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In mice implanted with an osmotic pump filled with the superantigen (SAG) staphylococcal enterotoxin A (SEA), the Vß3(+)CD4(+) T cells exhibited a high level of expansion whereas the Vß11(+)CD4(+) T cells exhibited a mild level of expansion. In contrast, in mice implanted with an osmotic pump filled with SE-like type P (SElP, 78.1% homologous with SEA), the Vß11(+)CD4(+) T cells exhibited a high level of expansion while the Vß3(+)CD4(+) T cells exhibited a low level of expansion, suggesting that the level of the SAG-induced response is determined by the affinities between the TCR Vß molecules and SAG. Analyses using several hybrids of SEA and SElP showed that residue 206 of SEA determines the response levels of Vß3(+)CD4(+) and Vß11(+)CD4(+) T cells both in vitro and in vivo. Analyses using the above-mentioned hybrids showed that the binding affinities between SEA and the Vß3/Vß11 ß chains and between SEA-MHC class II-molecule complex and Vß3(+)/Vß11(+) CD4(+) T cells determines the response levels of the SAG-reactive T cells both in vitro and in vivo.

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We witnessed outbreaks of multidrug-resistant (MDR) and drug-resistant Pseudomonas aeruginosa at a hospital in Tokyo, Japan, during the period September 2004 through May 2005. The first outbreak occurred in September and October 2004. Three isolates of MDR P. aeruginosa were identified from urine samples obtained from three nonambulatory immunodeficient patients in one ward. After 3 weeks, another outbreak of P. aeruginosa occurred in the hematology ward on the same floor of the hospital. During the outbreaks, environmental surveys were conducted twice in each of the two wards, at 2-week intervals, to identify the sources of the pathogens. A total of 23 P. aeruginosa isolates, including 11 from environmental sources, were analyzed for chromosomal DNA typing by pulsed-field gel electrophoresis, for O-antigen serotyping, and for other typing. Results revealed two causative clones, as well as environmental contamination by P. aeruginosa clones on the surfaces of urine volume-measuring devices in rooms where urine is handled, which may have been sources of the pathogens during the outbreaks. To prevent further outbreaks, we performed the following: (a) environmental surface monitoring for drug-resistant P. aeruginosa, (b) active surveillance of specimens, (c) strict isolation of infected patients or carriers of MDR P. aeruginosa, (d) rigorous contact precautions, and (e) disinfection with 70% alcohol on the surfaces of apparatuses contaminated by MDR or drug-resistant P. aeruginosa and in the rooms where urine is handled. As a result, the outbreaks were contained.

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We previously reported an outbreak in a neurosurgery ward of catheter-associated urinary tract infection with multidrug-resistant (MDR) Pseudomonas aeruginosa strain IMCJ2.S1, carrying the 6'-N-aminoglycoside acetyltransferase gene [aac(6')-Iae]. For further epidemiologic studies, 214 clinical isolates of MDR P. aeruginosa showing resistance to imipenem (MIC >or= 16 microg/ml), amikacin (MIC >or= 64 microg/ml), and ciprofloxacin (MIC >or= 4 microg/ml) were collected from 13 hospitals in the same prefecture in Japan. We also collected 70 clinical isolates of P. aeruginosa that were sensitive to one or more of these antibiotics and compared their characteristics with those of the MDR P. aeruginosa isolates. Of the 214 MDR P. aeruginosa isolates, 212 (99%) were serotype O11. We developed a loop-mediated isothermal amplification (LAMP) assay and a slide agglutination test for detection of the aac(6')-Iae gene and the AAC(6')-Iae protein, respectively. Of the 212 MDR P. aeruginosa isolates, 212 (100%) and 207 (98%) were positive in the LAMP assay and in the agglutination test, respectively. Mutations of gyrA and parC genes resulting in amino acid substitutions were detected in 213 of the 214 MDR P. aeruginosa isolates (99%). Of the 214 MDR P. aeruginosa isolates, 212 showed pulsed-field gel electrophoresis patterns with >or=70% similarity to that of IMCJ2.S1 and 83 showed a pattern identical to that of IMCJ2.S1, indicating that clonal expansion of MDR P. aeruginosa occurred in community hospitals in this area. The methods developed in this study to detect aac(6')-Iae were rapid and effective in diagnosing infections caused by various MDR P. aeruginosa clones.

