title: Direct detection by real-time PCR of ftsI gene mutations affecting MICs of β-lactam agents for Haemophilus influenzae isolates from meningitis.
abstract: One resistance mechanism of Haemophilus influenzae to ampicillin involves decreased affinity of penicillin-binding protein (PBP) 3 for β-lactam antibiotics reflecting amino acid substitutions in PBP3 encoded by the ftsI gene. Three amino acid substitutions, Ser385Thr, Arg517His, and Asn526Lys, are especially responsible for β-lactam resistance. We constructed a new real-time polymerase chain reaction (PCR) to directly detect these substitutions in addition to 16S ribosomal RNA (rRNA), cap, and bla(TEM) genes. Our real-time PCR was evaluated using 206 clinical H. influenzae strains isolated from pediatric patients with meningitis. Relative sensitivities and specificities of real-time PCR were 90.5-100% and 96.3-100% for all resistance classes compared with our previously reported conventional PCR. In addition, real-time PCR shortened time required from 3 h by conventional PCR to 1.5 h. When correlations between combinations of amino acid substitutions in the ftsI gene detected by real-time PCR and minimum inhibitory concentrations (MICs) of β-lactam antibiotics were evaluated, MIC(90)s of ampicillin for β-lactamase-nonproducing ampicillin-intermediate-resistant strains with Asn526Lys, β-lactamase-nonproducing, ampicillin-resistant strains with Ser385Thr, and β-lactamase-nonproducing ampicillin-resistant strains with both Asn526Lys and Ser385Thr, respectively, were two, four, and eight times higher than those for sensitive strains. Similarly, MIC(90)s of cephalosporins for these strains, respectively, were two, 16-32, and 16-32 times higher than those for sensitive strains. Thus, real-time PCR can guide antibiotic use.
authors.affiliation: Laboratory of Molecular Epidemiology for Infectious Agents, Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Kozue
authors.initials: K
authors.lastname: Kishii
authors.forename: Miyuki
authors.initials: M
authors.lastname: Morozumi
authors.forename: Naoko
authors.initials: N
authors.lastname: Chiba
authors.forename: Akiko
authors.initials: A
authors.lastname: Ono
authors.forename: Kimiko
authors.initials: K
authors.lastname: Ubukata
mesh_terms: D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002648:Child; D004269:DNA, Bacterial / Q000032:analysis; D005798:Genes, Bacterial; D006193:Haemophilus influenzae / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D008583:Meningitis, Haemophilus / Q000382:microbiology*; D008826:Microbial Sensitivity Tests / Q000379:methods; D009154:Mutation; D046915:Penicillin-Binding Proteins / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D018440:beta-Lactam Resistance; D047090:beta-Lactams / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D046915:Penicillin-Binding Proteins; D047090:beta-Lactams