Over a 2 1 ⁄2-month period in 1999, 37 ceftazidime-resistant nonrepetitive enterobacterial isolates were collected from 37 patients in a Bangkok hospital, Thailand. Eighty-one percent of these strains expressed a clavulanic acid-inhibited extended-cephalosporin resistance profile. An identical extended-spectrum ␤-lactamase (ESBL), VEB-1, was found in 16 unrelated enterobacterial isolates (Escherichia coli, n ‫؍‬ 10; Enterobacter cloacae, n ‫؍‬ 2; Enterobacter sakazakii, n ‫؍‬ 1; and Klebsiella pneumoniae, n ‫؍‬ 3) and in two clonally related E. cloacae isolates. The bla VEB-1 gene was located on mostly self-conjugative plasmids (ca. 24 to 200 kb) that conferred additional non-␤-lactam antibiotic resistance patterns. Additionally, the bla VEB-1 gene cassette was part of class 1 integrons varying in size and structure. The bla VEB-1 -containing integrons were mostly associated with bla OXA-10 -like and arr-2-like gene cassettes, the latter conferring resistance to rifampin. These data indicated the spread of bla VEB-1 in Bangkok due to frequent transfer of different plasmids and class 1 integrons and rarely to clonally related strains. Plasmid-and integron-mediated resistance to rifampin was also found in enterobacterial isolates.

The beta-lactamase gene content and epidemiology of ceftazidime-resistant Pseudomonas aeruginosa isolates (24% of the total number of P. aeruginosa isolates) were investigated at a University Hospital in Thailand during a 4-month period in 1999. Of 33 nonrepetitive clinical isolates, 31 produced a VEB-1-like clavulanic acid-inhibited extended-spectrum beta-lactamase (ESBL). These isolates belonged to different pulsed-field gel electrophoresis types and subtypes. In 1 case, the bla(VEB-1)-like gene was plasmid located. The bla(VEB-1)-like genes were present as a gene cassette on class 1 integrons that varied in size and structure. In most cases, the veb-1 cassette was associated with an arr-2 cassette (rifampin resistance), aminoglycoside resistance gene cassettes, and an oxa-10-like cassette encoding a narrow-spectrum oxacillinase-type beta-lactamase. The present study indicates that ESBLs may be endemic in P. aeruginosa and illustrates that integrons are efficient means for their spread.

Summary. The occurrence of resistance to antiseptics and disinfectants in clinical isolates of coagulase-negative staphylococci (CNS) was examined. Of 164 clinical strains of CNS isolated in the early 1980s, 65 were resistant to cationic antimicrobial compounds such as cetyltrimethylammonium bromide. Further characterisation of 40 resistant isolates by DNA-DNA hybridisation analysis and phenotypic resistance studies revealed that this resistance was mediated by the multidrug export genes qacA and qacC, characterised previously in Staphylococcus aureus. Of the resistant CNS isolates, 50 YO contained only qacA, 10% contained only qacC, and the remaining 40% contained both qacA and qacC. Both qacA and qacC genes resided on plasmids in all cases, with qacA located on plasmids of > 10 kb, whereas qacC was located primarily on plasmids of 2-3 kb. Representative qacA and qacC plasmids were characterised by restriction endonuclease mapping, and were found to be similar in some cases, but different in others, to those plasmids on which these genes are found in S. aureus.

The 46.4-kb nucleotide sequence of pSK41, a prototypical multiresistance plasmid from Staphylococcus aureus, has been determined, representing the first completely sequenced conjugative plasmid from a gram-positive organism. Analysis of the sequence has enabled the identification of the probable replication, maintenance, and transfer functions of the plasmid and has provided insights into the evolution of a clinically significant group of plasmids. The basis of deletions commonly associated with pSK41 family plasmids has been investigated, as has the observed insertion site specificity of Tn552-like β-lactamase transposons within them. Several of the resistance determinants carried by pSK41-like plasmids were found to be located on up to four smaller cointegrated plasmids. pSK41 and related plasmids appear to represent a consolidation of antimicrobial resistance functions, collected by a preexisting conjugative plasmid via transposon insertion and IS257-mediated cointegrative capture of other plasmids.

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Analyses of the Staphylococcus epidermidis multiresistance plasmids pSK697 and pSK818 have revealed them to be closely related to the trimethoprim resistance plasmid pSK639, also isolated from S. epidermidis. pSK697 and pSK818 were found to contain a cointegrated copy of a second plasmid related to the S. epidermidis multidrug antiseptic and disinfectant resistance plasmid pSK108 and the S. aureus tetracycline resistance plasmid pT181, respectively. In contrast to pSK639, both plasmids were found to contain a third copy of IS257, such that the integrated plasmids in both cases are flanked by a copy of this element. This organization and the presence of duplicated sequences at the extremities of the integrated plasmids implicate IS257 in the formation of these cointegrate plasmids. Sequence analysis of the IS257 elements from these plasmids has provided insights into the probable mechanism of cointegration, viz., nonresolved replicative transposition of IS257.

The transposon-like structure Tn4003 and related elements were found to encode high-and low-level trimethoprim resistance (Tpr) in Staphylococcus aureus and coagulase-negative staphylococci. By using transcriptional fusions in Escherichia coli, the variation in resistance levels was found to correlate with the transcriptional activity of the region presumed to carry the promoter for the operon containing the Tpr dihydrofolate reductase gene, dfrA, encoded by these elements. The reduced transcriptional activities exhibited by elements encoding low-level Tpr appear to be a consequence of deletions adjacent to the copy of IS257 which normally encodes the -35 sequences of these promoters. The data obtained not only support the involvement of IS257 in the transcription of the proposed thyE-dfrA-orf-140 operon of Tn4003 but may also implicate this insertion sequence in the mechanisms resulting in the variation in Tpr levels observed in staphylococci.

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