Indian Journal of Medical Microbiology, (2015) 33(1): 136-138 1
Brief Communication
Phenotyping of Salmonella serotypes isolated from natural sources of water in rural areas of East Sikkim *S Poonia, TS Singh, DC Tsering
Abstract 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. Key words: Natural water sources, phenotypes, salmonella serotypes, Sikkim
Introduction
Materials and Methods
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]
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.
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. *Corresponding author (email: Department of Microbiology (SP, TSS, DCT), Sikkim Manipal University, Gangtok, Sikkim, India Received: 21-09-2013 Accepted: 23-02-2014 Access this article online Quick Response Code:
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.148413
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),
January - March 2015
Poonia, et al.: Salmonella serotypes from natural sources of water in East Sikkim
trimethoprim/sulfamethoxaole chloramphenicol (30 μg).
(1.25/23.75
μg)
and
Results 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. Discussion 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 E1 (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 E1 in India.[9] Another study conducted Table 1: Distribution of phage types of isolated S. typhi Village Season Phage types Sajong Summer A (22.2%) Rakdong Monsoon Singaneybas Summer Dikling Summer Rapdang Summer Deg Vi (27.7%) Khese Summer Linzey Summer Pheri Winter Kopchey Monsoon Tumblabong Monsoon Vi Neg (5.5%) Parbing Summer UVS (44.4%) Martam Monsoon Samsing Winter Aho Summer Nimtar Summer Latuk Summer Aritar Summer Markang Monsoon
137
by Lady Hardinge Medical College for the period 1990– 1992, showed Phage E1 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. Acknowledgment
T
UT
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.
References 1.
UT UT
2.
3. 4.
5.
UVS: Untypable Vi strains, T: Typable, UT: Untypable, Values in parentheses indicate percentage
6.
www.ijmm.org
Crump JA, Luby SP, Mintz ED. The global burden of typhoid fever. Bull World Health Organ 2004;82:346-53. Central Bureau of Health Intelligence. National Health Profile. Ministry of Health and Family Welfare, Government of India; 2011. Integrated disease survillence project (IDSP), Health care, Human Services and Family welfare department, Sikkim. In: Collee JG, Marmion BP, Fraser AG, Simmons A, editors. Mackie and McCartney Practical Medical Microbiology. 14th ed. London: Churchill Livingstone; 1996. p. 889-92. Clinical Laboratories Standards Institute (CLSI). Performance of standards for antimicrobial disk susceptibility tests; approved standards. 10th ed. Vol. 29. M02-A10. Wayne: CLSI; 2009. Krishnan P, Stalin M, Balasubramanian S. Changing trends
138
Indian Journal of Medical Microbiology
in antimicrobial resistance of Salmonella enterica serovar typhi and salmonella enterica serovar paratyphi A in Chennai. Indian J Pathol Microbiol 2009;52:505-8. 7. Sahiba ST, Nilekar SL. Phage types of multidrug resistant Salmonella species in a rural area of Maharashtra, India. Bangladesh J Med Sci 2012;11:37-9. 8. Das A, Hari SS, Shalini U, Ganeshkumar A, Karthikeyan M. Molecular characterisation of salmonella enterica serovar typhi isolated from typhoidial humans. Malays J Microbiol 2012;8:148-55. 9. Prakash K, Pillai PK. Vi – Phage types of S. typhi isolated in various regions of India 1982-87. Indian J Med Microbiol 1988;6:241-50. 10. Pillai PK, Prakash K. Current status of drug resistance and phage types of Salmonalla typhi in India. Indian J Med Res 1993;97:154-8. 11. Chande C, Shrikhande S, Kapale S, Agarwal S, Fule RP.
vol. 33, No. 1
Change in antimicrobial resistance pattern of Salmonella Typhi in central India. Indian J Med Res 2002;115:248-50. 12. Manchanda V, Bhalla P, Sethi M, Sharma VK. Treatment of enteric fever in children on the basis of current trends of antimicrobial susceptibility of Salmonella enteric serovar typhi and paratyphi A. Indian J Med Microbiol 2006;24:101-6. 13. Sood S, Kapil A, Das B, Jain Y, Kabra SK. Re-emergence of chloramphenicol-sensitive Salmonella typhi. Lancet 1999;353:1241-2.
How to cite this article: Poonia S, Singh TS, Tsering DC. Phenotyping of Salmonella serotypes isolated from natural sources of water in rural areas of East Sikkim. Indian J Med Microbiol 2015;33:136-8. Source of Support: Nil, Conflict of Interest: None declared.
www.ijmm.org
Copyright of Indian Journal of Medical Microbiology is the property of Medknow Publications & Media Pvt. Ltd. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
Brief Communication
Phenotyping of Salmonella serotypes isolated from natural sources of water in rural areas of East Sikkim *S Poonia, TS Singh, DC Tsering
Abstract 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. Key words: Natural water sources, phenotypes, salmonella serotypes, Sikkim
Introduction
Materials and Methods
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]
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.
