Keiji Fujihara

Genetic Characteristics and Clonal Dissemination of β-Lactamase-Negative Ampicillin-Resistant Haemophilus influenzae Strains Isolated from the Upper Respiratory Tract of Patients in Japan

ABSTRACT We evaluated the recent prevalence of antimicrobial-resistant Haemophilus influenzae isolated from the upper respiratory tracts (URT) of patients in Japan. Mutations in the ftsI gene, which encodes penicillin binding protein 3 (PBP3), and the clonal dissemination of the resistant strains were also investigated. A total of 264 H. influenzae isolates were collected from patients with URT infections. According to the criteria of the Clinical and Laboratory Standards Institute for the susceptibility of H. influenzae to ampicillin (AMP), the isolates were distributed as follows: 161 (61.0%) susceptible strains (MIC ≤ 1 μg/ml), 37 (14.0%) intermediately resistant strains (MIC = 2 μg/ml), and 66 (25.0%) resistant strains (MIC ≥ 4 μg/ml). According to PCR-based genotyping, 172 (65.1%) of the isolates had mutations in the ftsI gene and were negative for the β-lactamase (bla) gene. These 172 isolates were thus defined as genetically β-lactamase-negative ampicillin-resistant (gBLNAR) strains. The ftsI mutant group included 98 (37.1%) strains with group I/II mutations in the variable mutated region (group I/II gBLNAR) and 74 (28.0%) strains with group III mutations in the highly mutated region (group III gBLNAR). Eighty-seven (33.0%) of the isolates were genetically β-lactamase-negative ampicillin-susceptible (gBLNAS) strains. The group III gBLNAR strains showed resistance to β-lactams. Only five strains (1.9%) were positive for a bla gene encoding TEM-type β-lactamase. The three clusters consisting of 16 strains found among the 61 BLNAR strains (MIC ≥ 4 μg/ml and without the bla gene) showed identical or closely related DNA restriction fragment patterns. Those isolates were frequently identified among strains with a MIC to AMP of 16 μg/ml. The current study demonstrates the apparent dissemination and spread of a resistant clone of H. influenzae among medical centers in Japan. The gBLNAR strains show a remarkable prevalence among H. influenzae isolates, with the prevalence increasing with time. This fact should be taken into account when treating URT infections.

Formation of biofilm by Haemophilus influenzae isolated from pediatric intractable otitis media

OBJECTIVES The aims of this study are to evaluate biofilm formation by nontypeable Haemophilus influenzae (NTHi) isolated from children with acute otitis media (AOM) and its relation with clinical outcome of the disease. METHODS Biofilm formations by NTHi clinical isolates from pediatric AOM patients were evaluated by a crystal violet microtiter plate and a 98 well pin-replicator assay with a confocal laser scanning microscopy (CLSM). Optical density values of clinical isolates were compared with a positive control and the ratio of clinical isolates to a positive control was defined as biofilm formation index (BFI). RESULTS 84.3% clinical isolates of NTHi were biofilm forming strains (BFI> or =0.4). The BFI represented the levels of biofilm formation and adherence on the surface. The identical strains isolated from both middle ear fluids (MEFs) and nasopharynx showed biofilm formation at the same level. The prevalence of biofilm forming isolates was significantly higher among the susceptible strains than resistant strains. The level of biofilm formation of NTHi isolated from AOM cases who was not improved by amoxicillin (AMPC) was significantly higher than that of NTHi isolated from AOM cases who was improved by AMPC. CONCLUSION We clearly showed the biofilm formation of clinical NTHi isolates from AOM children. In addition, the biofilm formed by NTHi would play an important role in persistent or intractable clinical course of AOM as a result of lowered treatment efficacy of antibiotics.

Cost-effectiveness of tonsillectomy for recurrent acute tonsillitis.

Objectives: We used a retrospective case series to perform a preliminary study to determine the clinical effectiveness and cost-effectiveness of tonsillectomy for recurrent acute tonsillitis. Methods: We studied 25 children and 16 adults who had tonsillectomy for recurrent acute tonsillitis. The adult patients and the childrens caregivers were asked to respond to a questionnaire regarding the efficacy of their tonsillectomy. The cost of medical care and the work disability cost for tonsillitis and for tonsillectomy were calculated. We then applied the technique of break-even time analysis to assess when the total health care cost savings from surgery overtook the total cost of tonsillectomy. Results: In children, the overall economic costs (medical costs and work-related costs) were recovered at 1.6 years after tonsillectomy (break-even point). In adults, the overall economic costs (medical costs and work-related costs) were recovered at 2.5 years after tonsillectomy (break-even point). Conclusions: Tonsillectomy for recurrent acute tonsillitis is both clinically effective and cost-effective for children and adults in Japan.

