| Abstract|| |
Purpose: This study was conducted to study the prevalence of diarrheagenic Escherichia coli (DEC) in dysentery cases with special reference to Shiga-like toxin producing Escherichia coli (STEC). Methods : During a two-year period, 1066 stool samples were collected from hospitalized patients with diarrhea and dysentery. After taking detailed clinical history and observing the gross and microscopic findings of the stool samples, they were cultured on MacConkey and Sorbitol MacConkey agars and E.coli isolates were identified by standard biochemical tests. Results: Of the 100 E.coli strains isolated in pure culture and sent for sero typing to Central Research Institute (CRI), Kasauli, 43% were found to be DEC, giving an isolation rate of 4.03%. Results of sero typing showed 37.21% STEC which were more common in children. Abdominal pain and stool with mucus flakes were statistically significant parameters (p less than 0.01) in patients with dysentery due to E.coli strains. Though E.coli O157 was not encountered, it was seen that 25% of STEC did not ferment sorbitol. The DEC strains showed maximum in vitro sensitivity to amikacin (83.72%) and all strains were resistant to nalidixic acid. Antibiotics along with ORS and intravenous fluids had to be given in 68.42% patients. As complications, about 16.67% of children developed hemolytic uremic syndrome (HUS),and 10.53% of patients developed acute renal failure. No mortality was reported. Conclusion: Though Enterohemorrhagic E.coli (EHEC) O157:H7 was not encountered in this study, STEC caused by E.coli non O157 was reported. STEC is also known to cause hemorrhagic colitis (HC) and HUS. In this study HUS was reported in 16.67% children. Therefore, proper isolation and identification of STEC is essential in a tertiary care centre, to initiate prompt management and reduce morbidity and mortality in children.
Keywords: Dysentery, Shiga- like toxin producing Escherichia coli
|How to cite this article:|
Lanjewar M, De AS, Mathur M. Diarrheagenic E. coli in hospitalized patients: Special reference to Shiga-like toxin producing Escherichia coli. Indian J Pathol Microbiol 2010;53:75-8
|How to cite this URL:|
Lanjewar M, De AS, Mathur M. Diarrheagenic E. coli in hospitalized patients: Special reference to Shiga-like toxin producing Escherichia coli. Indian J Pathol Microbiol [serial online] 2010 [cited 2014 Jul 25];53:75-8. Available from: http://www.ijpmonline.org/text.asp?2010/53/1/75/59188
| Introduction|| |
Infectious diarrhea is one of the greatest causes of morbidity and mortality worldwide. Bacterial gastroenteritis is very common in developing countries like India. The usual manifestations are vomiting, diarrhea and abdominal discomfort. Amongst the various enteric bacterial pathogens, diarrheagenic strains of Escherichia More Details coli (E. coli) are considerably responsible for causing bacterial diarrhea.  DEC contain and express virulence factors that enable them to exhibit pathogenicity. The causative agents of bacillary dysentery are the different species of Shigella (Shigella flexneri, Shigella dysenteriae, Shigella boydii and Shigella sonnei) and less frequently the closely related entero pathogens - Enteroinvasive E. coli (EIEC) and also Enterohemorrhagic E. coli (EHEC). Reports on the etiological role of E. coli O157:H7 in causing individual infections and outbreaks in developing countries like India and Bangladesh are sparse.  Hence this study was conducted to study DEC in cases of dysentery with special reference to Shiga-like toxin producing E. coli (STEC), to study the antibiotic susceptibility pattern of DEC and correlate the same with clinical findings.
| Material and Methods|| |
A prospective study over a period of two years (August 2004 - July 2006) was conducted at the department of Microbiology of a tertiary care hospital in Mumbai. A total of 1066 stool samples were collected from patients hospitalized with diarrhea and dysentery which showed either macroscopic evidence of frank blood and/or mucus flakes and/or microscopic evidence of red blood cells (RBCs) and/or pus cells. All the samples were collected in sterile wide-mouthed containers with tight-fitting leak proof lids and transported immediately to the laboratory to be processed within two hours of receipt. The samples showing darting motility or parasitic elements macroscopically and/or microscopically and samples from HIV infected patients were excluded from the study.
