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Summary: The indigenous plasmid pIJ101 is the parent of many cloning vectors used in Streptomyces. One early pIJ101 derivative, pIJ702, has been particularly widely used. pIJ702 lacks sti:cop/korB and accumulates single-stranded DNA (ssDNA). The 1.2 kb BclI-BclI sti:cop/korB and 0.7 kb SpeI-BclI sti regions were isolated from pIJ101 and cloned into pIJ702 at the PstI site in both orientations. No ssDNA was detected in constructs containing sti present in its correct orientation with respect to the basic replicon, with or without cop/korB. Constructs which contained sti in the reverse orientation did accumulate ssDNA. Thus, sti is only active as the site for second-strand synthesis in its natural orientation. Furthermore, sti inserted in either orientation into the structurally unstable pIJ702-pUC8 shuttle vectors prevented them from rearranging in S. lividans. The sti function was defined to a 0.53 kb SpeI-SacII fragment and the probable site for second-strand initiation (ssi) was identified.

Nosocomial isolates of Pseudomonas aeruginosa exhibit high rates of resistance to antibiotics, and are often multidrug resistant. P. aeruginosa clinical isolates (n = 56) were obtained from ICU patients in a hospital in Pakistan over a 3-y period. Antimicrobial susceptibility of the 56 P. aeruginosa clinical isolates was investigated using 7 antibiotics and the resistance rates were as follows: aztreonam (68% resistant), ceftazidime (67%), imipenem (66%), ofloxacin (59%), amikacin (56%), gentamicin (44%), and piperacillin-tazobactam (27%) (p0.01). In addition, 55% of the P. aeruginosa clinical isolates were resistant to 4 or more antibiotics. Imipenem-resistant strains were frequently associated with ceftazidime, ofloxacin, aztreonam, and more strikingly, amikacin resistance (p0.05). PCR (using P. aeruginosa-specific primers VIC1 + VIC2 and P1 + P2, respectively) was highly specific and sensitive, and was positive for all 56 P. aeruginosa isolates tested. Automated ribotyping was used to investigate the clonal diversity of the 56 P. aeruginosa isolates. Automated ribotyping indicated that the clinical isolates were clonally related and could be clustered into 4 major ribogroups based on their similarity index, with ribogroup II being the dominant one. The P. aeruginosa isolates in ribogroup II were correlated with their antibiotic resistance pattern and, interestingly, there seemed to be a gradual acquisition of multiple antibiotic resistance associated with the isolates within this group over time. The ribotyping data, together with the antibiotic resistance profile, provide valuable molecular epidemiology information for the control of hospital-acquired P. aeruginosa infections.

Seventeen pairs of published primer sets were compared for their relative sensitivity to detect malaria DNA extracted from blood samples, which were obtained from Pakistani patients suffering from malaria. The primer sets investigated consisted of: (i) 9 pairs of direct primers and 3 sets of nested primers for detecting Plasmodium falciparum, (ii) 2 pairs of direct primers and 2 sets of nested primers for detecting P. vivax, and (iii) 1 set of multiplex primers for detecting both P. falciparum and P. vivax, simultaneously. After a miniscreen of 9 DNA-extracted blood samples using the 17 primer sets stated above, 5 primer sets were short-listed (based on their superior sensitivity) and used for a maxi-screen of DNA extracted from 126 microscopy-positive blood samples from Pakistan, with the following results. (i) For the detection of P. falciparum, the direct primer pair 'PF1 + PF2' gave a sensitivity of 95% and the nested primer set 'RIT405 + RIT406/RIT371 + RIT372' gave a sensitivity of 97%. (ii) For the detection of P. vivax, the direct primer pair 'Forward + Reverse' and the nested primer set 'PLF + UNR/PLF + VIR' both gave a sensitivity of 94%. (iii) The nested multiplex primer set 'rPLU5 + rPLU6/rFAL1 + rFAL2 + rVIV1 + rVIV2' gave a sensitivity of 97% and 96% for P. falciparum and P. vivax, respectively. It was concluded that the nested multiplex primer set was the most optimal primer set to use for the detection of malaria DNA extracted from blood samples. Furthermore, the nested multiplex primer set has the advantage of simultaneously detecting and differentiating between P. vivax and P. falciparum.

The minimal replicon of pIJ101 was defined to a 2.0-kb BalI-SacII fragment containing the rep ORF and a non-coding region of DNA, but not orf56 (pQR435). This non-coding region of DNA was found to be crucial for plasmid replication in Streptomyces lividans and may contain either the origin of replication or the rep promoter (or both). A 1.4-kb NotI-SacII fragment containing only the rep ORF produced a 54-kDa protein in vitro when expressed from the lacZ promoter. pQR435 was structurally unstable in S. lividans and accumulated single-stranded (ss)DNA. The insertion of the site for second-strand initiation (ssi) into pQR435 did not prevent the plasmid from rearranging or from accumulating ssDNA.