MOOC 4, Module 28

Antibiotic Resistance in Bacteria

Main Text

Antibiotics can be in broad sense defined as the variety of substances derived from bacterial sources

(microorganisms) that deters the growth of other microorganisms or kill the other group of bacteria. They

are generally used to kill infections which are caused by a group of pathogenic microorganisms. They can

be prevented through the use of certain antibiotics. The word antibiotic came from the word antibiosis a

term coined in 1889 by Louis Pasteur's student Paul Vuillemin which means a process “by which life could

be used to destroy life”.

Fig. 1. Action of antibiotic on bacterial growth curve (Taken from

http://faculty.smu.edu/jbuynak/Medicinal_Outline_11_4_04.pdf).

Classification of antibiotics

A common scheme of classifications for antibiotics is shown below:

Fig. 2.The scheme of classification of antibiotics (Taken from http://www.experiment-

resources.com/history-of-antibiotics.html).

Antibiotics can also be classified based on their chemical structure (Fig. 2). A similar level of effectiveness,

toxicity and side-effects is rendered by the antibiotics of same structural group. Broad spectrum antibiotics

are effective against a broad range of microorganisms in comparison to narrow spectrum antibiotics.

Bactericidal antibiotics kill the bacteria whereas bacteriostatic antibiotics deter the growth of bacteria.

History of Antibiotics

Early history

During ancient times

Greeks and Indians used moulds and other plants to treat infections.

In Greece and Serbia, mouldy bread was traditionally used to treat wounds and infections.

Warm soil was used in Russia by peasants to cure infected wounds.

Sumerian doctors used patients beer soup mixed with turtle shells and snake skins.

In Babylon doctors healed the eyes using a mixture of frog bile and sour milk.

Sri Lankan army used oil cake (sweetmeat) to be used as desiccant and antibacterial.

Modern history

Late 1800s

The search for suitable antibiotics began in the late 1800s with the growing acceptance of germ theory of

disease; a theory which proved those bacteria and other microbes led to the causation of different types of

ailments. Scientists started their search for drugs for controlling the disease causing microbes.

1871

The surgeon Joseph Lister made careful observations that urine contaminated with mould would not allow

the growth of bacteria.

1890s

German doctors, Rudolf Emmerich and Oscar Low were the first to make an effective drug from microbe.

The drug was called pyocyanase and the first antibiotic to be discovered but often the drug did not work.

1928

Sir Alexander Fleming observed that colonies of Staphylococcus aureus could be destroyed by the mold

Penicilliumnotatum, demonstrating the antibacterial properties of the mold. It was one of the classical

experiments to be done in the history of antibiotics.

1935

Prontosil, the first sulfa drug, was discovered in 1935 by German chemist Gerhard Domagk (1895–1964).

1942

The manufacturing process for Penicillin G Procaine was invented by Howard Florey (1898–1968) and

Ernst Chain (1906–1979). Penicillin could now be sold as a drug. Fleming, Florey, and Chain were awarded

the 1945 Nobel Prize for medicine for their work on penicillin.

1943

In 1943, American microbiologist Selman Waksman (1888–1973) made the drug streptomycin from soil

bacteria, the first of a new class of drugs called aminoglycosides. Streptomycin could treat diseases like

tuberculosis; however, the side effects were often too severe.

1955

Tetracycline was patented by Lloyd Conover, which became the most prescribed broad spectrum antibiotic

in the United States.

1957

Nystatin was patented and used for curing many disfiguring and disabling fungal infections.

1981

SmithKline Beecham patented Amoxicillin or amoxicillin/clavulanate potassium tablets, and first sold the

antibiotic in 1998 under the tradenames of Amoxicillin, Amoxil, and Trimox. Amoxicillin is a

semisynthetic antibiotic.

Thus a brief outline of the developments in the modern history of antibiotics is given in Fig. 3 (Taken from

http://www.experiment-resources.com/history-of-antibiotics.html).

