Aetiology of shigellosis in northern Pakistan.

People of northern Pakistan face health hazards because of poor sanitation practices. Bacterial gastrointestinal infections are very common, and sometimes outbreaks occur. The present study was aimed at evaluating and analyzing infestation of Shigella spp. in patients with suspected gastroenteritis and ascertaining the status of antibiotic therapy. Five hundred and eighty-five faecal samples of patients with suspected gastroenteritis, referred to the District Headquarter Hospital Gilgit, were investigated for common enteropathogenic bacteria from July 1997 to September 1999. Seventy-seven (13.2%) of the faecal specimens were infected with different strains of Shigella spp., 61% of which were Shigella dysenteriae, 15.6% were S. flexneri, and 23.4% were Shigella sp. All Shigella strains were sensitive to ceftriaxone, cefotaxime, ciprofloxacin, and enoxacin. Sixty-one percent of the strains were resistant to both ampicillin and chloramphenicol, and 3.9% to ampicillin and nalidixic acid, while 10.4% were resistant to ampicillin alone and 14.3% to chloramphenicol only. Only 10.4% of the strains were sensitive to all the antibiotics tested. Sixty strains of Shigella spp. were processed for isolation of plasmids, and 58 (97%) of these antibiotic-resistant bacteria harboured at least one plasmid. The number of plasmids varied from 1 to 9. Escherichia coli C600 were transformed with the isolated plasmids. Transformants, containing 23-kb plasmid, resisted growth in media containing antibiotics, thereby indicating that antibiotic resistance is plasmid-borne. Based on the findings of the study, it is concluded that the infestation of Shigella spp. is high in northern Pakistan, the aetiological agents are highly resistant to chloramphenicol and ampicillin, and the antibiotic resistance is mediated by the 23-kb plasmid.

Factors affecting transformation efficiency of BCG with a Mycobacterium-Escherichia coli shuttle vector pYUB18 by electroporation

BCG has been one of the vehicles for multi-recombinant vaccine. However, low transformation efficiency of BCG with plasmid DNA hampered studies involving expression of foreign antigens in BCG. In an effort to determine the optimal conditions, this study was initiated to investigate factors involved in the transformation of BCG with a Mycobacterium-Escherichia coli shuttle vector, pYUB18, by electroporation. Mycobacterium bovis BCG (strain 1173P2) was grown in Middlebrook (M) 7H9 broth containing albumin-dextrose-catalase and 0.05% tween 80, and transformed BCG was grown in M7H10 agar containing kanamycin for counting viable cells. Pretreatment of BCG with 10 mM CaCl2 improved the transformation efficiency, but overnight incubation of BCG with 1% glycine did not. The transformation efficiency in BCG also varied depending on voltage, resistance, and DNA concentration. The maximum transformation efficiency was obtained when the infinity resistance, 12.5 Kv/cm, and 100 ng of DNA were used, and reached 1.4 x 10(5) CFU/microgram of plasmid DNA, which is about 3-100 times greater than those from previous reports. The transformation conditions described in this study, therefore, will give us a better position for employing BCG as a vehicle for developing multi-recombinant vaccines.