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Molecular Epidemiology of Ampicillin-resistant Haemophilus influenzae Causing Acute Otitis Media in Japanese Infants and Young Children

Kakuta, Risako MD, PhD; Yano, Hisakazu MD, PhD; Hidaka, Hiroshi MD, PhD; Kanamori, Hajime MD, PhD; Endo, Shiro MD, PhD; Ichimura, Sadahiro PhD; Ogawa, Miho MPAS; Shimojima, Masahiro PhD; Ozawa, Daiki MD; Inomata, Shinya MD, PhD; Tanouchi, Ayako MPAS; Kaku, Mitsuo MD, PhD; Katori, Yukio MD, PhD

Author Information
The Pediatric Infectious Disease Journal: May 2016 - Volume 35 - Issue 5 - p 501-506
doi: 10.1097/INF.0000000000001066


Acute otitis media (AOM) is the most common bacterial infection of early childhood, with over 80% of children having at least 1 episode before 3 years of age and up to 50% suffering from recurrent episodes.1 Because most episodes are treated with antibiotics,2 AOM is considered to be one of the most important reasons for emergence of microbial resistance.1 Nontypeable Haemophilus influenzae is a particularly important cause of AOM and recurrent AOM, and several reports have emphasized the problem of antibiotic resistance among these strains.2,3

There are 2 major mechanisms of β-lactam resistance, which are enzymatic hydrolysis and alterations in the transpeptidase domain of penicillin-binding protein 3 (PBP3).4 Enzymatic hydrolysis is mainly because of production of TEM-1 β-lactamase and sometimes ROB-1 β-lactamase.5 Amino acid substitutions in PBP3 result in β-lactamase–nonproducing ampicillin resistant (BLNAR) strains.5,6 In addition, strains with both mechanisms of resistance (β-lactamase–producing amoxicillin/clavulanic acid–resistant: BLPACR) have been described.7 Unlike the high prevalence of β-lactamase–producing ampicillin-resistant (BLPAR) strains, the prevalence of BLNAR strains remains low in many other countries. In contrast, BLNAR isolates are particularly frequent in Japan, which is one of the reasons that pediatric AOM often shows recurrence or requires prolonged treatment in this country.8,9 There have been few molecular epidemiological studies of BLNAR H. influenzae isolates from children with AOM focusing on patients younger than 3 years. Also, most studies of H. influenzae in pediatric patients have been based on various kinds of samples, including nasopharyngeal secretions, nasal discharge specimens and tonsillar swabs. Epidemiological studies of H. influenzae causing AOM in children younger than 3 years are important for determining the prevalence and mechanisms of antimicrobial resistance.

The aims of the present study were (i) to determine the current state of antimicrobial susceptibility of clinical isolates of H. influenzae obtained from infants and young children with AOM in Japan; (ii) to assess amino acid substitutions in the transpeptidase domain of PBP3 in these isolates and (iii) to determine the molecular epidemiology of ampicillin resistance in these H. influenzae strains.


Bacterial Isolates

A total of 157 consecutive and nonduplicate clinical isolates of H. influenzae obtained from the middle ear fluid (MEF) of pediatric patients (0–3 years old) with AOM during the period from October 2011 to March 2012 at 72 institutions in various part of Japan including 69 clinics and 3 diagnostic laboratories were studied (a maximum of 30 isolates were collected from 1 clinic). MEF samples were collected from the pediatric AOM patients by puncture or incision of the tympanic membrane, and otorrhea was also included. AOM was diagnosed from examination of the tympanic membrane by an otolaryngologist or by the presence of purulent otorrhea. The specimens of MEF and otorrhea were immediately sent for routine bacterial examination at Department of Bacteriology, BML Inc. All isolates were screened by determining the morphology of colonies on chocolate agar and confirming lack of haemolysis on horse blood agar, and the isolates were then sent to Department of Infection Control and Laboratory Diagnostics, Tohoku University Graduate School of Medicine. After we received them, the isolates were frozen at −80°C in Microbank vials (IWAKI & CO., Ltd, Tokyo, Japan) until susceptibility testing and molecular procedures were performed. Polymerase chain reaction (PCR) for the fuculokinase (fucK) gene was done for species identification as described previously.10–12 Isolates that were negative for this gene were confirmed to be H. influenzae by analysis of almost the entire 16S ribosomal RNA gene sequence.13 The EzTaxon-e Database was employed for sequence analysis (

