Genetic characteristics and antibiotic resistance of Haemophilus influenzae isolates from pediatric patients with acute otitis media after introduction of 13-valent pneumococcal conjugate vaccine in Japan.

Acute otitis media (AOM) occurs commonly in pediatric populations. We examined resistance genotype, antibiotic susceptibility, quinolone (QL) resistance, and multilocus sequence type (MLST) among Haemophilus influenzae isolates causing AOM following introduction of pneumococcal conjugate vaccines in Japan. The AOM surveillance group included 69 participating otolaryngologists. Causative pathogens isolated from middle ear fluid (MEF) samples collected from 582 children with AOM were identified using both bacterial culture and real-time PCR. H. influenzae isolates among these pathogens were characterized by capsular type, resistance genotype, antibiotic susceptibility, QL resistance, and MLST. In 2016, H. influenzae was identified in 319 samples (54.8%), among which 72.4% (n = 231) tested positive by both culture and PCR; remaining H. influenzae cases were only PCR-positive. This proportion of H. influenzae positivity has increased significantly from 41.2% in 2006 (p < 0.001). Among culture-positive strains, genotypic ß-lactamase-nonproducing ampicillin (AMP)-resistant (gBLNAR) strains were frequent (63.2%), with ß-lactamase-nonproducing AMP-susceptible (gBLNAS) strains accounting for only 24.2%. Susceptibilities of gBLNAR to oral antimicrobials were best for tosufloxacin, followed by cefditoren and tebipenem; MIC90s were 0.031 µg/mL, 0.5 µg/mL, and 1 µg/mL, respectively. In 7 gBLNAR isolates (3.0%), QL susceptibility was low, owing to amino acid substitutions in GyrA and/or ParC. Sequence types identified numbered 107, including 28 that were new. Prevention of further increases in resistance to antimicrobial agents will require antibiotic selection based on characterization of causative pathogens in clinical practice.

Etiology of Acute Otitis Media and Characterization of Pneumococcal Isolates After Introduction of 13-Valent Pneumococcal Conjugate Vaccine in Japanese Children.

BACKGROUND: Acute otitis media is a leading cause of childhood morbidity and antibiotic prescriptions. We examined etiologic changes in acute otitis media after introduction of 13-valent pneumococcal conjugate vaccine as routine immunization for Japanese children in 2014. Serotypes, resistance genotypes, antibiotic susceptibilities and multilocus sequence typing of pneumococcal isolates were also characterized. METHODS: Otolaryngologists prospectively collected middle ear fluid from 582 children by tympanocentesis or sampling through a spontaneously ruptured tympanic membrane between June 2016 and January 2017. Causative pathogens were identified by bacterial culture and real-time polymerase chain reaction for bacteria. Serotypes, resistance genotypes, sequence types and susceptibilities to 14 antimicrobial agents were determined for pneumococcal isolates. RESULTS: At least 1 bacterial pathogen was identified in 473 of the samples (81.3%). Nontypeable Haemophilus influenzae (54.8%) was detected most frequently, followed by Streptococcus pneumoniae (25.4%), Streptococcus pyogenes (2.9%) and others. Pneumococci of current vaccine serotypes have decreased dramatically from 82.1% in 2006 to 18.5% (P < 0.001). Commonest serotypes were 15A (14.8%), 3 (13.9%) and 35B (11.1%). Serotype 3 was significantly less frequent among children receiving 13-valent pneumococcal conjugate vaccine compared with 7-valent pneumococcal conjugate vaccine (P = 0.002). Genotypic penicillin-resistant S. pneumoniae accounted for 28.7%, slightly less than in 2006 (34.2%; P = 0.393); the penicillin-resistant serotypes 15A and 35B had increased. Serotypes 15A, 3 and 35B most often belonged to sequence types 63, 180 and 558. CONCLUSIONS: Our findings are expected to assist in development of future vaccines, and they underscore the need for appropriate clinical choice of oral agents based on testing of causative pathogens.

Therapeutic effect of meropenem on an experimental guinea pig model of meningitis with type b ß-lactamase-nonproducing ampicillin-resistant Haemophilus influenzae.