Year Origin Description

1640 England John Parkington recommended

using mold for treatment in his

book on pharmacology

1870 England Sir John Scott Burdon-Sanderson

observed that culture fluid

covered with mould did not

produce bacteria

1871 England Joseph Lister experimented with

the antibacterial action on human

tissue on what he called

Penicilliumglaucium

1875 England John Tyndall explained

antibacterial action of the

Penicillium fungus to the Royal

Society

1877 France Louis Pasteur postulated that

bacteria could kill other bacteria

(anthrax bacilli)

1897 France Ernest Duchesne healed infected

guinea pigs from typhoid using

mould (Penicilliumglaucium)

1928 England Sir Alexander Fleming

discovered enzyme lysozyme

and the antibiotic substance

penicillin from the fungus

Penicilliumnotatum

1932 Germany Gerhard Domagk discovered

Sulfonamidochrysoidine

(Prontosil )

Sir Alexander Fleming

Alexander Fleming(Fig. 4) was born in Loudon, Scotland on 6 August, 1881 in a farming family. He went

for his schooling to Regent Street Polytechnic after his family moved to London in 1895. He joined St.

Mary's Medical School and became research assistant to renowned Sir Almroth Wright after he qualified

with distinction in 1906. He completed his degree (M.B.B.S.) with gold medal in 1908 from the University

of London and was at St. Mart till 1914. He served as Captain during the World War I and worked in

battlefield hospitals in France. After the war he returned to St. Mary in 1918 and got elected Professor of

Bacteriology in 1928.

Following World War I, Fleming actively kept on searching for anti-bacterial agents, having witnessed the

death of many soldiers from sepsis resulting from infected wounds. Antisepticshampered the patients'

immunological defenses more efficiently than they killed the invading bacteria. It was then in the year 1927

that Fleming was working on Staphylococcus.On 3rd September 1928, Fleming returned to his laboratory

after a brief holiday with his family found that one of his stacked cultures of staphylococci had become

contaminated with a fungus and that colonies of staphylococci surrounding the fungus were destroyed

whereas other colonies of bacteria far away from fungus were normal. Fleming grew the fungus in a pure

culture and found that it produced a substance that killed a wide variety of pathogenic bacteria (Fig. 5). He

identified the fungus as Penicillium and the named the substance secreted from the fungus as penicillin on

7th march 1929.

Sir Alexander Fleming, a Scottish biologist made new horizons for modern antibiotics with his discovery

of enzyme lysozyme and the important antibiotic penicillin. The discovery of penicillin made easy the

treatment of bacterial infections as syphilis, gangrene and tuberculosis. He also contributed immensely

towards the other fields of medical sciences as bacteriology, immunology and chemotherapy.

Fig. 4. Sir Alexander Fleming

(Taken from http://en.wikipedia.org/wiki/Antibacterial)

Fig. 5. Fleming’s original petri dish that had discovered the drug penicillin (Taken from

http://www.onthisdeity.com/28th-september-1928-%E2%80%93-alexander-fleming-discovers-penicillin/)

Some of the important organisms cause pathogenic diseases to human beings. The list of which is shown

in Fig. 6.

Fig. 6.List of some important pathogenic bacteria (Taken from

http://faculty.smu.edu/jbuynak/Medicinal_Outline_11_4_04.pdf).

Types of antibiotics

Antibacterial antibiotics are commonly classified based on their mechanism of action, chemical structure,

or spectrum of activity. Most target bacterial functions or cell growth processes. Those that target the

bacterial cell wall (penicillins and cephalosporins) or the cell membrane (polymixins), or interfere with

essential bacterial enzymes (quinolones and sulfonamides) have bactericidal activities. Those that target

protein synthesis (aminoglycosides, macrolides, and tetracyclines) are usually bacteriostatic.Moreover

categorization is based on their target specificity. "Narrow-spectrum" antibacterial antibiotics target

specific types of bacteria, such as Gram-negative or Gram-positive bacteria, whereas broad-spectrum

antibiotics affect a wide range of bacteria. Other group of three new classes of antibacterial antibiotics have

been brought into clinical use: cyclic lipopeptides (such as daptomycin), glycylcyclines (such as

tigecycline), and oxazolidinones (such as linezolid).

