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The Antibiotic Resistance Era

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INTRODUCTION

The greatest threat of the 21st century is the dawn of the anti-biotic resistance era. In the last decade along with increased nosocomial infections, there is the increased cases of MDR and XDR in pathogens in community and hospitals. A greater number of MDR pathogens are the ESKAPE pathogens- which were so named by infectious Diseases Society of America because of their propensity to escape attack by antimicrobial agents. These pathogens are responsible for majority of the nosocomial infections and have the ability to escape the clutches of the immune system.

Due to the unrestricted use of anti-biotics especially in the aquaculture/poultry and over the counter prescription of antibiotics and lowered rate of innovation towards this form of MDR targetting, paving the way for mankind’s entry into post antibiotic era, wherein even minor infections can assume life threatening proportions. Their alarming rate of mutation and their ability of horizontal gene transfer is of very high concern, thus exhibiting a sudden need for more efficient combination of anti-biotics or other forms of resistance modulatory agents that can tackle MDR bacteria.

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Gram negative bacteria, which have the capability to elicit septic shock in some people, and there is increasing proof that antibiotic-mediated release of endotoxins may lead to clinical deterioration (Lepper et al. 2002). Hence, there is a need for antimicrobial agents which do not cause side-effects.

The process of drug resistance falls under different categories as follows

  1. Drug inactivation/alteration- Bacteria secrete enzymes such as beta-Lactamases, chloramphenicol acetyltransferases. They act by hydrolyzing the beta-lactam ring : found in penicillin,monobactams etc (Santajit et al)
  2. Moderation of drug binding sites/targets- Some bacteria develop resistance by modifying the target of binding of the drug thus obtaining resistance. For example, if there is a mutation in the gene which encodes the region of penicillin binding in pencillin binding protein, it results in resistance of the organism towards penicillin.(Fluit et al. 2001)
  3. Change in cell permeability -If there is a modification to the permeability , there can be a reduction in the amount of antibiotic intake The various mechanisms include.
    • Porin Loss- prevents the influx of antibiotics
    • Efflux pumps- extrudes diverse antimicrobial agents
  4. Biofilm formation :- These are complex microbial communities in which microbial cells are embedded in a slimy extracellular matrix made up of EPS, which is composed of polysaccharides, polypeptides and extracellular DNA from the microbes.EPS, lowered metabolism, slow growth, physiological, chemical and genetic heterogeneity contributes to resilience exhibited by biofilms against antimicrobial agents (Fluit et al. 2001).

Alternate strategies

In the last decade, incidences of MDR and XDR as seen in Klebsiella spp, has grown at a staggering rate. The increased prevalence of bacteria with MDR phenotype both in hospital settings and in natural environments over the years has prompted the search for better strategies to combat the same which includes, peptide conjugated oligomers, predatory bacteria, materials with intrinsic antibacterial properties,etc.Relative to other resistance modulatory/novel antimicrobial agents., phages serve as a biological control measure which remains unaffected by MDR status of the bacteria.

In addition they serve as highly targeted and self amplifying antimicrobial agents. Although bacteria can evolve resistance to phages by altering its cell surface receptor, which is employed by the phage for entry into bacterial host, use of phage cocktail instead of monophages can help to circumvent this problem. In this study, phages isolated from water bodies are tested for the antibacterical and antibiofilm efficacy (Exner et al. 2017).

LITERATURE SURVEY

Bacteriophages as effective anti-microbial and anti-biofilm agent

The existence of these organisms have been known for since antiquity, known as the most profuse organisms on the planet. It wasn’t known until the publication by Félix d’Hérelle in 1917. There have been many instances and literature work stating the ability of these organism and their inclusion in medicinal therapy.

They have obtained more recognition by the start of the 21st century due to their innate ability to target bacteria without affecting the human system/gut microbial. Phages provide an additional advantage of reducing the probability of side effects which is frequented with the usage of antibiotics and reduced number of dosages due to their ability to replicate within host organism. The bactericidal effect of phage is through the mechanism of lysis which is the death and breakage of the host cell, leading to liberation of more virulent off-springs which further continue to target host organism.

Mechanism of infection

Lytic phages infect their target by the following steps

  1. Contact
  2. Pinning
  3. Penetration
  4. DNA/RNA injection

Lytic phages take control of the molecular mechanisms of the target group, to produce more phage components to generate and propagate off-springs for further infection and survival if optimum conditions are provided. Without target cell, the phage either dies or enters a phase of inactivation.