After completing the gross and microscopic examinations, direct plating was done on MacConkey agar (MA) and Sorbitol MacConkey agar (SMA). The plates were incubated overnight at 37 0 C. Isolates were identified as E. coli by standard biochemical reactions.  Motility was done by hanging drop. All non-motile E. coli were differentiated from Shigella species by acetate utilization test.  Agglutination was done for sorbitol non fermenting colonies by E. coli O157 anti sera obtained from Central Research Institute (CRI), Kasauli, India. One hundred E. coli strains isolated in pure culture from the stool samples of these patients were sent for sero typing at Central Research Institute (CRI), Kasauli, India.
An antibiotic susceptibility test was performed on Mueller Hinton agar (MHA) by Kirby-Bauer disc diffusion method using the antibiotics - amikacin (30µg), trimethoprim-sulfamethoxazole (1.25/23.75µg), norfloxacin (10µg), nalidixic acid (30µg) and cefotaxime (30µg) (HiMedia).
| Results|| |
Of the 100 E. coli isolates sent for sero typing to CRI, Kasauli, 43% were DEC strains. Thus a total of 43 DEC from 1066 samples were obtained with an isolation rate of 4.03%. Amongst all the entero pathogenic bacteria isolated during two years, 68.25% (43) were DEC. About 55.81% (24) DEC was reported in children and in adults it was 44.19% (19). The adult:children ratio was 0.8:1. Males encountered 55.81% (24) more DEC than females 44.19% (19), with the male: female ratio of 1.26:1. Amongst the 19 cases of E. coli causing dysentery, 16 were STEC and three were Enteroinvasive E. coli (EIEC) [Table 1]. STEC was more common in children i.e. 68.75% (11) than adults i.e. 31.25% (five). The sex distribution of STEC showed 56.25% (nine) positivity in males and 43.75% (seven) in females. EIEC was isolated from one female child and two adults (one male and one female). The report of sero typing from CRI, Kasauli, is shown in [Table 1].
Clinically, of the 19 cases of dysentery due to E. coli, 50% (6/12) of children had four to six motions/day. Majority of the patients, i.e. 84.21% (16/19) had a history of motions since five days or less. Fever was present in 68.42% (13/19) of patients. Vomiting was seen in 58.33% (seven/12) of children, oliguria was present in 36.84% (seven/19) patients, also showing predominance in children i.e. 50% (six/12). Abdominal pain was predominant in adults i.e. 71.43% (five/seven). Mild dehydration was seen in 68.42% (13/19) of the patients, 21% (four/19) had moderate dehydration and 10.5% (two/19) had severe dehydration. Among other clinical features, two patients had generalized edema and one had convulsion.
Fourteen of 19 patients (73.68%) had presence of blood in stool samples, of which 21.05% (four) had frank blood and the remaining 52.63% (10) had blood and mucus. Five patients (26.32%) had liquid stools with mucus flakes. Sixteen (84.21%) and fifteen (78.95%) patients had microscopic evidence of RBCs and pus cells respectively. Abdominal pain and stool with mucus flakes were statistically significant (p less than 0.01) by Student t test. Oliguria, severe dehydration and frank blood in stool were not encountered at all amongst E. coli strains not causing dysentery.
Biochemically, all the three EIEC strains were non lactose fermenting and non motile. Seven out of 16 (43.75%) STEC strains were non lactose fermenting. All entero pathogenic E. coli (EPEC) and entero toxigenic E. coli (ETEC) strains were lactose fermenting. Amongst 16 STEC strains, four were sorbitol non fermenting (25%); none were entero hemorrhagic E. coli O157 strains (as per sero typing report).
The antibiotic susceptibility pattern of the DEC strains is shown in [Figure 1]. Both dysentery causing and diarrhea causing E. coli showed maximum susceptibility to Amikacin (89.47 and 79.17% respectively). Nalidixic acid susceptibility was zero per cent in all the DEC strains. Overall cefotaxime, norfloxacin and trimethoprim-sulfamethoxazole susceptibility of DEC strains were 48.84, 13.95 and 9.30% respectively.
Of the 19 cases of dysentery, 13 patients (68.42%) received intravenous fluids and antibiotics. Intravenous fluids and oral rehydration solution (ORS) were given to three patients, while three patients could be managed with ORS alone. The antibiotics administered to children were IV cefotaxime, IV amikacin and/or IV metronidazole; whereas IV ciprofloxacin and/or IV metronidazole were administered to adults.
