التوصيف الجزيئي لبكتيريا الاشيرشيا القولونية المعزولة من الأطفال المصابين بالإسهال في محافظة كربلاء

Molecular characterization of E. coli isolated from children infected with diarrhea in Kerbala province

Abstract

The present study was performed to determine the bacterial causative agents of diarrhea in children and also the virulence factors possessed by Escherichia coli which isolated from the stool of children under 5 years of age with symptoms of diarrhea and its relationship to antibiotic resistance as the main causative factor in diarrhea. One hundred samples were collected from stool of children who attended the Children’s Teaching Hospital in the holy city of Karbala, for the period from 2/2/2022 to 15/6/2022. The samples were cultured on several culture media, and then the bacterial isolates were identified using the Vitek2 technique. After that an antibiotic sensitivity test was performed, then prior to detection by PCR technique 40 isolates of E. coli were selected based on examining a stool sample under a light microscope, which contained pus cells, or Red blood cells.

The results showed that the percentage of males infected with diarrhea was 46 (57.5%), which was higher than the percentage of females 34 (45%). Statistical analysis showed that there was a significant difference between infected males and females (P = 0.02). The infected children 11(13.75%) was found in the age group of (0-6) months, and the number of infected children 23 (28.75%)was in the age group (7-11) months and the age group (1-2) years had the number of infected children 21 (26.25%) . The number of affected children was 16 (20%) in the age group of (2-3) years, followed by 9 (11.25%) in the age group of (3-5) years .It is clear from the above that the number of affected children was higher in the age of groups between (7-11) months and (1-2) years, but no significant differences were appeared between the age groups (P = 0. 99) . The number of children who live in randimals areas 52 (65%) were significantly higher infected by bacterial causative agents of diarrhea in comparison to those children live in residential neighborhoods 28 (35%) (P = 0.001).

It was found that the number of children whose parents practiced free professions 61 (76.25%) which were significantly more likely to suffer from diarrhea compared to children whose fathers were employed 19 (23.73%) (P =0.009) .

The results from the sensitivity test showed that (100%) 40 bacterial isolates were resistant to the antibiotic Ampicillin, which is the highest percentage of resistance , followed by (85%) 34 bacterial isolates resistant to the antibiotic Co-Trimoxazole , then (82.5%) 33 bacterial isolates were resistant to the antibiotic Cefotaxime . In addition, (72.5%) 29 bacterial isolates were resistant to the antibiotic Ceftriaxone , 26 (65%) bacterial isolate resistant to the antibiotic Tetracycline . Results also showed that (52.5%) 21 bacterial isolates were resistant to the antibiotic Azthromycine followed by (50%) 20 bacterial isolates had resistance activity against to the antibiotic Doxycycline . Beside, 15 (37.5%) isolate were resistant to the antibiotic Ciprofloxacin and 6 (15%) isolate were resistant to the antibiotic Gentamycin. Finally (7.5%) 3 bacterial isolate resistant to the antibiotic Amikacin which is the lowest percentage of resistance.

Molecular detection which represented by polymerase chain reaction PCR results showed that (50%) of 20 bacterial isolates carrying genes (stx1, stx2, eae, bfpA), which belong to the pathotypes of E. coli bacteria causing diarrhea (DEC), and (50%) 10 isolates of enteropathogenic bacteria EPEC, (50%) 10 isolates carrying two genes together (stx1 & stx2) belong to the intestinal hemorrhagic bacteria EHEC .It was noted that there no significant differences were found between the two strains (P > 0.05) , On the other hand among the ten isolates of enteropathogenic bacteria EPEC were (80%) 8 bacterial isolates carrying the (eae) gene for the atypical enteropathogenic bacteria aEPEC, and two isolates (20%) carrying two genes together (eae & bfpA) belong to the typical enteropathogenic bacteria tEPEC . The statistical analysis showed that there was a clear significant difference between the two types of these bacteria (P < 0.05).

The results appeared that the number of bacterial isolates which carried the CTX-M gene were (66%) 12 and was significantly higher (P = 0.003) compared to the isolates carried the TEM gene, which were (33%) 6 isolates, and the SHV gene was not appeared in any bacterial isolates .

Moreover, the results of the current study showed that EPEC bacteria were present in a higher percentage in males (60%) 6 compared to their presence in females (40%) 4, with a non-significant manner ( P = 0.070). In contrast EHEC bacteria were present in females by (70%) 7 which significantly higher than their presence in males (30%) 3 and almost (P = 0.053) .

The results showed that EPEC bacteria were present in the age group of (1-6) months with (20%) 2 isolates, while (40%) 4 isolates were present in the age group of (7-11) months , followed by the age group of (1-2) years with a number of (20%) 2 isolates .Finally, (10%) 1 bacterial isolate was present in the age groups of (2-3) years and (3-5) years.

The results also revealed that EHEC bacteria were not present in the age group of (1-6) months and (11-7) months, and were present in the of (1-2) years with a number of (20%) 2 isolates , (30%) 3 bacterial isolates were found in the age category of (2-3) years. Finally, (50%) 5 isolates were recorded in the age category of (3-5) years. The statistical analysis regarding present of EPEC bacteria among age groups showed that there was no significant difference between the age , except for the age group of (7-11) months, which was the number of the bacteria in this group significantly higher compared to the two age groups of (2-3) and (3-5) years. On the contrary, the number of EHEC was significantly higher in the age group of (3-5) years compared to the rest of the age groups (P < 0.05) .

Finally, the current results appeared that (30%) of 3 EPEC isolates was significantly carried the CTX-M gene and (P = 0.0024) in comparison to other genes. In addition, the TEM gene was carried by one bacterial isolate at a rate of (10%) . CTX-M gene was carried by one isolate of EHEC with a percentage of (10%) and two isolates of EHEC carried the TEM gene in with a percentage of (20%).