Plasmid

A method for rapid selection of allelic replacement mutations in the chromosome of Staphylococcus aureus is described. Plasmid pKOR1, an Escherichia coli/S. aureus shuttle vector, permits rapid cloning via lambda recombination and ccdB selection. Plasmid transformation of staphylococci and growth at 43 degrees C, a non-permissive condition for pKOR1 replication, selects for homologous recombination and pKOR1 integration into the bacterial chromosome. Anhydrotetracycline-mediated induction of pKOR1-encoded secY antisense transcripts via the Pxyl/tetO promoter, a condition that is not compatible with staphylococcal growth, selects for chromosomal excision and loss of plasmid. Using this strategy, allelic replacements in S. aureus rocA were generated at frequencies that obviated the need for antibiotic marker selection.

The occurrence and antibiotic resistance of Escherichia coli in tropical seafood was studied. A 3-tube MPN method was used for determining the level of faecal contamination of fresh and processed seafood. Of the 188 samples tested which included finfish, shellfish, water and ice, 155 were positive for the presence of faecal coliforms following incubation at 44.5 °C. However, E. coli was isolated from only 47% of the samples positive for faecal coliforms. The antibiotic resistance of 116 strains isolated from seafood was tested using 14 different antibiotics including ampicillin, cephalothin, chloramphenicol, ciprofloxacin, gentamycin, nalidixic acid, streptomycin and vancomycin. Seven strains were resistant to more than five antibiotics of which one was resistant to eight antibiotics. The multiple drug resistant strains harboured plasmids of varying sizes. Antibiotic susceptibility studies revealed that seafood from India contains multiple antibiotic resistant strains of E. coli which may serve as a reservoir for antibiotic resistance genes in the aquatic environment. All the strains used in this study did not harbour any virulence genes commonly associated with pathogenic E. coli, when tested by polymerase chain reaction (PCR).

Temperature and pH are important parameters in food processing. Their effect on degradation of plasmid and plant DNA was investigated. Application of HPLC permitted us to quantitatively distinguish between covalently closed circular and nicked plasmid DNA. Nicking of plasmid DNA in buffer increased strongly at decreased pH (99% within 90 min at 65 °C/pH 4.0). A virtually identical degradation rate was observed in tomato serum. Temperature exerted only minor effects (15% within 90 min at 85 °C/pH 8.4). The combined effect of the parameters was additive. Degradation of plant DNA was monitored by determining the maximal detectable fragment length using polymerase chain reaction. With transgenic maize DNA in buffer, fragments up to 957 bp remained detectable for at least 90 min upon application of 65 °C at pH 4.0. Soymilk and tofu were produced from genetically modified soybeans. After boiling (10 min) of raw soymilk, the detectable fragment length decreased from 1,714 to 1,339 bp. The study of the final tofu product did not reveal any further DNA degradation. In commercial soy protein isolate, a rather highly processed food, DNA fragments of at least 714 bp were detected.

Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli has become increasingly recognized as an emerging opportunistic pathogen of clinical importance. A total of 18 isolates comprising of 10 (55.6%) S. aureus, 6 (33.3%) P. aeruginosa and 2 (11.1%) E. coli were recovered from 15 pus samples of wound patients attending Federal Teaching Hospital Abakaliki I (FETHA I) and Federal Teaching Hospital Abakaliki II (FETHA II). All the isolates were subjected to antibiotic susceptibility testing using Kirby-Bauer disk diffusion method on Mueller-Hinton agar. S. aureus showed the highest susceptibility percentage of 80.0% to ciprofloxacin, followed by 60.0% to augmentin and 50.0% to streptomycin, while the highest resistance percentage was obtained with lincocin (100.0%), followed by ampiclox (80.0%). P. aeruginosa showed the highest susceptibility percentage of 83.3% to ciprofloxacin, followed by 66.6% to streptomycin and gentamycin (66.6%), while the highest resistance percentage was obtained with streptomycin (33.3%) and gentamycin (33.3%). E. coli showed the highest susceptibility percentage to gentamycin and streptomycin with 100% activity. The antibiotics with reasonable resistant profile was observed in 10 isolates (5 S. aureus, 5 P. aeruginosa and 1 E. coli) with isolate code which showed resistance to atleast 5 antibiotics, hence this isolates were subjected to plasmid profile analysis. Only three isolates (S 1 , S 4 and Ec 1) showed the presence of plasmids within the range of 1.8 kbp to 10.4 kbp. Hence antibiotic resistance of an organism does not always confer the presence of plasmid.