Amongst the complications, HUS developed in 10.53% (two) patients, and both of them were children, who required peritoneal dialysis. Acute renal failure developed in 10.53% (two) of the patients (one child and one adult). None of the patients expired. Thus mortality was zero per cent in this study.
| Discussion|| |
Amongst the etiological agents of bacterial gastroenteritis, DEC remain an important cause of diarrhea, particularly in the developing countries.  Though ETEC and EPEC are the major bacterial enteric pathogens amongst DEC, the newer DEC increasingly reported in the recent years are the Shiga-like toxin producing E. coli (STEC) and entero aggregative E. coli (EAggEC). , The STEC/VTEC cause severe disease in humans such as hemorrhagic colitis (HC) and HUS by the production of Shiga toxin (Stx1, Stx2, and their variants). ,
STEC, though quite common in outbreaks and also sporadically in developed nations, has not been not been recognized as a significant cause of human disease in Bangladesh and India. ,
In India, Gupta et al. isolated E. coli O157 from sporadic cases of diarrhea.  In a two-year period, this study isolated 68.25% DEC strains. Isolation rate by Albert et al. was 58.28%; by Vargas et al. it reached 57.1%. , The STEC encountered in this study was 37.21% in DEC, which is almost double the isolation rate of Kehl et al. (18.52%).  In our study, STEC isolation was closely followed by ETEC (34.88%) and EPEC (20.93%). However, no EAgg EC was encountered, which is an emerging pathogen, especially in infants. , STEC was more common in children (68.75%) than adults (31.25%). This has also been reported by Beutin et al; STEC was reported more in children (65.9%) than in adults (34.1%). 
The frequency and duration of motions in dysentery cases is definitely less than in cases of acute watery diarrhea.  Abdominal pain was less pronounced in children (41.67%), as individual children differ greatly in their perception of and tolerance of abdominal pain. 
Pus cells were seen in the stool samples of 78.95% of patients and the presence of pus cells is a characteristic feature of dysentery which differentiates it from acute watery diarrhea. The study by Gupta et al. also shows that raised fecal leukocyte count is a useful indicator for invasiveness. 
Abdominal pain and stool with mucus flakes were statistically significant in this study (p less than 0.01) and can be considered definite predictors of dysentery due to E. coli. CDC advocates the investigation of sporadic cases of hemorrhagic colitis with symptoms of abdominal cramps, grossly bloody diarrhea, absent or low grade fever and stool specimens negative for the usual bacterial pathogens. Such cases should be reported to the Enteric Diseases Branch. 
In this study, 25% of STEC were sorbitol non fermenters as also seen in the studies by Gunzer et al. as well as others. , This study detected 16 STEC strains - all E. coli non O157. E. coli O157 was not detected at all. Chattopadhyay et al. report 1.33% STEC from feces of diarrheic children.  All of them possessed the Stx gene but none of them were O157:H7.  Non O157 STEC have also been reported from other countries like U.S. and New Zealand as a major cause of dysentery. ,
Serotype O111 was categorized into STEC as well as EPEC and serotype O25 was grouped under ETEC as well as STEC [Table 1], depending on the presence or absence of blood/ RBCs in stool samples. It is most commonly seen that E. coli O25 belongs to ETEC.  In this study, a total of five O25 serotypes were isolated of which four were grouped under ETEC as the stool samples were watery without blood. Only one stool sample had frank blood and hence was grouped under STEC. Similarly, O111 belongs to both EPEC and STEC.  Two O111 serotypes were isolated. One stool sample was hemorrhagic and hence grouped under STEC and the other being only watery was put under ETEC. In E. coli causing dysentery, susceptibility to amikacin was 89.47% as compared to 79.17% in E. coli causing diarrhea. Only 36.84% were susceptible to cefotaxime amongst E. coli causing dysentery in contrast to 58.33% amongst E. coli causing diarrhea (bar diagram 1). E. coli causing diarrhea showed a greater antibiotic susceptibility with respect to cefotaxime, trimethoprim-sulfamethoxazole and norfloxacin as compared to E. coli causing dysentery (bar diagram 1). In this study, nalidixic acid susceptibility was zero per cent and fluoroquinolone susceptibility was 13.95% amongst the DEC strains. In 2004, Mendez reported 77% susceptibility to nalidixic acid and 65.50% to fluoroquinolones amongst the DEC strains. This shows that the resistance of DEC strains to nalidixic acid and fluoroquinolones is definitely on the rise. 
