Isolation and identification of E-coli 0157:H7 amongst Sudanese patients with bloody diarrhea and in animals

E.coli 0157:H7 is present as a commensal in the intestine of animals and as a pathogen in human beings. It causes bloody diarrhoea due to the secretion of a verotoxin which may lead to lethal complications. Objective: This study is aimed at determining the presence of this organism in animals and patients. Methods: Rectal swabs were collected from 250 cows. Stool specimens were obtained from 200 patients presenting with macroscopically or microscopically bloody diarrhoea. All specimens were cultured on sorbitol MacConkey agar and incubated at 37C overnight. Non-sorbitol fermenting colonies were identified by different biochemical and serological tests as E.coli 0157:H7. Antibiotic sensitivity was done for the isolates using ampicillin, cephalexin, ciprofloxacin, co-trimoxazole, gentamicin, and tetracycline. Results: Patients (n = 10, 5%) with bloody diarrhoea were found to harbour E.coli 0157:H7 in their stools. All isolates (in humans) were resistant to ampicillin. Eight (80%) were resistant to tetracycline and cephalexin, six (60%) to co-trimoxazole, and four (40%) to gentamicin. All isolates were sensitive to ciprofloxacin. Sixty percent of the human isolates were resistant to three antibiotics and 40% to four antibiotics. Twenty of the cows (8%) were found to be carriers of E.coli 0157:H7. All isolates (in animals) were resistant to ampicillin, five (25%) to tetracycline and cephalexin and four (20%) to co-trimoxazole. All animal isolates were sensitive to gentamicin and ciprofloxacin. Thirty five percent of the animal isolates were resistant to two antibiotics and 25% to three antibiotics. Conclusion: The isolation of E.coli 0157:H7 from animals and patients should direct the attention of physicians and paediatricians to consider the possibility of infection and complications by this organism.

-coli is a member of the normal intestinal flora of both human and warm blooded animals and birds.Most of E-coli infections are opportunistic infections of the bladder, kidney, wounds and meningitis.It is one of the important nosocomial pathogens.The strains capable of causing such diseases possess one or more virulent factors that are not found in E-coli strains comprising the normal flora.Special strains of E-coli can cause diarrhea; one of which is the verotoxigenic E-coli 1 .
The verotoxin is encoded on the genome of bacteriophages which enter E-coli via phageconversion 1,2 .In 1982 one particular serovar of E-coli 0157:H7 was identified as a causative agent involved in two outbreaks of distinctive bloody diarrhoea syndrome 3,4 .Since then, the organism has received much attention, not only in the United States but in other countries as well, as a cause of sporadic or epidemic bloody diarrhea and non bloody diarrhea, Haemolytic Uremic Syndrome (HUS) and Thrombotic Thrombocytopenic Purpura (TTP).Different verotoxin-producing E-coli serovars have so far been isolated from human, animals and food stuff.E-coli 0157:H7 is believed to live in the intestine of cattle, chicken, sheep and swine 5 .The organism is able to survive in bovine feces, food, water, manure and crops for an extended period of time (weeks to months).Meat can become contaminated during slaughter 6 .Person to person is an important mode of transmission through the oral fecal route 7,8 .Although most patients recover from E-coli 0157:H7 infections, about 5-10% of the infected individuals develop HUS.E-coli 0157:H7 is also responsible for 90% of the cases of HUS.In fact some researchers believe that E-coli 0157:H7 is the only cause of HUS in children.Among the elderly, HUS is associated with fever and neurological symptoms usually due to TTP 9 .Among the survivors of HUS, 5% eventually develop end stage-renal-failure with the resultant need for dialysis or transplantation.The highest rate of isolation of E.coli 0157:H7 is from fecal specimens from children and elderly patients 10 .E-coli 0157:H7 can also be isolated by the culture of stools and the normal routine biochemical and serological tests 11 .The verotoxin can be detected using vitro cell assay, latex agglutination ELISA, multiplex PCR and randomly amplified polymorphic DNA 12,13 .The majority of the strains of E-coli 0157:H7 are susceptible to the commonly used antimicrobials but resistance is very high to azithromycin 14 .The present study was meant to look for the presence of E.coli 0157:H7 in patients presenting with bloody diarrhea as well as in cattle.