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To assess whether the occurrence of rifampicin (RFP) resistance in methicillin-resistant Staphylococcus aureus (MRSA) is related to treatment of tuberculosis, we determined the RFP susceptibility of MRSA isolates obtained from tuberculosis patients and screened for mutation(s) in the rpoB gene of these isolates. The MICs of RFP for 84 MRSA isolates obtained from two hospitals in Japan were determined. DNA was sequenced in the region 1318-1602 nucleotides (nt) of the rpoB gene, which includes RFP resistance-determining clusters I (1384-1464 nt, 462-488 amino acids). The majority of MRSA isolates from tuberculosis wards, i.e., 48 of 51 (94%) [33 of 34 in a Tokyo hospital (97%) and 15 of 17 in a Chubu hospital (88%)], were resistant to RFP. Meanwhile, no isolates of 33 from the other wards were resistant to RFP. All RFP-resistant MRSA isolates had a mutation(s), including novel mutation(s) such as Val453-->AEPhe, Asp471-->AEAsn, and Ile527-->AELeu, in rpoB. An emergence of RFP-resistant MRSAs in tuberculosis wards in Japan was strongly suggested.

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A novel gene, dfrG, encoding a trimethoprim (TMP)-resistant dihydrofolate reductase (DHFR, designated S3DHFR) was cloned from a clinical isolate of methicillin-resistant Staphylococcus aureus. Escherichia coli expressing dfrG was highly resistant to TMP. Recombinant S3DHFR exhibited DHFR activity that was not inhibited by TMP.

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Antibacterial activity of biapenem (BIPM) against clinical isolates of 8 species between 2000 and 2002 was compared with those of imipenem/cilastatin (IPM/CS), meropenem (MEPM), panipenem/betamipron (PAPM/BP) and ceftazidime (CAZ). The MICs of biapenem for Gram-positive bacteria were higher than those of IPM/CS and PAPM/BP, equal to those of MEPM and lower than those of CAZ. The MICs of BIPM for Gram-negative bacteria were higher than those of MEPM, equal to those of IPM/CS and PAPM/BP, and lower than those of CAZ. Antibacterial activity of BIPM against Pseudomonas aeruginosa was equal to those of IPM/CS and MEPM and superior to those of PAPM/BP and CAZ. In conclusion, BIPM showed broad antibacterial activity against both Gram-positive and Gram-negative clinical isolates. These results suggest that BIPM is useful for the treatment of various bacterial infections.

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Antimicrobial activity of fosfomycin (FOM), cefazolin (CEZ), cefmetazole (CMZ), cefotiam (CTM) and piperacillin (PIPC) against clinical isolates of methicillin-sensitive Staphylococcus aureus (MSSA) (beta-lactamase-producing or non-producing) and methicillin-sensitive coagulase-negative Staphylococci (MSCNS) (beta-lactamase-producing or non-producing) were determined to make clear the differences in antimicrobial activity of FOM and beta-lactam antibiotics. The antimicrobial activity of PIPC against beta-lactamase-producing strains of MSSA was lower than that against non-producing ones, judging from the distribution patterns of susceptibility of the strains to PIPC. There were no differences in the antimicrobial activity of FOM, CEZ and CMZ for the producing and non-producing strains. The activity of FOM against MSCNS was comparable to that against MSSA, although those of CEZ, CMZ, CTM and PIPC were decreased. FOM, CEZ, CMZ and CTM showed bactericidal activity against TH4278 (MIC [microgram/ml]: FOM, 1; CEZ, 0.5; CMZ, 1; CTM, 0.5; PIPC, 1) of beta-lactamase-producing MSSA at 1 microgram/ml for 6 h, but PIPC did not at the same condition. FOM and CMZ at MIC suppressed regrowth of the strain, but CEZ, CTM and PIPC did not. In conclusion, FOM, which is not affected by beta-lactamase, demonstrated strong bactericidal activity at low concentration against the beta-lactam-resistant strains due to beta-lactamase production.

