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E Coli Identification: Biochemical Tests

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In Canada, the research shows that at least one coliform or pathogenic bacterium present in 28% of water dispensed in workplaces when compared to 22% of contaminated tap water samples (Rim, 2009). The quality of drinking water dispensed from water cooler were complying with the WHO drinking water standard and Egyptian drinking water standard that concluded that microbial contamination had occurred inside the water cooler. The researchers recommended that the water dispenser must be cleaned and sanitised for every 2 months to limit the extent of contamination.

E.coli as a biological indicator for determination of water quality

E. coli can be used as biological indicator for water treatment safety (Mabel & Roymon, 2012). Biological indicator is required to monitor continued effectiveness of water treatment in vending machines. E. coli is one of genus of fecal coliforms. E. coli can survive in drinking water for long time and may exist in high numbers in the faeces of warm-blood animals. It can be easily detected in drinking water. Besides that, E. coli is more thermo tolerant than other fecal coliform bacteria for production of acid and gas and it is more sensitive to disinfection when compared with other pathogen (Hertin, 2011).There is only a small fraction of E. coli that can cause diseases such as diarrhoea. The common strain of E. coli that cause severe cases of breaches in public health is E. coli O157:H7 (Neil et al., 2012) and a rare serotype of E. coli O157:H4. (Soon et al., 2013).

Most strains of E. coli produce β-glucuronidase at 37 °C. But stains that do not express them at 44 °C may also be E. coli. Biochemical tests can be used to detect the presence of E. coli in water samples from vending machines (Hertin, 2011). E. coli are oxidase-negative that produce acid from lactose after incubation for 48 hours. With presence of E. coli, production of indole from tryptophan can turn the indicator dye methyl red to yellow by lowering the pH to less than 4.5. The coliform bacteria that have this particular characteristic will be considered as E.coli (Shar et al., 2008).

According to Ahmed et al., (2009), E. coli ranging from 43 to >2400/100 ml was found in different types of squeezed fruit juices dispersed from vending machines in Dhaka city, Bangladesh. Besides that, Peters et al., (2008) examined different variety of drinks vending machines including semi and fully automatic machines and fresh-brew machines within 6 months. The reports shows that 30% swab samples collected from these vending machines's nozzle had a unacceptable total viable count (over 106 CFU/swabs) and 23 swab samples had type I E. coli. Thus, E. coli can be used as microbial indicator for drinking water samples from water vending machines.

Sources of cross contamination

Cross contamination of water samples can occur when consumers or pests contact with the outer parts of machines (Brooke et al., 2009). Insufficient hand washing handles hygiene can lead to microbial contamination of water from vending machines when bacteria are introduced from the outer part of machines into the dispensed water. Previous research showed that customers touch the spout from which water are dispensed from vending machines can allow coliform bacteria to be transmitted from the customer to the spout (White et al., 2010).

According to Hertin (2011), there are highly variable concentration of bacterial colonies present in the samples from soda machines at different sites of collection. This variability indicates the machines maintenance and sanitation performed by vending machine contractor or the age of the vending machine. Age of vending machines used can affect the variability of bacterial survival and reproduction. From time to time, machinery parts will deteriorate and possibly create biofilm formation as a microhabitats for microorganisms (Bloomfield et al., 2012).

Most probable number (MPN) test

Most probable number (MPN) test is one of the most widely methods used for enumeration of coliforms per 100 ml of drinking water in order to prevent transmission of waterborne diseases (Mudili, 2007). MPN test take 72 hours to produce result from the experiment. This test is a simple and reliable field test that provide significant result on international drinking water supply. MPN test can also used to determine the efficiency of a treatment process in water vending machine in order to detect the presence of coliform bacteria in drinking water ( Suthar et al., 2009).

MPN test is involved in inoculation of three set of selective broth with a known amount of diluted water samples as the standard protocol in order to determine sanitary quality of drinking water from vending machines. To identify the population density of viable coliform bacteria in a water samples, it is based on the probability of the number of positive result in each test tube as the series of diluted water samples inoculated in a set number of Macconkey broth. High dilutions of the samples will show fewer positive result in a series of culture tube with culture medium. When high population density of coliform bacteria present in water sample, the water samples must be diluted to the detected range where the MPN result can be detected. The method of tenfold serial dilution are used in three, five or ten tube MPN series. When decreasing the interval of the dilution factors, the accuracy of the MPN series increase dramatically (Sutton, 2010). High number of tubes are lowered the confident limit of MPN value that showed significant result.

16S rRNA analysis

Identification of strains of E. coli is a molecular method to detect fecal contamination in food and water samples and as a measurement for sanitary quality (Casarez, 2007). Presence of E. coli in the water samples can be pathogenic strains and non-pathogenic strains. Generally, the strains of E. coli present in the water samples are mainly non-pathogenic. According to Verma et al., (2007), the study shows that pathogenic bacteria such as enterotoxigenic and shiga-toxic producing E. coli can be detected in the water samples. Pathogenic bacteria mainly cause intestinal and systematic illness of human. 16S rRNA analysis are used to determine the genetic diversity and the probability of clone similarities among the strains of E. coli. Ribosomal RNA molecules pose different regions consists of nucleic acid sequence allow the identification of bacteria at the species and strain level (Elgaml et al., 2013). 16S rRNA analysis is a technology that provided new possibility to enhance selectivity and specificity of E.coli without requirement for complex cultivation and additional confirmation procedures.

16S rRNA for identification of strains of E. coli present in water samples focused on the use of PCR gene probe technology against the lacZ and the malB operon. According to Bej et al., (1990), the first target sequence proposed is the region encoding the maltose transport protein. E. coli specific bacteriophage recognise the surface protein encoding the lamB gene in order to detect specificity of E. coli. Presence of nine hyper-viable region in bacteria 16S rRNA indicate that the considerable sequence among different bacteria species can be used for detection of species and allowed PCR amplification of target sequence. The study had been focus on DNA sequences encoding invasion proteins, toxins, catabolic enzyme, structure lipoprotein and the V3 and V6 region of the 16S rRNA for detection of pathogenic and non-pathogenic strains of E. coli. E. coli belong to the Enterobacteriaceae family. To design the specific detection of all strains of E. coli, PCR primers such as 16E1, 16E2 and 16E3 are selected for 16S rRNA analysis. PCR primers applied on the identification of strains of E. coli in drinking water were successful. 16 S rRNA can be determined by Genbank database. Genbank is the largest database that has over 90000 nucleotide sequence to compare the sequence of unknown strain of E. coli (Jiii, 2004). Sequence identification can provide degree of the similarity sequence to define target region in term of taxonomic levels. High level of rRNA molecules is the interest in targeting rRNA molecules which is linked to the ribosome per cell.


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