Antibiotics should be given only if diarrhea is moderate or severe, as antibiotic treatment increases the likelihood of development of HUS.  Trimethoprim-sulfamethoxazale is the first line drug to be used in enteric infections. However, in this study, trimethoprim- sulfamethoxazole susceptibility (in vitro) amongst DEC strains was only 9.30% (4/43). Therefore, the other alternative is using a parenteral second generation or third generation cephalosporin for systemic complications.  Dehydration may precipitate renal or cerebral damage. Hence it is important to start ORS and/or intravenous fluids immediately on admission and appropriate antibiotics, if required. 
Non O157 STEC infection was not a notifiable disease in the United States until 2000. Only since 2000 the public health departments report STEC infections to the National Notifiable Diseases Surveillance System.  This reporting is not mandatory in India, nor is it a notifiable disease. Hence, very few documented evidences are available from India. ,
In this study, most common STEC reported was E. coli O1 (37.5%), followed by O153 (18.75%) [Table 1]. E. coli O153 is known to be involved in HUS cases without outbreaks.  E. coli O111 is known to cause HUS outbreaks and is also involved in HUS cases without outbreaks. This study also reported O111 [Table 1]. ,
The overall prevalence of HUS in this study was 10.53%. However, HUS was seen only in children (16.67%). Gunzer et al. and Brooks et al. reported prevalence of HUS as 25% in O157:H7 and 21.88% in non O157 STEC strains respectively. , All children who developed HUS required peritoneal dialysis. This was similar to the study by Caprioli et al. - 88.9% of children with HUS required peritoneal dialysis.  They were also managed with transfusion of fresh blood or plasma, as required. Acute renal failure was another complication encountered in this study in 10.53% of patients.
Srivastava et al. reported acute renal failure in 34% children with HUS;  about 11.11% mortality in HUS cases was reported bv Caprioli et al.  However, none of the patients expired in this study.
| Acknowledgement|| |
We acknowledge Central Research Institute (C.R.I.), Kasauli for sero typing Esherichia coli isolates.
| References|| |
|1.||Nataro JP, Kaper JB. Diarrhoeagenic Escherichia coli. Clin Microbiol Rev 1998;11:142-201. [PUBMED] [FULLTEXT] |
|2.||Chattopadhyay UK, 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. [PUBMED] |
|3.||Sonnenwirth A C. Media, tests and reagents. In: Sonnenwirth A C, Jarett L, editors. Gradwohl′s Clinical Laboratory Methods and Diagnosis. 8 th ed. The C.V. Mosby Company: London; 1980. p. 1391-450. |
|4.||Molbak K and Schleutz F. Verocytotoxin producing Escherichia coli and other diarrhoeagenic Escherichia coli. In: WHO. Waterborne Zoonoses: Identification, causes and control. 1 st ed. London: IWA Publishing; 2004. p. 213-27. |
|5.||Dutta S, Deb A, Chattopadhyay UK, Tsukamoto T. Isolation of Shiga toxin-producing Escherichia coli including O157: H7 strains from dairy cattle and beef samples marketed in Calcutta, India. J Med Microbiol 2000;49:765-7. [PUBMED] [FULLTEXT] |
|6.||Gupta S, Soni NK, Kaur P, Sood DK. Verocytopathic activity of Escherichia coli O157 and other ′O′ serogroups isolated from patients of diarrhoea. Indian J Med Res 1992;95:71-6. [PUBMED] [FULLTEXT] |
|7.||Albert MJ, Faruque SM, Faruque AS, Neogi PK, Ansaruzzaman M, Bhuiyan NA, et al. Controlled study of Escherichia coli diarrhoeal infections in Bangladeshi children. J Clin Microbiol 1995;33:973-7. [PUBMED] [FULLTEXT] |
