To Study Drug Resistance in Bacteria Using Antibiotics

TO STUDY DRUG RESISTANCE IN BACTERIA USING ANTIBIOTICS A BIOLOGY PROJECT REPORT SUBMITTED BY ANUSHA PRASAD IN PARTIAL FULFILMENT OF THE CBSE GRADE XII IN BIOLOGY AT

AECS MAGNOLIA MAARUTI PUBLIC SCHOOL #36/909, ARAKERE, BANNERGHATTA ROAD, BANGALORE- 560076. 2013-2014

CERTIFICATE

This is to certify that ANUSHA PRASAD of Grade XII, AECS MAGNOLIA MAARUTI PUBLIC SCHOOL, BANGALORE with register number ____________________ has satisfactorily completed the project in Biology on TO STUDY DRUG RESISTANCE IN BACTERIA USING ANTIBIOTICS in partial fulfillment of the requirements of All India Secondary School Certificate Examination (AISSCE) as prescribed by CBSE in the year 2013-2014.

Signature of the Candidate

Signature of the Principal

Signature of the Teacher In-Charge

Signature of the External Examiner

Table of Contents

INTRODUCTION........................................................................................................................ 1 HISTORY.................................................................................................................................... 3 OBJECTIVE................................................................................................................................ 9 SCOPE AND LIMITATIONS.....................................................................................................10 THEORY................................................................................................................................... 11 EXPERIMENT.......................................................................13 PROCEDURE...........................................................................18

OBSERVATION......................................................................................................................... 22 RESULT.................................................................................................................................... 23 BIBLIOGRAPHY...................................................................................................................... 24

ACKNOWLEDGEMENT I would like to thank my teachers, Mrs. Neelam and Mrs. Nikhila for guiding me through this project and for their valuable inputs which provided me with a constant nudge for improvement. It is imperative to thank our Principal, Mrs. Seema Goel for providing me the opportunity to work on this project. It goes without saying that my classmates, especially Madhumathi Mandal, Arathi Nair and Sushrutha Sricharan for their help in due course of this project. My parents have also played a part in helping me in this project. My thanks goes out to them also. This project and reading-up on the same has provided me with an in depth understanding of the topic. It has nurtured my scientific temperament and curiosity.

Signature of the Candidate

ABBREVIATION Sl.No 1 2 3 4 5 6 7 8

Abbreviation MDR MRSA HAQs SR MDRTB ml mg g

Expansion Multidrug Resistance Methicillin Resistant Staphylococcus Aureus 4-hydroxy-2-alkylquinolines Stringent response Multidrug Resistant TB Milliliter Milligram Gram

INTRODUCTION In this project we will study and attempt to cultivate bacteria that are resistance to drugs. According to external sources, Antibiotic resistance is a type of drug resistance where microorganism is able to survive exposure to an antibiotic. While a spontaneous or induced genetic mutation in bacteria may confer resistance to antimicrobial drugs, genes that confer resistance that transferred between bacteria in a horizontal fashion by conjugation, transduction, or transformation. Thus, a gene for antibiotic resistance that evolves via natural selection may be shared. Evolutionary stress such as exposure to antibiotics then selects for the antibiotic resistance trait. Many antibiotic resistance genes reside on plasmids, facilitating their transfer. If a bacterium carries several resistance genes, it is called multidrug resistance (MDR) or, informally, a super bug or super bacterium. What this tells us is that drug resistance bacteria can be a product of natural selection. For example, bacteria are cultivated in the lab and the drug that can kill these bacteria is added to the culture. Most of the bacteria will di, but the ones that survive have genetic mutation that enables them to survive in the presence of the antibiotic. The bacteria are ‘drug resistance.’

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Schematic representation on how antibiotic resistance develops under natural condition. As we can see from the above explanation, drug resistance bacteria are caused due to the overuse of antibiotics. In some countries like India antibiotics are sold over the counter. This results in misuse or overuse of antibiotics which cause drug resistance bacteria to proliferate. Once a bacterium becomes drug resistance it is very hard to get rid of it as it is a new drug and it has not been exposed to work against it.

