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Original Studies

Haemophilus influenzae Prevalence, Proportion of Capsulated Strains and Antibiotic Susceptibility During Colonization and Acute Otitis Media in Children, 2019–2020

Fuji, Naoko PhD; Pichichero, Michael MD; Kaur, Ravinder PhD

Author Information
The Pediatric Infectious Disease Journal: September 2021 - Volume 40 - Issue 9 - p 792-796
doi: 10.1097/INF.0000000000003171


Haemophilus influenzae is a pathogen able to cause a wide spectrum of diseases in children, ranging from respiratory tract infections to invasive disease.1 The species H. influenzae comprises 6 capsular types (types af) and noncapsule types, commonly referred to as nontypeable H. influenzae (NTHi).2 Introduction of H. influenzae type b (Hib) conjugate vaccine dramatically reduced the incidence of invasive and noninvasive diseases caused by type b in children. Although NTHi currently accounts for most of the childhood diseases caused by H. influenzae in Hib vaccinated children. Emergence of other encapsulated H. influenzae types causing invasive diseases, especially H. influenzae type a (Hia), has been noted in the United States.3

Acute otitis media (AOM) and recurrent AOM1,4–7 are common diagnoses in children and a common reason for antibiotic prescription.8,9 NTHi and Streptococcus pneumoniae are the 2 main bacterial otopathogens responsible for AOM. Pneumococcal conjugate vaccines have had a major impact on reducing the incidence of AOM caused by S. pneumoniae, especially strains expressing vaccine serotypes.10 Consequently, NTHi has emerged as the most common bacterial pathogen causing AOM in children.6 Currently, there is no licensed vaccine in the United States that targets NTHi causing AOM4,11 or other diseases caused by NTHi such as conjunctivitis,12 chronic obstructive pulmonary disease13 and invasive disease.14 Determining evolving antibiotic susceptibility of H. influenzae is important to guide appropriate antibiotic selection.

In the present study, the prevalence of H. influenzae in the nasopharynx (NP) at times of health and onset of AOM as well as in the middle ear fluid (MEF) during was investigated in young children in the Rochester, NY, area. Capsule types and antibiotic susceptibility among the H. influenzae strains isolated were determined.


Study Population

Children enrolled in this study were part of an ongoing prospective, longitudinal study of NP colonization and AOM in young children, funded in part by the Centers for Disease Control and Prevention from September 2019 to September 2020. Children were enrolled at 6–30 months of age from 2 pediatric clinical practices within the Rochester, NY, area. Written informed consent was obtained from parents before enrollment in the study as approved by the Rochester Regional Health Institutional Review Board.

Sample Processing

NP wash samples (instilling and withdrawing ~2 mL of saline in each nostril with bulb syringe) were collected during health visits of children at 6, 9, 12, 15, 18, 24 and 30–36 months of age. Clinical diagnosis of AOM of children with AOM symptoms between 6 and 36 months of age was made by validated otoscopist clinicians and then confirmed based on tympanocentesis with collection of MEF. NP wash samples were also obtained at AOM visits. Standard microbiology processing and identification techniques were used in detecting H. influenzae, S. pneumoniae and Moraxella catarrhalis.15H. influenzae isolates were confirmed using HAEMOPHILUS ID QUAD (Remel, Lenexa, KS). The number of culture-positive cases was used to calculate the prevalence of H. influenzae in this study. Stocks of all strains were maintained in brain heart infusion media with 20% glycerol and stored at −80 °C until further testing. H. influenzae isolates from healthy and AOM visits were tested for antibiotic susceptibility and typed by polymerase chain reaction (PCR). In cases where H. influenzae was isolated from both ears, only 1 ear sample was tested because previous results have shown that the same strain is isolated from both ears.16,17 PCR testing was performed on culture-negative MEF samples that were stored in TRI-reagent (Sigma-Aldrich, MO) after microbiologic processing. A multiplex PCR procedure was used for the simultaneous detection of H. influenzae, S. pneumoniae, M. catarrhalis and Alloiococcus otitidis using primers after DNA extraction from MEF.18

DNA Preparation and PCR

The H. influenzae isolates were cultured on Chocolate II Agar (BectonDickinson, MD) plates overnight at 37 °C, 5% CO2 and then suspended in 500 μL of phosphate-buffered saline. The suspension was centrifuged at 12,000 revolutions per minute for 5 minutes, and the supernatant discarded. DNA was extracted from the pellet using PureLink Genomic DNA Mini kit (Invitrogen, CA). Six different encapsulated H. influenzae strains (American Type Culture Collection number: A:9006, B: 9795, C:9007, D:9008, E: 8142, F: 9833) were used as control. Reference strains were kindly provided by Dr. Dayle Danies, Old Dominion University.

