e.,
chickens, pigs and cattle than are used in human medicine.

13. Use of Antimicrobials----Conti--
 Fifty million pounds of antibiotics are produced in the U.S. each year.
Twenty million pounds are given to animals, of which 80% (16 million
pounds) is used on livestock merely to promote more rapid growth..
American Medical News, "FDA Pledges to Fight Overuse of Antibiotics in Animals",
February 15, 1999.
 FDA (2010) reported use of antibiotics totals for food-producing
animals in 2009 were 13,068 tonnes of antibiotics for domestic use
and a further 1,632 tonnes exported.
 Who, 2011. In several parts of the world, more than 50% in tonnage of
all antimicrobial production is used in food-producing animals. In
addition.
 Veterinarians in some countries earn at least 40% of their income from
the sale of drugs, creating a strong disincentive to limit their use.
 In Australia, 500,000kg antibiotics are used in animals and 300,000kg
in humans per year (JETACAR 1999).
 In 1992, over 120,000kg of avoparcin (10% active ingredient by weight)
was used in animals in Australia (predominantly as a growth
promoter), while only 68kg of vancomycin was used in people
(JETACAR 1999).

14. From: Edquist and Pederson, 2000

15. Legalities of Antibiotic use in animals
 Penicillin Act and the Therapeutic Substances (Prevention of Misuse) Acts in
the UK, FDA in USA and similar acts in most of the developed countries
restricted the use of antibiotics to therapeutic use on prescription by a doctor,
veterinarian or dentist. (1947).
 1960s scientists discovered that antibiotic resistance could be transferred from
one bacterial species to another. In the UK, the Netherthorpe Committee in
1962, the Swann Committee in 1969 and the Lamming Committee in 1992
sounded the alarm.
 In Denmark, The Feedingstuffs Act was passed by parliament in November
1985 and came into force in January 1986. Antibitotic consumtipn got reduced
by 60% in 1996. Ban was full effective in 1998.
 In 1997 the World Health Organization recommended that the use of any
antibiotic for ‘growth promotion’ in animals should be terminated if that antibiotic
is used for human medicine or if its use in animals increases resistance to other
antibiotics used in human medicine.
 Between 1999 and January 1st 2006 the EU banned the use of 8 antibiotics for
‘growth promotion’: virginiamycin, tylosin phosphate, bacitracin zinc, spiramycin,
avilamycin, flavophospholipol, monensin and salinomycin, plus the drugs
carbadox and olaquindox.
 2000, WHO recommended total ban on feed antibiotics used as growth
promoter.
 In 2010, in USA, FDA limited the use of antibiotics for growth promotion.

17. Trends in Tylosin use in growth promotion and Erythromycin
Resistance in Enterococcus faecalis isolated from pigs.
(Aarestrup et al al., 2001, WHO, 2003)

18. Antibiotic in feed had effect on production: A Myth
Denmark Experience, totally stopped antibiotics in feed in 1998
After ban, poultry production was unaffected other than a one percent increase in feed intake (there were
no effects on weight gain or mortality). In finisher pigs there were also no important detrimental effects.
No detrimental effects on overall pork production which continued to rise.
(WHO, 2003)

19. Effect of Antibiotic use on Salmon production
(Source: Norvegian directorate of Fisheries)

20. Why AMDR is essential?
 For survival of Bacteria.
 It is Natural.
 If there was no antibiotic resistance, there
would be no need for a microbiology lab!
 Infections could be treated “syndromically”.
 It actually stimulate innovation in drug
discovery (Outterson, 2010).

21. Survival of Bacteria in Nature
 The ancestors of modern bacteria appeared on Earth, about 4
billion years ago. For about 3 billion years, all organisms were
microscopic, and bacteria and archaea were the dominant forms
of life.
 Bacteria are present in most habitats on Earth, growing in soil,
acidic hot springs, radioactive waste, water, and deep in the
Earth's crust, as well as in organic matter and the live bodies of
plants and animals, providing outstanding examples of mutualism
in the digestive tracts of humans, termites and cockroaches
(Fredrickson et al., 2004).
 There are typically 40 million bacterial cells in a gram of soil and a
million bacterial cells in a millilitre of fresh water; in all, there are
approximatelyfive nonillion (5×1030
) bacteria on Earth (Whitman et
al., 1998)].
 Bacteria form a biomass that exceeds that of all plants and
animals (Hogan, 2010). Bacteria are vital in:
 recycling nutrients,
 fixation of nitrogen.
 Provide the nutrients needed to sustain life by converting
dissolved compounds such as hydrogen sulphide and methane.

