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  • Published on: 15th October 2019
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1. Introduction

Water is a vital component in chemical metabolism which plays an important role in the digestion, food absorption, and nutrient transportation in the body. It eliminates the waste products via urine. The role of water in spreading communicable disease is much evident due to combined sources of water available commonly to all.

  Chlorination is the most widely used method of disinfection for community water distribution system and reservoir (Ludovici et al. 1977). It is primarily adopted to destroy or deactivate the disease producing microorganisms in the public water supplies, swimming pools, rivers etc. Chlorination has been practiced for several years.

  On addition of chlorine in water, some of the chlorine first reacts with organic material and metals present in the water and is not available for disinfection, this is called as chlorine demand of water. The remaining chlorine concentration after the chlorine demand is accounted for in disinfection. The chlorine residual is usually tested when the water is finally ready to be released into the distribution system.

   Although the pros and cons of disinfection with chlorine have been extensively debated, it remains the most widely used chemical for disinfection of water (IS: 3025, part 26- Reafirrmed 2003). Chlorine is the most common disinfectant; it is effective in killing most pathogenic bacteria and viruses. It is a strong oxidant that rapidly kills many harmful microorganisms. Large number of microorganisms which are known to be human opportunistic pathogen can be recovered from potable water distribution system maintaining free chlorine residual of  0.5-1.0mg/l.

   The concentration of chlorine in drinking water supplied should be in the ranges from 0 to about 1.5 mg/L (www.hunterwater.com.au). To maintain the purity of water, chorine is added into it. When chlorine is added in water, it breaks down into two chemicals- first almost instantly kills the bacteria and the second remains in water killing any bacteria which is brought in by new bathers. When chlorine is used in right amount it is not dangerous to health. With the correct amount of chlorine added, a pool’s pH level should be in between 7.4-7.6 which also is the pH of human tears.

  If the level of chlorine in pool water is too high it can cause sore throats, skin irritation and red eyes etc. that can be a high risk for the children who regularly swim in chlorinated water.

 In high level of chlorine, sweat and other organic compounds from the skin can react with chlorine as a result generating a harmful fume that causes irritation to the eyes, skin and respiratory system. Since chorine has been used for a century for its powerful sanitizing potential, it is rendered a threat to the lives of people now-a-days.

  There are certain bacteria which are responsible for water contamination such as Salmonella, Camphytobacter spp, Clostridium botulium, Staphylococcus aureus, Pseudomonas aeruginosa, Vibrio cholerae, and Escherichia coli. etc.

  A large number of viable microorganisms have been shown to be human secondary opportunistic pathogens. These microorganisms can be recovered from potable water distribution system. To kill the bacteria present in the swimming pool and drinking water for the safety of people water is chlorinated by the addition of chlorine chemicals such as Sodium hypochlorite and bleaching powder (Calcium hypochlorite). There are some alternatives to chlorine for disinfection process they are UV filtration, ozone etc. Inspite of all, there are some bacteria which can resist in chlorinated water of in the range of about 10ppm.  .

    There are some diseases and infections which are developed from the exposure to chlorinated water in swimming pools: Respiratory defects, Neurological dysfunction, Epiglottitis, Cardiovascular Defects, Eye infection, Skin infection, gastrointestinal issues, Kidney cancer, Liver infection, colorectal cancer.

Pseudomonas:

   Pseudomonas is a Greek word, it comprises of two words- Pseudo means ´False´ and monas means ´unit´ so literally this term meaning is ´false unit´. Pseudomonas is mostly saprophytic in nature. It is found almost every moist place in environment such as soil, water etc. It is rod shaped, slender, gram negative bacteria. It is an important cause of health care associated  opportunistic infections. Most of the isolates of  Pseudomonas  are resistant to many antibiotics.

Scientific classification

Kingdom   : Bacteria

Order   : Pseudomonadales

Family : Pseudomonadaceae   

Genus  : Pseudomonas

Species  : P. aeruginosa

There are eight groups of Pseudomonas: P.aeruginosa, P.chlororaphis, P.fluorescens, P.pertucinogena, P.pudida, , P.stutzeri, , P. syringae, , and  P. incertaesedis.

Pseudomonas aeruginosa:

  Pseudomonas aeruginosa is gram negative, rod shaped, opportunistic bacterial pathogen. It is  present in diverse environment that includes water, soil, and plants. This bacterium is able to infect different organisms including plants, animals and humans (Pereira SG et al., 2014). This opportunistic bacterium is one of the most common cause of health care- associated diseases including urinary tract infections, blood stream infections, pneumonia and surgical site infections especially in patients with compromised host defenses (Pereira SG et al., 2014). It typically infects the pulmonary tract, urinary tract, burns, wounds and it also causes other blood infections. ( Todar’stextbook of biotechnology).  

    P. aeruginosa can also be a common cause of hot- tub rash (Dermatitis) caused by lack of proper attention to water quality. Patients with cystic fibrosis are predisposed to P. aeruginosa infection of lungs.

P. aeruginosa infections are also known to commonly spread through swimming pools and such recreational activity. P. aeruginosa …robust..able to survive in chlorinated water…reference. it

    Specific mechanisms may exist for the survival of certain bacteria and viruses in waters containing relatively high concentrations of chlorine (Hass et al., 1979).

This is particularly important since Pseudomonas infections that were earlier generally treated with antibiotics have progressively developed resistance to several antibiotics that are presently used. Most of the infections caused by P. aeruginosa are strikingly difficult to treat using conventional antibiotic therapies because this microorganism shows high intrinsic resistance to wide range of antibiotics.   