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HISTORY Before the early 20th century, treatments for infections were based primarily on medicinal folklore. Mixtures with antimicrobial properties that were used in treatment of infections were described over 2000years ago. Many ancients’ cultures, including the ancient Egyptians and ancient Greeks, used specially selected mold and plant materials and extracts to treat infections. More recent observation made in the laboratory of antibiotics between microorganisms led to the discovery of antibacterial produced by microorganisms. Louis Pasteur observed, “If we could intervene between antagonism observed between some bacteria it would offer perhaps the greatest hopes of therapeutics”. The terms, “antibiosis”, meaning, “Against life”, was introduced by French bacteriologist Vuillemin as descriptive name of the phenomenon by these early antibacterial drugs. 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.

Bacillus anthracis. Synthetic antibiosis chemotherapy as a science of antibacterial began in Germany by Paul Ehrlich in 1880’s. Ehrlich observed that certain dyes could color humans, animal, or bacterial cells, while other did not. He then proposed the idea that it would be possible to create chemicals that would act as a selective drug that would bind and would kill the bacteria without harming the human host. After screening hundreds to dyes against various organisms, he 3

discovered medicinally useful drug, the synthetically antibacterial Salvarsan now called arspheamine. In 1895, Vincenzo Tiberio, of university of Naples discovered that a mold in water has antibacterial action. After this initial chemotherapeutic compound proved effective other perused similar lines of inquiry, but it was not until 1928 that Alexander Fleming observed antibiosis against bacteria by a fungus genus of the Penicillium. Fleming postulated that the effect was mediated by an antibacterial compound named penicillin. He initially characterized some of its properties, but he did not pursue its further development. The first sulfonamide and first commercially available antibacterial antibiotic, Pronstosil, was developed by a research team led by Gerhard Domagk in 1932 in Bayer’s laboratory Germany. Domagk received the 1939 Noble Prize for Medicine for his efforts.

Penicillin, the first natural antibiotic discovered by Alexander Fleming

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SOME RESISTANT PATHOGENS Staphylococcus aureus Staphylococcus aureus is one of the major resistant pathogens. Found on the mucous membranes and the human skin of around a third of the population, it is extremely adaptable to antibiotic pressure. It was one of the earlier bacteria in which penicillin resistance was found—in 1947, just four years after the drug started being mass-produced. Methicillin was then the antibiotic of choice, but has since been replaced by oxacillin due to significant kidney toxicity. Methicillin resistant Staphylococcus aureus (MRSA) was first detected in Britain in 1961, and is now "quite common" in hospitals. MRSA was responsible for 37% of fatal cases of sepsis in the UK in 1999, up from 4% in 1991. Half of all S. aureus infections in the US are resistant to penicillin, methicillin, tetracycline and erythromycin.

Streptococcus and Enterococcus Streptococcus and Enterococcus infections can usually be treated with many different antibiotics. Early treatment may reduce the risk of death from invasive group A streptococcal disease. However, even the best medical care does not prevent death in every case. For those with very severe illness, supportive care in an intensive care unit may be needed. For persons with necrotizing fasciitis, surgery often is needed to remove damaged tissue. Strains of S. pyogenes resistant to macrolide antibiotics have emerged; however, all strains remain uniformly sensitive to penicillin. Pseudomonas aeruginosa Pseudomonas aeruginosa is a highly prevalent opportunistic pathogen. One of the most worrisome characteristics of P. aeruginosa is its low antibiotic susceptibility, which is attributable to a concerted action of multidrug efflux 5

pumps with chromosomally encoded antibiotic resistance genes and the low permeability of the bacterial cellular envelopes. Pseudomonas aeruginosa has the ability to produce HAQs and it has been found that HAQs have prooxidant effects, and over expressing modestly increased susceptibility to antibiotics. The study experimented with the Pseudomonas aeruginosa biofilms and found that a disruption of relA and spot genes produced an inactivation of the Stringent response (SR) in cells who were with nutrient limitation which provides cells be more susceptible to antibiotics.

