Isolation and identification of Escherichia coli from diarrhoeic calf faeces by biochemical tests, antibiogram pattern and PCR based detection of toxigenic genes
Arya, Gitanjali (2004) Isolation and identification of Escherichia coli from diarrhoeic calf faeces by biochemical tests, antibiogram pattern and PCR based detection of toxigenic genes. Masters thesis, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand.
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The present study was undertaken to investigate biochemical characters, serotypes, biotypes, multiple drug resistance, colicinogeny, haemolytic activity and detection of toxigenic genes of E. coli from diarrhoeic calf faecal samples. In all 91 E. coli isolates obtained from 46 faecal samples revealed typical cultural characters on the MacConkey agar and eosin methylene blue agar. Some of the E. coli isolates showed variation from the standard biochemical pattern viz. H2S production (1.09% isolates), urea hydrolysis (21.97% isolates), citrate utilization (3.29 % isolates) and fermentation of adonitol (2.19% isolates) and raffinose (35.1% isolates). All the 91 isolates were serotyped, of which 82 typable isolates belonged to 36 different ‘O’ serogroups, while 6 isolates were untypable and 3 were rough isolates. In all 17 different serogroups obtained from Livestock Research Station- Anand Agricultural University (LRS-AAU), Anand were in decreasing frequency viz. O86 (7 isolates), four isolates each of O128, O171 and O172, O62 (3 isolates), serogroups O22, O26, O32, O55, O110, O157 having 2 isolates each and one isolate each belonging to serogroups O11, O18, O24, O126, O131 and UT (untypable). Out of six isolates obtained from Veterinary College Clinics (VC), AAU, Anand 4 different serogroups were recorded viz. O2 and O167 two isolate each and one isolate each of O12 and O197. Out of twenty seven isolates obtained from other unorganized farms (OF), in and around Anand twenty different serogroups detected were O5, O9, O11, O22, O66, O78, O86, O100, O101, O108, O126, O157, O161, O162, O171 and O172 one isolate each while two isolates each of serogroup O98 and O131, 3 rough and four untypable isolates. Eighteen isolates obtained from Livestock Research Station-Junagadh Agricultural University (LRS-JAU), Junagadh comprised of twelve different serogroups viz. O18 (5 isolates), two isolates each of O109, O128, while one isolate each of O3, O15, O22, O68, O76, O131, O168, O171 and UT. The most common serogroups were O22, O131 and O171 followed by O11, O18, O86, O126, O128, O157 and O172 among the E. coli isolates obtained from various locations. Twenty four different biotypes of E. coli were obtained on the basis of fermentation reactions of sugars viz. dulcitol, raffinose, rhamnose, salicin, starch and sucrose. In vitro antibiotic resistance pattern against 12 antibiotics were detected. Higher percent (72-100%) of E. coli isolates showed resistance against kanamycin, cepahlexin, amikacin, cephaloridine, enrofloxacin and ampicillin. Moderate numbers (28-57%) of isolates were found to be resistant to ciprofloxacin, ceftiofur and tetracycline. Lesser percent (8.79-15.38%) of isolates were resistant to nalidixic acid, colistin and co-trimoxazole. Among the 91 E. coli isolates studied for colicinogeny 32 (35.16%) were colicin producers. PCR based detection of toxigenic genes with the primer sets LT1-LT2 and microST1-microST2 gave products of expected size i.e. 132 bp for LT (heat labile) enterotoxin and 171 bp for ST (heat stable) enterotoxin genes. Out of 91 E. coli isolates one (1.09%) possessed ST gene and two isolates (2.19%) harboured LT gene. The primer directed amplification of the verotoxin (VT1 and VT2) genes gave the products of expected size i.e. 130 bp for VT1 gene and 228 bp for VT2 gene. Total 41 (45.05%) isolates were found to be positive for VT genes. Among the isolates 32 (35.16%) isolates harboured both VT1 and VT2 genes while 6 (6.5%) isolates possessed only VT2 gene and 3 (3.25%) had VT1 gene only. Overall 38 (41.75%) isolates were positive for VT2 gene and 35 (38.46%) isolates were positive for VT1 gene.
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