Antimicrobial resistance of Haemophilus influenzae isolated from the nasopharynx of Japanese children with acute otitis media

Conclusion. A high prevalence of penicillin-binding protein gene-mutated (PGM) strains of Haemophilus influenzae should be taken into account when treating otitis media in children. Objective. To evaluate the prevalence of β-lactamase-non-producing ampicillin-resistant strains of H. influenzae with mutations in the ftsI gene encoding penicillin-binding protein 3 (PBP3) among children with otitis media. Material and methods. A total of 644 nasopharyngeal isolates of H. influenzae were collected from pediatric acute otitis media patients with or without otitis media with effusion at the clinics of the Department of Otolaryngology—Head and Neck Surgery, Wakayama Medical University Hospital and 6 affiliated hospitals in Wakayama Prefecture between January 1999 and December 2003. MICs to ampicillin (AMP), cefdinir (CFD), cefaclor (CCL), cefpodoxime (CPD) and cefcapene (CFPN) were determined by a microbroth dilution method according to the recommendations of the National Committee for Clinical Laboratory Standards. Types of mutations in the PBP3 gene (ftsI) were evaluated by means of a polymerase chain reaction (PCR)-based genotyping method. The β-lactamase gene (bla) was also identified by means of PCR. Results. β-lactamase-producing (BLP) strains having the bla gene were identified in 16 isolates (2.5%). PGM strains were identified in 279 isolates (43.3%). There were 242 PGM1-non-BLP strains (37.6%) with mutations in the variable mutated locus of ftsI, 35 PGM2-non-BLP strains (5.4%) with mutations in the highly mutated locus of ftsI and 2 BLP-PGM strains (0.3%) with mutations in ftsI that produced β-lactamase. BLP-non-PGM strains producing β-lactamase without mutations in ftsI were identified in 14 isolates (2.2%). MICs of PGM1-non-BLP strains to AMP were 0.5–2.0 µg/ml. The MIC90 of CDN to the PGM1-non-BLP strains was the lowest (0.06 µg/ml). The proportion of PGM1-non-BLP strains increased rapidly during 1999–2002 and then decreased in 2003. In contrast, the proportion of PGM2-non-BLP strains increased in 2003.

Determination of pneumococcal serotypes/genotypes in nasopharyngeal secretions of otitis media children by multiplex PCR.

The appropriate clinical applications of pneumococcal polysaccharide vaccines against recent increases in antimicrobial resistant Streptococcus pneumoniae (S. pneumoniae) urgently require accurate analytical methodologies for determining and characterizing the serotypes. The results of current immunological determinations of serotypes with anti-capsular polysaccharide-specific sera are difficult to interpret in terms of quellung changes of the pneumococci. In this study, we applied the multiplex PCR technique for the rapid identification of pneumococci and simultaneous rapid determinations of their serotypes and genotypes that directly correlated with antimicrobial susceptibilities from nasopharyngeal secretions (NPS). Serogroups 6, 19F and 23F were the predominant capsular types of S. pnuemoniae in the NPS samples. Strains of serotypes 19F and 23F frequently had mutations in pbp1a, pbp2x and pbp2b and expressed ermB and mefA; they also were mostly resistant to both penicillin G (PCG) and clarithromycin (CAM). Two NPS samples contained the strain of serotype 19F together with the strain of serotype 23F, although only the strain of serotype 19F was identified by a conventional bacterial culture. Pneumococci were identified in six NPS samples and their serotypes determined by the multiplex PCR, while a conventional bacterial culture failed to identify the pathogens. Our findings suggest that PCR-based serotyping and genotyping can provide an accurate and rapid distribution of pneumococcal serotypes and antimicrobial resistance. The relatively minor populations in the nasopharynx may be determined using molecular techniques.

Increase of Macrolide‐Resistant Streptococcus pneumoniae‐Expressing mefE or ermB Gene in the Nasopharynx among Children with Otitis Media

Objective: To evaluate prevalence of macrolide resistant strains and the genotypes of the resistance among Streptococcus pneumoniae isolated from the nasopharynx of children with otitis media.

High Prevalence of Streptococcus pneumoniae with Mutations in pbp1a, pbp2x, and pbp2b Genes of Penicillin-Binding Proteins in the Nasopharynx in Children in Japan

Objective: To evaluate the resistances of Streptococcus pneumoniae to β-lactams developed by stepwise alterations in high-molecular-weight penicillin-binding proteins (PBPs) with a reduced binding affinity of β-lactams. Among the numerous mutations in pbp genes that alter the affinity for β-lactams, the decreased affinity of PBP1A, 2X and 2B is especially important in the development of resistances to β-lactams. Study Design: Retrospective review. Methods:In this study, we investigated the mutations in pbp1a, pbp2x, and pbp2b genes evaluated by polymerase chain reaction (PCR) in 866 pneumococcal isolates collected from the nasopharynx of Japanese children with acute otitis media. Results: 210 strains (24.3%) exhibited no mutations in the three pbp genes. 333 strains (38.5%) had mutations in the three pbp genes, 78 (9.0%) in two pbp genes, whereas 245 (28.3%) displayed mutations in only one pbp gene. Among the 656 strains with mutations in pbp genes, 620 (94.5%) strains had mutations in pbp2x. The annual prevalence of antimicrobial-resistant S. pneumoniae showed a gradual increase in strains with mutations in the three pbp genes and a parallel decrease in strains without mutations. Conclusions: PCR-based genotyping can characterize the antimicrobial resistances in pneumococci along with minimal inhibitory concentrations (MICs). Physicians should pay attention to the recent increase in antimicrobial-resistant S. pneumoniae when treating pediatric acute otitis media.

