Acute otitis media (AOM), one of the most common pediatric infections, is a leading indication for antibiotic prescriptions in children.1,2 Judicious antimicrobial use has been recommended by guidelines for treating AOM.3–6 Major bacterial causes of AOM include Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi), which account for some 50%–90% of cases.3,7–10 Less frequent bacterial causes are Moraxella catarrhalis7,8 and Streptococcus pyogenes.10–12 Additionally, respiratory viral infection often is confirmed in AOM.13,14
Among these pathogens, penicillin (PEN)-nonsusceptible S. pneumoniae commonly present clinical problems, but β-lactamase–nonproducing, ampicillin (AMP)-resistant H. influenzae (BLNAR) is particular concern in certain countries such as Japan and Spain. The relative frequency of PEN-nonsusceptible S. pneumoniae in various countries has ranged from 25% to 50%; more than 20% of these S. pneumoniae also show macrolide (ML) resistance.15,16 In Asia, the rate of PEN resistance may exceed 50%.17–19 Moreover, pneumococcal strains with multidrug-resistance phenotypes are becoming common, attaining prevalence of 40% or more.15,17,20
Prevention of AOM is an important public health objective. Pediatric pneumococcal conjugate vaccines (PCVs), which prevent AOM caused by S. pneumoniae, also are important in reduction of antibiotic resistance. PCVs have decreased community-acquired pneumonia21–23 and invasive pneumococcal diseases (IPDs),24–32 as well as AOM33–39 and chronic sinusitis.40 However, pneumococcal serotype replacement from vaccine types (VTs) to nonVTs (NVTs) occurred after PCV introduction.28,29,41–44 Although NVT serotypes are now more prevalent in patients presenting with AOM, the overall reduction of AOM suggests that this represents mostly a reduction of VT serotypes rather than a true increase in NVT.38,39
In Japan, 7-valent PCV (PCV7) was introduced for use in children below 5 years of age in November 2010 by the Provisional Fund for the Urgent Promotion of Vaccination. The rate of inoculation for PCV7 exceeded 90% 2 years later throughout Japan, and PCV7 was incorporated into the immunization schedule in April 2013. Almost immediately, in November of the same year, PCV7 was replaced by 13-valent PCV (PCV13). These PCVs have impressively decreased IPD in children,45,46 as well as exerting a herd effect against adult IPD.47 Regrettably, serotype replacements from VT to NVT serotypes have been observed in pneumococcal isolates from children and adults with IPD, as has occurred in other countries.
Before PCV7 introduction in 2006, we identified causative agents in middle ear fluid (MEF) from patients with AOM using comprehensive real-time polymerase chain reaction (PCR) and bacterial cultures.48 Pneumococcal isolates among these agents were analyzed further concerning capsular serotypes, resistance genotypes and susceptibility to various agents.49
In this study, we examined changes of etiologic agents and characteristics of pneumococcal isolates in MEF from children with AOM after introduction of PCV13. In particular, serotype distribution, resistance genotypes, results of multilocus sequence typing (MLST) and susceptibilities to 14 antimicrobial agents were analyzed.
MATERIALS AND METHODS
Patients and Sample Collection
This prospective study, designated an AOM Surveillance Study, involved participation of 69 private otolaryngologic clinics throughout Japan (Fig., Supplemental Digital Content 1, http://links.lww.com/INF/D13). Between June 2016 and January 2017, a total of 582 AOM patients 15 years of age or younger were enrolled in this study after informed consent was obtained from parents or guardians. AOM was diagnosed in each patient according to guidelines determined in 2013 in a joint effort by 3 Japanese otolaryngologic societies6 (http://minds4.jcqhc.or.jp/minds/otitis/CPG_AOM_JPN.pdf). After sterilization of the external auditory canal, MEF obtained by tympanocentesis or sampling through a spontaneously ruptured tympanic membrane was collected by otolaryngologists using sterile transport swabs (Transystem; Copan Italia, Brescia, Italy). All samples were sent immediately to the Department of Infectious Diseases at Keio University School of Medicine.
