|Year : 2003 | Volume
| Issue : 1 | Page : 17-20
Search for shiga toxin producing escherichia coli (STEC) including 0157: H7 strains in and around Kolkata
UK Chattopadhyay, S Gupta, S Dutta
Department of Microbiology, All India Institute of Hygiene & Public Health, Kolkata - 700 073, India
All India Institute of Hygiene & Public Health, Kolkata - 700 073, India
PURPOSE: An attempt was made to isolate and characterize Shiga toxin producing E. coli (STEC) from animals handlers, animal products and admitted diarrhoeic children in and around Kolkata. METHODS: A total of 415 samples were processed for detection of STEC by PCR and colony hybridization techniques. RESULTS: 0 (4.81%) samples were found to be positive for STEC. Diarrhoeic cattle accounted for maximum (22.1 %) isolation. CONCLUSIONS: The study showed PCR to be more sensitive than hybridization technique for detection of STEC.
|How to cite this article:|
Chattopadhyay U K, Gupta S, Dutta S. Search for shiga toxin producing escherichia coli (STEC) including 0157: H7 strains in and around Kolkata. Indian J Med Microbiol 2003;21:17-20
|How to cite this URL:|
Chattopadhyay U K, Gupta S, Dutta S. Search for shiga toxin producing escherichia coli (STEC) including 0157: H7 strains in and around Kolkata. Indian J Med Microbiol [serial online] 2003 [cited 2014 Jul 28];21:17-20. Available from: http://www.ijmm.org/text.asp?2003/21/1/17/8309
The role of Escherichia More Details coli in causing enteric infections in human beings is well known. Enterohaemorrhagic Escherichia More Details coli (EHEC) is one of the recently encountered diarrhoegenic strains of E. coli. EHEC is also known as verocytotoxin producing E. coli (VTEC). Since VTEC also produce a Shiga like toxin (SLT) an alternative nomenclature “Shiga toxin producing E. coli” (STEC)has also been proposed. EHEC/VTEC/STEC are often incriminated to possess zoonotic association particularly of bovine species.5 Since data relating to the occurrence of STEC amongst animals, animal handlers and some of the animal products are very sparse in and around Kolkata, the present study was carried out with the following objectives: 1) To study the presence of Shiga toxin producing Escherichia More Details coli (STEC) in domestic animals, dairy cattle and their handlers. 2) To study the occurence of 0157:H7 in fresh beef, pork, goat meat from slaughter houses and grocery stores in Kolkata. 3) To identify the prevalence of STEC in hospitalized cases of bloody diarrhoea in Kolkata. 4) Phenotypic and genotypic characterization of STEC strains after isolation.
| ~ Material and Methods|| |
A total of 415 samples belonging to different categories viz. animal faeces (77 diarrhoeic, 78 healthy), faeces from 50 animal handlers, 60 food samples (50 beef, 10 pork) and 150 faeces samples from diarrhoeic children admitted to a hospital in Kolkata were collected from December, 1999 to May, 2000. Animals belonged to Government owned farms like intensive cattle breeding project (ICBP) farm from Haringhata, West Bengal, state livestock farm and private dairies located around Kolkata. Raw meat samples were also collected from Kolkata Municipality Corporation slaughter house and also from local retail shops in the City. Visits were made twice a week during entire period of the study.
Faecal samples from adult animals were collected aseptically per rectum while from calves by rectal swab. Samples from animal handlers were collected in sterile wide mouthed containers. Stool samples from the children admitted to the hospital were also collected in the same way as of animal handlers. Rectal swab were transported to the laboratory in Cary-Blair transport media while stool samples were capped tightly in the containers and transported to the laboratory in ice packed container. Ten gram of raw minced meat pieces were collected in sterile MacCartney bottles containing ringer lactate solution and kept in ice packed container for transportation to the laboratory for further processing.
All the food and faecal samples were homogenised for one minute and enriched in Luria broth incorporated with cefixime (0.05 mg/litre) and /or peptone water for enrichment and then incubated overnight for 12 - 18 hours.
DNA was extracted from overnight enriched cultures. Polymerase chain reaction was performed using crude DNA extracts as template DNA. For food samples, peptone water culture was used as template DNA. Samples which gave positive results in PCR were diluted 100 fold with phosphate buffer saline (PBS), and then were incubated onto sorbitol MacConkey agar (SMAC) containing cefixime for isolated colonies.
The colony hybridization was performed by stx1 and stx2 DNA probes to detect the presence of stx1 and stx2 genes in the respective colonies.7 Non-radioactive nucleic acid labelling and detection system (ECL, Amersham Biosciences) was used for autoradiography. The DNA probes consisted of 1142-bp Bam HI fragment cloned into pUC 19 for stx1 and 842-bp SmaI-PstI fragment cloned to pUC 18 for stx2 probe.
The isolated stx gene containing strains (STEC) were characterized by; a) Serotyping with “0” and “H” antiserum (Denka Seiken Co. Japan) using slide and tube agglutination method, b) biotyping using fermentation test with sorbitol, dulcitol and raffinose and decarboxylase test with lysine, arginine and ornithine, and c) antimicrobial susceptibility test by disc diffusion method.8 Mueller-Hinton agar plates were used to test the susceptibility of STEC isolates against the following antimicrobial agents (HI MEDIA): amoxycillin (30 µg), amikacin (30 µg), cephalexin (30 µg), cefotaxime (30 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), co-trimoxazole (25 µg), furoxone (100 µg), gentamicin (10 µg), nalidixic acid (30 µg), norfloxacin (10 µg), tetracycline (30 µg).
| ~ Results|| |
A total of 20 STEC strains were positive by PCR test out of 415 samples of different categories screened. The isolation rate is highest in diarrhoeic cattle where 17 (22.07 %) were found positive for STEC out of 77 samples screened. One out of 78 healthy cattle and 2 out of 150 diarrhoeic children admitted to the hospital were also found positive for STEC [Table - 1]. No STEC could be recovered from animal handlers as well as food samples.