|8.||Vargas M, Gascon J, Casals C, Schellenberg D, Urassa H, Kahigwa E, et al. Etiology of diarrhoea in children less than five years of age in Ifakara, Tanzania. Am J Trop Med Hyg 2004;70:536-9. [PUBMED] [FULLTEXT] |
|9.||Kehl KS, Havens P, Behnke CE, Acheson DW. Evaluation of the Premier EHEC Assay for detection of Shiga toxin producing Escherichia coli. J Clin Microbiol 1997;35:2051-4. [PUBMED] [FULLTEXT] |
|10.||Nguyen TV, Le Van P, Le Huy C, Gia KN, Weintraub A. Detection of characterization of diarrhoeagenic Escherichia coli from young children in Hanoi, Vietnam. J Clin Microbiol 2005;43:755-60. [PUBMED] [FULLTEXT] |
|11.||Beutin L, Zimmermann S, Gleier K. Human infections with Shiga toxin producing Escherichia coli other than serogroup O157 in Germany. Emerg Infect Dis 1998;4:635-9. [PUBMED] [FULLTEXT] |
|12.||Larry K. Pickering, John D. Snyder. Gastroenteritis. In: Richard E. Behrman, Robert M. Kleigman and Hal B. Jenson. Nelson Textbook of Pediatrics. 17 th ed. Saunders: Philadelphia; 2004. p.1198-204,1272-6. |
|13.||Gupta DN, Saha DR, Sengupta PG, Mondal SK, Ghosh S, Saha MR, Value of faecal leucocyte count as an indicator of invasiveness in mucoid diarrhoea. J Commun Dis 1997;29:329-33. |
|14.||Centers for Disease Control (CDC). Outbreak of hemorrhagic colitis, Ottawa, Canada. MMWR Morb Mortal Wkly Rep 1983;32:133-4. [PUBMED] [FULLTEXT] |
|15.||Gunzer F, Böhm H, Rüssmann H, Bitzan M, Aleksic S, Karch H. Molecular detection of sorbitol fermenting Escherichia coli O157 in patients with hemolytic uremic syndrome. J Clin Microbiol 1992;30:1807-10. |
|16.||Centers for Disease Control and Prevention (CDC). Community outbreak of Hemolytic Uraemic Syndrome attributable to Escherichia coli O111: NM - South Australia, 1995. MMWR Morb Mortal Wkly Rep 1995;44:550-8. [PUBMED] [FULLTEXT] |
|17.||Brooks J T, Sowers E G, Wells J G, Greene K D, Griffin P M et al. Non-O157 Shiga toxin producing Escherichia coli infection in United States 1983 to 2002. J Infect Dis 2005;192:1442-9. |
|18.||Microbial Pathogen Data Sheet (Ministry of Health by ESR Ltd.) [Non - O157 Shiga toxin producing Escherichia coli (STEC)]. Available from: http://www.nzfsa.govt.nz/science/">http://www.nzfsa.govt.nz/science/ datasheets/non O157 stec [last accessed on May 2001] |
|19.||J.J.Farmer III, Michael T. Kelly. Enterobacteriaceae. In: Balows A, Hauster W J Jr., Herrmann K L, Isenberg H D, Shadomy H J. Manual of Clinical Microbiology. 5 th ed. American Society For Microbiology: Washington; 1991. p. 360-73. |
|20.||Mendez A E. Comparison of antimicrobial resistance in diarrhoeagenic Escherichia coli isolates causing traveller′s diarrhoea between two periods, 1994-1997 and 2001-2003. European Society of Clinical Microbiology and Infectious Diseases: 14 th European Congress of Clinical Microbiology and Infectious Diseases; Praque, Czech Republic; 2004. |
|21.||Richard E Frye. Bacterial gastroenteritis [last updated on 11 th August 2004]. Available from: http:// www.emedicine.com / Bacterial gastroenteritis. |
|22.||Caprioli A, Luzzi I, Rosmini F, Resti C, Edefonti A, Perfumo F, et al. Community-wide outbreak of hemolytic uremic syndrome associated with non-O157 verocytotoxin-producing Escherichia coli. J Infect Dis 1994;169:208-11. [PUBMED] [FULLTEXT] |
|23.||Srivastava RN, Moudgil A, Bagga A, Vasudev AS. Hemolytic uremic syndrome in children in Northern India. Pediatr Nephrol 1991;5:284-8. [PUBMED] [FULLTEXT] |
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