Clinical isolates of Haemophilus influenzae from Japan (n = 296) and the United States (n = 100) were tested by the microdilution method for susceptibility in vitro to 10 beta-lactam antibiotics and molecular mechanisms of beta-lactam resistance. For all isolates, PCR was used to identify six elements, including beta-lactamase-producing ampicillin (AMP)-resistance (BLPAR) and beta-lactamase-nonproducing AMP-resistance (BLNAR) genes as follows: (1) TEM-1 type beta-lactamase gene, (2) ROB-1 type beta-lactamase gene, (3) part of normal ftsI gene encoding PBP3, which is involved in septal peptidoglycan synthesis, (4) a portion of the ftsI gene possessing some amino acid substitutions commonly detected in BLNAR strains, (5) p6 gene encoding P6 membrane proteins specific to H. influenzae, and (6) serotype b capsule gene. In Japanese and U.S. isolates, respective prevalences of each resistance class in Japan and the United States were 55.1% and 46% for beta-lactamase-nonproducing, AMP-susceptible (BLNAS); 3.0% and 26% for the TEM-1 type beta-lactamase gene; 0% and 10% for the ROB-1 type; 26.4% and 13% for low-BLNAR with a low degree of AMP resistance; and 13.2% and 0% for BLNAR strains. A few remaining isolates were beta-lactamase-producing strains with a mutation in the ftsI gene. MICs of all beta-lactam agents against low-BLNAR strains were 2-8 times higher than against BLNAS. MICs of cephalosporin antibiotics against BLNAR strains were 16-32 times higher than against BLNAS. The rank order of beta-lactam MIC90 values against BLNAR strains was piperacillin = ceftriaxone = cefditoren (0.25 microg/ml), meropenem (0.5), cefotaxime (1), AMP = cefpodoxime (8), cefdinir (16), amoxicillin (16), and cefaclor (64). Serotype b isolates were few in both countries (2.4% in Japan, 3% in the United States). Differences in proportions of respective AMP-resistant genes in H. influenzae isolates between the two countries might reflect differences in antibiotic agents ordinarily given to outpatients with community-acquired bacterial infections.
A total of 395 Haemophilus influenzae strains from 226 Japanese institutions participating in the Nationwide Surveillance Study Group for Bacterial Meningitis were received from 1999 to 2002. All strains were analyzed by PCR to identify the resistance genes, and their susceptibilities to -lactam agents were determined. Of these strains, 29.1% were -lactamase nonproducing and ampicillin (AMP) susceptible (BLNAS) and lacked all resistance genes; 15.4% were -lactamase producing and AMP resistant and had the bla TEM-1 gene; 30.6% were -lactamase nonproducing and AMP resistant (low-BLNAR) and had a Lys-526 or His-517 amino acid substitution in ftsI encoding PBP 3; 13.9% were -lactamase nonproducing and AMP resistant (BLNAR) and had an additional substitution of Thr-385 in ftsI; 9.1% were amoxicillin-clavulanic acid resistant (BLPACR I) and had the bla TEM-1 gene and a Lys-526 or His-517 amino acid substitution in ftsI; and 1.8% showed resistance similar to that of the BLPACR I group (BLPACR II) but had bla TEM-1 gene and ftsI substitutions, as was the case for the BLNAR strains. All but three strains were serotype b. The prevalence of BLNAR strains has increased rapidly: 0% in 1999, 5.8% in 2000, 14.1% in 2001, and 21.3% in 2002. The MICs at which 90% of BLNAR isolates were inhibited were as follows: AMP, 16 g/ml; cefotaxime, 1 g/ml; ceftriaxone, 0.25 g/ml; and meropenem, 0.5 g/ml. All of these values were higher than those for the BLNAS counterpart strains. The relatively wide distributions of the -lactam MICs for BLNAR strains presumably reflect variations in ftsI gene mutations. Pulsed-field gel electrophoresis suggested the rapid spread of specific H. influenzae type b strains throughout Japan. Expedited vaccination, rapid identification, and judicious antibiotic use could slow their spread.Penicillin-intermediate Streptococcus pneumoniae and penicillin-resistant S. pneumoniae isolates from patients with respiratory tract infections (RTIs) have emerged and increased in number in Japan since 1988 (1). In contrast to the phenotypes of S. pneumoniae isolates from the United States (15), strains with an abnormal PBP 2X and strains for which penicillin G MICs at which 50% of isolates are inhibited (MIC 50 s) were 0.06 g/ml and cefotaxime (CTX) MIC 50 s were 0.125 to 0.5 g/ml accounted for 20% of all strains collected throughout Japan between 1998 and 2000 (22). The high prevalence of these resistant organisms is attributed to the frequent use of oral and intravenous cephem antibiotic agents in Japan (15,22). A similar situation is now evident among Haemophilus influenzae isolates from patients with RTIs, in which a rapid increase in the number of -lactamase-nonproducing, ampicillin (AMP)-resistant (BLNAR) H. influenzae strains has been detected (11,23). Resistance in BLNAR strains results from mutations in the ftsI gene encoding PBP 3, which mediates septal peptidoglycan synthesis (24). Amino acid substitutions identified at three positions in the ftsI gene are very important for resistance phenotypes: (i) Asn-...
