ABSTRACT

Multi drug resistant bacteria are found in our surroundings and it is not restricted only to

clinical samples but also seen on fruits, vegetables, soil and waste water. MDR bacteria

were isolated on EMB medium from suspected patients urine sample and subjected

to Bio-Disc 12 analysis followed by the determination of MIC of the organism

towards ampicillin and cefotaxime. A set of biochemical tests performed paved

way for the identification of bacteria in urine as Citrobacter freundii whose

genomic DNA was isolated and amplified using adapters such as EcoRi and Msel

with the help of AFLP technique and their genetic variation was studied.
 INTRODUCTION

Bacteria existed on earth for 3 billion years and became adapted for

protecting themselves against toxic chemicals. Antibiotics were in clinical use for

more than six decades and overuse of those antibiotics resulted in multiple drug

resistance in microbes and thus none of the antibiotics killed microbes (Varsha

Shriram et al., 2013). Bacteria are found in every possible habitat on the planet

and they adapt to changes in external factors which is essential for their survival

and growth. In order to adapt to environmental threats bacteria become resistant to

all classes of antimicrobial drugs. Antimicrobial drug development is focused on

targeting essential proteins that prevent growth of micro-organism or which are

bactericidal (Alekshum, 2005).

According to World Health Organization (WHO) the resistance of bacteria

towards antibiotics enables them to survive the effect of inhibitory concentration

(Paramasivam et al., 2007). A bacterium of normally susceptible species might

become resistant by mutation or due to the effect of new genes. Some bacterial

species are inherently resistant to certain antibiotics, whereas others are sensitive.

Sensitivity has three requirements: a target for reaction, a mechanism for transport

into the cell before the antibiotic action takes place and absence of enzymes that

could inactivate or modify the antibiotic. A change in any of these requirements

could render an antibiotic-sensitive bacterium resistant to the drug (Levy, 1992). In

1928, Alexander Fleming discovered the bactericidal property of penicillin in a

Staphylococcus aureus plate and that it had particular group of compounds which

could be used to treat infection effectively. This kind of compounds were later

named antibiotic. The need of antimicrobials in treating wounded soldiers in

World War II stimulated the beginning of the antibiotic era (Weiss and Michael,

2004). Till 1960s, more than 100 antibiotics became commercially available and

were used extensively in the treatment of infectious disease. This period of time

was considered as the golden age of antibiotics.

Based on the mechanism of action, antibiotics are categorized into several

classes. Antibiotics can be bactericidal or bacteriostatic through inhibiting the

synthesis of cell wall, DNA, RNA, protein, cell growth, and cell division (Mingeot

Leclereq et al., 1999). Antibiotics isolated from microorganisms were used earlier

and later compounds derived or synthesized from natural products were used.

Large scale usage of antibiotics in animal faming led to its accumulation in food

chain and organisms developed resistance to drugs. Such organisms are called

multi drug resistant organisms which created a threat in public health (Koehn and

Carter, 2005).

Multi-drug resistant micro-organisms (MDRMOs) are micro-organisms that are

resistant to one or more therapeutic classes of anti-microbial agents. The number

of microorganisms will increase if the selective antibiotic use continues and the

resistant organism is able to spread from one person to another. Gram-positive and
Gram-negative bacteria are both affected by the effect of antimicrobial resistance

(Chakrabarti et al., 2000).

Salmonella typhi strains are resistant to ampicillin, chloramphenicol,

trimethoprim sulphamethoxazole streptomycin, sulfonamides and tetracycline.

Cephalosporin-resistant Eseherichia coli, Klebsiella spp, methicillin resistant

Staphylococcus aureus, vancomycin resistant Enterococci and carbapenem

resistant Acinetobacter spp. Corynebacterium from the urine of a comatose patient

is resistant to furazole, trimethroprion, nalidixic acid, cefazolin and floxacin

(Bauer et al., 2003).

