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Nontypeable Haemophilus influenzae isolated from intractable acute otitis media internalized into cultured human epithelial cells
Auris Nasus Larynx 37 (2010) 137–144 www.elsevier.com/locate/anl
Nontypeable Haemophilus influenzae isolated from intractable acute otitis media internalized into cultured human epithelial cells§ Muneki Hotomi a, Jun Arai a, Dewan S. Billal a, Shin Takei a, Yorihiko Ikeda a, Masashi Ogami a, Masamitsu Kono a, Levent B. Beder a, Kazuo Toya b, Michio Kimura b, Noboru Yamanaka a,* a
Department of Otolaryngology-Head and Neck Surgery, Wakayama Medical University, 811-1 Kimiidera, Wakayama-shi, Wakayama, Japan b Department of Anatomy, Kansai University of Health Science, Osaka, Japan Received 2 January 2009; received in revised form 26 February 2009; accepted 22 March 2009 Available online 7 June 2009
Abstract Objectives: The aim of this study is to examine the internalization of nontypeable Haemophilus influenzae (NTHi) into human epithelial cells. Methods: Bactericidal assay was applied to examine the effects of antibiotics against cell-adherent NTHi using HEp-2 cells. A trans-well chamber assay was applied to examine the internalization and penetration of NTHi using Detroit562 cells. Results: The adherence of NTHi to HEp-2 cells was noted after 2 h of incubation. Azithromycin had a strong bactericidal effect against both cell-associated and non-adherent NTHi, while ceftriaxone did not show bactericidal effects on NTHi adhered to the HEp-2 cells. Three (60.0%) out of five NTHi isolates from the nasopharynx of children with intractable acute otitis media (AOM) internalized into and subsequently penetrated through the epithelial cells at various degrees. Azithromycin had a strong bactericidal effect against the cell-internalized NTHi, while ceftriaxone was bactericidal only against extracellular NTHi. Conclusion: The potential of NTHi as the intracellular pathogen may contribute to the persistent existence of this pathogen that result in the prolonged and intractable clinical course of AOM. Azithromycin may be a therapeutically significant antibiotic for patients with prolonged respiratory tract infections due to NTHi. # 2009 Published by Elsevier Ireland Ltd. Keywords: Nontypeable Haemophilus influenzae; Internalization; Human epithelial cell
1. Introduction Acute otitis media (AOM) is one of the leading infectious diseases during childhood [1]. Nontypeable Haemophilus influenzae (NTHi) is an important causative pathogen responsible for AOM [2]. The clinical outcomes of AOM by antimicrobial treatments have long been favorable. However, recent increases in intractable cases of AOM against antimicrobial treatment become serious problem worldwide
§ This work was presented at 17th European Congress of Clinical Microbiology and Infectious Disease, Munich, Germany, 2007 (abstract no. P746) and 9th International Symposium on Recent advances in Otitis Media, St Petersburg, USA, 2007. * Corresponding author. Tel.: +81 73 441 0651; fax: +81 73 446 3846. E-mail address: [email protected] (N. Yamanaka).
0385-8146/$ – see front matter # 2009 Published by Elsevier Ireland Ltd. doi:10.1016/j.anl.2009.03.012
[3]. Several factors are involved in the pathogenesis of intractable clinical course of AOM [4]. An alarming increase of b-lactamase non-producing ampicillin resistant (BLNAR) strains, especially in Japan, is a leading cause for the increase of intractable AOM [5]. Production of biofilm by NTHi also results in the persistent clinical course of AOM [6,7]. In addition to these two factors, internalization of NTHi into epithelial cells has been suggested to have important roles for persistent infections of NTHi [8,9]. Following the adhesion onto epithelial cells, some NTHi can internalize into epithelial cells in vitro. The relatively high frequency of persistent infections occurring in spite of intensive antimicrobial therapy will be consistent with the notion that NTHi could exist intracellularly and thereby evade immunological defenses of host and bactericidal effects of antimicrobial agents.
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In the current study, we attempted to gain insight into the mechanisms by which H. influenzae attaches to and persists within the nasopharynx. We examined the interactions between clinical isolates of H. influenzae from AOM patients and cultured human epithelial cells. In the process, we evaluated the internalization and penetration of NTHi into human epithelial cells. Furthermore, we studied the bacteriological efficacy of azithromycin against NTHi under a variety of conditions in a trans-well chamber assay.
