Plasmid pDGO100 contains a second integron with the trimethoprim resistance gene dfrA7 as the inserted cassette.

Southern hybridization analysis of the IncC plasmid pDGO100 showed that, in addition to the well-characterized integron In7, there is a second integron which is located on a 3.6-kb BamHI fragment. This integron also possesses the qacE delta l and sulI genes typically found as part of the 3'-conserved segment of integrons. The 3.6-kb BamHI fragment, when cloned into pUC19 to form pDGO301, conferred resistance to trimethoprim as well as sulfamethoxazole. The DNA sequence of the trimethoprim resistance gene in pDGO301 was determined and it was shown that the gene was precisely inserted as a cassette in an integron with 100% identity to the trimethoprim resistance gene dfrA7.

Nucleotide sequence of the AAD(2'') aminoglycoside adenylyltransferase determinant aadB. Evolutionary relationship of this region with those surrounding aadA in R538-1 and dhfrII in R388.

The nucleotide sequence of the aadB gene which confers resistance to kanamycin, gentamicin, and tobramycin has been determined. The size of the longest reading frame is 747 bases encoding a protein of predicted size 27,992 daltons. A segment of the aadB gene sequence (including the promoter region) was found upstream of the aadA gene in R538-1 and of the dhfrII gene in R388 and the proposed promoters for these genes coincide with the aadB promoter region. The sequence homology extends upstream to the end of the sequenced regions of R388 and R538-1. Almost perfect homology was also found between the sequences 3'- to the aadB gene and 3'- to the aadA genes of R538-1 and pSa. This segment includes a 59 base element previously found flanking the Tn7 aadA gene. A model is presented for the evolution of this region of the plasmid genomes in which the 59- base element functions as an insertional "hot spot" and the possibility that this region is analogous to the aadA/aadB region of the Tn21- like transposon family is considered.

Construction of a gentamicin resistance gene probe for epidemiological studies.

A 7.7-kilobase BamHI fragment was cloned from the transconjugant of a clinical isolate of Escherichia coli containing a 120-kilobase multiresistance IncC plasmid. The recombinant plasmid conferred resistance to kanamycin, gentamicin, tobramycin, sulfamethoxazole, and trimethoprim. This clone was used to generate a series of subclones from which a 2.0-kilobase BamHI-HindIII probe containing a gentamicin 2''-O-adenylyltransferase [AAD(2'')] gene was obtained. This probe hybridized specifically in both colony and Southern hybridizations with the AAD(2'') gene but not with other resistance genes, including other aminoglycoside-modifying genes, or with a reference IncC plasmid lacking the AAD(2'') gene. The AAD(2'') gene may be part of a transposon, since hybridization occurred with both nonconjugative plasmids and the chromosome in some isolates.

A replicator method for the combined determination of minimum inhibitory concentration and minimum bactericidal concentration.

Minimal bactericidal concentrations (MBCs) of nine antimicrobial agents were determined for clinical isolates by a replica plating method. Membranes were placed on the antibiotic-containing plates and the organisms replicated onto the membranes. After 18 h incubation the minimal inhibitory concentrations (MICs) were determined, the membranes were transferred to antibiotic-free plates and incubated a further 18 h and the MBCs determined. MICs and MBCs were also determined in broth. The reproducibility of the 'membrane' method and the agreement of these results for MIC and MBC with the agar and/or broth methods was satisfactory for most antibiotics, within one two-fold dilution. With sulphamethoxazole, trimethoprim and co-trimoxazole the results were less satisfactory, especially with Gram-negative rods, but agreement within two dilutions could be achieved.

Transferable antibiotic resistance in a general hospital: a two year survey.

For two years transferable antibiotic resistance (TAR) was studied by replicator methods in strains of Enterobacteriaceae isolated in a 900-bed hospital. Transfer to an Escherichia coli recipient was demonstrated in 21% of 7,800 Enterobacteriaceae. It was most common in Klebsiella (37% of isolates) and least in Acinetobacter (6%). The mean number of phenotypic resistance markers (RMs) transferred was higher from Klebsiella pneumoniae or Enterobacter cloacae than from E. coli or P. mirabilis. K. pneumoniae and E. cloacae especially transferred 7 or more RMs much more often than other species. Some RMs were associated with a particular species, e.g. streptomycin with E. coli, kanamycin or gentamicin with E. cloacae and kanamycin with P. mirabilis. The same was true of certain patterns of resistance transfer, e.g. Ap-Km and P. mirabilis, Ap-Sm and E. coli, Ap-Km-Gm-Tm-Cm and K. pneumoniae. However, many of the commonest resistance phenotypes were transferred from several species and from several biotypes within any given species. Resistance patterns transferred from E. coli were detected sporadically throughout, whereas many of those transferred from Klebsiella, Enterobacter or Proteus were found only for a limited time.

Reporting practices of microbiology laboratories.

Results of investigations on typical specimens were circulated to Australian microbiologists, who were asked to draft reports on the basis of the data provided. Many laboratories were found simply to report the results of their activities without explanations. This was true whether the finding was that of a Gram-negative rod in a postoperative sputum or an anaerobic diphtheroid in a blood culture. There was diversity of views as to what constituted probable contamination in a urine specimen. Often no clearcut verdict was given, nor did the report indicate when no conclusion was possible. Remedial measures are discussed.