Multidrug-resistant gram-negative rods such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae have presented problems.1,2) In particular, P. aeruginosa is an important species causing nosocomial infection, 3) and its development of multidrug-resistance has become a major social issue. [4][5][6] In Japan, multidrug-resistant P. aeruginosa was defined as isolates resistant to all of the following drugs: imipenem, MIC (minimum inhibitory concentration) Ն16 mg/ml; amikacin, MIC Ն32 mg/ml; and ciprofloxacin, MIC Ն4 mg/ ml. However, some of these isolates resistant to imipenem, ceftazidime, and amikacin are also resistant to all other commonly used antipseudomonal drugs such as piperacillintazobactam, aztreonam, and ciprofloxacin. For such isolates, drugs for treatment are not available at present. Our previous studies have shown that three drug combinations such as the combination of ceftazidime, aztreonam, and amikacin are more effective against multidrug-resistant P. aeruginosa than the combinations of b-lactam and aminoglycoside antibiotics, such as ceftazidime and amikacin. 7,8) Therefore, in this study, we evaluated the effects of three drug combinations of ceftazidime, aztreonam, and amikacin and ceftazidime, aztreonam, amikacin, or colistin alone at a concentration three times the breakpoint concentration on four strains of P. aeruginosa that were isolated from patients in two hospitals in 2002-2006 and resistant to all commonly used antipseudomonal drugs.
MATERIALS AND METHODS
Bacterial StrainsOf a total of 1720 P. aeruginosa strains isolated from clinical materials in two hospitals 733 and 315 beds, respectively in Yamaguchi Prefecture between January 2002 and December 2006, four strains that were resistant to all of piperacillin, piperacillin-tazobactam, imipenem, meropenem, ceftazidime, aztreonam, amikacin, and ciprofloxacin were used. The sources of the four strains were blood (two strains), bile (one), and frank hip pus (one).Random Amplified Polymorphic DNA (Deoxyribonucleic Acid) Analysis [9][10][11] Genomic DNA was purified by the phenol-chloroform method. The primers 272 (5Ј-AGC-GGGCCAA-3Ј) and ERIC2 (enterobacterial repetitive intragenic consensus; 5Ј-ATGTAAGCTCCTGGGATTTCA-3Ј) were employed. The reaction took place in 1 ml 10ϫPCR (polymerase chain reaction) buffer (Ex Taq, Takara, Tokyo, Japan), 0.8 ml dNTP (deoxyribonucleoside triphosphates) mixture (Ex Taq, Takara, Tokyo, Japan), 0.06 ml Ex Taq (Ex Taq, Takara, Tokyo, Japan), 0.2 ml primer, 80 ng DNA template, 6.94 ml water, and 10 ml mineral oil. Amplification was performed in a DNA thermal cycler (Perkin ELMER 9600) under the following conditions: primer 272; 94°C for 7 min and 94°C for 1 min, 35°C for 1 min, 72°C for 1 min (40 cycles), and 72°C for 16 min. Primer ERIC2 was used under the same conditions except for the following: the temperature was changed from 35 to 52°C, and the number of cycles from 40 to 35. Amplification products were resolved by electrophoresis in a 3% agarose gel and were detected by staining ...