Mode of action of antibiotics

Fig.7. Major sites for antibiotic actions (Taken from

http://faculty.smu.edu/jbuynak/Medicinal_Outline_11_4_04.pdf).

Fig. 8.Sites of antibacterial action (Taken from

http://faculty.smu.edu/jbuynak/Medicinal_Outline_11_4_04.pdf).

Synthetic drugs

There are certain compounds like Salvarasan and Protonsil, which are made in the pharmaceutical

laboratory are called synthetic drug. Synthetic agents like sulfadiazine produces a bacteriostatic effect on

microorganisms by an action called competitive inhibition. The synthetic and antibiotic drugs have certain

important properties which need to be considered when a doctor prescribes them for medical use.

1) Selective toxicity. The toxic dose refers to the concentration of the drug that causes harm to the

host while therapeutic dose refers to the concentration of the drug that eliminates the pathogen from

the host. Together these two terms constitute the chemotherapeutic index. It is the highest

concentration of the drug tolerated by the host divided by the lowest concentration of the drug that

will kill the infection agent (Fig. 9).

Fig. 9.Chemotherapeutic index.

2) Antimicrobial spectrum. Those drugs that affect many taxonomic groups of bacteria are considered

as broad spectrum of action while those which are specific in their action against few bacteria are

said to have a narrow spectrum of action.

Antibiotic resistance

Since the mid 1900s an increasing number of bacteria have become resistant to antibiotics. Resistance

towards antibiotics develops in the following two ways:

Horizontal gene transfer.The resistance from one bacterial cell is transmitted to another bacterial cell

through aid of horizontal gene transfer. The transfer mechanisms include conjugation, transformation or by

transformation where bacteriophages pass resistance genes between bacterial cells.

Mutations. Spontaneous mutations and other kinds of mutations induce resistance towards antibiotics in

bacteria. Such resistant bacteria are increasingly responsible for human diseases of the tract, lungs, skin and

urinary tract. Common diseases like bacterial pneumonia, tuberculosis, streptococcal sore throat are

difficult to treat because of increasing number of antibiotic resistant bacteria.

Altered metabolic pathway. Resistant to sulfonamides may develop when the drugs fail to bind with

enzymes that synthesize folic acid. The microbes utilize an altered metabolic pathway which the enzyme is

not able to inhibit.

Reduced permeability. Certain microbes pump out the antibiotic from the bacterial cell before it reaches

the cytoplasm and mediates its action on the various cellular components.

Target alteration. Some streptomycin resistant bacteria can modify the structure of ribosomes so that the

antibiotic cannot bind with the ribosomes. Other targets are DNA and RNA polymerase which are important

enzymes of cellular metabolism.

Antibiotic inactivation. Certain microbes synthesize enzymes which break down the antibiotics.

Aminoglycosides which binds with ribosomes cannot show their mode of action in certain bacteria as the

bacteria have developed ways to chemically modify aminoglycosides.

Thus, the different pathways are shown in Fig. 10.

Fig. 10.Different antibiotic resistant mechanisms of bacteria (Taken from Alcamo’s fundamentals of

Microbiology by Pommerville, 8th edition, 2007, Jones and Bartlett Publishers Canada).

Determination of antibiotic resistance

The Kirby-Bauer Antibiotic Test Procedure

Theory

The Kirby Bauer test named after its inventor is the most widely used method for determination of antibiotic

reaction to a bacterium. It is the widely used method to determine what treatment of antibiotic should be

used to an infection. Most of the bacteria are resistant to one or more antibiotics. The drug susceptibilities

of many pathogenic bacteria are still unknown and needs to be found out by this method.

A standardized filter paper-paper disc agar diffusion procedure, known as the Kirby-Bauer method is

frequently used to monitor the reaction of antibiotic to bacteria, i.e. whether they are susceptible, resistant

or intermediate. This method became the method of choice when in 1966, significant progress in

standardization of the disc method occurred when Bauer, Kirby and co-workers published their experiments

as a practical method of testing with a broad application to clinical laboratories.