Laminins are the major cell-adhesive proteins in the basement membrane, consisting of three subunits termed ␣, , and ␥. The putative binding site for integrins has been mapped to the G domain of the ␣ chain, although trimerization with  and ␥ chains is necessary for the G domain to exert its integrin binding activity. The mechanism underlying the requirement of  and ␥ chains in integrin binding by laminins remains poorly understood. Here, we show that the C-terminal region of the ␥ chain is involved in modulation of the integrin binding activity of laminins. We found that deletion of the C-terminal three but not two amino acids within the ␥1 chain completely abrogated the integrin binding activity of laminin-511. Furthermore, substitution of Gln for Glu-1607, the amino acid residue at the third position from the C terminus of the ␥1 chain, also abolished the integrin binding activity, underscoring the role of Glu-1607 in integrin binding by the laminin. We also found that the conserved Glu residue of the ␥2 chain is necessary for integrin binding by laminin-332, suggesting that the same mechanism operates in the modulation of the integrin binding activity of laminins containing either ␥1 or ␥2 chains. However, the peptide segment modeled after the C-terminal region of ␥1 chain was incapable of either binding to integrin or inhibiting integrin binding by laminin-511, making it unlikely that the Glu residue is directly recognized by integrin. These results, together, indicate a novel mechanism operating in ligand recognition by laminin binding integrins.Laminins are a family of glycoproteins present in the basement membrane (1-3). All laminins are large heterotrimeric glycoproteins composed of ␣, , and ␥ chains that assemble into a cross-shaped structure. To date, five ␣ chains (␣1-␣5), three  chains (1-3), and three ␥ chains (␥1-␥3) have been identified, combinations of which yield at least 15 isoforms with distinct subunit compositions (4). Laminins contribute to basement membrane architecture and influence cell adhesion, spreading, and migration through binding to their cell surface receptors, particularly the integrin family of cell adhesion receptors (5-9).Integrins play important roles in cell-matrix adhesion and signaling events regulating proliferation and differentiation of cells. Among the various integrin family members, ␣61, ␣64, ␣31, and ␣71 have been shown to be the major laminin receptors expressed in many cell types (10). Binding sites for these integrins have been mapped to the C-terminal globular (G) 3 domain of the laminin ␣ chains (6, 11-15). The G domain consists of five tandemly repeated LG modules of ϳ200 amino acid residues, designated LG1 through LG5. By analogy with the identification of the Arg-Gly-Asp (RGD) cell-adhesive motif in fibronectin, many attempts have been made to identify specific sequences mimicking the integrin binding activity of laminins. However, neither recombinant fragments of the G domain nor synthetic peptides modeled after the sequences in the G domain ...
A total of 195 Mycoplasma pneumoniae strains were isolated from 2,462 clinical specimens collected between April 2002 and March 2004 from pediatric outpatients with respiratory tract infections. Susceptibilities to six macrolide antibiotics (ML), telithromycin, minocycline, levofloxacin, and sitafloxacin were determined by the microdilution method using PPLO broth. A total of 183 M. pneumoniae isolates were susceptible to all agents and had excellent MIC 90 s in the following order: 0.00195 g/ml for azithromycin and telithromycin, 0.0078 g/ml for clarithromycin, 0.0156 g/ml for erythromycin, 0.0625 g/ml for sitafloxacin, 0.5 g/ml for minocycline, and 1 g/ml for levofloxacin. Notably, 12 ML-resistant M. pneumoniae strains were isolated from patients with pneumonia (10 strains) or acute bronchitis (2 strains). These strains showed resistance to ML with MICs of >1 g/ml, except to rokitamycin. Transition mutations of A2063G or A2064G, which correspond to A2058 and A2059 in Escherichia coli, in domain V on the 23S rRNA gene in 11 ML-resistant strains were identified. By pulsed-field gel electrophoresis typing, these strains were classified into groups I and Vb, as Mycoplasma pneumoniae is one of the main pathogens in respiratory tract infections (RTI) acquired in the community. In school-aged children and young adults with communityacquired pneumonia (CAP), M. pneumoniae accounts for as many as 10 to 30% of cases (4,6,13).In recent years, the clinical diagnosis for M. pneumoniae infection has relied on serological methods such as passive agglutination (Serodia-Myco II kit, Fujirebio, Tokyo, Japan) and complement fixation, even though a PCR method was partially applied (3,22,26). Accordingly, culture methods for this pathogen that require up to 1 week or more are rarely carried out in the laboratory. Thus, the current status of susceptibility to macrolide antibiotics (ML) used as the firstchoice agent for M. pneumoniae is unclear.In Japan, in parallel with the increase in usage of oral 14-membered ring ML (14-ML) and azithromycin for RTI, M. pneumoniae showing resistance to 14-ML has been isolated from clinical samples from pediatric outpatients with CAP (12, 16). We suspected an increase in cases with ML-resistant M. pneumoniae infection from prolonged clinical symptoms. In such cases, PCR positivity with rapid identification of M. pneumoniae persisted after the administration of clarithromycin or azithromycin for several days.We isolated causative M. pneumoniae by cultivation from clinical specimens collected from pediatric outpatients with RTI to determine susceptibilities to 10 oral antibiotics and identified a transition mutation on the 23S rRNA gene in ML-resistant isolates. MATERIALS AND METHODSMicroorganisms. M. pneumoniae was isolated from clinical specimens from patients with RTI. The specimens were collected from pediatric outpatients who visited 10 medical Japanese institutions participating in the study group on acute respiratory diseases (ARD). A total of 2,462 samples were sent to our laboratory (Kitasa...
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