Methods for sampling of MEF from AOM patients described above, and for testing of causative microorganisms, pneumococcal serotyping, antimicrobial resistance genotyping and antibiotic susceptibility described below, also were used in our 2006 study48,49 under the direction of the same author (K.U.).
Bacterial cultures and real-time PCR were conducted immediately after arrival of samples, which were suspended in 500 µL of Mueller-Hinton broth (Becton Dickinson, Franklin Lakes, NJ) and centrifuged at 2000g for 5 minutes at 4°C to collect bacterial cells. After discarding the supernatant, the pellet remaining (150 µL) was stirred and used for direct DNA extraction (100 µL) and for culture (20 µL).
For PCR, each 100 µL sample was placed in 44 µL of a lysis solution that included SimplePrep reagent for DNA (Takara Bio, Shiga, Japan). The lytic reaction was carried out for 6 minutes at 37°C, followed by 3 minutes at 95°C. The lysate was added to each of the tubes containing PCR mixtures to be used for identifying the following 4 bacterial species by detection of 2 distinctive genes: lyt A, which encodes autolysin enzyme specific to S. pneumoniae; and each of 3 16S ribosomal RNA (rRNA) genes specific to H. influenzae, S. pyogenes or Mycoplasma pneumoniae. Total volume (30 µL) of the PCR mixture included 20 pmol of each primer, 25 pmol of each probe, and reagents from the Cycleave PCR Core kit (Takara Bio, Shiga, Japan). Real-time PCR was carried out for 45 cycles as follows: 95°C for 5 seconds, 55°C for 15 seconds and 72°C for 20 seconds.
For bacterial cultures, 5 µL of each sample was inoculated on a sheep blood agar plate, a chocolate agar plate, a mannitol salt agar plate and a modified Drygalski agar plate (all from Nippon Becton Dickinson, Tokyo, Japan). Colonies grown on each agar plate after overnight culture were picked up and subjected to identification of bacterial species by routine methods.
Serotype and Resistance Genotype
Serotypes were determined by the capsular quellung reaction using antiserum purchased from the Statens Serum Institute (Copenhagen, Denmark). Alterations in 3 PEN-binding protein (PBP) genes mediating β-lactam resistance in S. pneumoniae—pbp1a (encoding PBP1A), pbp2x (encoding PBP2X) and pbp2b (encoding PBP2B)—were identified by real-time PCR methods described previously.50 The mefA and ermB genes mediating ML resistance also were identified by real-time PCR.
Results of genotypic (g) analysis were represented as follows: PEN-susceptible S. pneumoniae (gPSSP) possessing 3 normal pbp genes; PEN-intermediate S. pneumoniae (gPISP) subclassified as gPISP (pbp2x), gPISP (pbp2b), gPISP (pbp1a+pbp2x) or gPISP (pbp2x+pbp2b) or PEN-resistant S. pneumoniae (gPRSP) possessing all 3 abnormal pbp genes.45
Minimum inhibitory concentrations (MICs) of 10 oral agents including PEN, AMP, amoxicillin (AMX), cefcapene (CFN), cefdinir (CDR), cefditoren (CDN), tebipenem (TBM), clarithromycin, azithromycin, tosufloxacin (TFX) and 4 intravenous agents including tazobactam/piperacillin, cefotaxime, ceftriaxone and meropenem were determined for all pneumococcal isolates by well-controlled agar dilution methods, with the reference strains R6 and ATCC49619 tested at the same time.
Multilocus Sequence Typing
Primers used for MLST were based on sequences listed on the Centers for Disease Control and Prevention website (http://www.cdc.gov/streplab/alt-mlst-primers.html). MLST and enhanced based upon related sequence types (eBURST) analyses were performed according to the MLST website (http://spneumoniae.mlst.net).