This method on the basis of zone of inhibition that results from the diffusion of the drug into the medium

surrounding the disc measures the efficacy of a drug. In this process filter paper of uniform size are absorbed

with different concentrations of antibiotic of different nature and placed on the surface of an agar plate that

has seeded with the organism to be tested. The medium of choice recommended by National Committee on

Clinical Laboratory Standards (NCCLS) is Mueller-Hinton Agar. The medium is poured into plates to a

uniform depth of 5 mm and refrigerated well. Mueller-Hinton agar is recommended due to:

1. It results in good reproducibility,

2. It is low in sulfonamide, trimethoprim, and tetracycline inhibitors

3. It results in satisfactory growth of most bacterial pathogens.

The susceptibility of an organism to a drug is determined by the size and diameter of this zone, which in

turn is dependent upon the variables:

1. The ability and rate of diffusion of the antibiotic into the medium and its reaction with the organism.

2. The number of organism inoculated.

3. The growth rate of the organism.

4. The degree of susceptibility of the organism to the antibiotic.

Through the measurement of the zone of the inhibition in mm the size is compared with the standardized

size given in charts and accordingly the test organism is said to be resistant, intermediate or susceptible.

Materials

Any standard bacterial culture. To standardize the inoculum density, 0.5 McFarland standards is used.

Media:

Standard Mueller-Hinton Agar plates are used.

Antimicrobial sensitivity discs are used containing different antibiotics like streptomycin 10µg, tetracycline

30 µg, vancomycin 30µg, Penicillin G 10µg (Fig. 11).

Fig. 11. Structure of penicillin G and tetracycline in capsules form (Taken from

http://www.drsfostersmith.com/product/prod_display.cfm?pcatid=10326 and

http://www.onlinepharmacycatalog.com/category/common-drugs-and-medications/antibiotics/penicillin-

g-bicillin-pfizerpen-wycillin/).

Equipment:

Sterile Forceps, Bunsen burner, sterile cotton swabs, glassware marking pencil and millimeter scale.

Procedure

Place agar plates in an incubator heated to 37ᵒC for 10-20 mins.

Label the petri plates with the names of the test organism to be inoculated.

Using sterile technique, inoculate all agar plates with the respective test organisms as follows:

a. Take a sterile cotton swab and place it into the well mixed saline test culture or culture adjusted to

suitable conditions and remove the excess inoculum by pressing the saturated swab against the

walls of the culture tube.

b. Using the swab streak the entire plate with in different directions to cover the entire surface area of

the petri plate with inoculum.

Allow the culture plates to become air dry for about 5 min.

Apply the antibiotic discs by using a sterile forceps. Do not press the antibiotic discs too hard on

the agar. The discs should be placed at equal distances.

Gently press the disc down and ensure that they adhere to the agar surface.

Incubate the plates for 24 to 48 hours at 37ᵒC (Fig. 12).

Fig. 12.Procedure for performing Kirby-Bauer Test Procedure (Taken from Microbiology; A Lab Manual

by Cappuchino, 9th edition, 2011, Pearson Education, Inc).

Result

After 24 to 48 hours of incubation there are zones of inhibition found in the plate. Those antibiotics which

have no effect on the growth of bacteria produce no zone of inhibition (Fig. 13). Those antibiotics which

have produced zone of inhibition are said to be effective against bacteria. Based on the diameter of zone of

inhibition the bacteria are said to be resistant, susceptible or intermediate to that particular antibiotic (Fig.

14).

Fig. 13. Disc diffusion assay performed with different antibiotics showing the presence of zones of

inhibition or no zone of inhibition (Taken from

http://blogs.nature.com/drjim/2010/08/bugs_and_drugs.html and

http://www.visualphotos.com/image/1x6348956/disk_diffusion_assay).

Fig. 14.Table of zone of diameter standards for organisms other than Haemophilus and Neisseria (Taken

from Taken from Microbiology; A Lab Manual by Cappuchino, 9th edition, 2011, Pearson Education, Inc).