Isolates were serotyped by slide agglutination with antisera (H. influenzae Antisera “SEIKEN” Set: Denka Seiken Co., Ltd., Tokyo, Japan), and the serotypes were further confirmed by a PCR reference method.14

Antimicrobial Susceptibility Testing

Production of β-lactamase was confirmed by the nitrocefin test (Showa Chemical, Tokyo, Japan). The minimum inhibitory concentrations (MICs) of ampicillin, amoxicillin/clavulanic acid (2:1 ratio), cefditoren, ceftriaxone, meropenem and tosufloxacin were determined by the broth microdilution method according to the Clinical Laboratory Standards Institute guidelines.15 The quality control strains were ATCC 49247 (BLNAR phenotype) and ATCC 49766 (ampicillin-susceptible). All H. influenzae isolates were classified according to the Clinical Laboratory Standards Institute criteria.15 Thus, β-lactamase–nonproducing strains were classified into β-lactamase–nonproducing ampicillin-susceptible (BLNAS) strains (MIC of ampicillin ≤ 1 mg/L), β-lactamase–nonproducing ampicillin-intermediately resistant (low-BLNAR)16 strains (MIC of ampicillin = 2 mg/L) and BLNAR strains (MIC of ampicillin ≥ 4 mg/L). In addition, BLPAR strains were defined as those for which the MIC of amoxicillin/clavulanic acid was ≤4 mg/L, whereas the MIC of amoxicillin/clavulanic acid was ≥8 mg/L for BLPACR strains.

PCR and DNA Sequencing

The presence of β-lactamase–encoding genes (blaTEM-1 and blaROB-1) was investigated in all of the β-lactamase–producing isolates by PCR amplification, as described previously.7

Alterations of PBP3 were investigated in ampicillin-resistant H. influenzae isolates (MIC of ampicillin ≥ 4 mg/L) by sequencing the region of the ftsI gene encoding the transpeptidase domain of PBP3, as described previously.4 PCR products were purified with a QIA quick PCR Purification kit (Qiagen, Tokyo, Japan), followed by DNA sequencing using the ABI BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA) and ABI3730xl Analyser (Applied Biosystems). BLAST was used for sequence analysis ( The ftsI sequences were analyzed by comparison with those from H. influenzae Rd KW20 (accession number NC_000907). Amino acid substitution patterns were classified into 9 groups (groups I, IIa, IIb, IIc, IId, III and III-like and others, and no change) according to the criteria of Ubukata et al,4 Dabernat et al17 and García-Cobos et al.6 Using PCR-based genotyping, the strains were classified into the following 4 genotypes: genetically BLNAS (gBLNAS) strains without amino acid substitutions in the PBP3 or β-lactamase (bla) genes; genetically BLNAR (gBLNAR) strains with an amino acid substitution in PBP3 but without the bla gene; genetically BLPAR (gBLPAR) strains with the bla gene but without an amino acid substitution in PBP3 and genetically BLPACR (gBLPACR) strains with the bla gene and an amino acid substitution in PBP3.

Multilocus Sequence Typing

Ampicillin-resistant H. influenzae isolates (MIC of ampicillin ≥ 4 mg/L) were analyzed by MLST that included 7 housekeeping genes (adk, atpG, frdB, fucK, mdh, pgi and recA) to identify relatedness among the strains.18 The allele number and ST were assigned using the H. influenzae MLST website ( The total database was analyzed using eBURST v3 to define groups available on the H. influenzae MLST website.19 STs were only included in a group if they shared alleles at a minimum of 6 of the seven loci (single-locus variants). The neighbor-joining method was employed to generate a phylogenetic tree with MLST data analysis (