Clostridium difficile Clostridium difficile is a nosocomial pathogen that causes diarrheal disease in hospitals worldwide. Clindamycin-resistant C. difficile was reported as the causative agent of large outbreaks of diarrheal disease in hospitals in New York, Arizona, Florida and Massachusetts between 1989 and 1992. [87] Geographically dispersed outbreaks of C. difficile strains resistant to fluoroquinolone antibiotics, such as ciprofloxacin and levofloxacin, were also reported in North America in 2005. Salmonella and E. coli Escherichia coli and Salmonella come directly from contaminated food. When both bacteria are spread, serious health conditions arise. Many people are hospitalized each year after becoming infected, with some dying as a result. By 1993, E. coli resistant to multiple fluoroquinolone variants was documented.

Acinetobacter baumannii On November 5, 2004, the Centers for Disease Control and Prevention (CDC) reported an increasing number of Acinetobacter baumannii bloodstream infections in patients at military medical facilities in which service members 6

injured in the Iraq/Kuwait region during Operation Iraqi Freedom and in Afghanistan during Operation Enduring Freedom were treated. Most of these showed multidrug resistance (MRAB), with a few isolates resistant to all drugs tested.

Mycobacterium tuberculosis Tuberculosis is increasing across the globe, especially in developing countries, over the past few years. TB resistant to antibiotics is called MDR TB (Multidrug Resistant TB). The rise of the HIV/AIDS epidemic has contributed to this.

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OBJECTIVE

Our objective is to identify these drug resistant bacteria by cultivating the bacteria and placing then in an environment of bacteria.

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SCOPE AND LIMITATIONS In our experiment we plan to take antibiotics in a dish and selectively grow bacteria that are resistant to drugs. The main limitations are the equipments. For us to correctly identify the drug resistant bacteria out of the mass of the normal bacteria, we need powerful microscopes that are not easily available. After culturing the bacteria, we also need to be able to verify these bacteria are truly drug resistant. For this, we need to be able to count the number of bacteria in the sample oe at least know that their density in a given area. Once again this will be difficult without the required equipment. This field of bacteria has immense scope and can provide more efficient healthcare system. Today a lot of people are suffering from drug resistant bacteria because none of the drugs are working to cure them. With more research in this field, we provide more targeted approach to kill these harmful bacteria. Existing Scientific Literature on new medication for drug resistant bacteria: Until recently, research and development (R&D) has provided new drugs in time to treat bacteria that became resistant to older antibiotics. That is no longer the case. The potential crisis at hand is the marked decrease in industry R&D, the increasing prevalence of resistant bacteria. Infectious diseases physicians are alarmed by the prospect that effective antibiotics may not be available to treat seriously ill patients in the near future. As bacterial antibiotic resistance continues to exhaust the supply of effective antibiotics, a global public health disaster appears likely. Poor financial investment in antibiotic research has exacerbated the situation. A call to arms raised by several prestigious scientific organizations a few years ago rallied the scientific community, and now the scope of antibacterial research has broadened considerably.

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THEORY Antibiotics are the chemical substances produces by microorganisms to kill other organisms or retard their growth. Tetracycline, Streptomycin, Penicillin are a few examples of the antibiotics which have been useful in treating various bacterial diseases. Continuous use of particular antibiotic against any microorganism reduces its effect because of a few bacterial cells develop resistance to antibiotic, may be due to mutation and thus such resistant stains keep on growing even in the presence of antibiotic and do not respond to treatment. Genes for resistance to antibiotics, like the antibiotics themselves, are ancient. However, the increasing prevalence of antibiotic-resistant bacterial infections stem from antibiotic in medicine. Any use of antibiotics can increase selective pressure in a population of bacteria to allow the resistant bacteria to thrive and the susceptible bacteria to die off. As resistance towards antibiotics becomes more common, a greater need for alternative treatments arises. However, despite a push for new antibiotic therapies there has been a continued decline in the number of newly approved drugs. Antibiotic resistance therefore poses a significant problem.