Six primers set specific for capsular types af were modified from Falla et al19 based on the references (Table, Supplement Digital Content 1, Primer sequence homology was confirmed using nucleotide-nucleotide BLAST against available H. influenzae sequences at the National Center for Biotechnology Information ( that had both query coverage and percent identity higher than 90%. Detailed primer sequences and references are described in Table (Supplement Digital Content 1, Multiplex PCR Primer sets (Set1: Type A, C, E, Set2: Type B, D, F) were optimized using Multiple Primer Analyzer software (Thermo Scientific Web Tools available at Two primary multiplex PCRs were carried out for each isolate. The PCR reaction, 30 μL, contained 1× TaqMaster Mix (ACCURIS, NJ), 1 μM (each) oligonucleotide primers (Integrated DNA Technologies, IA) and 1 μL of template DNA. Annealing temperature was set at 53 °C for 35 cycles. PCR product was checked on a 1% agarose gel and compared with positive control. Confirmation of the primary product was performed by a seminested PCR for 25 cycles under the same conditions except annealing at 55 °C and 0.5 µL of first PCR product as the template. PCR negative samples were recorded as NTHi.

Antibiotic Susceptibility

β-lactamase production of H. influenzae was determined using Cefinase-Disc (BectonDickinson). Antibiotic susceptibility of H. influenzae isolates to 13 different antibiotics (ampicillin, trimethoprim/sulfamethoxazole, cefaclor, cefuroxime, cefprozil, cefdinir, cefixime, cefpodoxime, ceftriaxone, erythromycin, azithromycin, clarithromycin, amoxicillin-clavulanate) was determined with the Sensi-Disc (BectonDickinson) by agar disc diffusion test using media recommended by Clinical and Laboratory Standard Institute (CLSI).15H. influenzae isolates were grown on Chocolate II Agar (BectonDickinson) overnight at 37 °C, 5% CO2. The inoculum was suspended in phosphate-buffered saline and adjusted to 0.15–0.20 OD600 (equivalent to 0.5 on the McFarland scale). Haemophilus test medium agar was prepared with Difco Mueller-Hinton broth (BectonDickinson), Bacto Agar (BectonDickinson), Yeast Extract (BectonDickinson), Hemin (BectonDickinson, NJ) and nicotinamide-adenine-dinucleotide (Roche, Mannheim, Germany). The adjusted inoculum was plated on Haemophilus test medium agar within 15 minutes from adjusting OD600 inoculums by a sterile swab and then each antibiotic disc was placed. The inoculated plates were incubated for 16–18 hours at 37 °C, 5% CO2. The interpretive zone sizes were measured. The isolates were classified as susceptible, intermediate or resistant based on the size of the interpretive zone by referring to current CLSI breakpoints for Haemophilus.20 Erythromycin disc breakpoints are not provided in CLSI-2018 M100 guidelines for H. influenzae. Therefore, we used arbitrary cut off values (zone diameter: ≤15 mm = R; ≥21 mm = S). Although there is no uniform definition,21 β-lactamase positive and ampicillin resistant (AR), β-lactamase negative and ampicillin sensitive (AS) and β-lactamase negative, AR (BLNAR) phenotypes were defined by the susceptibility of β-lactamase and AR according to the CLSI definition. For AR, we differentiated ampicillin-intermediate strains (zone diameter: 19–21 mm) as low-BLNAR and ampicillin-resistant strains (zone diameter: ≤18 mm) as BLNAR as described in previous studies.22,23 β-lactamase–positive amoxicillin-clavulanate resistant (BLPACR) strains were defined as amoxicillin-clavulanate–resistant (zone diameter ≤19 mm) that excluded intermediate resistant isolates.21

Statistical Analysis

GraphPad Prism 8.2.1 (CA) was used for all statistical analyses. Nominal variables were compared using Fisher exact tests except gender and antibiotic history that were tested by Pearson χ2 tests. Continuous variables were compared using student t test for 2 independent groups.