22. Survival of Bacteria in Nature
 Most bacteria have not been characterized, and only about half
of the phyla of bacteria have species that can be grown in the
laboratory (Rappé and Giovannoni, 2003)
 Eukaryotes resulted from ancient bacteria entering into
endosymbiotic associations with the ancestors of eukaryotic
cells, which were themselves possibly related to the Archaea.
 Due to their small size, commensal bacteria are ubiquitous and
grow on animals and plants exactly as they will grow on any
other surface.
 They degrade a variety of organic compounds thus used in
waste processing and bioremediation of industrial wastes.
Bacteria capable of digesting the hydrocarbons in petroleum are
often used to clean up oil spills.
 Bacteria a highly social organisms with an uncanny ability to
protect themselves, as a group (Wassenaar, 2011).

23. Secrets of survival
 Altruism or selflessness is the principle or practice of concern
for the welfare of others and Bacteria practice it. A traditional
virtue in many cultures.
 Lee et al (2010) observed that most individual bacteria within an antibiotic-
resistant population can be significantly more sensitive to the antibiotic than the
global population, then how they survive in presence of antibiotic.
 In a 2010 Collins et al., periodically analyzed the levels of drug resistance in an
E. coli the colony, they saw something unexpected: although the entire
population was thriving in the presence of the drug, only a few individual bacteria
were actually resistant. Further analysis revealed that the resistant mutants were
secreting a molecule called indole that thwarts their own growth but helps the
rest of the population to survive by activating drug-export pumps on the bacterial
cell membranes.
 Tanouchi et al (2012) while studying programmed death in bacterial cells they
reported an altruistic trait, whereby some cells trigger the cell death program and
release stress-relieving substances that increase the chances of survival of
other cells within the population.

24. Why Survival of Bacteria Important for
life?
 The bacteria is:
 First and the last in the food-web.
 Life forms perform as their microbes evolved.
 Ruminant digest fibrous food, Rumen and caecum: the fermenters
 Termites digest lignin
 Over 1,000 bacterial species in the normal human gut flora of the
intestines and contribute to:
 gut immunity,
 synthesize vitamins such as folic acid, vitamin K and biotin
 convert sugars to lactic acid
 fermenting complex indigestible carbohydrates
 inhibit the growth of potentially pathogenic bacteria (usually
through competitive exclusion)
 Life is not possible without bacteria (L. Pasteur, 1885).
 Nencki (1986) disagreed with Pasteur's thought, gnotobiotics.
 Gnotobiotics in not natural.
 Life without bacteria if even possible is worse (Wassenaar, 2011).
 Bacteria: The Benign, the Bad, and the Beautiful. (Wassenaar,
2011).

25. Life within life
 There are approximately ten times as many bacterial cells in the
human flora as there are human cells in the body, with large
numbers of bacteria on the skin and as gut flora (Sears, 2005).
 In human body, the Living mass of enteric flora accounts for 1
kg to 2 kg of body weight.
 It represents a combined genetic microbiome exceeding the
human genome by one hundred-fold.
 Magnitude and complexity of the alimentary ecosystem is ill
understood.
 It seems surprising that the rediscovery of the importance of the
gut flora is only a latter-day occurrence.
 It exhibits a collective metabolic activity that eclipses that of the
liver. The gut flora is tantamount to a hidden organ.
 The vast majority of the bacteria in the body are rendered
harmless by the protective effects of the immune system.
 A few are beneficial and still fewer are pathogenic and cause
infectious diseases (WHO, 2002).

26. Lesson from germfree life (Shanahan, 2004)
 Life without bacteria is associated with reductions in:
 mucosal cell turnover, digestive enzyme activity, cytokine
production, lymphoid tissue, lamina propria cellularity, vascularity,
muscle wall thickness and motility.
 Absence of bacteria is associated with an increase in
 enterochromaffin cell area, larger GIT.
 caloric intake to sustain a normal body weight.
 Thus, the normal host–flora relationship is not one in which the host
is in nutritional deficit because of the presence of bacterial guests
within the gut.
 Important metabolic effects of the flora include:
 the production of short chain fatty acids—a major energy source
for colonic epithelia—from dietary fermentable carbohydrates.
 Breakdown dietary carcinogens.
 synthesis of biotin, folate, and K vitamins.
 Understanding of the activity of the intestinal microbiome, may offer
strong potential for therapeutic exploitation in health and disease.

27. Lesson from germfree life
 The fertility of germfree animals is very low, but the reasons for this
are not known (Levenson et al., 1959).
 Antibodies can be produced by the germfree animal, but seemingly
at a slower rate (Miyakawa et al., 1957).
 The presence of a commensal flora is necessary for fine tuning of T
cell repertoires and TH1/TH2 cytokine profiles (Rook and Stanford,
1998).
 The germfree animal, generally is highly susceptible to pathogenic
(and at times to ordinarily nonpathogenic) bacteria (Levenson et al.,
1959).
 About 70% germfree animals never reach the adulthood. Almost 30
per cent of germfree animals have spontaneous deaths of due to
volvulus of the enlarged caecum. Forty per cent dies due to asphyxia
(Wardcet al., 1958).

