A total of 621 clinical isolates of Haemophilus influenzae collected in Japan between 1995 and 2003 were studied for their susceptibilities to several antimicrobial agents, ␤-lactamase production, and amino acid substitutions in penicillin-binding protein 3 (PBP 3). Over the four study periods (first period, 1995 to 1996; second period, 1997 to 1998; third period, 2000 to 2001; fourth period, 2002 to 2003), the susceptibilities to ␤-lactam agents decreased and the incidence of isolates with substitutions at positions 377, 385, 389, 517, and/or 526 in PBP 3 increased from 28.8% to 52.0%. Five hundred seventy-one ␤-lactamase-nonproducing isolates were grouped into 18 classes, based on the pattern of the five mutations in PBP 3. The Asp526Lys substitution led to 6.0-, 4.3-, 2.4-, and 5.4-fold increases in amoxicillin-clavulanic acid, cefdinir, cefditoren, and faropenem resistance, respectively. PBP 3 with multiple substitutions (Met377Ile, Ser385Thr, and/or Leu389Phe) together with Asp526Lys resulted in increased resistance compared to that for PBP 3 with the Asp526Lys substitution alone. These results indicate that mutations at these five positions increased resistance to most ␤-lactams. Although a significant change in the prevalence of ␤-lactamase-producing strains was not observed, the proportions of those possessing both PBP 3 alterations and ␤-lactamase production have slightly increased (from 1.4% to 5.0%). The ROB-1 ␤-lactamase was rare, but this is the first report of this ␤-lactamase in Japan.Haemophilus influenzae is an important pathogen that causes community-acquired infections such as pneumonia, otitis media, and meningitis and has become increasingly resistant to ␤-lactam antibiotics (5, 18). There are two major mechanisms involved in ␤-lactam resistance, one enzymatic and the other nonenzymatic. The enzymatic resistance mechanism is mainly mediated by the hydrolysis of ␤-lactams due to the production of TEM-1 ␤-lactamase (17, 24) and, in some cases, to a ROB-1 ␤-lactamase (10, 13). The nonenzymatic mechanism involves a decreased affinity of penicillin-binding protein 3 (PBP 3) for ␤-lactam antibiotics due to amino acid substitutions (17, 23). The ␤-lactam resistance phenotype mediated by the nonenzymatic mechanism is called "␤-lactamase-nonproducing ampicillin resistance (BLNAR)" in H. influenzae.Recently, the isolation frequency of BLNAR strains has been increasing exponentially among clinical isolates from patients with community-acquired infections in Japan (21, 25). A recent report also described an increased prevalence of the BLNAR phenotype in Europe (4). Strains with both resistance mechanisms were also found among clinical isolates, and such strains are termed ␤-lactamase-producing ampicillin-clavulanic acid-resistant (BLPACR) H. influenzae (6,12,22).Studies of ␤-lactam resistance caused by H. influenzae PBP 3 mutations have been reported by several investigators (1,6,16,20,23). From the genetic analysis of the ftsI gene encoding PBP 3 in BLNAR strains, the amino acid substitutions surroundi...

All six penicillin-binding protein (PBP) genes, namely, pbp1a, pbp1b, pbp2a, pbp2b, pbp2x, and pbp3, of 40 Streptococcus pneumoniae clinical isolates, including penicillin-resistant S. pneumoniae isolates collected in Japan, were completely sequenced. The MICs of penicillin for these strains varied between 0.015 and 8 g/ml. In PBP 2X, the Thr550Ala mutation close to the KSG motif was observed in only 1 of 40 strains, whereas the Met339Phe mutation in the STMK motif was observed in six strains. These six strains were highly resistant (MICs м 2 g/ml) to cefotaxime. The MICs of cefotaxime for 27 strains bearing the Thr338Ala mutation tended to increase, but the His394Leu mutation next to the SSN motif did not exist in these strains. In PBP 2B, the Thr451Ala/Phe/Ser and Glu481Gly mutations close to the SSN motif were observed in 24 strains, which showed penicillin resistance and intermediate resistance, and the Thr624Gly mutation close to the KTG motif was observed in 2 strains for which the imipenem MIC (0.5 g/ml) was the highest imipenem MIC detected. In PBP 1A, the Thr371Ser/Ala mutation in the STMK motif was observed in all 13 strains for which the penicillin MICs were м1 g/ml. In PBP 2A, the Thr411Ala mutation in the STIK motif was observed in one strain for which with the cefotaxime MIC (8 g/ml) was the highest cefotaxime MIC detected. On the other hand, in PBPs 1B and 3, no mutations associated with resistance were observed. The results obtained here support the concept that alterations in PBPs 2B, 2X, and 1A are mainly involved in S. pneumoniae resistance to ␤-lactam antibiotics. Our findings also suggest that the Thr411Ala mutation in PBP 2A may be associated with ␤-lactam resistance.