Pulsed-field Gel Electrophoresis

After digestion of DNA with SmaI restriction enzyme (Takara Bio Inc., Otsu, Japan), genetic relatedness among highly resistant isolates (MIC of ampicillin ≥ 8 mg/L) was examined by pulsed-field gel electrophoresis (PFGE) according to the procedures described previously with some modifications.20 Electrophoresis was performed on 1% PFGE agarose gel with a CHEF-DR III system (Bio-Rad Laboratories, Richmond, CA) for 19.5 hours at 6.0 V/cm and 14°C with a ramped pulse time of 5.3–34.9 seconds. A Lambda DNA ladder (Lambda DNA Ladders; Lonza, Tokyo, Japan) was used as the size standard. PFGE bands were analyzed with GelCompar II v.3.0 (Applied Maths, Sint-Martens-Latem, Belgium) and the unweighted-pair group method using arithmetic averages. The similarity of the PFGE patterns was estimated with the Jaccard coefficient after setting the optimization and tolerance at 0.87%. Isolates that showed ≥80% relatedness were defined as being highly genetically related.21


Features of the Isolates

Among 157 H. influenzae isolates, 79 (50.3%) were obtained from boys and 78 (49.7%) were from girls. Only 1.3% (2/157) of the strains were encapsulated, with both 2 isolates being type b. The remaining 98.7 % (155/157) of the isolates were nontypeable H. influenzae.

Antimicrobial Susceptibility

Among the 157 H. influenzae isolates, the MIC of ampicillin ranged from 0.125 to >16 mg/L. Testing for β-lactamase with a nitrocefin disk showed 13 positive strains (8.3%) among the isolates, including 12 strains with blaTEM-1 genes and 1 strain with blaROB-1. There were 49 BLNAS (31.2%) strains, 42 low-BLNAR (26.7%) strains, 53 BLNAR (33.8%) strains, 7 BLPAR (4.5%) strains and 6 BLPACR (3.8%) strains. Of the 2 encapsulated isolates, 1 was BLNAS strain and the other was low-BLNAR strain.

Table 1 shows the MIC50, MIC90 and MIC ranges of 6 antibiotics for the 157 H. influenzae isolates. The MIC50 values for the BLNAR strains were 2-fold to more than 32-fold higher than those for the BLNAS strains for all antibiotics, with the exception of tosufloxacin. Compared with the β-lactams, tosufloxacin (an oral fluoroquinolone used for children in Japan) showed strong activity against H. influenzae, including β-lactam–resistant strains.

Antimicrobial Susceptibility Profile of Haemophilus influenzae Isolates to 6 Antimicrobial Agents According to the Resistance Class

Amino Acid Substitutions in PBP3

The ftsI sequence encoding the transpeptidase domain of PBP3 was determined in 66 isolates for which the MIC of ampicillin was ≥4 mg/L, including 53 BLNAR, 7 BLPAR and 6 BLPACR strains. All BLNAR strains, 5 of 7 (71.4%) BLPAR strains, and all BLPACR strains showed amino acid substitutions in PBP3 related to antimicrobial resistance, with all 53 BLNAR being gBLNAR, 5 of 7 BLPAR, all 6 BLPACR strains being gBLPACR and 2 of 7 BLPAR strains being gBLPAR. There were no gBLNAS strains among the 66 isolates analyzed. Table (Supplemental Digital Content 1, shows the amino acid substitutions in the transpeptidase domain of PBP3. None of the isolates were classified into groups I, IIc or IId. Among the 66 isolates for which the MIC of ampicillin was ≥4 mg/L, the main substitutions (Asp350Asn, Ser357Asn, Met377Ile, Ser385Thr, Leu389Phe, Asn526Lys, Val547Ile, Val562Leu and Asn569Ser) accounted for 49 of the 66 strains (74.2%), and 15 different substitution patterns were identified. Of the 53 BLNAR isolates, 47 (88.7%) were in group III and only 3.8% (2/53) belonged to group II.