2. Materials and methods 2.1. Bacterial strains Five NTHi strains isolated from the nasopharynx of the five children with intractable AOM were used in this study. Intractable AOM was defined as the cases having persistent middle ear effusion and frequent episodes of AOM caused by NTHi in spite of the antimicrobial treatment. Escherichia coli K-12 (ATCC 10798) and Pseudomonas aeruginosa PAO1 (ATCC BAA-47) were used as positive and negative controls for internalization and penetration, respectively. All strains were susceptible to gentamycin (MIC 0.5 mg/ml). 2.2. Minimal bactericidal concentrations of antibotics Approximately 2 105 CFUs of NTHi were pre-cultured in 1 ml of modified Eagle medium (MEM) with Earle’s salt (Gibco BRL, Grand Island, NY) supplemented with 10% fetal calf serum (FCS) (Hyclone, Logan, TU) and 1% Supplement B in 24-well culture plates for 2 h at 37 8C under a 5% CO2 environment. Then antibiotics were added to each well and incubated for 24 h. After the incubation, samples were plated on chocolate agar plates and the numbers of CFUs were evaluated. The minimal concentration of antibiotics that inhibited the growth of NTHi was designated as minimal bactericidal concentrations (MBC). 2.3. Bactericidal effects of antibiotics against cell-associated NTHi Approximately 1 105 cells/0.5 ml of HEp-2 cells, the human laryngeal epithelial cell delivered from human laryngeal cancer, at semi-confluent growth in MEM with Earle’s salt (Gibco BRL) supplemented with 10% FCS (Hyclone), 2 mM L-glutamine, 0.1 mM nonessential amino acids, and 1.0 mM sodium pyruvate (Gibco BRL) were incubated with NTHi strain I-197 for 2 h at 37 8C under a 5% CO2 environment. After incubation, the epithelial cell monolayer was rinsed with sterilized PBS. The PBS rinse for non-adherent NTHi and the HEp-2 cells for cellassociated NTHi were saved separately for the bactericidal assay. Both specimens were incubated with a 1 minimal
bactericidal concentration (MBC) of azithromycin (4 mg/ ml) (Pfizer Japan Inc., Tokyo, Japan), ceftriaxone (200 mg/ ml) (Chugai Pharmaceutical Co., Ltd., Tokyo, Japan), or gentamicin (4 mg/ml) (Sigma Chemical Co.) for 4, 8, and 12 h. The HEp-2 cells were further treated with 1 ml of 1% saponin for 15 min. Both specimens were plated on HI agar plates with 2% digested horse blood and incubated overnight at 37 8C. The viability of cell-internalized NTHi was also evaluated. Briefly, 1 105 cells/0.5 ml of HEp-2 cells at semi-confluent growth were incubated with NTHi strain I197 for 2 h at 37 8C under a 5% CO2 environment. The infected epithelial monolayers were rinsed with sterilized PBS to remove non-adherent bacteria and treated with 200 mg/ml gentamicin for 2 h which was sufficient to kill all of the bacterial inocula. After washing, the epithelial cell monolayer was incubated with 1 MIC of azithromycin (2 mg/ml) or 2 MIC of ceftriaxone (8 mg/ml) for 12 h. The HEp-2 cells were monitored after smashing on agar films under a video scope. 2.4. Trans-well chamber assay Approximately 3.3 104 cells/well of Detroit562 cells (ATCC CCL-138), the human pharyngeal epithelial cell delivered from human pharyngeal carcinoma, were cultured in 24 trans-well chambers (Corning International K.K., Tokyo, Japan) in MEM with Earle’s salt (Gibco BRL) supplemented with 10% FCS (Hyclone), 2 mM Lglutamine, 0.1 mM nonessential amino acids, and 1.0 mM sodium pyruvate (Gibco BRL) at 37 8C under a 5% CO2 environment until monolayer growth. To inhibit nonspecific penetration of bacteria through cell-to-cell junctions, the growth of epithelial cells at the confluent phase was confirmed by trans-epithelium electrical resistance (TER) at 20 V/m2 measured by MillicellERS (Millipore, Billerica, MA). The NTHi strains grown at late log phase were inoculated onto the epithelial cell monolayers at 1 107 CFUs per 10 ml. After incubation at 37 8C under a 5% CO2 environment for 3 h, the infected epithelial monolayers were rinsed with sterilized PBS to remove non-adherent bacteria and treated with 200 mg/ml gentamicin for 2 h which was sufficient to kill all of the bacterial inocula. After washing with sterilized PBS, the cells were lysed with saponin and the diluents were plated on HI agar plates to test for internalized NTHi. The diluents of culture media in the chamber plates were also plated onto HI agar plates to test for penetration of NTHi. Accumulation of azithromycin in cytoplasm and bactericidal efficacies of azithromycin against NTHi internalized into the epithelial cells were further evaluated. The Detroit562 cell monolayer prepared for trans-well chamber assay as mentioned above was incubated in culture media containing various concentrations of azithromycin (1 mg/ml, 5 mg/ml, 10 mg/ml, 50 mg/ml, 100 mg/ml) for 2 h. The
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monolayers in that cytoplasm azithromycin were accumulated and were then rinsed with fresh culture media 5 times with gentle rocking to remove residual azithromycin. The NTHi strains were inoculated onto the epithelial cell monolayer treated with azithromycin at 5 107 CFUs per 10 ml. After incubation at 37 8C under a 5% CO2 environment for 3 h, the infected epithelial monolayers were rinsed with sterilized PBS to remove non-adherent bacteria. After washing with sterilized PBS, the cells were lysed with saponin and the diluents were plated on HI agar plates to test for internalized NTHi. The diluents of culture media in the chamber plates were also plated onto HI agar plates to test for penetration of NTHi. 2.5. Microscopy (i) Confocal laser scanning microscopy: The NTHi infected monolayers of HEp-2 cells were stained with 1 mg/ml acridine orange and 5 mg/ml ethidium bromide for 5 min at room temperature after rinsing with sterilized PBS and treating with GM. The specimens were viewed with a Nikon TE-300 microscope attached to a BIO-RAD LSM Radiance 2100 confocal imaging system. (ii) Transmission electron microscopy: The infected epithelial monolayer was fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4 8C for 2 h. After being washed with the buffer, the samples were postfixed with 2% OsO4 in 0.1 M cadodylate buffer (pH 7.2) for 2 h. The samples were then dehydrated through a graded ethanol series and embedded in epoxy resin. The samples were sectioned by using an ultramicrotome, mounted on grids, and contrast stained with uranyl acetate and lead citrate for viewing with a JEM1230 transmission electron microscope (JEOL, Tokyo, Japan). (iii) Scanning electron microscopy (SEM): The infected epithelial monolayer were fixed with 4% paraformaldehyde (Sigma) in 0.1 M sodium phosphate buffer (pH 7.4) at 4 8C for 2 h and stained with 1% OsO4 and 1% tannic acid (Wako Pure Chemical Industries, Ltd., Osaka, Japan). They were dehydrated with a series of graded ethanol solutions in a critical-point apparatus and lyophilized with t-butyl alcohol. After an ionspatter coating with gold–palladium, the samples were viewed with a JSM-5410LV JEOL Co., scanning electron microscope. 2.6. Statistical analysis Statistical comparisons were carried out on logtransformed data by the ANOVA test with the Dunnet method and Student’s t test. A p-value of <0.05 was considered statistically significant. Calculations were performed using the statistical software package Prism4 (GraphPad Software, Inc.).