28. Bad Effects of Antibiotic use
 Change in our food (antibiotic load) may alter the microbial education and
fine tuning of the immune system.
 With increase in use of antibiotics the following disorders have shown an
increasing trend (Bach, 2002; Ekbom, 2003; Anthony Gucciardi , 2011):
 Asthma, Autoimmune disorders and allergies (Penicillins, Carbapenems, cefalosporins
and Sulpha drugs),
 Mutagenicity and carcinogenicity (Sulphamethazine, Oxytetracycline, Furazolidone)
 Nephrotoxicity (Aminoglycosides, Penicillins, sulphonamides)
 Bone-marrow toxicity (Chloramphenicol, Tetracyclines, Linezolid, Sulphonamides)
 Hepatotoxicity (Macroloides, Telithromycin, Penicillins)
 Brain and Nerve damage (Carbapenems, penicillins, polypeptides, quinolones)
 Hearing impairment –Ototoxicity (Aminoglycosisdes)
 Visual disturbances (Telithromycin)
 Foetotoxic (Tetracyclines)
 Tendinosis (Quinolones)
 Photosensitivity (Sulphonamides, tetracyclines)
 Change in Taste (metallic by metronidazole, bitter by tinidazole)
 Multiple sclerosis
 Soaring obesity rates around the globe
 Increased cases of mental illness
 Insulin-dependent diabetes mellitus.
 Autoimmune disorders
 Obesity

29.  Autoimmune disorder: About 75 percent of humans host a
microorganism called Bacteroides fragilis which helps hold the immune
system in check by producing a protein that restrains T-cells. When T-
cells get too high, it can lead to inflammatory and autoimmune
diseases.
 Obesity: Human stomach contains bacteria called Helicobacter pylori.
This bacterium regulates the amount of stomach acid that the stomach
produces. Acidity inturn stimulate ghrelin, secretion which signals to
the brain that the stomach is empty and the body needs to eat. When
after eating acidity is low ‘leptin’ is secreted and brain sense that now it
is to stop eating. Eradication of Helicobacter pylori Increases Ghrelin
mRNA expression in the Gastric Mucosa and decrease in ‘leptin’ mRNA
expression. Lack of ghrelin and leptin regulation after Helicobacter
pylori eradication (after antibiotic use) may lead to over-eating and
obesity (Roper et al., 2008).

30. Unwanted effects of Antibiotics
(Blaser, 2011)
 Reproductive disorders: Dr. Wassenaar (2011) explains
how the antibiotic use alters intestinal bacterial microflora of a
fruit fly which in-turn drives mating preference. If this applies
to humans!
 Mating preference was achieved by dividing a population of Drosophila melanogaster and rearing one part
on a molasses medium and the other on a starch medium. When the isolated populations were mixed,
“molasses flies” preferred to mate with other molasses flies and “starch flies” preferred to mate with other
starch flies. The mating preference appeared after only one generation and was maintained for at least 37
generations. Antibiotic treatment abolished mating preference, suggesting that the fly microbiota was
responsible for the phenomenon (Sharon et al., 2010).
 Gastro-intestinal problems
 IBD (Krohn’s disease)
 Peptic ulcers
 Antibiotic Associated Pseudo-Membranous Colitis (AAPMC)
due to Clostridium difficile.
 Antibiotic-associated diarrhea (although all antibiotics may
be associated with AAD, cephalosporins, extended
coverage penicillins, and clindamycin are the main)
 Superinfection (or Suprainfection):
 Candida albicans vaginal infections
 Suppression of Normal Microbiota

31. Estimates of Adverse effects of Antibiotic
chemotherapy
 It is estimated that 10% to 15% of all hospitalized
patients treated with antibiotics will develop AAD.
 Most important, twice as many will become
asymptomatic carriers. Risk factors include:
 compromised immune status
 advanced age,
 abdominal surgery
 Comorbidity
 types and length of use of antibiotics, and
 the length of hospitalization (infection rates for C.
difficile are reported to be around 10% after 2 weeks of
hospitalization but may reach 50% after 4 or more
weeks

32. Conclusion
 Many of the emerging health problems are
associated with change/ destruction of our
microbiota.
 Bacteria are necessary for healthy life on
earth thus the antibiotic resistance.
 Antibiotic is to destroy the life while ADR
is for life.
 Most life on earth perform due to life
within (bacteria).