Molecular typing was done by MLST for 66 ampicillin-resistant H. influenzae isolates (MIC of ampicillin ≥ 4 mg/L), including 53 BLNAR strains, 7 BLPAR strains and 6 BLPACR strains. In addition, PFGE was employed for 43 highly ampicillin-resistant H. influenzae isolates (MIC of ampicillin ≥8 mg/L), including 30 BLNAR strains, 7 BLPAR strains, 6 BLPACR strains. MLST showed 39 different STs, with 12 of them being novel types containing two novel alleles (Table 2). The 12 novel STs and 2 alleles were added to the MLST database ( as ST1413-ST1422, ST1428, ST1429, adk183 and mdh250 (Table 2). The phylogenetic tree that was obtained is shown in Figure (Supplemental Digital Content 2, Twenty-seven STs were found in a single strain and 12 STs were found in 2 or more strains. The β-lactamase producing isolates (n = 13) showed 8 different STs. Analysis with eBURST v3 revealed 4 groups and 31 singletons. Group 3 was the largest group containing 5 isolates.

Allelic Profiles of New Isolates in This Study

The PFGE patterns of 43 isolates from which the MIC of ampicillin was ≥8 mg/L were diverse and the isolates fitted into 40 patterns and 38 singletons with a similarity of ≥80 % (see Figure, Supplemental Digital Content 3, Among the BLNAR isolates, there were 2 clusters contained 2 or 3 genetically related isolates (clusters 1 and 2). Cluster 1 consisted of 3 isolates (strains 21, 32 and 40). All of them belonged to ST161 and had the same amino acid substitution pattern (group III). These 3 isolates had an identical substitution pattern in PBP3 (Asp350Asn, Ser357Asn, Met377Ile, Ser385Thr, Leu389Phe, Asn526Lys, Val547Ile, Val562Leu and Asn569Ser) and showed similar MICs for β-lactams (Table 3). Cluster 2 consisted of 2 isolates (strains 44 and 84). They belonged to ST549 and had a group III amino acid substitution pattern, but their substitutions were not completely identical. All of the BLNAR strains in the 2 clusters were isolated from different locations in Japan.

Profiles of the 2 Pulsed-field Gel Electrophoresis Clusters of Haemophilus influenzae Isolates Among the Strains with MICs of Ampicillin ≥8 mg/L


In this study, we analyzed the molecular epidemiology of H. influenzae causing AOM in Japanese children younger than 3 years. Reduced susceptibility to ampicillin was found in 68.8% of the isolates (26.7% were low-BLNAR, 33.8% were BLNAR, 4.5% were BLPAR and 3.8% were BLPACR), which is similar to the findings of a recent Japanese study performed in pediatric patients by Hoshino et al.8 In Japan, the prevalence of β-lactamase–producing isolates is lower and that of BLNAR isolates is higher compared with other countries because the percentage of β-lactamase–positive/BLNAR (including low-BLNAR) isolates in the US, France and Korea was reported to be 28.3/0.7, 27.3/16.9 and 52.4/6.1 %, respectively.22–24

This study showed that amino acid substitutions of PBP3 were the most prevalent mechanism of ampicillin resistance for H. influenzae. According to the PCR-based genotyping of H. influenzae demonstrated that all of the BLNAR strains had amino acid substitutions related to ampicillin resistance (gBLNAR).25 Amino acid substitutions, including Arg517His or Asn526Lys (Group I and II), are commonly found in isolates that are susceptible or show intermediate resistance to ampicillin, whereas additional amino acid substitutions (group III: Met377Ile, Ser385Thr and/or Leu389Phe) are found in isolates with higher levels of resistance to ampicillin.4,5,7 In a number of European countries and the US, BLNAR isolates are less common and most strains with PBP3 substitutions are group I or II.6,22,23,26,27 In contrast, BLNAR isolates in Japan tend to be group III strains that mainly have PBP3 substitutions.5,28,29 In fact, 88.7% of gBLNAR strains were classified as group III in this study. This high prevalence of gBLNAR strains in Japan has been attributed to the widespread use of oral and intravenous cephem antibiotics.28,29 In 2006, Sanbongi et al found the Val329Ile/Ala and Val511Ala amino acid substitutions in Japan,5 and suggested that a change of antibiotic use from oral cephalosporins to amoxicillin may have promoted the emergence of these 2 substitutions.28 Both of these substitutions (Val329Ile and Val511Ala) were also detected in this study. In Japan, the guideline for treating AOM in children published in 2006 emphasized the use of amoxicillin,30 and this change of the antimicrobial agents recommended for AOM might have led to these substitutions of PBP3.