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3. Results 3.1. Bacteriological effects of antimicrobial agents on NTHi interacted with cultured human epithelial cells The adherence of strain I-197 to HEp-2 cells was first noted after 2 h of incubation, increased after 2–4 h incubation, and maintained for 12 h after incubation (Fig. 1A). Treatments with azithromycin, ceftriaxone, and gentamicin reduced the numbers of both adherent and nonadherent NTHi to various degrees depending on the time point. Gentamicin, which is recognized as a bactericidal antibiotic but which is unable to enter eukaryotic cells, showed a strong bactericidal effect against non-adherent NTHi (Fig. 1B). In contrast to the non-adherent bacteria, a very small number of NTHi associated with HEp-2 cell were identified even after treatment with a sufficient concentration of gentamicin for 4–8 h ( p < 0.05). When treating the cell-associated NTHi with gentamicin for 12 h, both cellassociated and non-adherent NTHi were killed. Ceftriaxone also reduced the numbers of non-adherent NTHi between 4 and 12 h of incubation. However, a number of NTHi survived even after the treatment for 12 h (Fig. 1C). Comparing against planktonic cells, ceftriaxone showed reduced bactericidal activities against NTHi adhered to the HEp-2 cells during a course of study. Azithromycin showed a marked bactericidal effect on both the adherent and nonadherent NTHi after treatment for 4 h (Fig. 1D). When the infected Hep-2 cells were monitored over time, the viable cells grew again even after the treatment with ceftriaxone (Fig. 2). In contrast, the NTHi in the cytoplasm of the HEp-2 cells showed spherocytic changes and did not grow anymore after the treatment with azithromycin (Fig. 2). The interaction between NTHi strain I-197 and HEp-2 cell was further demonstrated by microscopes. The scanning electron microscope depicted the general sequences of events during the course of adherence. It was apparent that the initial contact between the bacteria and the cell surface occurred via attachment with microvilli (Fig. 3A and B). Microvilli of HEp-2 cells wrapped around the individual NTHi cells. Some bacteria sporadically internalized into the HEp-2 cells by the process of micropinocytosis. The transmission electron microscope also demonstrated that NTHi contacted with the epithelium cells by micropinocytosis (Fig. 3C). The confocal laser scanning microscope showed the association between NTHi and the HEp-2 cells (Fig. 3D). Adherence of the bacteria to the Hep-2 cells is confirmed by the color shift from green to yellow as the adhering bacteria became internalizing ones. 3.2. Internalization of NTHi into the cultured human epithelial cells NTHi strains adhered to Detroit562 cell monolayer and subsequently internalized into and penetrated through the epithelial cells to various degrees. Three strains (60.0%), I-
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Fig. 1. Bacteriological effects of antimicrobial agents on NTHi interacted with HEp-2 cells and planktonic organisms. Values represent the mean S.D. log 10 CFUs/ml of triplicate measurements. Bacteriological effects of antimicrobial agents on NTHi interacted with HEp-2 cells are shown by solid bars. Bacteriological effects on non-adherent, planktonic NTHi are shown in open bar. (A) Controls without antimicrobial treatments, (B) gentamicin (4 mg/ ml), (C) ceftriaxone (200 mg/ml), (D) azithromycin (4 mg/ml). *p < 0.05 by Student’s t test.
168, I-193, and I-270, out of five NTHi isolated from nasopharynx of the five children with intractable AOM internalized into Detroit562 cells and were identified from the epithelial cell lysates (Fig. 4A). The three strains also penetrated through the epithelial monolayer and came out into the chamber medium (Fig. 4B). The I-270 strain most frequently internalized into and penetrated through the Detroit562 cells. Strain I-168 quickly penetrated through the epithelial cells. There were very few of the bacteria in the cells, but a considerable number of NTHi were found in the chamber medium. In contrast to the two strains, strain I-193 could internalize into and subsequently penetrate through the epithelial cells a little. The other two strains, I-189 and I221, did not internalize into and penetrate through the epithelial cells after 3 h incubation. There were no difference in the clinical course between the internalized strains and non-internalized strains. P. aeruginosa PAO1 strain as a positive control highly internalized into and
penetrated through Detroit562 cells. E. coli K-12 strain as negative control did not show any internalization and transcytosis. We further evaluated the bactericidal efficacies of azithromycin against NTHi I-270 internalized into Detroit562 cells. The concentration of azithromycin in the Detroit562 cells depended on that of azithromycin used to treat the culture epithelial cells. The concentration of azithromycin in the cytoplasm increased with time when the cells were incubated with 10 mg/ml of azithromycin, while those when the cell were incubated with 1 mg/ml did not increased with time. Amoxicillin did not accumulate into cytoplasm (Fig. 5). The concentrations of AZM in cytoplasm of Detroit562 cells treated with 1 mg/ml of azithromycin were below 1 mg/ml. There were no significant differences in the internalization and penetration between Detroit562 cells treated with 1 mg/ml of azithromycin and the controls (Fig. 6). On the other hand, when the
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Fig. 2. Bacteriological effects of antimicrobial agents on NTHi internalized into HEp-2 cells. NTHi internalized in HEp-2 cells grew after treatment with ceftriaxone over time (closed arrow). NTHi internalized into the HEp-2 cell showed spherocytic changes and did not grow anymore after the treatment with azithromycin (open arrow).
Fig. 3. Histological findings of NTHi internalized into HEp-2 cells. (A and B) SEM, (C) TEM, (D) CLSM. Arrows show NTHi internalized into HEp-2 cells.
Detroit562 cells were treated with 5 mg/ml of azithromycin, the internalization and penetration of strain I-270 were significantly reduced compared with the controls. When the Detroit562 cells were treated with 100 mg/ml of azithromycin, the internalization and penetration of NTHi were completely inhibited.