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. *Corresponding author (email: Department of Microbiology (SP, TSS, DCT), Sikkim Manipal University, Gangtok, Sikkim, India Received: 21-09-2013 Accepted: 23-02-2014 Access this article online Quick Response Code:
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.148413
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),
January - March 2015
Poonia, et al.: Salmonella serotypes from natural sources of water in East Sikkim
trimethoprim/sulfamethoxaole chloramphenicol (30 μg).
(1.25/23.75
μg)
and
Results 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. Discussion 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 E1 (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 E1 in India.[9] Another study conducted Table 1: Distribution of phage types of isolated S. typhi Village Season Phage types Sajong Summer A (22.2%) Rakdong Monsoon Singaneybas Summer Dikling Summer Rapdang Summer Deg Vi (27.7%) Khese Summer Linzey Summer Pheri Winter Kopchey Monsoon Tumblabong Monsoon Vi Neg (5.5%) Parbing Summer UVS (44.4%) Martam Monsoon Samsing Winter Aho Summer Nimtar Summer Latuk Summer Aritar Summer Markang Monsoon
137
by Lady Hardinge Medical College for the period 1990– 1992, showed Phage E1 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. Acknowledgment
T
UT
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.
References 1.
UT UT
2.
3. 4.
5.
UVS: Untypable Vi strains, T: Typable, UT: Untypable, Values in parentheses indicate percentage
6.
www.ijmm.org
Crump JA, Luby SP, Mintz ED. The global burden of typhoid fever. Bull World Health Organ 2004;82:346-53. Central Bureau of Health Intelligence. National Health Profile. Ministry of Health and Family Welfare, Government of India; 2011. Integrated disease survillence project (IDSP), Health care, Human Services and Family welfare department, Sikkim. In: Collee JG, Marmion BP, Fraser AG, Simmons A, editors. Mackie and McCartney Practical Medical Microbiology. 14th ed. London: Churchill Livingstone; 1996. p. 889-92. Clinical Laboratories Standards Institute (CLSI). Performance of standards for antimicrobial disk susceptibility tests; approved standards. 10th ed. Vol. 29. M02-A10. Wayne: CLSI; 2009. Krishnan P, Stalin M, Balasubramanian S. Changing trends
138
Indian Journal of Medical Microbiology
in antimicrobial resistance of Salmonella enterica serovar typhi and salmonella enterica serovar paratyphi A in Chennai. Indian J Pathol Microbiol 2009;52:505-8. 7. Sahiba ST, Nilekar SL. Phage types of multidrug resistant Salmonella species in a rural area of Maharashtra, India. Bangladesh J Med Sci 2012;11:37-9. 8. Das A, Hari SS, Shalini U, Ganeshkumar A, Karthikeyan M. Molecular characterisation of salmonella enterica serovar typhi isolated from typhoidial humans. Malays J Microbiol 2012;8:148-55. 9. Prakash K, Pillai PK. Vi – Phage types of S. typhi isolated in various regions of India 1982-87. Indian J Med Microbiol 1988;6:241-50. 10. Pillai PK, Prakash K. Current status of drug resistance and phage types of Salmonalla typhi in India. Indian J Med Res 1993;97:154-8. 11. Chande C, Shrikhande S, Kapale S, Agarwal S, Fule RP.
vol. 33, No. 1
Change in antimicrobial resistance pattern of Salmonella Typhi in central India. Indian J Med Res 2002;115:248-50. 12. Manchanda V, Bhalla P, Sethi M, Sharma VK. Treatment of enteric fever in children on the basis of current trends of antimicrobial susceptibility of Salmonella enteric serovar typhi and paratyphi A. Indian J Med Microbiol 2006;24:101-6. 13. Sood S, Kapil A, Das B, Jain Y, Kabra SK. Re-emergence of chloramphenicol-sensitive Salmonella typhi. Lancet 1999;353:1241-2.
How to cite this article: Poonia S, Singh TS, Tsering DC. Phenotyping of Salmonella serotypes isolated from natural sources of water in rural areas of East Sikkim. Indian J Med Microbiol 2015;33:136-8. Source of Support: Nil, Conflict of Interest: None declared.
www.ijmm.org
Copyright of Indian Journal of Medical Microbiology is the property of Medknow Publications & Media Pvt. Ltd. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