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...

Horizontal gene transfer has been identified in only a small number of genes in Haemophilus influenzae, an organism which is naturally competent for transformation. This report provides evidence for the genetic transfer of the ftsI gene, which encodes penicillin-binding protein 3, in H. influenzae. Mosaic structures of the ftsI gene were found in several clinical isolates of H. influenzae. To identify the origin of the mosaic sequence, complete sequences of the corresponding gene from seven type strains of Haemophilus species were determined. Comparison of these sequences with mosaic regions identified a homologous recombination of the ftsI gene between H. influenzae and Haemophilus haemolyticus. Subsequently, ampicillin-resistant H. influenzae strains harboring identical ftsI sequences were genotyped by pulsed-field gel electrophoresis (PFGE). Divergent PFGE patterns among ␤-lactamase-nonproducing ampicillin-resistant (BLNAR) strains from different hospitals indicated the potential for the genetic transfer of the mutated ftsI gene between these isolates. Moreover, transfer of the ftsI gene from BLNAR strains to ␤-lactamase-nonproducing ampicillin-susceptible (BLNAS) H. influenzae strains was evaluated in vitro. Coincubation of a BLNAS strain (a rifampin-resistant mutant of strain Rd) and BLNAR strains resulted in the emergence of rifampin-and cefdinir-resistant clones at frequencies of 5.1 ؋ 10 ؊7 to 1.5 ؋ 10 ؊6 . Characterization of these doubly resistant mutants by DNA sequencing of the ftsI gene, susceptibility testing, and genotyping by PFGE revealed that the ftsI genes of BLNAR strains had transferred to BLNAS strains during coincubation. In conclusion, horizontal transfer of the ftsI gene in H. influenzae can occur in an intraspecies and an interspecies manner.

A total of 99 clinical isolates of metallo-ß-lactamase-negative Pseudomonas aeruginosa collected in Japan between 1998 and 2001 were studied for their susceptibilities to carbapenem agents and corresponding oprD gene mutations. The OprD sequence of each strain was grouped into two major classes, based on the pattern of alterations. Eighty strains (80.8%) were so-called 'full length type', whose OprD proteins were fully encoded. The remaining 19 strains (19.2%) were so-called 'defective type', which possessed deletions or major alterations that might cause conformational changes in the OprD porin protein. The changes in 'defective type' strains led to 15-, 17-and 23-fold increases in the geometric mean MIC for imipenem, meropenem and biapenem compared with 'full length type' strains, respectively. 'Full length type' strains were further classified into six carbapenem susceptible types with the exception of four carbapenemresistant subtypes with additional amino acid substitutions at D43, G183, R154, G314, G316. However, 'defective type' strains were classified into four types as follows: 10 strains which contained a stop codon within the coding region; six strains which contained IS; one strain with a short deletion near the C-terminal domain; and two strains without a stop codon in the sequenced region. Western blot analysis using OprD antibody showed that binding abilities of OprD proteins against 'full length type' strains were normal, whereas those against 'defective type' strains were lost without exception. These results indicate that OprD structure and antimicrobial activities for carbapenem agents proved to be highly correlated in P. aeruginosa Key words carbapenem resistance, OprD, porin protein, Pseudomonas aeruginosa.Carbapenem antibiotics are recognized as one of the most potent agents with activity against Pseudomonas aeruginosa and remain important for the therapy of serious infections due to these pathogens. Recently, however, the strains with resistance to carbapenems have been increasing among clinical isolates of P. aeruginosa (1).Resistance to carbapenems in P. aeruginosa is often due to the impermeability that arises via the loss of the OprD porin, the overproduction of an active efflux pump system, and the production of carbapenem-hydrolyzing ß-lactamase.Carbapenem-hydrolyzing enzymes are divided into two types based on molecular classification: serine enzymes, which are derivatives of class A or class D enzymes, and metallo-enzymes, which belong to class B enzymes. The production of carbapenem-hydrolyzing enzymes lead to