4. Discussion Nontypeable H. influenzae is frequently colonizes in the human nasopharynx and is traditionally regarded as an
extracellular bacterium [10–12]. The primary aim for a satisfactory outcome of antimicrobial chemotherapy is to achieve a sufficient drug concentration at the site of infection for an adequate length of time to ensure bacterial eradication and optimize clinical success [13–15]. In this study, we have first examined the interactions between a clinical isolates of NTHi and cultured human epithelial cells and evaluated the bactericidal efficacies of azithromycin, ceftriaxone, and gentamicin against cell-associated NTHi. Although ceftriaxone, which is frequently prescribed for treatment of respiratory tract infections, has been shown to exhibit bactericidal efficacy against NTHi in vitro, the antimicrobial
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Fig. 4. Internalization and penetrations of NTHi via Detroit562 cells. Values represent the mean S.D. log 10 CFUs/ml of triplicate measurements by a trans-well chamber assy. (A) Internalization of NTHi into Detroit562 cell, (B) penetrations of NTHi through Detroit562 cell.
Fig. 5. Accumulation of antibiotics in Detroti562 cells. Values represent the concentrations of antibiotics in cytoplasm after incubation with antibiotics. Concentrations of azithromycin in cytoplasm after incubation at 1 mg/ml (closed triangle) and at 10 mg/ml (closed circle). Concentration of ampicillin in cytoplasm after incubation at 10 mg/ml (open square).
Fig. 6. Bacteriological effects of azithromycin on NTHi interacted with Detroti562 cells. Values represent the mean S.D. log 10 CFUs/ml of triplicate measurements. The MIC of NTHi strain I-270 to azithromycin was 1.0 mg/ml. Bacteriological effects of azithromycin against NTHi are shown by solid bars. Controls without treatments are shown in open bar. (A) Bacteriological effects of azithromycin against NTHi internalized into Detroti562 cells. (B) Bacteriological effects of azithromycin against NTHi penetrated through Detroit562 cells. *p < 0.05 by ANOVA test with the Dunnet method.
reagent could not kill the NTHi associated with HEp-2 cells. After treatments with ceftriaxone, the NTHi strain persisted in the cytoplasm of HEp-2 cells and actually exhibited regrowth. Even though gentamicin is frequently used for studies of bacteria internalized into eukaryotic cells, small numbers of NTHi were still alive even after treatment with gentamicin [16]. Recent observations suggest that NTHi can enter a variety of eukaryotic cells [8,9,17–19]. Persistent colonization of NTHi is identified in the lower respiratory tract of 33% of clinically stable patients who were previously diagnosed with respiratory infections and 87% of patients with acute infections, whereas healthy volunteers have no NTHi in
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their lower respiratory tracts [20]. Molecular studies of culture-negative sputum have revealed persistent colonization by NTHi in patients with chronic obstructive pulmonary disease [21]. Histopathological studies demonstrated the presence of H. influenzae in the epithelium and submucosa of bronchi and respiratory tissues [22]. Intracellular localization of NTHi in the epithelial cells seems to play a role in the persistent existence of this pathogen which may result in the pathogenesis of intractable AOM. In this report, we applied a trans-well chamber assay to evaluate NTHi internalized into the epithelial cells separating from planktonic NTHi. NTHi clinical isolates showed various degree of internalization into Detroit562 cells and penetration through the cell monolayer. Holmes and Bakaletz showed that NTHi cells adhered to epithelial cells induced cytoskeletal changes [23]. Other studies also suggested that Hap protein or gag-dependent binding of the high molecular weight proteins on bacterial surfaces was associated with the internalization of NTHi into epithelial cells [13,24]. The present study showed a wide spectrum of internalization and penetration of NTHi clinical isolates. A number of NTHi were capable of entering into epithelial cells by the process of micropinocytosis. The possibility that NTHi enter mammalian cells and thereby escape the local immune system suggests that the host cells act as a reservoir for NTHi. Although we did not evaluate the internalization and subsequent penetration of NTHi via cultured human epithelial cells of NTHi isolated from simple AOM, the intracellular existence of NTHi might be a feasible explanation for the recurrent or prolonged clinical course of infections. For the treatment of infections caused by facultative intracellular pathogens, both the accumulation of antibiotics and their magnitude become important. b-Lactams do not accumulate in the cytoplasm and cannot kill the NTHi internalized into the epithelial cells although most of the antimicrobial reagent has been shown to exhibit bactericidal efficacy against NTHi in vitro [14]. We further evaluated the bactericidal efficacies of azithromycin against intracellular NTHi. Azithromycin can kill both cellassociated and planktonic NTHi. Although the b-lactams do not accumulate in the cytoplasm, azithromycin can accumulate intracellularly [25]. The unique pharmacokinetics of AZM will bring the advanced efficacies of AZM against intracellular NTHi. The administration of azithromycin, which is effective over an excellent magnitude of intracellular concentrations, results in enhanced phagocytic killing [26]. Azithromycin may indeed be of high therapeutic significance in the treatment of prolonged respiratory tract infections due to NTHi internalized in to the epithelial cells. In conclusion, the present study showed a wide spectrum in the level of NTHi internalization and penetration. The possibility that NTHi could enter mammalian cells and thereby escape the local immune system suggests that the host cells act as a reservoir for NTHi.
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Azithromycin showed a marked bacteriological efficacy against cell-internalized NTHi.
Conflict of interest All of the authors have no conflict of interest.
Acknowledgements We greatly thank Miss Yuki Tatsumi (Department of Otolaryngology-Head and Neck Surgery, Wakayama Medical University, Wakayama, Japan), Sekie Saito and Tsutomu Tomita (Cine Science Ltd., Tokyo, Japan) for their technical assistance. This work was supported by national grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology (Grant Nos. 19791224 and 19791226).