A scoring system for management of acute pharyngo-tonsillitis in adults

Abstract Objectives The aim of this study was to develop and evaluate a scoring system for the management of acute pharyngo-tonsillitis. Methods We conducted a prospective study between May 2004 and June 2005. Patients with acute pharyngo-tonsillitis were evaluated for causative pathogens and were assessed clinical symptoms and pharyngo-tonsillar finding by a clinical scoring system. Results A total 214 adult patients were enrolled in this study. Streptococcus pyogenes were identified at 13.6%. Thirty-one viruses were also identified by PCR. They were adenovirus (4.8%), influenza virus (1.0%), RS virus (6.3%), and human metapneumovirus (2.9%). Numbers of total white blood cells and levels of C-reactive protein showed a significant positive correlation with clinical scores (p <0.001) and were also higher in cases with S. pyogenes. The clinical scores rapidly improved after the antimicrobial treatments in moderate cases and severe cases. Conclusion The current study strongly suggested that the clinical scoring system reflected disease severity well and would be very useful for evaluating clinical course and decision making for the antimicrobial treatment of acute pharyngo-tonisllitis.

Distribution of fibronectin-binding protein genes ( prtF1 and prtF2 ) and streptococcal pyrogenic exotoxin genes ( spe ) among Streptococcus pyogenes in Japan

Two hundred and seventy-two strains of Streptococcus pyogenes isolated from patients with invasive and noninvasive infections in Japan were evaluated for the prevalence of fibronectin-binding protein genes (prtF1 and prtF2). The possible associations of the genes with streptococcal pyrogenic exotoxin genes, macrolide resistance genes, and emm types were also evaluated. Overall, about 50% of S. pyogenes isolates carried fibronectin-binding protein genes. The prevalence of the prtF1 gene was significantly higher among isolates from noninvasive infections (71.4%) than among isolates from invasive infections (30.8%; P = 0.0037). Strains possessing both the prtF1 and prtF2 genes were more likely to be isolates from noninvasive infections than isolates from invasive infections (50.6% vs 15.4%; P = 0.019). S. pyogenes isolates with streptococcus pyrogenic exotoxin genes (speA and speZ) were more common among isolates without fibronectin-binding protein genes. The speC gene was more frequently identified among isolates with fibronectin-binding protein genes (P = 0.05). Strains belonging to emm75 or emm12 types more frequently harbored macrolide resistance genes than other emm types (P = 0.0094 and P = 0.043, respectively). Strains carrying more than one repeat at the RD2 region of the prtF1 gene and the FBRD region of the prtF2 gene were more prevalent among strains with macrolide resistance genes than among strains negative for macrolide resistance genes. These genes (i.e., the prtF1, prtF2, and spe genes) may enable host-bacteria interaction, and internalization in the host cell, but may not enable infection complications such as invasive diseases.

Evaluation of mutations in penicillin binding protein-3 gene of non-typeable Haemophilus influenzae isolated from the nasopharynx of children with acute otitis media

Conclusion Younger children tend to harbor more resistant strains because they are exposed to these pathogens more often through contacts with siblings or attendance at day-care centers and are frequently treated with antibiotics. The high prevalence of BLNAR strains should be taken into account in the treatment of AOM in young children. Objective Non-β-lactamase-producing ampicillin-resistant (BLNAR) strains with mutations in penicillin-binding protein (PBP) genes of Haemophilus influenzae have been prevalent recently among younger children. Material and methods We investigated mutations in the ftsI gene encoding PBP-3 of H. influenzae isolated from the nasopharynx of children with acute otitis media (AOM) using polymerase chain reaction (PCR). Results Strains containing the bla gene (β-lactamase-producing ampicillin-resistant) were identified in 4.7% of cases. Strains with mutations in the ftsI gene (BLNAR) were identified in 23.3% of cases. Strains without mutations in the ftsI gene and that did not contain the bla gene (non-β-lactamase-producing ampicillin-susceptible) were identified in 70.7% of cases. Strains with both expression of the bla gene and mutations in the ftsI gene (β-lactamase-producing amoxicillin–clavulanate-resistant) were identified in 1.3% of cases. The MICs of ampicillin against the strains evaluated in this study were 0.5–2.0 μg/ml. Cefditoren-pivoxil had the lowest MIC90 against the strains (0.06 μg/ml). Strains with mutations in the ftsI gene (BLNAR) were broadly identified among young children.