A total of 611 healthy visits and 130 AOM visits (Table 1) occurred among the 334 study children from September 2019 to September 2020. Two-hundred forty-four (73%) children were Caucasian, 11 (3%) African American, 13 (4%) Hispanic and 66 (20%) mixed/other race. Forty-two percent of the children were female. No significant racial or gender differences in H. influenzae detection during healthy colonization or AOM were identified. Median age at time of AOM visit was 16 months.

TABLE 1. - Prevalence of Haemophilus influenzae Detection and β-Lactamase Production From NP of Healthy and AOM Visits Along With H. influenzae Detection in MEF During AOM of Children Isolated in September 2019 to September 2020
Healthy (n = 611) AOM (n = 130) P Healthy vs. AOM Total Taps (n = 104) Total AOM (n = 70) P AOM NP vs. MEF (Total AOM)
H. influenzae, n (%) 36 (5.9) 35 (27) <0.0001 42 (40) 30 (43) 0.03
H. influenzae β-lactamase production, n (%) 15 (42) 12 (34) 0.63 18 (43) 13 (43) 0.45
The prevalence of H. influenzae was based on the isolation by culture method. The PCR results on culture negative cases were not included in the data tabulated. P < 0.05 was considered as significant in bold.

The prevalence of H. influenzae in the NP at healthy visits was 5.9%. At onset of AOM, H. influenzae was isolated in 27% of the NP samples, significantly higher compared with healthy visits (Table 1, P < 0.0001). β-lactamase positivity was 42% among H. influenzae isolates collected at healthy visits and 34% when isolates were collected at onset of AOM (P = 0.63, not significant). Overall, 39.7% of isolates were β-lactamase producing. H. influenzae was isolated from MEF in 43% of AOM cases. At onset of AOM, the detection of H. influenzae from the NP was 27% of cases, significantly lower than the isolation rate from MEF (P = 0.03). In the MEF, 43% of all AOM cases caused by H. influenzae were β-lactamase producing (Table 1). Thirty-three MEF samples were culture negative. In addition to the H. influenzae culture-positive AOM cases (43%), H. influenzae DNA was detected by PCR in 21% of the culture-negative MEF samples. H. influenzae DNA was detected by PCR in 21% of the culture-negative MEF samples.

The frequency of β-lactamase–producing H. influenzae strains occurring among children with risk factors, that is, day-care center attendance, number of siblings and antibiotic prescription history, was analyzed. Antibiotic prescription history in the 30 days before collection of a H. influenzae isolate was identified as a risk factor for β-lactamase producing (52.3%) versus not producing (34.2%) H. influenzae (odds ratio 2.1 [95% CI, 1.0–4.4]).

H. influenzae isolates were tested for capsular type. Eight of 101 H. influenzae isolates were encapsulated, and of these, 1 isolate was type e (isolated from NP during AOM) and 7 isolates were type f (4 from NP during AOM, 2 from MEF, 1 from a healthy visit). β-lactamase–producing strains and nonsusceptibility to ampicillin were not observed in encapsulated isolates (Table 2); therefore, β-lactamase–producing strains (P = 0.02) and nonsusceptibility to ampicillin (P = 0.02) among encapsulated isolates were significantly lower compared with NTHi.

TABLE 2. - Antibiotic Susceptibility Among Haemophilus influenzae Isolates
% Non susceptible (I + R)
NTHi Encapsulated (n = 8)
Antibiotics Healthy (n = 35) AOM (NP) (n = 28) AOM (MEF) (n = 28)
Ampicillin 42.9 39.3 39.3 0.0*
TMP/SMX 34.3 35.7 35.7 37.5
Cefaclor 2.9 21.4 7.1 12.5
Cefuroxime 0.0 3.6 7.1 0.0
Cefprozil 5.7 17.9 7.1 25.0
Cefdinir 2.9 10.7 7.1 0.0
Cefixime 2.9 3.6 10.7 0.0
Cefpodoxime 2.9 3.6 3.6 0.0
Ceftriaxone 2.9 3.6 7.1 0.0
Erythromycin 80.0 67.9 58.6 62.5
Azithromycin 5.7 7.1 3.4 0.0
Clarithromycin 28.6 21.4 27.6 37.5
Amoxicillin-clavulanate 0.0 7.1 3.4 0.0
*Ampicillin nonsusceptibility in total NTHi and encapsulated H. influenzae P =0.02.
P = 0.04.