References [1] Berman S. Otitis media in children. N Eng J Med 1995;332:1560–5. [2] Bluestone CD, Stephenson JS, Martin LM. Ten-year review of otitis media pathogens. Pediatr Infect Dis J 1992;11(Suppl.8):7–11. [3] Hotomi M, Yamanaka N, Samukawa T, et al. Treatment and outcome of severe and non-severe acute otitis media. Eur J Pediatr 2005;164: 3–8. [4] Hotomi M, Yamanaka N, Shimada J, Ikeda Y, Faden H. Factors associated with clinical outcomes in acute otitis media. Ann Otol Rhinol Laryngol 2004;113:846–52. [5] Hotomi M, Fujihara K, Billal DS, et al. Genetic characteristics and clonal dissemination of b-lactamase non-producing ampicillin resistant (BLNAR) Haemophilus influenzae isolated from the upper respiratory tract in Japan. Antimicrob Agents Chemother 2007;51: 3969–76. [6] West-Barnette S, Rockel A, Swords WE. Biofilm growth increases phosphorylcholine content and decreases potency of nontypeable Haemophilus influenzae endotoxins. Infect Immun 2006;74:1828–36. [7] Jurcisek JA, Bookwalter JE, Baker BD, et al. The PilA protein of nontypeable Haemophilus influenzae plays a role in biofilm formation, adherence to epithelial cells and colonization of the mammalian upper respiratory tract. Mol Microbiol 2007;65:1288–99. [8] St Geme III JW, Falkow S. Haemophilus influenzae adhered to and enters cultured human epithelial cells. Infect Immun 1990;58: 4036–44. [9] van Schilfgaarde M, Eijk P, Regelink A, et al. Haemophilus influenzae localized in epithelial cell layers is shilded from antibiotics and antibody-mediated bactericidal activity. Microb Pathog 1999;26: 249–62. [10] Murphy TF, Apicella MA. Nontypeable Haemophilus influenzae: a review of clinical aspects, surface antigens and the human response to infection. Rev Infect Dis 1987;9:1–15. [11] Murphy TF. Respiratory infections caused by nontypeable Haemophilus influenzae. Curr Opin Infect Dis 2003;16:129–34. [12] Foxwell AR, Kyd JM, Cripps AW. Nontypeable Haemophilus influenzae: pathogenesis and prevention. Microbiol Mol Biol Rev 1998;62:294–308. [13] Ahren IL, Williams DL, Rice PJ, Forsgren A, Riesbeck K. The importance of a b-glucan receptor in the nonopsonic entry of nontypeable Haemophilus influenzae into human monocytic and epithelial cells. J Infect Dis 2006;184:150–8.
144
M. Hotomi et al. / Auris Nasus Larynx 37 (2010) 137–144
[14] Ahren IL, Karlsson E, Forsgren A, Riesbeck K. Comparison of the antibacterial activities of ampicillin, ciprofloxacin, clarithromycin, telithromycin and quinupristin/dalfopristin against intracellular nontypeable Haemophilus influenzae. J Antimicrob Chemother 2002;50: 903–6. [15] Kratzer C, Graninger W, Macfelda K, Buxbaum A, Georgopoulos A. Comparative activities of antibiotics against intracellular non-typeable Haemophilus influenzae. Wien Klin Wochenschr 2007;119:297–302. [16] Kihlstrom E, Andaker L. Inability of gentamicin and fosfomycin to liminate intracellular Enterobacteriaceae. J Antimicrob Chemother 1985;15:723–8. [17] Ketterer MR, Shao JQ, Hornick DB, Buscher B, Bandi VK, Apicella MA. Infection of primary human bronchial epithelial cells by Haemophilus influenzae: macropinocytosis as a mechanism of airway epithelial cell entry. Infect Immun 1999;67:4161–70. [18] Jonhson PDR, Oppedisano F, Bennett-Wood V, Gilbert GL, RobinsBrowne R. Sporadic invasion of cultured epithelial cells by Haemophilus influenzae type b. Infect Immun 1996;64:1051–3. [19] Ahren IL, Janson H, Forsgren A, Riesbeck K. Protein D expression promotes the adherence and internalization of non-typeable Haemophilus influenzae into human monocytic cells. Microb Pathog 2001;31: 151–8.
[20] Bandi V, Apicella MA, Mason E, et al. Nontypeable Haemophilus influenzae in the lower respiratory tract patients with chronic bronchitis. Am J Respir Cirt Care Med 2001;164:2114–9. [21] Murphy TF, Brauer AL, Schiffmacher AT, Sethi S. Persistent colonization by Haemophilus influenzae in chronic obstructive pulmonary disease. Am J Respir Cirt Care Med 2004;170:266–72. [22] Forsgren J, Samuelson A, Ahlin A, Jonasson J, Rynnel-Dagoo B, Lindberg A. Haemophilus influenzae resides and multiplies intracellularly in human adenoid tissue as demonstrated by in situ hybridization and bacterial viability assay. Infect Immun 1994;62:673–9. [23] Holmes KA, Bakaletz LO. Adherence of non-typeable Haemophilus influenzae promotes recognization of the actin cytoskelton in human or chinchilla epithelial cells in vitro. Microb Pathog 1997;23:157–66. [24] Fink DL, Green BA, St Geme III JW. The Haemophilus influenzae Hap autotransporter binds to fibronectin, laminin, and collagen IV. Infect Immun 2002;70:4902–7. [25] Gladue RP, Sinder ME. Intracellular accumulation of azithromycin by cultured human fibroblast. Antimicrob Agents Chemother 1990;34: 1056–60. [26] Wildfeuer A, Laufen H, Zimmermann T. Uptake of azithromycin by various cells and its intracellular activity under in vitro conditions. Antimicrob Agents Chemother 1996;40:75–9.
