Serotype Distribution and Antimicrobial Resistance Profile of Haemophilus influenzae Isolated from School Children with Acute Otitis Media

Haemophilus influenzae is a Gram-negative opportunistic bacterial pathogen of the human respiratory tract. This study describes the prevalence, serotype distribution, and susceptibility profiles of H. influenzae strains isolated from the nasopharynx of school children with acute otitis media (AOM) in Banyumas Regency, Central Java, Indonesia. H. influenzae was isolated from nasopharyngeal swab specimens using chocolate agar plates supplemented with IsoVitaleX and bacitracin. Serotyping was performed using quantitative polymerase chain reaction. Antimicrobial susceptibility profiles were determined using a microdilution broth assay. H. influenzae was present in 69.7% of samples (85/122). Nontypeable H. influenzae (NHTi) was the most common serotype (95.3%), followed by H. influenzae type b (3.5%) and H. influenzae type f (1.2%). All the H. influenzae isolates were susceptible to levofloxacin, ceftriaxone, imipenem, meropenem, cefuroxime, and cefixime. Most isolates were susceptible to sparfloxacin (99%), cefepime (99%), amoxicillin/clavulanic acid 2 : 1 (99%), ampicillin/sulbactam 2 : 1 (96%), chloramphenicol (94%), tetracycline (93%), ampicillin (87%), and clarithromycin (82%). Nineteen percent of the isolates were resistant to cotrimoxazole, and 11% of the isolates were resistant to ampicillin. This study showed that H. influenzae carriage among samples was dominated by NTHi and less susceptible to cotrimoxazole.


Introduction
Haemophilus influenzae is a Gram-negative opportunistic bacterial pathogen of the human respiratory tract. is bacterium is grouped into capsulated and noncapsulated bacteria (nontypeable Haemophilus influenzae; NTHi) [1,2]. Noncapsulated bacteria have been reported as the most common pathogenic bacteria causing invasive disease since the implementation of the Hib vaccine. H. influenzae contributes to approximately 21,000 otitis media-associated deaths annually [3]. e NTHi strain mainly colonizes the mucosal surfaces of the upper respiratory tract and is highly associated with acute otitis media (AOM), sinusitis, bronchitis, exacerbations, and chronic persistent infections in older patients with chronic obstructive pulmonary disease [4]. NTHi is one of the three dominant bacterial otopathogens causing otitis media, which has been reported globally along with other pathogens such as Streptococcus pneumoniae and Moraxella catarrhalis [5]. e presence of H. influenzae in the nasopharynx was associated with older age and recurrent AOM. e proportion of NTHi-causing otitis media has trended upward in the postpneumococcal conjugate vaccine (PCV) era, and the majority of NTHi isolates were nonsusceptible to ampicillin in Taiwan [6]. Recently, NTHi strains isolated from the nasopharynx of HIV-infected patients were less susceptible to ampicillin (62%) and trimethoprim/sulfamethoxazole (cotrimoxazole) (41%) in Indonesia [7]. More than half of the H. influenzae strains isolated from pediatric patients with AOM in Japan are genotypic β-lactamase-nonproducing ampicillin-resistant strains [8]. Studies on H. influenzae among school-aged children with AOM in Indonesia are limited. In this study, we investigated the serotype distribution and susceptibility profiles of H. influenzae strains isolated from the nasopharynx of school children with AOM in Banyumas Regency, Central Java, Indonesia.  [9,10]. However, only 122 nasopharyngeal (NP) swab specimens were available for the present study. e NP swab specimens were collected with sterile FLOQSwabs (Copan) and inoculated into 1 mL STGG (skim milk (BD), tryptone (BD), dextrose (BD), and glycerol (Sigma)) as a transport medium. e specimens were then stored at −70°C before further testing.

Haemophilus influenzae Isolation and Identification.
Briefly, the isolation was performed as follows: a 100 µL of inoculated STGG media with nasopharyngeal swab specimens were streaked on chocolate agar plate supplemented (sCAP) with IsoVitaleX (BD) with addition of bacitracin (20 U ml −1 ) (Sigma), followed by incubation at 37°C with 5% CO 2 for 20 h. Extended incubation for 48 h was performed for no-growth plates. All suspected H. influenzae isolates were identified by Gram staining (BD) for Gram-negative coccobacillus, oxidase test, and XV factor-dependent test (Oxoid) [7].
All suspected H. influenzae isolates were further confirmed using quantitative polymerase chain reaction (qPCR) targeting hpd encoding H. influenzae protein D (Table 1), as described previously [11]. All isolates were subcultured onto a sCAP and incubated at 37°C in a 5% CO 2 atmosphere for 20 h. e DNA was extracted using a boiling method as follows: a fine touch of single colony was transferred into 200 µL Tris-EDTA (Sigma), then homogenized, and heated at 100°C for 5 min. e suspension was then immediately incubated at −20°C for 5 min, followed by centrifugation at 13,000 × g for 10 min. e qPCR reaction mixture consisted of TaqMan Universal Master Mix (Cat. No. 4304437), ROX 1 : 10 (25 μM), paired hpd primers, and probe. e DNA template used for each reaction was 2.5 µL. e H. influenzae ATCC 49247 strain was used as the positive control. e qPCR conditions were set as follows: 2 min at 50°C, followed by 95°C for 10 min as predenaturation, and 40 cycles at 95°C for 15 s and 60°C for 1 min. e hpd was considered positive if the Ct was ≤35; Ct ranging from 36 to 40 was repeated with dilution, and negative if Ct was >40 or defined as undetermined by the instrument.

