Molecular Evolution of β-Lactam-Resistant Haemophilus influenzae : 9-Year Surveillance of Penicillin-Binding Protein 3 Mutations in Isolates from Japan

Abstract

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...

Genetic Characteristics and Clonal Dissemination of β-Lactamase-Negative Ampicillin-ResistantHaemophilus influenzaeStrains Isolated from the Upper Respiratory Tract of Patients in Japan

“…Furthermore, a prospective prevalence study in Japan in 1999 showed that 55.1% of the H. influenzae strains were BLNAS, 3% were BLPAR, 26.4% were intermediateresistant strains, and 13.2% were BLNAR (35). The increase in the percentage of BLNAR strains has led to serious problems in the treatment of infectious disease in Japan (26,(31)(32)(33). Unfortunately, there is still limited information about the precise prevalence and dissemination of antimicrobial-resistant pathogens.…”

Can the Etest Correctly Determine the MICs of β-Lactam and Cephalosporin Antibiotics for Beta-Lactamase-Negative Ampicillin-Resistant Haemophilus influenzae ?

“…MICs of the Etest were rounded up to the next log 2 concentration, and agreement was defined as the results of broth microdilution and those of Etest in the range of Ϯlog 2 1 of each other. The mutation status of the ftsI gene in all strains was checked by using primers described previously (3,8). MICs of BLNAS and beta-lactamase-positive ampicillinresistant (BLPAR) strains showed consistent results by both tests, and the agreement was 92.59 to 100% for beta-lactams and 90 to 92.59% for meropenem, respectively.…”

Prevalence and Genotypic Characteristics of ß-lactamase Negative-Ampicillin-Resistant (BLNAR) Haemophilus Influenzae in a Children's Hospital in Turkey

“…Low BLNAR strains are infrequently isolated, and their rates are increasing all of the world. [18][19][20] Occasionally low BLNAR strains will be classified as resistant because recurrent MIC determinations can be expected to be different by a size of positive or negative a fold concentration. 21 In pediatric isolates nontypeable H. influenzae was the predominant seroytpe.…”