  P. aeruginosa also has an ability to adhere to surfaces, such as those of medical devices or epithelial cells, and to form biofilms consisting of microbial communities embedded in a matrix of extracellular polymeric substances (EPS) ( Garcia-Medina R et al., 2005). Biofilms are frequently associated with chronic infections, since their structure confers protection of bacterial cells from antimicrobial agents (Garcia Medina R et al., 1999). Multidrug – resistance Pseudomonas can be deadly for patients in critical care. Hence, it is important to study the survivability and spread of Pseudomonas aeruginosa and find ways to control it.

Principal of chlorine estimation

    The starch-iodide titration method is one of the oldest method for determination of chlorine. It is very non specific for oxidants and it is generally used for total chlorine testing at levels above 1 mg/l chlorine.

   Chlorine librates free iodine from potassium iodide (KI) solution at the pH 8 or less. The librated iodine is titrated with standard solution of sodium thiosulphate (Na2S2O3) with starch as an indicator. This method is based on the reaction with sodium thiosulphate solution. The end point of the titration is indicated by the disappearance of the blue colored starch iodide complex.

DNA purification and isolation

   DNA is a nucleic acid that carries genetic information from generation to generation which is used in the growth, development and normal functioning of all living organisms. DNA stores bacterial information that can be used in many molecular biology applications. It comprises of two polymeric strands that includes nitrogenous bases viz, Adenine, Cytosine, Guanine and Thymine (or Uracil in place of thymine in case of RNA) and deoxyribose sugar and phosphate group.

The sugar and phosphate groups are located on the surface of DNA which forms the backbone of each strand, while the bases are present on the inside of the structure (Swigon et al., 2009).

Bacteriology molecular techniques usually start with bacterial DNA extraction and purification. A number of DNA extraction methods, which is performed manually or by automation, are still being developed. Each method have their own advantages and disadvantages (Zweifel et al., 2012).

  In general, bacterial genomic DNA isolation includes three main steps viz, cell disruption, DNA extraction and DNA purification. Bacterial DNA is usually extracted with an extraction buffer and is further purified with phenol-chloroform followed by ethanol or isopropanol precipitation (Dashti et al.,2009). Standard methods for the isolation of bacterial DNA mostly rely on cell lysis using combination of heat, detergents and enzymes followed by extraction of the DNA with phenol-chloroform (Ahmed et al., 2014).

Polymerase chain reaction

   Polymerase chain reaction (PCR) is a nucleic acid amplification method. It was developed in the mid 80s and has been used in the detection of waterborne pathogens. Each PCR assessment requires the presence of primers, nucleotides, template DNA and DNA polymerase. DNA polymerase is the key enzyme that connects individual components together to form the PCR product (Ibrahim et al.,2014). Denaturation, annealing, and extension are the three major steps involved in the PCR technique. In first step, DNA is denatured at high temperature. In the second step primers are annealed for primer extension to the DNA template strand. In the third step extension occurs at the end of annealed primers creating a complementary copy strand of DNA (Garibyan  et al.,2010).

   

  The enumeration and isolation of water borne pathogens are commonly performed using culture based methods. These methods are inexpensive, sensitive and provide both quantitative and qualitative evaluation of microorganisms present in the water sample but they are time consuming (Moore et al., 2004). Various rapid methods have been developed with high sensitivity and specificity to overcome the constraints of conventional methods for the specific identification and detection of bacterial water borne pathogens (Valderram et al., 2013).

Fig1.2: polymerase chain reaction (courtesy: en.wikipedia.org)

Bacteriophages as a Biocontrol Agent  

   Bacteriophages are bacterial viruses that infect bacteria and replicate within their specific host bacterium. Felix  d´Herelle, was the first who introduced the use of phages as biocontrol agent for bacteria (Curtis et et al.,2014). Bacteriophages can be isolated by: Coal bed method, Enrichment method and Membrane enrichment technique. These bacteriophages are considered to be the most ubiquitous and diversified organisms on the earth and are believed to exist wherever their host thrive (Ceyssens P-J et al., 2010)  

 Phages primarily consist of a nucleic acid molecule that is surrounded by protein coat (capsid). Capsid is made up of capsomeres and consists of a number of protein molecules or subunits, called as protomers. Some phages also contain lipid and additional structures such as tails, spikes etc.

    According to their life cycle, phages can be distributed into two classes: virulent phages and temperate phages (Guttam et al.,2004). Lytic phages adsorb to the host cell surface, inject and replicate their DNA, and then induce host cell lysis, resulting in the release of progeny phages that can start another round of infection. Temperate phages generally integrate their genome into the host chromosome or sometimes maintain it as a plasmid which is transmitted by cell division to the daughter cell (Guttam et al., 2004).

    

Fig1. 3 : Structure of bacteriophage  Fig1.4 : Life cycle of Bacteriophage

    

  The use of phages as antimicrobial agents was proposed by Félix d’Hérelle  in 1917 (d’Hérelle F. et al., 1917). The inadequate use of antibiotics has significantly increased the emergence of multidrug-resistant (MDR) bacteria, and new alternative strategies for antibiotherapy are highly desired by the worldwide scientific community (Levy SB  et al 2014). Consequently, a new interest was given to phages and they have been again come to be considered a good option for the treatment of bacterial infections (Kutateladze M et al., 2010).

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