Statistical analyses were performed using Ekuseru-Toukei 2012 software for statistics (Social Survey Research information, Tokyo, Japan). The χ2 test and Fisher exact test were used as appropriate. A P value below 0.05 was considered to indicate statistical significance.
This study was approved by the Hakujikai Memorial Hospital Ethics Committee (approval number: 17) and by the Keio University School of Medicine Ethics Committee (approval number: 20140432).
Otolaryngologists explained the purpose of the study to parents or guardians of eligible patients, who signed an informed consent form just before sample collection.
Table 1 shows causative bacterial pathogens isolated from MEF collected by tympanocentesis (n = 264) or by sampling through a ruptured tympanic membrane (n = 318) in 582 patients. At least 1 bacterial pathogen was identified in 473 samples (81.3%). The most frequent pathogen was H. influenzae (n = 319; 54.8%), followed by S. pneumoniae (n = 148; 25.4%). Samples positive for these pathogens by both culture methods and real-time PCR were 73.4% (n = 234) for H. influenzae and 73.0% (n = 108) for S. pneumoniae. Other pathogens identified included Staphylococcus aureus (3.1%), S. pyogenes (2.9%), M. catarrhalis (1.2%), M. pneumoniae (1.0%) and other bacteria (2.4%).
Isolation frequency for S. pneumoniae was significantly higher in tympanocentesis than ruptured tympanic membrane specimens. In contrast, S. pyogenes and S. aureus were isolated predominantly from patients with a ruptured tympanic membrane.
In comparison with our similar 2006 study (n = 399) which found bacteria to be responsible 78.2% cases,48 the present one showed a significant decrease in frequency for S. pneumoniae from 39.3% in 2006 to 25.4% presently (P < 0.001), while frequency of H. influenzae increased significantly from 4 1.1% to 54.8% (P < 0.001).
Pathogens by Patient Age
A breakdown of pathogens by age of the present patients with AOM is shown in Table 2. Patients 1 year of age or younger accounted for 58.6%, while patient numbers decreased overall at 2, 3 and 4 years to 15.1%, 6.4% and 7.6%, respectively.
Coinfections with S. pneumoniae and H. influenzae and infection with H. influenzae alone were most frequent at 1 year, with a decrease at 2 years and beyond. Pneumococcal infection was particularly frequent at 1 year, increasing again at 4 years or more, particularly at and beyond 5 years. S. pyogenes was more frequent at 4 years or more than earlier in life. In sum, bacterial pathogens clearly varied according to age at onset of infection.
Serotypes and Resistance Genotypes
In Figure 1, results of this study (n = 108) and findings in 2006 (n = 117) obtained before introduction of PCV7 were compared with respect to serotypes and genotypes in pneumococcal isolates among MEF samples collected from patients with AOM. While occurrence of PCV13 VTs was high with 82.1% in 2006, their share among pneumococcal isolates decreased dramatically to 18.5% after routine vaccination with PCV13. In contrast, 6 NVTs, 15A (P = 0.023), 15B (P = 0.001), 15C (P = 0.011), 10A (P = 0.029), 23A (P = 0.031) and 35B (P = 0.001), increased significantly in the present study. Serotype 3, which is VT and prevalent among AOM isolates, decreased to 13.9% in 2016 compared with 23.1% in 2006, but the difference fell short of significance (P = 0.088). In particular, serotype 3 was infrequent among children receiving PCV13 (6.7%, 5/75) compared with frequency among children receiving PCV7 (38.9%, 7/18; P = 0.002).
Genotypically resistant pneumococci were frequent: gPISP (pbp2x), 45.4%; gPRSP (pbp1a+2x+2b), 28.7%; gPSSP, 9.3%; gPISP (pbp1a+pbp2x), 8.3%; gPISP (pbp2x+pbp2b), 7.4% and gPISP (pbp2b), 0.9%. Among NVT, most serotypes 15A and 35B were gPRSP. Accordingly, gPRSP decreased only slightly (34.2% in 2006 vs. 28.7% in this study; P = 0.393), reflecting impact of serotypes 15A and 35B. Most pneumococcal strains (88.8%) had the ML resistance genes ermB, mefA or both (details not shown).