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The susceptibility to arbekacin (ABK) of methicillin-resistant Staphylococcus aureus (MRSA) was investigated to find out how it related to aac(6')/aph(2") gene. In 49 isolates of MRSA for which MIC of ABK ranged from 0.125 to 64 micrograms/ml, the MICs of ABK for 38 strains carrying aac(6')/aph(2") gene were widely distributed from 0.25 to 64, whereas those for 11 strains without that gene were all < or = 0.5 microgram/ml. Residual rate of ABK activity was higher than that of gentamicin after the reaction with each crude enzyme preparation extracted from 3 isolates of MRSA, carrying aac(6')/aph(2") and aad(4',4") genes. Furthermore, 97 strains of MRSA isolated at Kanagawa prefecture in Japan in 1999 were all sensitive to ABK, although 28 strains of them carried aac(6')/aph(2") gene. These results showed that ABK resistance was not necessarily related to carrying aac(6')/aph(2") gene in clinical isolates of MRSA.

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The bactericidal activity of biapenem, a new carbapenem, against various efflux-mutants of Pseudomonas aeruginosa was compared with those of imipenem, panipenem, meropenem and ceftazidime. The bactericidal activity of biapenem against P. aeruginosa KG5001, a strain deficient in MexAB-OprM, MexCD-OprJ and MexXY-OprM, was very strong compared with those of imipenem and meropenem. In terms of bactericidal activities, biapenem and imipenem had similar activities against P. aeruginosa KG5003, a strain overexpressing MexAB-OprM, as against P. aeruginosa KG5001, however meropenem and ceftazidime had weaker activities against KG5003 than KG5001. The bactericidal activity against P. aeruginosa KG5007, a strain overexpressing MexCD-OprJ, was observed only by biapenem. The bactericidal activity of biapenem was strong and not influenced by all of these three efflux systems.

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We compared antibacterial activity of NM394, which is the active metabolite of a prodrug of new fluoroquinolone prulifloxacin (PUFX), against clinical isolates of bacteria with those of ciprofloxacin (CPFX), levofloxacin (LVFX), gatifloxacin (GFLX), tosufloxacin (TFLX) and fleroxacin (FLRX). 1. NM394 showed a broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria. 2. MIC80 of NM394 for methicillin-sensitive Staphylococcus aureus, Streptococcus pneumoniae and Enterococcus faecalis were 0.5 microgram/ml, 2 micrograms/ml and 4 micrograms/ml, respectively. MIC80 of NM394 for Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae was lower than 0.06 microgram/ml. MIC80 of NM394 for Serratia marcescens and Pseudomonas aeruginosa were 0.25 microgram/ml and 2 micrograms/ml, respectively. 3. Short-time bactericidal activity of NM394 against P. aeruginosa was stronger than those of CPFX, GFLX, LVFX and TFLX. 4. Short-time bactericidal activity of NM394 at Cmax concentration against 12 strains of P. aeruginosa was stronger than those of CPFX, LVFX, GFLX and TFLX.

