To clarify the relationship between mutations commonly found for penicillin-binding protein 3 (PBP 3) of ␤-lactamase-nonproducing ampicillin-resistant (BLNAR) Haemophilus influenzae isolates and ␤-lactam resistance, single and multiple amino acid mutations at positions 377, 385, 389, 517, and 526 were introduced into PBP 3 of a ␤-lactam-susceptible Rd strain by site-directed mutagenesis. Twelve isogenic recombinant strains were challenged with nine ␤-lactam antibiotics. Replacement of the asparagine at position 526 with lysine (N526K) increased the resistance to imipenem eightfold and increased the resistance to various cephalosporins two-to eightfold. Substitution of threonine for serine at position 385 (S385T) and/or substitution of phenylalanine for leucine at position 389 (L389F), in addition to the N526K mutation, led to two-to fourfold additional increases in cephalosporin resistance. An isoleucine-to-methionine substitution at position 377 did not change the antibiotic sensitivity of any of the recombinant strains also carrying other PBP 3 mutations tested. Thirty-six clinical isolates carrying a PBP 3 gene (ftsI) with the S385T, L389F, R517H, and/or N526K mutation were chosen from among 279 clinical isolates collected in Japan, and the isolates were grouped into six classes on the basis of the patterns of the four mutations in PBP 3. Rd recombinants were made with each of the ftsI genes. The levels of resistance to ␤-lactams varied between recombinants of different classes but were comparable for those of the same class. The levels of resistance to cephalosporins of these recombinants were similar to those of the parent clinical isolates, while those to ampicillin and carbapenems were lower. These results indicate that resistance to ␤-lactams, at least to cephalosporins, depends in large part on the PBP 3 mutations R517H, N526K, S385T, and L389F.Haemophilus influenzae has two ␤-lactam resistance mechanisms. One involves enzymatic hydrolysis of ␤-lactams by the TEM-1 or the ROB-1 ␤-lactamase (7). The other mechanism involves decreased ␤-lactam affinities for penicillin-binding protein 3 (PBP 3) (11). The ␤-lactam resistance phenotype mediated by the second mechanism is named ␤-lactamasenonproducing ampicillin resistance (BLNAR). Since 1992 the frequency of BLNAR strains has increased exponentially among Japanese isolates from community-acquired infections (9, 12). Recently, strains with both resistance mechanisms were found among clinical isolates, and such H. influenzae strains are termed ␤-lactamase-producing ampicillin-clavulanic acid resistant (2,5,10).From the genetic analysis of the ftsI gene, which encodes PBP 3 in BLNAR strains, the amino acid mutations surrounding the conserved KTG (Lys512-Thr-Gly) and SSN (Ser379-Ser-Asn) motif would be relevant to ␤-lactam resistance (1, 2, 8, 11). Amino acid substitutions, such as N526K and R517H, near the KTG motif are commonly found in isolates with cefotaxime intermediate resistance (MICs, 0.063 to 0.25 g/ ml). Additional mutations (M377I, S385T, and/or L38...

A previous study recommended "Please do not eat salads" as a note for immunocompromised patients, and the contamination of raw vegetables by microorganisms such as Pseudomonas aeruginosa has long been known. [1][2][3][4][5] Therefore, the consumption of canned fruit and cooked vegetables has been promoted among immunocompromised patients. However, many immunocompromised patients prefer to eat raw fruit and vegetables, which may also be nutritionally desirable.The cultivation methods of vegetables and fruit have changed markedly; the main vegetable cultivation method has shifted from open-field to greenhouse cultivation. However, there have been no recent studies on the microbial contamination of fruit and vegetables. In addition, the types of fruit and vegetable which are relatively safe microbiologically for immunocompromised patients have not been fully clarified. Therefore we evaluated the microbial contamination of fruit and vegetables and the effects of sodium hypochlorite disinfection on it. MATERIALS AND METHODS Fruit and VegetablesWe examined 17 types of vegetable [cabbage, lettuce, sunny lettuce, leek, perilla, spinach, komatsuna (Japanese mustard spinach), napa cabbage, parsley, tomato, minitomato, eggplant, cucumber, carrot, sweet pepper, onion, and Japanese radish] and 10 types of fruit (apple, pear, persimmon, grape, mandarin orange, orange, grapefruit, lemon, kiwi, and banana) served in meals in a university hospital (736 beds) between September 20 and December 20, 2006. Investigation of the microbial contamination of these fruit and vegetables was performed 3-5 times at intervals of 14 d or longer. Among the vegetables evaluated, peeled samples were used only for onion. Among fruit, unpeeled samples were used for grape and lemon, and both peeled and unpeeled samples were used for the other 8 types of fruit. The cores of apples and pears and calyces of persimmons were also evaluated. Washing and DisinfectionFruit and vegetables were washed without scrubbing under flowing tap water (flow rate, about 7 l/min) for 30 s. Disinfection was performed by 10-min immersion in 0.01% (100 ppm) sodium hypochlorite prepared by dilution of Milton (Kyorin Pharmaceutical Co., Tokyo, Japan) with tap water. These procedures were performed while wearing sterilized rubber gloves. During immersion in sodium hypochlorite, a lid was placed directly on the samples to ensure adequate contact with the disinfecting agent.Microbiological Analysis After washing alone or washing followed by disinfection, each sample was cut (5-20 g) using a knife wiped with ethanol for disinfection and placed in bottles containing 30 ml of sterile solution (25 ml of physiologic salineϩ5 ml of broth). The bottles were then vibrated with a recipro shaker (SR-1, Taiyo Industry Ltd., Tokyo, Japan) at a frequency of 250 cycles/min and an amplitude of 50 mm and ultrasonicated (Sine Sonic 100, Ikemoto Rikagaku Co., Tokyo, Japan) at 36 kHz for 5 min. Each sample was diluted 10-fold, 100-fold, and 1000-fold in sterile saline; four aliquots (0.5 ml each) ...

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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 ...

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