MLST by Serotypes and Genotypes
Clonal complexes (CCs) and sequence types (STs) for all pneumococcal strains are shown in Table 3 according to serotype and PEN resistance genotype. These isolates were divided into 21 serotypes, 23 CCs and 32 STs. Phylogenetic analyses of MLST are illustrated in the Figure, Supplemental Digital Content 2, http://links.lww.com/INF/D14, where a solid line indicates a single-locus variant, indicating that only 1 allelic difference existed between the connected STs. Thus, the CC was the same in serotypes 15A (CC63), 15B (CC199), 23A (CC156) and 24B (CC2572), even when ST and genotype were different in strains belonging to the same serotype. Two CCs were recognized within each of 6 serotypes: 35B, 10A, 15C, 22F, 6C and 23B. These CCs were distant from each other.
Antimicrobial Susceptibility by Genotype
Susceptibilities (MIC range, MIC for 50% of isolates [MIC50] and MIC for 90% of isolates [MIC90]) to 6 oral agents in all pneumococcal strains are shown according to genotype in Table 4, and the remaining 4 oral agents used mostly in Japan are shown in the Table, Supplemental Digital Content 3, http://links.lww.com/INF/D15.
MIC90s of oral agents for all strains, in order of most to least active, were as follows: TBM (0.125 μg/mL), CDN and TFX (each 0.5 μg/mL), CFN (1 μg/mL), PEN and AMX (each 2 μg/mL) and AMP and CDR (each 4 μg/mL). Relationships between susceptibilities and alterations in 3 pbp genes (pbp1a, pbp2x and pbp2b) that affect MICs of β-lactam agents (AMP, AMX, CDN and TBM) are shown in the Figure, Supplemental Digital Content 4, http://links.lww.com/INF/D16. About half of strains in this study were gPISP (45.4%), having pbp2x gene alterations that reduced susceptibility to cephalosporins, such as CDR, CFN and CDN, with less effect on PENs. Strains of gPRSP having pbp1a, pbp2x and pbp2b gene alterations showed reduced susceptibilities to β-lactam agents, even when degree of reduction varied. MIC90 against gPRSP was superior for TBM (0.125 μg/mL) and CDN (0.5 μg/mL). Strains tested were highly resistant to clarithromycin and azithromycin, with both MIC50 and MIC90 exceeding 64 μg/mL.
MIC90s of parenteral agents for all strains (Table, Supplemental Digital Content 3, http://links.lww.com/INF/D15) showed efficacy in this order of greater to lesser: cefotaxime and ceftriaxone (each 0.5 μg/mL), meropenem (1 μg/mL) and tazobactam/piperacillin (4 μg/mL).
In addition, no individual pneumococcal multidrug-resistance strain possessed 3 or more resistance mechanisms, such as characteristics mediating PEN resistance, ML resistance and quinolone resistance.
To our knowledge, this report presents the largest etiologic surveillance study in Japanese pediatric patients with AOM after PCV13 implementation for children younger than 5 years of age. The study characterized pneumococcal isolates with respect to serotype, genotypic PEN resistance and MLST. Bacterial pathogens were identifiable in 81.3% of these patients, who were diagnosed with AOM by otolaryngologists who strictly followed Japanese AOM guidelines.6
The authors have performed a similar etiologic study in AOM using cultures in combination with real-time PCR in 2006, before introduction of PCV7.48 Comparing results of the earlier and present studies, isolation frequency of S. pneumoniae decreased significantly from 39.3% in 2006 to 25.4% in 2016, while the rate for Hflu increased significantly from 41.4% to 54.8%. These changes as well as serotype replacements increasing the proportion of NVT, discussed further on, appear to be consequences of introduction of PCV7 since 2011 and replacement with PCV13 in 2014.