Nontypeable Haemophilus influenzae isolated from intractable acute otitis media internalized into cultured human epithelial cells§ Muneki Hotomi a, Jun Arai a, Dewan S. Billal a, Shin Takei a, Yorihiko Ikeda a, Masashi Ogami a, Masamitsu Kono a, Levent B. Beder a, Kazuo Toya b, Michio Kimura b, Noboru Yamanaka a,* a
Department of Otolaryngology-Head and Neck Surgery, Wakayama Medical University, 811-1 Kimiidera, Wakayama-shi, Wakayama, Japan b Department of Anatomy, Kansai University of Health Science, Osaka, Japan Received 2 January 2009; received in revised form 26 February 2009; accepted 22 March 2009 Available online 7 June 2009
Abstract Objectives: The aim of this study is to examine the internalization of nontypeable Haemophilus influenzae (NTHi) into human epithelial cells. Methods: Bactericidal assay was applied to examine the effects of antibiotics against cell-adherent NTHi using HEp-2 cells. A trans-well chamber assay was applied to examine the internalization and penetration of NTHi using Detroit562 cells. Results: The adherence of NTHi to HEp-2 cells was noted after 2 h of incubation. Azithromycin had a strong bactericidal effect against both cell-associated and non-adherent NTHi, while ceftriaxone did not show bactericidal effects on NTHi adhered to the HEp-2 cells. Three (60.0%) out of five NTHi isolates from the nasopharynx of children with intractable acute otitis media (AOM) internalized into and subsequently penetrated through the epithelial cells at various degrees. Azithromycin had a strong bactericidal effect against the cell-internalized NTHi, while ceftriaxone was bactericidal only against extracellular NTHi. Conclusion: The potential of NTHi as the intracellular pathogen may contribute to the persistent existence of this pathogen that result in the prolonged and intractable clinical course of AOM. Azithromycin may be a therapeutically significant antibiotic for patients with prolonged respiratory tract infections due to NTHi. # 2009 Published by Elsevier Ireland Ltd. Keywords: Nontypeable Haemophilus influenzae; Internalization; Human epithelial cell
1. Introduction Acute otitis media (AOM) is one of the leading infectious diseases during childhood [1]. Nontypeable Haemophilus influenzae (NTHi) is an important causative pathogen responsible for AOM [2]. The clinical outcomes of AOM by antimicrobial treatments have long been favorable. However, recent increases in intractable cases of AOM against antimicrobial treatment become serious problem worldwide
§ This work was presented at 17th European Congress of Clinical Microbiology and Infectious Disease, Munich, Germany, 2007 (abstract no. P746) and 9th International Symposium on Recent advances in Otitis Media, St Petersburg, USA, 2007. * Corresponding author. Tel.: +81 73 441 0651; fax: +81 73 446 3846. E-mail address: [email protected] (N. Yamanaka).
0385-8146/$ – see front matter # 2009 Published by Elsevier Ireland Ltd. doi:10.1016/j.anl.2009.03.012
[3]. Several factors are involved in the pathogenesis of intractable clinical course of AOM [4]. An alarming increase of b-lactamase non-producing ampicillin resistant (BLNAR) strains, especially in Japan, is a leading cause for the increase of intractable AOM [5]. Production of biofilm by NTHi also results in the persistent clinical course of AOM [6,7]. In addition to these two factors, internalization of NTHi into epithelial cells has been suggested to have important roles for persistent infections of NTHi [8,9]. Following the adhesion onto epithelial cells, some NTHi can internalize into epithelial cells in vitro. The relatively high frequency of persistent infections occurring in spite of intensive antimicrobial therapy will be consistent with the notion that NTHi could exist intracellularly and thereby evade immunological defenses of host and bactericidal effects of antimicrobial agents.
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In the current study, we attempted to gain insight into the mechanisms by which H. influenzae attaches to and persists within the nasopharynx. We examined the interactions between clinical isolates of H. influenzae from AOM patients and cultured human epithelial cells. In the process, we evaluated the internalization and penetration of NTHi into human epithelial cells. Furthermore, we studied the bacteriological efficacy of azithromycin against NTHi under a variety of conditions in a trans-well chamber assay.
2. Materials and methods 2.1. Bacterial strains Five NTHi strains isolated from the nasopharynx of the five children with intractable AOM were used in this study. Intractable AOM was defined as the cases having persistent middle ear effusion and frequent episodes of AOM caused by NTHi in spite of the antimicrobial treatment. Escherichia coli K-12 (ATCC 10798) and Pseudomonas aeruginosa PAO1 (ATCC BAA-47) were used as positive and negative controls for internalization and penetration, respectively. All strains were susceptible to gentamycin (MIC 0.5 mg/ml). 2.2. Minimal bactericidal concentrations of antibotics Approximately 2 105 CFUs of NTHi were pre-cultured in 1 ml of modified Eagle medium (MEM) with Earle’s salt (Gibco BRL, Grand Island, NY) supplemented with 10% fetal calf serum (FCS) (Hyclone, Logan, TU) and 1% Supplement B in 24-well culture plates for 2 h at 37 8C under a 5% CO2 environment. Then antibiotics were added to each well and incubated for 24 h. After the incubation, samples were plated on chocolate agar plates and the numbers of CFUs were evaluated. The minimal concentration of antibiotics that inhibited the growth of NTHi was designated as minimal bactericidal concentrations (MBC). 2.3. Bactericidal effects of antibiotics against cell-associated NTHi Approximately 1 105 cells/0.5 ml of HEp-2 cells, the human laryngeal epithelial cell delivered from human laryngeal cancer, at semi-confluent growth in MEM with Earle’s salt (Gibco BRL) supplemented with 10% FCS (Hyclone), 2 mM L-glutamine, 0.1 mM nonessential amino acids, and 1.0 mM sodium pyruvate (Gibco BRL) were incubated with NTHi strain I-197 for 2 h at 37 8C under a 5% CO2 environment. After incubation, the epithelial cell monolayer was rinsed with sterilized PBS. The PBS rinse for non-adherent NTHi and the HEp-2 cells for cellassociated NTHi were saved separately for the bactericidal assay. Both specimens were incubated with a 1 minimal