Serotype Determination.
Serotyping was performed using qPCR, as described previously [11]. ere were six single reactions for the detection of serotypes a (acsB), b (bcsB), c (ccsD), d (dcsE), e (ecsH), and f (bexD) using six pairs of primers and different probes according to the target gene to be amplified [11]. e qPCR conditions were similar to those for hpd detection.

Discussion
In this study, it was observed that 69.7% of the school children with AOM in Banyumas Regency, Central Java, Indonesia, tested positive for H. influenzae in their nasopharynx. NTHi was the major type (95.3%) observed in this study. e carriage prevalence of H. influenzae in this study was higher than that reported in previous studies (with an interval of 9-32%) in Indonesia [7,14,15]. Many previous studies reported correlation between bacteria colonizing the nasopharynx and otitis media cases [16][17][18]. Some bacteria colonizing the nasopharynx, including H. influenzae, S. pneumoniae, and Moraxella catarrhalis [16][17][18], and others such as S. pyogenes and Staphylococcus aureus were reported to be in concordance with those isolated from the middle ear fluid of patients with otitis media [18]. S. pneumoniae and H. influenzae were the most prevalent pathogenic bacteria that showed a positive association with otitis media infection. NTHi was reported as a common type of otitis media in many regions, including South America, North America, Germany, and Asia [18][19][20]. is is also concordant with the findings in this study, which defined 95.3% of H. influenzae isolates as NTHi. e implementation of the Hib vaccine in the national vaccine program might explain the high prevalence of NTHi among children in Indonesia. Detection of H. influenzae, which is dominated by NTHi, in the nasopharynx, showed 90.91% sensitivity for otitis media cases compared to middle ear fluid as the standard for determining otitis media etiological bacteria [17]. Furthermore, the development of otitis media due to the presence of pathogenic bacteria in the nasopharynx is also triggered by alterations in the nasopharynx environment caused by viral infection. e alteration of the nasopharyngeal environment, including ATP and glucose release from cell or tissue damage, norepinephrine release due to sympathomimetic response, and an increase in temperature, will induce dissemination and dispersal of pathogenic bacteria by inducing bacteria to produce bacteriocin and many virulence factors that upregulate the dispersal of bacteria [21]. Among 85 isolates of H. influenzae, it was discovered that 19% of isolates were resistant to cotrimoxazole followed by ampicillin (11%) while isolates resistant to tetracycline, chloramphenicol, ampicillin/sulbactam, cefaclor, cefepime, sparfloxacin, and AMC were less than 10%. is finding was in concordance with a study in ailand which reported that strains of H. influenzae isolated from patients with otitis media were commonly nonsusceptible to cotrimoxazole (33%), followed by ampicillin (20%), while fewer isolates were nonsusceptible to macrolides, represented by azithromycin (10%) [22]. Isolates resistant to chloramphenicol and tetracycline were reported to be less than 10% (9.5%), which is similar to our findings (6% and 7%, respectively) [22]. is is in contrast to a study from Taiwan, where the susceptibility to ampicillin was the lowest among the tested antimicrobials (19.7%), followed by cotrimoxazole (31.1%) [6].
In this study, among ampicillin nonsusceptible H. influenzae isolates, BLNAR strains were the most frequent compared to BLNAI and BLNASR. Ampicillin nonsusceptible strains have also been reported in various regions among ampicillin nonsusceptible isolates worldwide. In 2016, a study from Japan reported that BLNAR was the most prevalent (60%) among H. influenzae isolates, followed by BLNAS (17.5%) and BLPAR (10%) [13]. In Korea, one study reported that BLPAR was the most prevalent (47.2%), followed by BLNAS (41.5%) and BLNAR (6.1%) [23]. Meanwhile, a study in Spain reported that BLNAR was the most dominant (56%), followed by BLNAS (17.8%), BLPAR (15.8%), and BLPACR (10.4%) [24]. In this study, we found one isolate that showed resistance to amoxicillin/clavulanate (2 : 1) with an MIC value > 16/8 μg/mL, but was susceptible to ampicillin, defined as BLNACR. ree isolates were resistant to ampicillin/sulbactam, which were defined as BLNASR. In this study, β-lactamase-producing strains were defined according to their MIC values and were not confirmed by PCR detection of the gene encoding β-lactamase. In conclusion, the prevalence of H. influenzae carriage in the nasopharynx of school children with AOM in Indonesia was 69.7%. e isolates were predominantly NTHi. e H. influenzae isolates identified in this study were less susceptible to cotrimoxazole. In addition, BLNAR strains were the most prevalent among the nonsusceptible ampicillin strains.
Data Availability e serotyping and minimum inhibitory concentration data used to support the findings of this study are included within the article. Disclosure e contents are the sole responsibility of the authors and do not necessarily represent the official views of the Ministry of Research, Technology, and Higher Education, National Agency for Research and Innovation of the Republic of Indonesia. e funders had no role in the study design, data collection and analysis, decision to publish, or manuscript preparation.

Conflicts of Interest
e authors declare no conflicts of interest.