Antibiotic susceptibility results at healthy and AOM visits against 13 different drugs are shown in Table 2. Ampicillin, trimethoprim/sulfamethoxazole, erythromycin and clarithromycin nonsusceptibility were commonly found (> 25%). Cefuroxime, cefpodoxime, ceftriaxone and amoxicillin-clavulanate nonsusceptibility were detected in small numbers of isolates (<5%). Cefaclor nonsusceptibility was significantly higher in NP isolates at onset of AOM compared with healthy visit isolates (P = 0.04). Prior work has shown that NP and AOM isolates are concordant, consistent with pathogenesis.16,17 We secured more NP samples than MEF samples from the children at onset of AOM, and the additional NP samples led to differing percentages of isolates that we report as antibiotic susceptible. For susceptibility to cefaclor, comparing NP and MEF at onset of AOM, a significant difference was observed (Table 2). However, to confirm that NP and MEF isolates were concordant with regard to antibiotic susceptibility, we compared 17 NP and MEF paired isolates that were cefaclor nonsusceptible and found they were concordant for that antibiotic, and the other 12 antibiotics tested. Antibiotic susceptibility results of H. influenzae were further analyzed based on whether the child had prior exposure of antibiotics (30 days before the visit when H. influenzae was isolated). No significant difference was detected.

Based on β-lactamase production indicated as β-lactamase positive and β-lactamase negative and AR and AS, isolates were further categorized in 4 groups. 51.0% and 26.5% were β-lactamase negative and AS and β-lactamase positive and AR. 4.1% and 5.1% of isolates were defined as BLNAR and Low-BLNAR, respectively. One isolate (1%) was categorized as BLPACR based on β-lactamase production and amoxicillin-clavulanic acid-resistance and showed multidrug nonsusceptibility against trimethoprim/sulfamethoxazole, cefuroxime, cefpodoxime and ceftriaxone.


Understanding the current etiology as well as antibiotic susceptibility is very important to prescribe the most appropriate course of treatment in any clinical setting. In this study, H. influenzae isolates were collected from children at health and AOM visits from the NP as well as MEF at onset of AOM. Capsular types and antibiotic susceptibility were determined. Several epidemiologic studies have described H. influenzae being the major cause of AOM.24,25 Since 2015, we have observed that H. influenzae has become the predominant otopathogen detected in MEF.5 The results from this 1-year study support H. influenzae as the major cause of AOM (43% of all culture-positive isolates plus 21% of culture negative, polymerase chain reaction–positive MEF). Among all the H. influenzae isolates from health or AOM visits, >92% were characterized as NTHi which is consistent with previous reports (85.7–100%).26–28

In our population of 101 H. influenzae isolates, 8 were encapsulated: 7 H. influenzae type f and 1 H. influenzae type e. Since the introduction of the Hib vaccine in 1985, the incidence of invasive Hib disease dramatically decreased. Invasive disease has become mainly caused by NTHi (2018, 43/87 49.4%) among less than 5 years old children.29H. influenzae capsule type f has been reported to be the most prevalent type found in invasive diseases among children below 5 years old, followed by type e.3 Emergence of Hia has also been reported, especially in American Indian and Alaska Natives.3,30 Although some studies detected Hia from MEF at onset of AOM31,32 as well as during healthy colonization in children,33 we did not detect any Hia cases. This result might reflect that limited circulation of Hia in our cohort. We found that encapsulated H. influenzae isolates were more frequently β-lactamase negative and susceptible to ampicillin, a result similar to reports regarding invasive diseases occurring in France in 201734 and Italy in 2012–2016.35

The antibiotic recommended by American Academy of Pediatrics4 as the first-line treatment of AOM is amoxicillin. Amoxicillin is also the first-line antibiotic for children with AOM in most European countries.36 In our cohort study, 39.7% of all H. influenzae isolates and 43% of AOM-causing isolates were β-lactamase producing. We found recent antibiotic prescription was a risk factor of detection of β-lactamase–positive strains but not to specific antibiotics.