bactericidal concentration (MBC) of azithromycin (4 mg/ ml) (Pfizer Japan Inc., Tokyo, Japan), ceftriaxone (200 mg/ ml) (Chugai Pharmaceutical Co., Ltd., Tokyo, Japan), or gentamicin (4 mg/ml) (Sigma Chemical Co.) for 4, 8, and 12 h. The HEp-2 cells were further treated with 1 ml of 1% saponin for 15 min. Both specimens were plated on HI agar plates with 2% digested horse blood and incubated overnight at 37 8C. The viability of cell-internalized NTHi was also evaluated. Briefly, 1 105 cells/0.5 ml of HEp-2 cells at semi-confluent growth were incubated with NTHi strain I197 for 2 h at 37 8C under a 5% CO2 environment. The infected epithelial monolayers were rinsed with sterilized PBS to remove non-adherent bacteria and treated with 200 mg/ml gentamicin for 2 h which was sufficient to kill all of the bacterial inocula. After washing, the epithelial cell monolayer was incubated with 1 MIC of azithromycin (2 mg/ml) or 2 MIC of ceftriaxone (8 mg/ml) for 12 h. The HEp-2 cells were monitored after smashing on agar films under a video scope. 2.4. Trans-well chamber assay Approximately 3.3 104 cells/well of Detroit562 cells (ATCC CCL-138), the human pharyngeal epithelial cell delivered from human pharyngeal carcinoma, were cultured in 24 trans-well chambers (Corning International K.K., Tokyo, Japan) in MEM with Earle’s salt (Gibco BRL) supplemented with 10% FCS (Hyclone), 2 mM Lglutamine, 0.1 mM nonessential amino acids, and 1.0 mM sodium pyruvate (Gibco BRL) at 37 8C under a 5% CO2 environment until monolayer growth. To inhibit nonspecific penetration of bacteria through cell-to-cell junctions, the growth of epithelial cells at the confluent phase was confirmed by trans-epithelium electrical resistance (TER) at 20 V/m2 measured by MillicellERS (Millipore, Billerica, MA). The NTHi strains grown at late log phase were inoculated onto the epithelial cell monolayers at 1 107 CFUs per 10 ml. After incubation at 37 8C under a 5% CO2 environment for 3 h, the infected epithelial monolayers were rinsed with sterilized PBS to remove non-adherent bacteria and treated with 200 mg/ml gentamicin for 2 h which was sufficient to kill all of the bacterial inocula. After washing with sterilized PBS, the cells were lysed with saponin and the diluents were plated on HI agar plates to test for internalized NTHi. The diluents of culture media in the chamber plates were also plated onto HI agar plates to test for penetration of NTHi. Accumulation of azithromycin in cytoplasm and bactericidal efficacies of azithromycin against NTHi internalized into the epithelial cells were further evaluated. The Detroit562 cell monolayer prepared for trans-well chamber assay as mentioned above was incubated in culture media containing various concentrations of azithromycin (1 mg/ml, 5 mg/ml, 10 mg/ml, 50 mg/ml, 100 mg/ml) for 2 h. The
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monolayers in that cytoplasm azithromycin were accumulated and were then rinsed with fresh culture media 5 times with gentle rocking to remove residual azithromycin. The NTHi strains were inoculated onto the epithelial cell monolayer treated with azithromycin at 5 107 CFUs per 10 ml. After incubation at 37 8C under a 5% CO2 environment for 3 h, the infected epithelial monolayers were rinsed with sterilized PBS to remove non-adherent bacteria. After washing with sterilized PBS, the cells were lysed with saponin and the diluents were plated on HI agar plates to test for internalized NTHi. The diluents of culture media in the chamber plates were also plated onto HI agar plates to test for penetration of NTHi. 2.5. Microscopy (i) Confocal laser scanning microscopy: The NTHi infected monolayers of HEp-2 cells were stained with 1 mg/ml acridine orange and 5 mg/ml ethidium bromide for 5 min at room temperature after rinsing with sterilized PBS and treating with GM. The specimens were viewed with a Nikon TE-300 microscope attached to a BIO-RAD LSM Radiance 2100 confocal imaging system. (ii) Transmission electron microscopy: The infected epithelial monolayer was fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4 8C for 2 h. After being washed with the buffer, the samples were postfixed with 2% OsO4 in 0.1 M cadodylate buffer (pH 7.2) for 2 h. The samples were then dehydrated through a graded ethanol series and embedded in epoxy resin. The samples were sectioned by using an ultramicrotome, mounted on grids, and contrast stained with uranyl acetate and lead citrate for viewing with a JEM1230 transmission electron microscope (JEOL, Tokyo, Japan). (iii) Scanning electron microscopy (SEM): The infected epithelial monolayer were fixed with 4% paraformaldehyde (Sigma) in 0.1 M sodium phosphate buffer (pH 7.4) at 4 8C for 2 h and stained with 1% OsO4 and 1% tannic acid (Wako Pure Chemical Industries, Ltd., Osaka, Japan). They were dehydrated with a series of graded ethanol solutions in a critical-point apparatus and lyophilized with t-butyl alcohol. After an ionspatter coating with gold–palladium, the samples were viewed with a JSM-5410LV JEOL Co., scanning electron microscope. 2.6. Statistical analysis Statistical comparisons were carried out on logtransformed data by the ANOVA test with the Dunnet method and Student’s t test. A p-value of <0.05 was considered statistically significant. Calculations were performed using the statistical software package Prism4 (GraphPad Software, Inc.).
