A multiple-primer PCR was used to identify genes encoding aminoglycoside-modifying enzymes in 381 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). The technique used three sets of primers delineating specific DNA fragments of the aph(3)-III, ant(4)-I, and aac(6)-aph(2؆) genes, which influence the MICs of gentamicin, tobramycin, and lividomycin. Isolates with none of the three genes detected were susceptible to all three agents. Isolates with the aph(3)-III gene showed resistance to lividomycin (MIC > 1,024 g/ml), and those with the ant(4)-I gene were resistant to tobramycin (MIC > 8 g/ml). Isolates with only the aac(6)-aph(2؆) gene were resistant to gentamicin (MIC > 8 g/ml) and tobramycin in decreasing order; those with both the ant(4)-I and aac(6)-aph(2؆) genes also were resistant to gentamicin and tobramycin, but in increasing order. Susceptibility testing, then, could detect specific genes. In 381 Japanese MRSA isolates, the ant (4)

The gene bla IMP-10 of a variant metallo-β-lactamase, IMP-10, had a single base replacement of G by T at nucleotide 145, which led to an amino acid alteration of Val49 to Phe compared to the IMP-1 enzyme, indicating that IMP-10 was a point mutation derivative of IMP-1. Highly purified enzymes revealed that IMP-10 was different from IMP-1 in its extremely low hydrolyzing activities for penicillins, such as benzylpenicillin, ampicillin, and piperacillin.

To determine the persistence and spread of antibiotic-resistant strains in Gunma University Hospital, 83 Pseudomonas putida strains (each from a different patient) were isolated from January 1997 through December 2001. Of the 83 strains isolated, 27 were resistant to carbapenems. All 27 produced metallo-␤-lactamase and were found to be PCR positive for the bla IMP gene. Most (22 strains) were primarily isolated from the wards (W7 [9 strains] and W4 [8 strains]). Another five bla IMP -positive P. putida strains from wards W7 and W4 were obtained by swabbing around the water pipes. A total of 32 bla IMP -positive P. putida strains were assessed by pulsed-field gel electrophoresis (PFGE) and testing of drug susceptibility to 10 chemotherapeutic agents. Both PFGE and MIC patterns revealed that there were long-term resident strains among inpatients and hospital environments. The bla IMP genes of 22 of 32 strains were all transferable to a recipient strain of Pseudomonas aeruginosa by conjugation or transformation and conferred resistance to carbapenems and cephems. The bla IMP plasmids were conjugally transmissible among P. aeruginosa strains and mediated resistance to amikacin as well as ␤-lactams. Ten of the 22 plasmids mediated additional resistance to gentamicin and tobramycin. Plasmids with identical DNA and drug resistance patterns were found in P. putida strains with identical PFGE patterns and with different PFGE patterns. We presumed that P. putida was one of the resident species in inpatients and especially in hospital environments, spreading drug resistance genes via plasmids among P. putida strains and supplying them to more pathogenically important species, such as P. aeruginosa.Carbapenem-hydrolyzing metallo-␤-lactamases, especially IMP-type and VIM-type metallo-␤-lactamases, are clinically important, because these enzymes effectively hydrolyze almost all ␤-lactam antibiotics except monobactams, conferring resistance to penicillins and cephems in addition to carbapenems on pathogenic bacteria (12,13,18,21,23,32). Since genes encoding these metallo-␤-lactamases (bla IMP and bla VIM ) and their variant genes have become easy to detect using the PCR method, dissemination of these genes in clinical isolates has been widely observed in gram-negative bacteria, especially in Pseudomonas aeruginosa and other non-glucose-fermenting bacteria (3, 4, 6, 8-10, 26-30, 34, 35). Recently, nosocomial infections of Pseudomonas putida producing VIM-type metallo-␤-lactamase were reported in Italy (15). The bla VIM gene was plasmid-borne but was not transferable to either P. aeruginosa or Escherichia coli. We also observed metallo-␤-lactamase-producing P. putida strains in Gunma University Hospital, but the responsible gene was bla IMP , not bla VIM , and it was plasmid-borne in most of the isolates. In this paper, we report the existence of multiple-drug-resistant P. putida strains that harbored bla IMP plasmids easily transmissible to P. aeruginosa in Gunma University Hospital. MATERIALS AND METHODSBacterial strains. A to...

This study describes the use of the polymerase chain reaction (PCR) to detect the tet(K) and tet(M) tetracycline resistance genes in Staphylococcus aureus. Primers based on the DNAsequence of the tet(K) and tet(M) genes from S. aureus were used as primers in the PCRassay to detect the presence of genes for resistance to tetracycline and minocycline. Two-hundred and fifteen isolates of S. aureus from Asian countries as Japan, Indonesia, China, Korea and Thailand were examined, and the results confirm that tet(K) specifies resistance to tetracycline but not to minocycline and tet(M) specifies resistance to both tetracycline and minocycline. Weobserved two different types of clinical isolates of S. aureus strains resistant to minocycline and tetracycline: the first carried only the tet(M) gene, while the second carried both the tet(M) and the tet(K) genes. Almost all of the clinical isolates of S. aureus resistant to minocycline and tetracycline from Indonesia, China and Thailand carried both tet(M) and tet(K) genes, while most of clinical isolates of S. aureuss from Japan and Korea carried only tet(M) gene.

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