To our knowledge, this is the first study that has employed MLST to obtain information about the molecular epidemiology of ampicillin-resistant H. influenzae isolates from MEF in Japanese children with AOM. Molecular evaluation of ampicillin-resistant isolates by MLST demonstrated their genetic diversity and identified 12 new STs. MLST is a useful tool for assessing the genetic relations of isolates and allows comparison of the major genotypes associated with antimicrobial resistance, even across different medical settings and countries.12,18 LaCross et al12 reported an association of ST57 with AOM, and 3 out of 66 isolates were ST57 in this study. Several previous studies have indicated a relationship between ampicillin-resistant isolates and some STs. Although bla genes are generally carried by a plasmid, ST3, ST57 and ST165 have sometimes been reported in β-lactamase producers.21,26 In this study, 3/4 ST3 strains, 3/3 ST57 strains (1/3 was ROB-1) and 2/2 ST165 strains were β-lactamase producers. Skaare et al26 stated that ftsI alleles were linked to STs in H. influenzae, and they reported that the most prevalent PBP3 substitution pattern in Norway was mainly carried by ST367 and ST14. In this study, there were 3 or more strains that were ST549 (n = 8), ST161 (n = 6) or ST249 (n = 4) among BLNAR isolates, as well as 3 ST3 and 3 ST57 strains among β-lactamase producers, and 4 small eBurst v3 groups were detected. A limitation of this study is that we did not obtain detailed information about the patients, such as their living circumstances, complications, severity of AOM and outcomes. Therefore, although these isolates with the same STs and same eBurst v3 groups might be associated with occurrence of AOM or antimicrobial resistance of H. influenzae in Japanese children, there is still limited information about their relations and further investigations are needed.

Although PFGE demonstrated diversity among the isolates with an MIC of ampicillin ≥8 mg/L, 2 clusters (clusters 1 and 2) were detected and both of them had the same STs (ST161 and 549). All isolates of the same clusters showed a group III substitution of PBP3. Kishii et al28 reported that BLNAR strains isolated from children with AOM in Japan were highly diverse according to PFGE. In contrast, some reports have mentioned clonal strains among BLNAR isolates.6,21,27,29 García-Cobos et al6 detected clonal strains of BLNAR isolates that were associated with otitis media and conjunctivitis in children. Hotomi et al29 demonstrated the dissemination of resistant clones of H. influenzae isolates from the upper respiratory tract in Japanese patients of all ages. In our study, 2 clusters were found among highly resistant isolates. The main cluster was isolates of ST161 (cluster 1), with 3 of 4 ST161 isolates and 2 of 6 ST549 isolates detected by PFGE being in the same cluster. Skaare et al26 reported that combining MLST and PFGE for the typing of nontypeable H. influenzae may increase both the sensitivity and resolution of detecting clones. The diversity of MLST and PFGE findings in this study support the previous concept that BLNAR strains of H. influenzae appear independently on many occasions, possibly because of the widespread use of antibiotics.6,29 However, our detection of clusters among the highly resistant isolates is noteworthy. If these highly resistant H. influenzae isolates become more prevalent, a potential increase of recurrent AOM and treatment failure would be a concern. Investigation of the bacterial characteristics, such as antimicrobial susceptibility, pathogenicity, adherence, biofilm formation and invasive capacity, of these ST161 and ST549 clones is required to understand their characteristics and avoid the emergence of additional strains of highly resistant H. influenzae.

In conclusion, this study was the first to identify the ST clones associated with BLNAR H. influenzae strains isolated from Japanese infants and young children with AOM. Although most gBLNAR strains might arise independently, clusters of gBLNAR consisting of ST161 and ST549 were detected, which could lead to an increase of treatment failure in children with AOM. Continuous monitoring of BLNAR isolates and assessment of genetic relations among them are needed, and further studies are also required to clarify the clinical significance of ST161 and ST549.


We thank Qiu Hongli, clinical laboratory technologist at the Department of Bacteriology, BML Inc., for kindly supporting our work.


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β-lactamase-nonproducing ampicillin-resistant (BLNAR) Haemophilus influenzae; penicillin-binding protein 3; multilocus sequence typing; pulsed-field gel electrophoresis; genetic relatedness

Supplemental Digital Content

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