Klebsiella pneumoniae as target organism

Klebsiella pneumoniae is a gram negative organism. They are non-motile organisms which are rod shaped. They are found mainly in the skin and intestines, being innocuous here, can cause serious damage if aspirated, with bloody sputum, which can also be accompanied by peridonitits, meningitis etc.As a rule of thumb, Klebsiella spp infections can be seen in immune undermined patients,these being new-world or older people. The virulence factor is mainly attributed to the production of capsule around the bacteria, which substantiates its increased ability to cause upper respiratory tract infections.

Consensus data

It has a mortality rate of around 50% even with various antimicrobial therapy, which can also go upto 100% mortality in people with addiction to alcohol and bacteremia. These organisms are recently known for their MDR. Some studies have shown that plasmids are the source.These are resistant to all Beta-Lactam (Bhatty et al. 2012). The fast growth , complex biofilm mechanisms ,MDR phenotype, large prevalance makes this a suitable model organism to evaluate effect of phages against planktonic and biofilm mode of growth both In-vitro and In-Vivo.

SCOPE OF THE PROJECT

The scope of this project is to test the efficacy of the purified phage from different water bodies and to test it against the current ESKAPE pathogens both In-vitro and In-vivo. Since clinical isolates with MDR phenotype are being employed both for in vitro and in vivo studies, the present work would pave way for understanding virulence and host pathogen interactions of clinical isolates, in addition to testing phage therapy in higher animal models.

EXPERIMENTAL PROCEDURES

Bacterial strains and inoculum

Clinical strains of Klebsiella penumoniae ( U3790, BC2412, BC1994) were procured from Dr.Vaidhehi ,Sundaram Medical Foundation , Chennai. Bacterial strains were maintained on LB agar slants grown in static incubator at 37℃

Sample preparation

10mL of Water is incubated with 1mL of overnight inoculum and 1mL broth and left over night at 30 degrees celsius to enhance number of phages.The sample was centrifuged at 5000 RPM for 5 mins to pellet bacteria. Supernatant containing the putative phages of unknown concentration was stored at 4℃.

Phage isolation and purification

This method is used to enumerate number of phages. Here, 2 layers of different agars are poured on top of each other. The Basal agar and the top agar.(Basal agar-Nutrient agar, top agar- LB agarose -0.4%). The basal agar is the tougher base layer which is poured first. Precautions should be taken such that the nutrient basal agar medium is free of moisture to enhance the ability of the top agar to stick to it. A small dilution of the phages at 10-2,10-4,10-6,10-8,10-10 are prepared by serial dilution. These are incubated with the target bacteria at mid-log phase (OD-0.4) in a 2:1 ratio and are incubated for 1 hour. These are then mixed with 4mL of top agar and poured on top of the Basal agar medium, which forms a thin layer and immobilizes the phages. After the final purification, plaques phages from infecting neighbouring bacteria. (Mullan, W.M.A. (2002). Plaque formation.)

Each infected bacterium bursts after a specific duration and liberates progeny phages that infect adjacent bacteria which are in turn lysed. Plates which have less than 30 and more than 300 plaques cannot be used further. Also size and shape of plaque have to be taken into consideration for further use. This is used to obtain a purified phage culture. An isolated plaque is found and is picked off/up using a inverted micropipette tip. This is incubated in SM buffer and shredded to release phage from the plaque by vortexing at high speed. Using this as initial phage culture, double layer agar method was repeated once more. (Sambrook et al. 1989) Plaques with uniform morphology were washed and collected with 5mL of SM buffer. The plate was washed once more with 3mL SM buffer , the pooled SM buffer was stored at 4℃. This will serve as the phage bank.

Capsule staining

Capsules are produced by organisms such as K.pneumoniae. They can either be made of polysaccharides or polypeptides. Capsules encapsulate the bacteria and protect it from the attack of leukocytes and aid in invasion of the body. The capsules are non-ionic. Hence both acidic and basic stains do not stick to the capsule.The cell can be stained by a basic stain and the background with an acidic stain. If there is a capsule, would have a clear halo region. A drop of nigrosin stain was placed on the coverslip. K.pneumoniae was placed on nigrosin stain and was dispersed using the loop. The mixture was spread using a sterile slide it into a film.Allow the film to air dry. A drop of crystal violet stain was added. Excess stain was removed by washing gently against running water.The slide was observed under the microscope at 100X magnification.

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