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The in vitro short-term bactericidal activity and accumulation of NM394, the active metabolite of prulifloxacin, was compared with those of ciprofloxacin (CPFX), levofloxacin (LVFX) and gatifloxacin (GFLX), using Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Of the 4 fluoroquinolones examined, NM394 accumulated to the highest concentration in all three strains. The order of concentration of the fluoroquinolones accumurated in S. aureus 209P JC-1, E. coli NIHJ JC-2 and P. aeruginosa PAO1 were NM394 >> CPFX > GFLX > or = LVFX. The accumulation of fluoroquinolones into bacterial cells correlated with their MICs of the drugs for E. coli and P. aeruginosa, whereas there was no correlation between the accumulation and MICs of the drugs for S. aureus. We also studied the reduction of viable cells after addition of each fluoroquinolones to clarify relationship between the short-term bactericidal activity and the accumulation of the quinolones. The short-term bactericidal activity of NM394 against S. aureus 209P JC-1, E. coli NIHJ JC-2 and P. aeruginosa PAO1 were stronger than those of CPFX, LVFX and GFLX when compared at the same concentration. In conclusion, the strong short-term bactericidal activity of NM394 may be attributed to its high accumulation in bacterial cells.

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In vitro antibacterial activity of fosfomycin was evaluated by various methods. Strains of methicillin-sensitive Staphylococcus aureus, methicillin-sensitive coagulase-negative Staphylococci and Escherichia coli were much more susceptible when glucose-6-phosphate was added to the test medium, but strains of Serratia marcescens and Pseudomonas aeruginosa were not affected. Nutrient agar instead of Mueller-Hinton agar allowed to exhibiting the higher activity of fosfomycin against all the species tested. The activity of fosfomycin was as equivalent or superior to those of cefazolin, cefmetazole, cefotiam and piperacillin. The susceptibility of strains isolated during 2000 to 2001 to fosfomycin was almost the same as that of the isolates reported in 1975. Fosfomycin was considered to show high efficacy in several infections, since it has maintained its favorable antibacterial activities against several bacterial species for more than 20 years after the first application to clinical practice.

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The inhibitory activity of NM394, the active form of the prodrug prulifloxacin, against type II topoisomerase from Pseudomonas aeruginosa was compared with those of ciprofloxacin (CPFX), levofloxacin (LVFX) and gatifloxacin (GFLX). The 50% inhibitory concentrations (IC50S) of NM394 for supercoiling activity of DNA gyrase and the decatenation activity of topoisomerase IV were 1.21 and 21.1 micrograms/ml, respectively. The IC50 of NM394 was equal to that of CPFX and lower than those of LVFX and GFLX. The inhibitory activity of the four drugs for DNA gyrase was also corresponding to the antimicrobial activity of the drugs for P. aeruginosa PAO1. The IC50S of the drugs tested for the decatenation activity of topoisomerase IV were from 17.4 to 24.2 times higher than those for the supercoiling activities of DNA gyrase. These results show that DNA gyrase is more sensitive to quinolones than is topoisomerase IV and may be a primary target of quinolones in P. aeruginosa. We concluded that NM394 exerts the potent antimicrobial activity through its strong inhibitory activity for DNA gyrase.

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To determine whether mycoplasma infection produces airway hyper-responsiveness to tachykinins and bradykinin and, if so, to elucidate the role of neutral endopeptidase (NEP), isolated hamster tracheal segments were studied under isometric conditions in vitro. Nasal inoculation with Mycoplasma pneumoniae potentiated contractile responses to neurokinin A and bradykinin, causing a leftward shift of the dose-response curves to a lower concentration by 1 log unit for each agonist, whereas there was no response with acetylcholine. Pretreatment of tissues with the NEP inhibitor phosphoramidon augmented neurokinin A- and bradykinin-induced contractions in saline-treated control tissues, but did not further potentiate the responsiveness in M. pneumoniae-infected tissues. NEP activity in the tracheal epithelium, but not in epithelium-denuded tissues, was decreased in infected animals. These results suggest that M. pneumoniae infection causes airway bronchoconstrictor hyper-responsiveness to neurokinin A and bradykinin and that this effect may be associated with an inhibition of epithelial NEP activity.

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