Precautions

1. The inoculum density should be adjusted according to NCCLS standards.

2. The strain against which the antimicrobial assays are done should be pure.

3. The antibiotic discs should not be pressed too hard on the agar surface.

Determination of Minimum Inhibitory Concentration

Theory

Besides, Kirby-Bauer method the broth tube dilution method helps us to know about the resistance of

bacteria to an antibiotic. In this case, the dilutions of the antibiotic are made in broth medium is known as

minimum inhibitory concentration. The MIC helps us to know about the concentration of antibiotic which

the bacteria are able to tolerate.This data is important as it helps us to fix the dose of antibiotic to which the

bacteria is resistant (Fig. 15). Besides, the broth dilution method there is Etest also known as Epsilometer

test method also to determine the MIC. The Etest is basically an agar diffusion method.

The Etestuses a rectangular strip that has been adsorbed with the drug to be studied. A lawn of bacteria is

spread and grown on an agar plate, and the Etest strip is laid on top; the drug diffuses out into the agar,

producing an exponential gradient of the drug to be tested. An exponential scale is printed on the top of the

strip. After 24 hours of incubation, an elliptical zone of inhibition is seen and the point at which the ellipse

meets the strip gives a reading for the minimum inhibitory concentration (MIC) of the drug (Fig. 16).

Fig. 15.Antibiotic testing through the serial dilution method as seen through MIC method (Taken from

http://vads.vetmed.vt.edu/demos/Education/display.cfm?ShowMyFile=QuestionAnswer/VADSQA.cfm).

Fig. 16.Etest method (Taken from http://en.wikipedia.org/wiki/Epsilometer_test).

Materials

BHI broth dilutions of S. aureus , penicillin G (100 µg/ml), Erlenmeyer flask, sterile test tubes, sterile 2 ml

and 10 ml pipettes, spirit lamp, spectrophotometer, glassware marking pencil.

Procedure

Ten test tubes were taken and labeled. Using a sterile 10 ml pipette add 2 ml of broth to the sterile test tubes.

With a sterile pipette, added 2 ml of penicillin solution to tubes 1 and 2. Tube 1 served as control. Mix the

contents of the tube well.

Using a sterile 2 ml pipette transfer 2 ml from tube 2 to tube 3. Mix well and continue this procedure to

tube 9 by the method of serial dilution. Tube 10 receives no antibiotic and serves as positive control.

Using a sterile 2 ml pipette, added 2 ml of 1:1000 dilutions of S. aureus to all tubes.

Incubate both the tubes in incubator set at 37ᵒC for 12 to 18 hours (Fig. 17).

Fig. 17.Table showing the protocol followed for performing the Antibiotic Serial Dilution (Taken from

Microbiology; A Lab Manual by Cappuchino, 9th edition, 2011, Pearson Education, Inc).

Result

The dilution of the antibiotic in which there is no growth found in the test tubes is known as minimum

inhibitory concentration. It represents the maximum concentration of antibiotic which the bacteria can

endure (Fig. 18).

C

Fig 18. Diagram shows the determination of minimum inhibitory concentration of ampicillin against S.

aureus (A, B-Taken from Alcamo’s fundamentals of microbiology by Pommerville, 8th edition, 2007, Jones

and Bartlett Publishers Canada). (C taken from http://www.brass-asiapacific.com/c/services/test-

services/antimicrobial-

activity?page=shop.product_details&flypage=flypage.tpl&product_id=72&category_id=40).

Precautions

The concentration of the antibiotics should be carefully diluted.

The tubes should be mixed very well after the addition of antibiotic.

There should be no contamination in the broth during serial dilution step.

The pure culture of bacteria should be used for broth dilution test.

Thus, we can say that bacteria have evolved plenty of mechanisms to evade the toxic effects of antibiotics.

The antibiotics which we generally employ to kill bacteria are slowly helping in evolution of antibiotic

resistant bacteria. There are numerous experimental ways to study the antibiotic bacteria response. The

most common ones are disc diffusion assay using a single antibiotic disc or two discs showing synergistic

effect. Further, in order to know about the concentration dose response of an antibiotic MIC method is best

preferred.