In our present evaluation of AOM etiology in Japan using cultures and real-time PCR, S. pneumoniae were identified more frequently in samples obtained by tympanocentesis than in samples obtained through a ruptured tympanic membrane. In contrast, S. pyogenes and S. aureus frequently were isolated from samples obtained from patients with a ruptured tympanic membrane. In this study, sampling is believed to have been performed according to detailed guidelines6 followed closely by each otolaryngologist. Nevertheless, the possibility of S. aureus representing contamination from the external auditory canal could not be completely eliminated. Some variability in etiologic proportion associated with sample quality also may have resulted from limited sensitivity of bacterial culture used alone.
Another important concern is that frequencies of specific etiologic agents differ between AOM and complicated or recurrent otitis media cases. In particular, contributions of biofilms in cases of unknown etiology have been pointed out by Dagan et al.51 We intend to focus on AOM in the present study, but we also seek to make if relevant to complicated cases. We also would like to focus more closely on etiologic agents in complicated cases in the future.
Another important difference evident in our study is that bacterial pathogens varied appreciably according to age at onset of infection. The relatively high occurrence rate of S. pneumoniae in patients 4 years of age or older likely reflects absence of immunity for serotypes not covered by the vaccine that they received, specifically PCV7, as opposed to PCV13. For example, infection with serotype 3, which has a mucoid capsule, was significantly less frequent in patients receiving PCV13 than in PCV7 recipients. Another difference is that S. pyogenes also was detected more frequently in older children than in those 4 years of age or younger, and often was associated with perforation of the tympanic membrane. As reported previously, this rapid progression to tympanic membrane perforation appears to reflect particular severity of infection with S. pyogenes.10,11
Most patients enrolled in this study received PCV13 (78.6%) or PCV7 (16.8%), resulting in dramatic changes of serotype distribution among pneumococcal isolates from VT to NVT. VT isolates decreased after PCV7 immunization in many countries, but an overall increase in serotype19A, representing PRSP, was a significant clinical concern24,52 that led to development of PCV13. In our present study, the proportion of serotype 19A already had decreased at 3 years after implementation of PCV13 immunization. Emergence of serotypes 15A and 35B was reported in 200253; their proportions among isolates from patients with AOM or IPD have gradually increased worldwide since PCV13 replaced PCV7.25,28,29,34,42–44,54 In our previous study in 2006,49 serotypes 15A and 35B among PEN-susceptible S. pneumoniae were uncommon before vaccination with PCV7; after transition to PCV13, these PEN-susceptible S. pneumoniae were replaced rapidly by gPRSP showing the same serotype.45 Such overall increases of gPRSP belonging to 15A and 35B will pose problems worldwide. In addition, several serotypes identified as gPISP having pbp alterations, such as 10A, 11A, 15B, 15C, 22F and 23A, gradually increased. These strains can evolve to gPRSP through further pbp alteration followed by selection under antibiotic pressure.
In MLST analysis of our isolates, CCs and STs were confirmed to differ among serotypes and genotypes. Particularly, serotype 15A, representing gPRSP, had the same ST, ST63, that originated from Pneumococcal Molecular Epidemiology Network clone Sweden15A-25, which was described as PEN-susceptible S. pneumoniae. ST558 of serotype 35B, found to be gPRSP, similarly had an ST that initially was posted in the MLST database from the United States in 2002, and the resistant strains rapidly increased.44 Finally, an ST83 isolate of serotype 15C, identified among gPRSP, was posted to database from Japan in 2013. In sum, serotypes of 15A and 35B among gPRSP likely arose in other countries.