The purpose of this study was to investigate the relationship between efficacy and percentage of time above the MIC (%T>MIC) in the cerebrospinal fluid (CSF) for different dosing regimens of meropenem against an experimental lethal meningitis model in guinea pigs with type b ß-lactamase-nonproducing ampicillin-resistant Haemophilus influenzae (Hib BLNAR). Guinea pigs were intrathecally inoculated with 10(8) CFU/head of Hib BLNAR 8 h before the start of therapy. A single dose of 20, 40, or 80 mg/kg meropenem or multiple doses of 40 mg/kg meropenem were subcutaneously administered. Numbers of bacteria in CSF were counted 8 h after the start of therapy. Meropenem concentration in serum and CSF were determined in infected guinea pigs receiving a single dose of 40 mg/kg. In the single-dose regimen, 40 and 80 mg/kg meropenem significantly reduced the number of bacteria in CSF compared with the control, but 20 mg/kg meropenem did not. The %T>MIC for an 8-h period of 20, 40, and 80 mg/kg meropenem were 41, 52, and 62, respectively. Two and four doses of 40 mg/kg meropenem, for both of which %T>MIC was calculated as 100, had similar efficacy and were significantly superior to a single-dose of 40 mg/kg. In conclusion, meropenem had high efficacy when %T>MIC in the CSF was increased because of the high dose level and shortening of the dosing interval in a guinea pig meningitis model caused by Hib BLNAR, suggesting that high and frequent doses of meropenem are useful for treatment of meningitis with Hib BLNAR.

Rapid effectiveness of minocycline or doxycycline against macrolide-resistant Mycoplasma pneumoniae infection in a 2011 outbreak among Japanese children.

BACKGROUND: Mycoplasma pneumoniae is a major pathogen causing community-acquired pneumonia in children and young adults. Outbreaks typically occur at intervals of several years. In 2011, a widespread outbreak was associated with macrolide-resistant M. pneumoniae (MRMP) in Japanese children, often those of school age. METHODS: Two hundred fifty-eight children were diagnosed with M. pneumoniae-associated pneumonia based on chest radiography, real-time polymerase chain reaction (PCR), and antibody titers between January and December 2011. Mycoplasma pneumoniae cultures obtained from nasopharyngeal samples using appropriate broth were subjected to real-time PCR, by which decreases in M. pneumoniae in patients treated with minocycline (MIN), doxycycline (DOX), or tosufloxacin (TFX) were calculated. Mutations of the 23S ribosomal RNA gene that confer high resistance to macrolides in M. pneumoniae were identified by DNA sequencing. RESULTS: Among 202 M. pneumoniae isolates from M. pneumoniae-associated pneumonia patients, 176 (87.1%) were MRMP. Macrolide-resistant M. pneumoniae infection was significantly related to school age (P < .01) and initial administration of macrolides (P < .01). Minocycline or DOX (n = 125) or TFX or levofloxacin (n = 15) was used for definitive treatment of MRMP patients. Minocycline or DOX was significantly more effective than TFX (P ≤ .05) in achieving defervescence within 24 hours and in decreasing numbers of M. pneumoniae DNA copies 3 days after initiation. CONCLUSIONS: Macrolides are inappropriate as first-choice agents against MRMP in terms of shortening the clinical course and decreasing M. pneumoniae. Control and prevention of MRMP outbreaks in children require early decreases in M. pneumoniae as well as improvement of clinical findings.

Comparative efficacies of different antibiotic treatments to eradicate nontypeable Haemophilus influenzae infection.

BACKGROUND: Nonencapsulated and nontypeable Haemophilus influenzae (NTHi) is a major cause of human respiratory tract infections. Some strains of NTHi can cause invasive diseases such as septicemia and meningitis, even if H. influenzae is not generally considered to be an intracellular pathogen. There have been very few reports about the therapeutic efficacy of antibiotics against respiratory tract infection caused by NTHi in mice because it is difficult for H. influenzae to infect mice. Therefore, we evaluated the efficacy of antibiotics against NTHi in both a cell culture model and a mouse model of infection. METHODS: We used six strains of NTHi isolated from adult patients with chronic otitis media, namely three beta-lactamase-negative ampicillin (AMP)-resistant (BLNAR) strains and three beta-lactamase-negative AMP-susceptible (BLNAS) strains, to evaluate the efficacy of AMP, cefcapene (CFPN), levofloxacin (LVX), clarithromycin (CLR), and azithromycin (AZM) in both a cell culture infection model and a mouse infection model. In the cell culture infection model, strains that invade A549 human alveolar epithelial cells were treated with each antibiotic (1 mug/ml). In the mouse infection model, female C57BL/6 mice were intraperitoneally injected with cyclophosphamide (200 mg/kg) three days before intranasal infection with 1 x 109 colony-forming units (CFU) of NTHi and on the day of infection. After infection, the mice were orally administered each antibiotic three times daily for three days, except for AZM, which was administered once daily for three days, at a dose of 100 mg/kg/day. RESULTS: In the cell culture infection model, it was found that two BLNAR strains were able to enter the cell monolayers by the process of macropinocytosis, and treatment with LVX yielded good bactericidal activity against both strains inside the cells. In the mouse infection model, no bacteria were detected by means of plating the lung homogenates of LVX-treated mice at day 4 after infection, while more than 105 CFU of bacteria per tissue sample were detected in nontreated mice. CONCLUSION: Our findings show the outcome and rich benefits of fluoroquinolone treatment of respiratory infections caused by either invasive or noninvasive BLNAR strains of NTHi.