Citrobacter freundii belongs to Enterobacteriacece family and it is a facultative

anaerobic gram negative bacilli. It was discovered by Werkman and Gilen in 1932

and it is found in soil organism, water, sewage, food and in the intestinal tracts of

animals and humans. It causes infections such as nosocomial infection, Urinary

Tract Infection (UTI), blood etc. (Nada et al., 2004).

1.2.1. Mode of action of antibiotics

Inhibition of protein synthesis and nucleic acid: Many antibiotics interfere

in different stages of protein synthesis process like elongation of polypeptide

chains, peptidyl transferase reaction, and arrest translation. Antibiotics interfere in

the activities of the enzyme like gyrase and polymerases in DNA synthesis.

Inhibition of a metabolic pathway: Antibiotics target the inhibition of

peptidoglycan biosynthesis by targeting transglycosylation. An antibiotic also

attacks the channels on the membrane which are responsible for the creation

isotonic on the bacterial cell, leading to cell death.

1.2.2. Mode of transmission

MDROs are transmitted from one patient to another through contaminated

hands of direct care workers. Transmission from direct or indirect contact with

infected patients and their environment. Contact transmission is the primary mode

of spread for MDRO. Surfaces and equipment can also become reservoirs of

MDRO and contribute to spread within the healthcare environment. Droplet

transmission may be implicated in the spread of MDRO when the patient has a

respiratory tract infection where the MDRO is causative organism.

1.2.3. Impact of MDROs

Difficult to treat infections

Untreatable infections

Antibiotic use increases the spread of antibiotic-resistant bacteria

Costs

Antibiotic-resistant bacteria spread internationally.

 Increasing antimicrobial resistance is threatening due to limited number of

new antimicrobial agents that are in development. MDR is severe with patients

having high morbidity score and mortality rate often with unusual

complications (Bhutta, 1996).

1.2.4. AFLP (Amplified Fragment Length Polymorphism)-Molecular marker

Each gene has a particular place along the chromosome called locus. Due to

mutations, genes can be modified in several forms called alleles (or allelic forms).

Molecular markers are loci markers related to DNA (markers can also be

biochemical, or morphological) and AELP is one such molecular marker. They are

dominant markers and are capable of detecting high amount of polymorphism

making it an efficient marker.

This method is based on PCR amplification of selected restriction fragments of

a total digested genomic DNA. Amplified products are separated by

electrophoresis and the DNA fragments obtained range from 60 to 500 base pairs.

In order to visualize DNA polymorphism amplification is necessary. This

amplification is commonly done by Polymerase Chain Reaction. The PCR method

can amplify specific DNA fragment by using specific primers for polymerization

at each end of the target DNA. Primer (short oligonucleotide sequences) anneal to

the template DNA in the target zone. Primers are 18-24 base pairs long and

correspond to a complementary DNA sequence in the flanking regions of the

strand of the target DNA. PCR begins with a high temperature phase

(denaturation) and produce single-stranded DNA. Once temperature has reached

primers will bind to the template DNA. Taq polymerase will recognizes each

double-stranded DNA and continue the polymerization reaction in the direction

5-3 as soon as the temperature has reached 72C (optimal elongation

temperature). Hence to identify specific primers sequences of flanking regions of

the target DNA must be known. The AFLP primers are called adapters and

consist of a known sequence of 20 nucleotides. The target DNA sequences are

DNA fragments generated by restriction enzymes. Fragments are produced from

total genomic DNA by the combined action of two restriction enzymes. Adapters

are ligated at each end of a restriction fragment by a protein ligase. Finally,

adapters are used in a PCR as priming sites to amplify the restriction fragments.

AFLP marker is a dominant marker because homozygotes and heterozygotes of a

breed/pedigree are studied in order to determine inheritance patterns of each

fragment.

AFLF technique is inefficient in differentiating between strains because may be

only a subset of chromosomal DNA fragments is amplification and thus only a

portion of varying sites between different strains is examined (Mueller and

Wolfenbarger, 1999).