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Year : 2015  |  Volume : 33  |  Issue : 1  |  Page : 136--138

Phenotyping of Salmonella serotypes isolated from natural sources of water in rural areas of East Sikkim

S Poonia, TS Singh, DC Tsering 
 Department of Microbiology, Sikkim Manipal University, Gangtok, Sikkim, India

Correspondence Address:
S Poonia
Department of Microbiology, Sikkim Manipal University, Gangtok, Sikkim


The objective of the study was to conduct bacteriological analysis of water with special reference to Salmonella spp from natural sources of rural habitations of East Sikkim. A total of 28 Salmonella serovars isolated were biotyped, phage typed and tested for their anti-microbial susceptibility. All the isolates of Salmonella enterica serovar Typhi belonged to Biotype I. Four isolates of S. typhi belonged to phage type A. All S. paratyphi A isolates belong to phage 2. All the isolates were sensitive to chloramphenicol, cefixime and amikacin. Untreated natural water sources are unsafe for human consumption.

How to cite this article:
Poonia S, Singh T S, Tsering D C. Phenotyping of Salmonella serotypes isolated from natural sources of water in rural areas of East Sikkim.Indian J Med Microbiol 2015;33:136-138

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Poonia S, Singh T S, Tsering D C. Phenotyping of Salmonella serotypes isolated from natural sources of water in rural areas of East Sikkim. Indian J Med Microbiol [serial online] 2015 [cited 2021 Mar 2 ];33:136-138
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Enteric fever is a common worldwide illness, transmitted by the ingestion of food or water contaminated with faeces from an infected person. The World Health Organisation has estimated that annually typhoid fever accounts for 21.7 million cases and paratyphoid accounts for 5.4 million cases globally. [1] In India, Central Bureau of Health Intelligence data showed a continuous rise in number of enteric fever cases (488, 033-1, 062, 446) from 2002 to 2011. [2]

In the rural areas of Sikkim, untreated natural surface water is used for drinking and other domestic purposes. The data of the Integrated Disease Surveillance Project (IDSP), Sikkim for the period 2009-2011 have shown a continuous rise in enteric fever cases although there was slight decrease in 2012. [3] The objective was to determine the prevalent phage types and biotypes of Salmonella serotypes isolated from natural sources of water in rural areas of East Sikkim, India. The data of this study may provide some important information about public health risks associated with water quality in this region.

 Materials and Methods

A total of 225 water samples, 75 each in winter, summer and monsoon from the same source were collected from rural habitations of East district of Sikkim, India during the period from November 2011 to October 2012 from 75 villages. Sources of water in rural areas are springs and streams. The samples were collected in sterile screw capped bottles, kept in ice box and transported to microbiology laboratory within 6 h of collection for bacteriological analysis. [4] Microbial quality assessment was done using the standard membrane filtration technique. [4] Each sample (100 mL) was filtered through a 47 mm diameter, 0.45 μm pore size cellulose acetate MF grid (Millipore) held in a filtration unit. Then membrane, with upper side upwards with the help of sterile flat ended forcep was transferred to the surface of MacConkey agar. Plates were incubated at 37°C overnight. After incubation, colonies grown on membrane filter were sub-cultured on the MacConkey agar. The primary identification of the bacterial isolates was done by the conventional methods of morphological and the cultural characteristics. Confirmation of bacterial species was done on the basis of biochemical characteristics. All the biochemically identified Salmonella spp. were further confirmed by serotyping, which along with biotyping and phage typing was done at the National Salmonella Phage Typing Centre, Lady Hardinge Medical College, New Delhi.

Antimicrobial susceptibility was performed by the Kirby Bauer disk diffusion method and results were interpreted as per Clinical laboratory standard institute (CLSI) guidelines. [5] Anti-microbial disks were obtained commercially (Himedia, Mumbai, India). The anti-microbial agents tested were ampicillin (10 μg), cefixime (5 μg), tetracycline (30 μg), ofloxacin (5 μg), imipenem (10 μg), piperacillin/tazobactam (100/10 μg), trimethoprim/sulfamethoxaole (1.25/23.75 μg) and chloramphenicol (30 μg).


In the present study out of 28 (5.1%) Salmonella serovars isolated from natural sources of water, 18 (64.3%) were identified as Salmonella enterica serovar Typhi and 10 (35.7%) as Salmonella enterica serovar Paratyphi A. [Table 1] shows the phage typing and biotyping of Salmonella typhi. All the isolates of S. typhi showed sensitivity to all the antibiotics except one strain belonging to phage A that showed resistance to ampicillin, ofloxacin and tetracycline. All S. paratyphi A isolates belong to phage 2 and were sensitive to all the antibiotics tested except one strain that was resistant to ampicillin. S. paratyphi A was found in water samples of 10 places, in summer: Namin, Shyagyong, Navey, Linkey, Nimachen, Deoling and in monsoon: Sajong, Nampong, Mamring, Sisney.{Table 1}


All the isolates of S. typhi belonged to biotype I. This finding was similar to earlier reports from Chennai and Maharashtra, where all isolates belonged to biotype I. [6],[7] In contrast, a study in Tamil Nadu showed that all isolates belonged to biotype IV. [8] Among S. typhi phage A (22.2%) was isolated. In contrast, studies from Chennai and Maharashtra showed high prevalence of phage E 1 ( 80%). [6],[7] A study conducted by Lady Hardinge Medical College for the period 1982-1987, showed Phage A as the predominant phage followed by E 1 in India. [9] Another study conducted by Lady Hardinge Medical College for the period 1990-1992, showed Phage E 1 as the most common followed by O and A in India. [10]

In the present study Salmonella enterica serovars Typhi and Paratyphi A showed significant sensitivity to first line drugs for enteric fever, chloramphenicol (100%), ampicillin (96.4%) in contrast to other reports indicating up to 92% of multi-drug resistance. [11],[12] Similar to our study some studies showed high sensitivity to ampicillin and chloramphenicol. [6] S. typhi, which has been reported many times by the researchers as resistant to chloramphenicol, is now found 100% sensitive in this study along with cefixime and amikacin. This is in full agreement with the reports of re-emergence of sensitivity of S. typhi to chloramphenicol. [13]

Although chloramphenicol and other antibiotics showed 100% sensitivity, still continuous evaluation of sensitivity-resistance pattern of Salmonella spp. isolates is necessary to make rational use of antibiotics in the management of enteric fever in future.

Isolation of pathogenic microorganisms such as Salmonella spp. is of public health importance as it indicates that water is unsafe. The pathogens mostly transmitted via drinking water are predominantly of faecal origin. The occurrences of pathogenic bacteria in drinking water may increase the risks of water-related diseases and health problems in local residents of these areas, especially in children. The present study will be useful in water borne disease control and prevention strategy formulation. Educating people in rural communities about pathogenic waterborne bacteria and facility for safe drinking water are recommended.


The authors thank Dr Renu Dutta, Professor and Head and the technical staff, Department of Microbiology, Lady Hardinge Medical College, National Salmonella Phage Typing Reference Centre, New Delhi, for phage typing and biotyping of the Salmonella enterica serovars.


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