A report involving H. influenzae isolates from the NP at the time of AOM children collected from 2012 to 2014 described 30% β-lactamase–producing strains.37 In 2019, Wald and DeMuri38 reported that 31 (44%) of 71 NP isolates obtained from 228 children with presumptive sinusitis were β-lactamase positive.

We found an increase in β-lactamase–producing strains associated with recent antibiotic exposure, similar to the work by Eliasson et al.39 Two prior studies of H. influenzae did not find an increase in β-lactamase–producing strains associated with recent antibiotic exposure (2–3 months before the isolation of H. influenzae).40,41 This result differs from those observed with S. pneumoniae, where a more consistent association has been observed between recent antibiotic exposure and increased isolation of antibiotic nonsusceptible strains.42

In the case of penicillin allergies, cefdinir, cefuroxime, cefpodoxime and ceftriaxone are recommended.4 The overall rate of nonsusceptibility to these 4 antibiotics was low. However, there were notable differences in nonsusceptibility rates to cephalosporins among NP isolates when comparing healthy and AOM visits. Among NP isolates collected at health, rates of nonsusceptibility to cefaclor, cefuroxime, cefprozil, cefdinir and amoxicillin-clavulanate were consistently lower than among NP isolates collected at onset of AOM. Antibiotic nonsusceptibility rates for cephalosporins, amoxicillin and amoxicillin-clavulanate among NP isolates collected at onset of AOM matched better with MEF isolates than NP isolates collected at times of health. The further analysis on paired samples (NP and MEF at the time of AOM) confirmed concordance in antibiotic susceptibility. The results suggest that antibiotic susceptibility testing of NP isolates collected at onset of AOM better correlates with results of MEF cultures when MEF isolates are not available.

We detected 9.2% (BLNAR + low-BLNAR) in our cohort. In an early multicenter studies in the United States (1994–1995, 2000–2001), the prevalence rate of BLNAR H. influenzae strains was low (2.5% BLNAR + low-BLNAR and 0.9%/0.1% BLNAR/low-BLNAR, respectively).43,44 6.8% of invasive H. influenzae cases from 2013 to 2016 reported from European countries were caused by BLNAR strains45 and 6.9% in France from 2017.34 Perhaps, BLNAR strains have started circulating more commonly in the United States. Furthermore, we also detected 1 case of BLPACR that was from an AOM treatment failure case. This isolate showed multidrug resistance as defined by nonsusceptibility to 3 or more antibiotic categories.46,47 Continuous epidemiologic monitoring and antibiotic susceptibility of circulating strain of H. influenzae is required.

We have limitations to note. This study involved collection of H. influenzae isolates during 1 year and derived from a predominantly middle-class, suburban population of children in upstate NY. Therefore, our results may not be representative of other types of populations in the United States or those in other countries. During the second half of this study period, a NY State “stay at home” executive order because of ongoing coronavirus disease 2019 circulation was implemented that may have influenced the results. Another limitation is we adapted a phenotypic definition for BLNAR and BLPACR by following CLSI guidelines. Guidelines and interpretation criteria for ampicillin susceptibility set by different committees differ (CLSI: 10 µg disc content, >2 µg/mL minimum inhibitory concentration breakpoint, whereas European Committee on Antimicrobial Susceptibility Testing: 2 µg disc content, >1 µg/mL minimum inhibitory concentration breakpoint).15,48 Some groups have reported higher sensitivity to detect BLNAR (AR) with a reduced dose of disc22,49 that would favor European Committee on Antimicrobial Susceptibility Testing susceptibility methods.

In conclusion, this study provides novel data on the prevalence of H. influenzae at the time of health and AOM in children, the proportion of capsulated strains and antibiotic susceptibility among strains collected during colonization and onset of AOM in children during 2019–2020. While the results provide overall and up-to-date trends of antibiotic susceptibility of H. influenzae in children, the situation is dynamic, supporting the need for continuous monitoring of H. influenzae as a pathogen.


The authors thank physicians, the nurses and staff at Finger Lakes Medical Associates Pediatrics and Bay Creek pediatrics group in Rochester Regional Health, the parents who consented and the children who participated in this study. The authors thank Eduardo Gonzalez and Karin Pryharski who performed bacterial identifications on clinical samples. The authors thank Robert Zagursky, Ph.D., and Karl Yu, M.D., Ph.D., for their critical reading.


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Haemophilus influenzae; acute otitis media; colonization; antibiotic susceptibility

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