Thirteen strains out of 20 samples positive by PCR test could be isolated and characterized for biotyping, serotyping and antibiogram typing [Table - 2]. All strains were untypable against 0157:H7 antisera but all of them possessed stx gene. The strains also showed variations in sugar fermentation and decarboxylation test. Antimicrobial susceptibility pattern of STEC showed that 68.2%, 61.5%, and 46% were resistant to cotrimoxazole, furoxone and tetracycline/nalidixic acid respectively. Most of the strains were sensitive to norfloxacin, gentamicin and chloramphenicol.
In PCR test all the 20 samples showed presence of stx gene at 521 bp region when stained in ethidium bromide gel [Figure - 1], [Figure - 2]. On colony hybridization with stx1 [Figure - 3] and stx2 [Figure:4] gene probes, out of 20 samples that were positive by PCR test, 10 contained both stx1 and stx2 genes while 3 contained only stx2 gene.
| ~ Discussion|| |
STEC, an emerging zoonosis, has been associated clinically with a wide range of presentation extending from a symptomatic infection to severe bloody diarrhoea or haemorrhagic colitis, which can lead to life-threatening sequelae like haemolytic uraemic syndrome. The aetiological role of STEC in causing individual infections as well as outbreaks in the developed countries has been identified while reports from developing countries like India and Bangladesh are sparse. E.coli belonging to 0157 serogroup and possessing verotoxin gene have been isolated from India from animals, animal handlers and raw beef and a zoonotic correlation has also been worked out.
In this study, an attempt was made to identify, isolate and characterize STEC from animal, food and human sources with the help of available modern methods. The samples were collected from different sources like organized dairy farms, local dairy units, slaughter house and butcher shops in and around Kolkata. Out of 20 PCR positive samples, 13 were positive with stx1 and / or stx2 DNA probes on colony hybridization. This study showed PCR to be more sensitive than hybridization technique. The isolation of STEC was more in diarrhoeic calves in the present study which correlated with the study of Brunen's et al.
Rearing of cattle is a very common practice in West Bengal particularly in the rural areas. Again, proper hygienic practices and sanitary measures are lacking while handling of the cattle in these areas. Thus, it may be presumed that the diarrhoeic cattles, particularly the calves, can be an important source of STEC causing human enteritis in Kolkata. Therefore, emphasis should be given to screen the children suffering from diarrhoea for presence of STEC as a part of surveillance system. This will enable to reveal the actual magnitude of the problem caused by STEC and also give early warning regarding any outbreaks in future.
| ~ References|| |
|1.||Gross RJ. The pathogenesis of Escherichia coli diarrhoea. Rev Med Microbiol 1991;2:37-44. |
|3.||Riley LW, Remis RS, Helgerson SD, McGee HB, Wells JG, Davis BR, Bebert RJ, Olcott ES, Johnsosn LM, Hargrett NT, Blake PA, Cohen ML. Haemorrhagic colitis associated with a rare Escherichia coli serotype. N Engl J Med 1983;308:681-685. |
|5.||Konowalchuk J, Speris JI, Staveric S. Vero response to a cytotoxin of Escherichia coli. Infect Immun 1977;18:775-779. |
|7.||O'Brein AD, La Vech GD. Purification and characterization of a Shigella dysenteriae-I like toxin produced by Escherichia coli. Infect Immun 1983;40:675-683. |
|9.||Bentin LO, Geier D, Strinruck H, Zimmermann S, Schentry F. Prevalence and some properties of verotoxin (Shiga like toxin) producing Escherichia coli on seven different species of healthy domestic animals. J Clin Microbiol 1983;31:2483-2488. |
|11.||Yamasaki S, Lin Z, Shirai H. Typing of verotoxins by DNA colony hybridization with poly-and oligonucleotide probes, a bead-enzyme-linked immunosorbent assay and polymerase chain raction. Microbiol Immunol 1996;40:345-352. |
|13.||New land JW, Neill RJ. DNA probes for Shiga-like tomixs I and II for toxincoverting bacteriophages. J Clin Microbiol 1998;26:1292-1297. |
|15.||Baur AW, Kirby M. Antibiotic susceptibility testing by standardized single disc method. Am J Clin Pathol 1966;45:493-496. |
|17.||Dutta S, Deb A, Chattopadhyay UK. Isolation of Shigatoxin producing Escherichia coli including 0157:H7 strains from dairy cattle and beef samples marketed in Calcutta, Indian J Med Microbiol 2000;49:765-767. |
|19.||Armstrong GL, Hollingsworth J, Morris JG. Emerging foodborne pathogens: Escherichia coli 0157:H7 as a model of entry of a new pathogen into the food supply of the developed world. Epidemiol Rev 1996;18:29-51. |
|21.||Albert MJ, Farugue ASG. Controlled study of Escherichia coli diarrhoeal infections in Bangladeshi Children. J Clin Microbiol 1995;33:973-977. |
|23.||Chattopadhyay UK, Dutta SD, Pal D. Verotoxin - producing Escherichia coli - an environment-induced emerging zoonosis in and around Calcutta. Int J Environ Health Res 2001;11:107-112. |
|25.||Brunens AP, Frey A, Lior H, Nicolet J. Prevalence and clinical significance of verocytoxin producing Escherichia coli (VTEC) isolated from cattle in herds with and without diarrhoea. J Vet Med 1995;42:311-318. |