Materials and methods:
This study was carried out in Khartoum State from February until August 2008.It included 200 patients presenting with macroscopically or microscopically bloody diarrhea and 250 cattle from three farms in Khartoum State.All of which were investigated for the carriage of E-coli O157:H7.Fecal specimens were collected from the patients and rectal swabs from the cattle in clean containers.Direct microscopy of the stool specimens was done to exclude any protozoan infection.The specimens were cultured on 1% sorbitol MacConkey agar and incubated at 37 °C overnight.On the following day, non sorbitol fermenting colonies were identified using biochemical and serological tests as E-coli 0157:H7.Antibiotic susceptibility of the isolates was carried out using six antibiotics' discs which were: ampicillin 10[g, cephalexin 30 [g, ciprofloxacin 5 [g, cotrimoxazole 25[g, gentamicin 10[g and tetracycline 30[g and were reported according to the NCCL recommendations 15 .

Results:
Out of the 200 fecal specimens collected from the patients presenting with bloody diarrhea, ten (5%) were found to be positive for E-coli 0157:H7 (Fig 1).All isolates were resistant to ampicillin, eight (80%) to tetracycline and cephalexin, six (60%) to co-trimoxazole and four (40%) to gentamicin.Moreover, all isolates were sensitive to ciprofloxacin (Fig. 2).

Fig 2:
The percentage of antibiotic resistance of E.coli 0157:H7 isolates from patients and animals.Sixty percent of the human isolates were resistant to three antibiotics and 40% to four antibiotics.Out of the 250 cows, twenty (8%) were positive for E-coli 0157:H7 (Fig 1).The isolates were all resistant to ampicillin.Six (30%) of the isolates were resistant to tetracycline and cephalexin and four (25%) to co-trimoxazole.All of the isolates were sensitive to ciprofloxacin and gentamicin (Fig 2).Multi drug resistance was common amongst the isolates.Thirty five percent of the animal isolates were resistant to two antibiotics and 25 % to three antibiotics.

Discussion:
Verocytotoxin producing E-coli 0157:H7 emerged as a major food borne zoonotic pathogen in during the 1980s and 1990s.The disease was often severe with significant mortality in young and elderly patients 16 .It can cause haemorrhagic colitis, Haemolytic Uraemic Syndrome and Thrombotic Thrombocytopenic Purpura.The organism can be isolated from the intestine of different animals but it seems that the main reservoir is cattle 8 .The percentage of E-coli 0157:H7 isolated from patients suffering from bloody diarrhoea in the current study was 5% which is similar to findings by the MMWR 17 .Different results were reported by Elmer 18 .Furthermore, the current isolates showed resistance to the commonly prescribed antibiotics like ampicillin (100%), tetracycline and cephalexin (80%), cotrimoxazole (60%) and gentamicin (40%).The isolates were fully sensitive to ciprofloxacin.The increase in resistance to the commonly used antibiotics is well known and stated in previous studies.Most of the isolated E-coli 0157:H7 (97%) during the year 1988, were sensitive to ampicillin tetracycline and co-trimoxazole 19 .Alvaro and co-workers 20 found that resistance of the organism to ampicillin, tetracycline and cotrimoxazole was increasing.In 1999, Banerjee et al 21 found that most of the strains of E-coli 0157:H7 were resistant to ampicillin and tetracycline.
The present study documented the first report of the presence of carriers of this organism amongst cattle in Khartoum State.The percentage of the carriers in cattle was found to be 8%.Similar range of carriers among cattle was reported by different authors 3,13 .High percentage of carriers was reported by Lothar and colleagues in 1993 22 .The variability in the percentages of carriers can be attributed to the different methods of sampling and culturing techniques.The antibiotic susceptibility of the animal's isolates seems to be more sensitive than the human's isolates.This can be explained by the fact that antibiotics are commonly used and routinely prescribed in hospitals.

Conclusions:
The isolation of E-coli 0157:H7 from patients and animal carriers should direct the attention of the physicians and pediatricians to the possibility of the appearance of its complications among young and elderly patients.

Fig 1 :
Fig 1: The percentage of E-coli 0157:H7 isolates from patients and animals.