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EXPERIMENT

Aim: To study the drug resistance in bacteria using antibiotics.

Requirement: a.

Apparatus Requirement

 Petridish

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 Sterilized culture tubes

 Forceps

 Flasks

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 Beakers

 Burner

b.

Chemical Requirements

 Penicillin and other antibiotics

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 Agar

 Starch

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 Distilled water

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PROCEDURE Prepare pure culture of bacteria:  100ml distilled water; 4gms of agar and 1g of starch was added to a test tube and shaken well.

 The test-tube was placed over a burner ad boiled for 5minutes.  While the solution was boiled, 10pieces of dry hay were added.  The resulting mixture was then kept in a warm place for 5days for bacterial growth.

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Culture medium:  Beaker was taken and molten agar was prepared in it.

 Samples from the bacterial culture were taken in the petridish.

Transfer bacteria from agar test tube into petri dish containing antibiotics:  Petridish were taken. 18

 The warm culture medium prepared in the previuos step was poured into each petridish. Care was taken to sterilize the petridish so as to avoid any unnecessary growth.  The culture was spread evenly.

Antibiotics:  A solution of antibiotics was made by dissolving different antibiotic tablets in distilled water. Eg: penicillin solution, tetracycline solution.

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 Filter paper cut in circles were soaked in antibiotic solution ad placed in one part of the petriplate with culture.

 Another petridish with agar was taken and labeled ‘Control’. No antibiotic was added to this.

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OBSERVATION

A. After one week Sl.no.

Antibiotic used

1.

Levoflaxacin

Number of bacterial colonies 5

2.

Amoxicillin

8

3.

Cefixime

16

4.

Roxithromycin

29

5.

Ciprofloxacin

4

6.

Cefpodoxime

31

7. 8. 9.

Ofloxacin Azithromycin Control

34 14 0

Description Few medium colonies near the edge of the Petri dish Few medium sized colonies around the filter paper Small to medium size colonies Dense growth of small colonies of bacteria. Few medium to large sized colonies Large number of small to medium sized colonies around the edge. Small sized colonies Medium sized colonies N/A

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B. After two weeks Sl.no.

Antibiotic used

1.

Levoflaxacin

Number of bacterial colonies 3

2.

Amoxicillin

4

3.

Cefixime

9

4. 5.

Roxithromycin Ciprofloxacin

15 3

6.

Cefpodoxime

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7. 8. 9.

Ofloxacin Azithromycin Control

21 11 0

C. After three weeks Sl.no. Antibiotic used 1.

Levoflaxacin

Number of bacterial colonies 2

2.

Amoxicillin

3

3.

Cefixime

6

4. 5.

Roxithromycin Ciprofloxacin

12 3

6.

Cefpodoxime

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Description Medium colonies near the edge of the petri dish Medium sized colonies around the filter paper Small to medium size colonies Small sized colonies Medium to large sized colonies Large number of small to medium sized colonies around the edge. Small sized colonies Medium sized colonies N/A

Description Medium colonies near the edge of the petri dish Medium sized colonies around the filter paper Small to medium size colonies Small sized colonies Medium to large sized colonies Large number of small to medium sized colonies 22

7. 8. 9.

Ofloxacin Azithromycin Control

13 9 0

around the edge. Small sized colonies Medium sized colonies N/A

RESULT The observed trend was that the number of bacterial colonies decreased with time and family remained constant. We can infer that the decrease was because bacteria with the mutation survived. Over time as only the mutated bacteria remained, and the population stabilized. We can predict that after a few more weeks, the population may show an upward trend with mutations produce progeny with mutations.

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BIBLIOGRAPHY Wikipedia - The free encyclopedia - (http://en.wikipedia.org) Comprehensive Practical Chemistry

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