Susceptibility to oral and intravenous antimicrobial agents among S. pneumoniae isolates causing AOM have changed little with respond to resistance genotype since 2006.49 TBM,55 an oral carbapenem agent approved by the Japanese Ministry of Health, Labour and Welfare still achieved the best susceptibility against gPRSP, followed by CDN, a third-generation cephalosporin. In our country, either AMX or AMX/clavulanate also is the first-choice antibiotic according to the guidelines for AOM based on bioavailability.6 Use of TBM or TFX is limited to children with AOM or pneumonia caused by PRSP or β-lactamase-nonproducing ampicillin-resistant (BLNAR).
Unfortunately, 62% of Hflu isolated from respiratory infections in Japanese children now represent genetically BLNAR (gBLNAR).56 Susceptibilities to AMP and AMX of gBLNAR were less than those to TFX, CDN and TBM. Given this high prevalence of gBLNAR in Japan, AOM caused by gBLNAR has increased relatively, in contrast to S. pneumoniae–associated AOM. High prevalence of gBLNAR in the nasopharynx likely is related to the increase of H. influenzae as a causative pathogen in AOM; accordingly, choice of therapeutic agents has narrowed.
We concluded that new laboratory methods such as real-time PCR able to detect both bacterial and viruses are needed to determine causative microorganisms and guide selection of antimicrobial agents in AOM. Large-scale surveillance with participation of otolaryngologists also is needed, as is development of more extensive PCV covering NVT and new oral agents effective against resistant organisms.
The authors thank the otolaryngologists who participated actively in this study. The authors also thank their staff members, Madoka Naito and Shinji Masuyoshi, for their helpful assistance.
Mikiya Inagaki MD, Kiyoaki Kamakazu MD, Yoshihiro Atago MD, Shinichi Kakurai MD, Junichi Iwamoto MD, Shinji Usui MD, Seizou Ooyama MD, Sumio Sugano MD, Hisashi Kuroda MD, Yousuke Kamide MD, Michihiro Kamide MD, Yasuko Murakawa MD, Satoru Kaieda MD, Ippei Kawaziri MD, Akimitsu Kawai MD, Yasutake Kikuchi MD, Seiji Sawaki MD, Koujin Kyou MD, Shigeru Kimura MD, Yasuko Kimura MD, Yutaka Kimura MD, Hideyuki Sashiyou MD, Hirohide Yoneya MD, Kazumi Kosaka MD, Mototane Komeda MD, Katsuhiko Kobayashi MD, Yudzuru Sakaida MD, Shoichi Sawada MD, Tatsuaki Satoh MD, Masami Satoh MD, Toshiaki Shimizu MD, Osamu Kimura MD, Jun Shimada MD, Yasutaka Hori MD, Rinya Sugita MD, Yoshinobu Sugita MD, Akihiro Uchizono MD, Kazuhiro Soeda MD, Tomizou Tabuchi MD, Chiharu Matsuoka MD, Chiaki Suzuki MD, Hideki Chiba MD, Yasuhiro Tsuboi MD, Michio Tomiyama MD, Kouji Nakano MD, Tsutomu Nakazawa MD, Akira Fukumoto MD, Akiko Fukumoto MD, Mitsuyoshi Nagura MD, Ikuo Nagayama MD, Mutsumi Satoh MD, Katsuhiko Nakamura MD, Kenji Noguchi MD, Kyouko Shiiba MD, Kenichirou Nogami MD, Hideki Matsuda MD, Emiko Shiba MD, Keishi Hirabayashi MD, Masaaki Hiyoshi MD, Shigeo Yamagishi MD, Chika Odzu MD, Jun Maruyama MD, Shigenori Matsubara MD, Mitsuko Suetake MD, Motoaki Miyashita MD, Akira Mogami MD, Mikio Yamaguchi MD, Hideyuki Yamaoka MD, Atsushi Yuta MD, Takuma Yoshikawa MD, Meiwa Toyofuku MD, Sachiko Mori MD, Mitsuaki Inagaki MD
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