Presence of NDM-1 Gene in Bacteria in Delhi waters

Investigatory Project in Biology By, Avyaya Bhushan, Springdales School Roll No:

Acknowledgements
I would like to express profound gratitude to my teacher, Mrs. Alka Verma, for her valuable support, encouragement and useful suggestions throughout this project work. Her moral support and continuous guidance enabled me to complete my work successfully

I would like to also express my gratitude to Dr. (Prof.) (Mrs.) Rajni Gaind, for allowing me to use her laboratory premises for the conduction of my experiment, for without this, my project would have been impossible to produce. -Avyaya Bhushan

Index

S. No. 1. 2. 3. 4. 5. 6.

Contents Acknowledgments Apparatus Drugs Required Antibiotics Beta-Lactamases The NDM-1 Gene

Page no. 1

Apparatus
Petri Dishes Culture Medium (Liquid peptone-glucose, Solid agar gel) Incubation Chamber Samples of water

Drugs Required Beta-Lactam antibiotics o Penicillin derived antibiotic (Penam) o Cephalosporins (Cephems) o Monobactams o Carbapenems Polymixin Tigecycline

*Note: All aforementioned drugs are non-prescription antibiotics.

Antibiotics
Antibacterial, or otherwise known as antibiotic, is an agent that inhibits bacterial growth or kills bacteria. This happens mainly through their actions on the cell walls (penams and cephems) or their cell membranes (polymixin) and there are two main classifications of antibiotics, narrow range (Tigecycline) or wide range (penams, etc.) Widely used antibiotics are from the beta-lactam antibiotics. Out of which, carbapenams are considered to be the strongest variety Penam- These are a subclass of the broader -lactam family of antibiotics and related compounds. Penams contain a lactam ring fused to a 5-membered ring, where one of the atoms in the ring is sulphur and the ring is fully saturated. Penicillin is a member of this family of compounds (hence the name of the family).

Cephalosporins- These are a class of -lactam antibiotics originally derived from the fungus Acremonium, which was previously known as "Cephalosporium". Together with cephamycins, they constitute a subgroup of lactam antibiotics called cephems.

Monobactam- These are -lactam compounds wherein the lactam ring is alone and not fused to another ring (in contrast to most other -lactams, which have at least two rings). They work only against Gram-negative bacteria.

Carbapenem- These are a class of -lactam antibiotics with a broad spectrum of antibacterial activity. They have a structure that renders them highly resistant to most lactamases.

Beta Lactamases
These are enzymes produced by some bacteria and are responsible for their resistance to beta-lactam antibiotics. these antibiotics have a common element in their molecular structure: a four-atom ring known as a beta-lactam. The lactamase enzyme breaks the -lactam ring open, deactivating the molecule's antibacterial properties.

NDM-1 Gene (New Delhi Metallo-beta-lactamase 1)

Metallo-beta-lactamase-1 (NDM-1) is an enzyme that makes bacteria resistant to a broad range of beta-lactam antibiotics. These include the antibiotics of the carbapenem family, which are a mainstay for the treatment of antibiotic-resistant bacterial infections. The gene for NDM-1 is one member of a large gene family that encodes beta-lactamase enzymes called carbapenemases. Bacteria that produce carbapenemases are often referred to in the news media as "superbugs" because infections caused by them are difficult to treat. Such bacteria are usually susceptible only to polymyxins and tigecycline. Also, it is noted, that this gene, was first detected in an Indian origin Swedish patient after he was infected with Klebsiella pneumoniae after which, it was found that it was rampant in waters in hospital, possibly arising due to excessive use of antibiotics and their concentrations in waters in hospitals.

Theory
Liquid Culture: Overnight culture/growth of a source of bacteria in a liquid culture medium to concentrate the bacteria for later analysis Perti-Dish Culture: Some sample taken from the liquid culture is put on the agar plates (Innoculation of petri dish) and allowed to grow for a set amount of time at 37oC for human/animal cultures In my case, I shall be using a peptone-glucose culture solution for my liquid culture and a solid agar gel for my gel plate culture. For each of my water samples, I shall be making 7 petri dish cultures, 1 control culture 1 penam based agar gel culture 1 cepham based agar gel culture 1 carbapenem based agar gel culture 1 monobactam based agar gel culture 1 tygecycline based agar gel culture 1 polymixin based agar gel culture

for a total of 70 petri dish cultures.

Antibiotic Resistance: Antibiotic resistance is a form of drug resistance whereby some (or, less commonly, all) subpopulations of a microorganism, usually a bacterial species, are able to survive exposure to one or more antibiotics. Accordingly, pathogenic species which have become resistant cause infections which can not be treated with the usual, formerly efficacious antibiotic drugs and/or their usual, formerly efficacious, dosages and concentrations. Resistance may be instrinsic or acquired. Some clinically relevant pathogens have developed resistance to multiple antibiotics and are dubbed multidrug resistant. More recently, the colloquial term superbug has become widespread in both popular and technical accounts of the phenomenon with which it is synonymous.

Procedure
1. Water samples taken in a sterile manner (as far as possible) and transferred to the primary liquid culture medium. 2. Liquid medium placed in an incubation chamber at 37oC for approximately 24-26 hours 3. Small amount of solution taken from aforementioned liquid medium and inoculated on the petri dishes 4. Petri dishes then incubated for another 24-26 hours and then observations recorded. 5. Contents of the petri dishes then transferred into a biohazard disposal box and discarded in a safe way (burned)

Observations
Presence of bacterial colonies in dishes containing following antibiotics S.No. Penam Cephem Carbapenem Monobactam Polymixin Tigecycline Control

1 2 3 4 5 6 7 8 9 10 Water samples and their locations. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Control sample: UV, RO and chlorine treated water Water from Najafgargh Drain Water from main raw water supply- NDMC area Water from main raw water supply- DDA are Water from underground wells Water from AIIMS Hospital Water from Safdarjung Hospital Water from tanker supplies Water from Yamuna river banks (before treatment) Water from Yamuna river banks (after treatment)

Result
It was observed that while the control experiment had a few colonies of bacteria in the control dish, and more or less none in the dishes with antibiotics, there was an alarmingly large concentration of colonies in water sample #2, #6, #7 and #9, while the rest had a varying range from almost nil to low, to medium high concentration of bacterial colonies* However, in the case of dishes containing polymixin and tigecycline, there was almost nil bacterial growth. We have thus showed that bacteria with NDM-1 gene is present in Delhi waters, and this is a risk that should be met with a lot of carefulness as it can lead to severe harm in human health.

Bibliography