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3. Results 3.1. Bacteriological effects of antimicrobial agents on NTHi interacted with cultured human epithelial cells The adherence of strain I-197 to HEp-2 cells was first noted after 2 h of incubation, increased after 2–4 h incubation, and maintained for 12 h after incubation (Fig. 1A). Treatments with azithromycin, ceftriaxone, and gentamicin reduced the numbers of both adherent and nonadherent NTHi to various degrees depending on the time point. Gentamicin, which is recognized as a bactericidal antibiotic but which is unable to enter eukaryotic cells, showed a strong bactericidal effect against non-adherent NTHi (Fig. 1B). In contrast to the non-adherent bacteria, a very small number of NTHi associated with HEp-2 cell were identified even after treatment with a sufficient concentration of gentamicin for 4–8 h ( p < 0.05). When treating the cell-associated NTHi with gentamicin for 12 h, both cellassociated and non-adherent NTHi were killed. Ceftriaxone also reduced the numbers of non-adherent NTHi between 4 and 12 h of incubation. However, a number of NTHi survived even after the treatment for 12 h (Fig. 1C). Comparing against planktonic cells, ceftriaxone showed reduced bactericidal activities against NTHi adhered to the HEp-2 cells during a course of study. Azithromycin showed a marked bactericidal effect on both the adherent and nonadherent NTHi after treatment for 4 h (Fig. 1D). When the infected Hep-2 cells were monitored over time, the viable cells grew again even after the treatment with ceftriaxone (Fig. 2). In contrast, the NTHi in the cytoplasm of the HEp-2 cells showed spherocytic changes and did not grow anymore after the treatment with azithromycin (Fig. 2). The interaction between NTHi strain I-197 and HEp-2 cell was further demonstrated by microscopes. The scanning electron microscope depicted the general sequences of events during the course of adherence. It was apparent that the initial contact between the bacteria and the cell surface occurred via attachment with microvilli (Fig. 3A and B). Microvilli of HEp-2 cells wrapped around the individual NTHi cells. Some bacteria sporadically internalized into the HEp-2 cells by the process of micropinocytosis. The transmission electron microscope also demonstrated that NTHi contacted with the epithelium cells by micropinocytosis (Fig. 3C). The confocal laser scanning microscope showed the association between NTHi and the HEp-2 cells (Fig. 3D). Adherence of the bacteria to the Hep-2 cells is confirmed by the color shift from green to yellow as the adhering bacteria became internalizing ones. 3.2. Internalization of NTHi into the cultured human epithelial cells NTHi strains adhered to Detroit562 cell monolayer and subsequently internalized into and penetrated through the epithelial cells to various degrees. Three strains (60.0%), I-
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Fig. 1. Bacteriological effects of antimicrobial agents on NTHi interacted with HEp-2 cells and planktonic organisms. Values represent the mean S.D. log 10 CFUs/ml of triplicate measurements. Bacteriological effects of antimicrobial agents on NTHi interacted with HEp-2 cells are shown by solid bars. Bacteriological effects on non-adherent, planktonic NTHi are shown in open bar. (A) Controls without antimicrobial treatments, (B) gentamicin (4 mg/ ml), (C) ceftriaxone (200 mg/ml), (D) azithromycin (4 mg/ml). *p < 0.05 by Student’s t test.
168, I-193, and I-270, out of five NTHi isolated from nasopharynx of the five children with intractable AOM internalized into Detroit562 cells and were identified from the epithelial cell lysates (Fig. 4A). The three strains also penetrated through the epithelial monolayer and came out into the chamber medium (Fig. 4B). The I-270 strain most frequently internalized into and penetrated through the Detroit562 cells. Strain I-168 quickly penetrated through the epithelial cells. There were very few of the bacteria in the cells, but a considerable number of NTHi were found in the chamber medium. In contrast to the two strains, strain I-193 could internalize into and subsequently penetrate through the epithelial cells a little. The other two strains, I-189 and I221, did not internalize into and penetrate through the epithelial cells after 3 h incubation. There were no difference in the clinical course between the internalized strains and non-internalized strains. P. aeruginosa PAO1 strain as a positive control highly internalized into and
penetrated through Detroit562 cells. E. coli K-12 strain as negative control did not show any internalization and transcytosis. We further evaluated the bactericidal efficacies of azithromycin against NTHi I-270 internalized into Detroit562 cells. The concentration of azithromycin in the Detroit562 cells depended on that of azithromycin used to treat the culture epithelial cells. The concentration of azithromycin in the cytoplasm increased with time when the cells were incubated with 10 mg/ml of azithromycin, while those when the cell were incubated with 1 mg/ml did not increased with time. Amoxicillin did not accumulate into cytoplasm (Fig. 5). The concentrations of AZM in cytoplasm of Detroit562 cells treated with 1 mg/ml of azithromycin were below 1 mg/ml. There were no significant differences in the internalization and penetration between Detroit562 cells treated with 1 mg/ml of azithromycin and the controls (Fig. 6). On the other hand, when the
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Fig. 2. Bacteriological effects of antimicrobial agents on NTHi internalized into HEp-2 cells. NTHi internalized in HEp-2 cells grew after treatment with ceftriaxone over time (closed arrow). NTHi internalized into the HEp-2 cell showed spherocytic changes and did not grow anymore after the treatment with azithromycin (open arrow).
Fig. 3. Histological findings of NTHi internalized into HEp-2 cells. (A and B) SEM, (C) TEM, (D) CLSM. Arrows show NTHi internalized into HEp-2 cells.
Detroit562 cells were treated with 5 mg/ml of azithromycin, the internalization and penetration of strain I-270 were significantly reduced compared with the controls. When the Detroit562 cells were treated with 100 mg/ml of azithromycin, the internalization and penetration of NTHi were completely inhibited.
4. Discussion Nontypeable H. influenzae is frequently colonizes in the human nasopharynx and is traditionally regarded as an
extracellular bacterium [10–12]. The primary aim for a satisfactory outcome of antimicrobial chemotherapy is to achieve a sufficient drug concentration at the site of infection for an adequate length of time to ensure bacterial eradication and optimize clinical success [13–15]. In this study, we have first examined the interactions between a clinical isolates of NTHi and cultured human epithelial cells and evaluated the bactericidal efficacies of azithromycin, ceftriaxone, and gentamicin against cell-associated NTHi. Although ceftriaxone, which is frequently prescribed for treatment of respiratory tract infections, has been shown to exhibit bactericidal efficacy against NTHi in vitro, the antimicrobial
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Fig. 4. Internalization and penetrations of NTHi via Detroit562 cells. Values represent the mean S.D. log 10 CFUs/ml of triplicate measurements by a trans-well chamber assy. (A) Internalization of NTHi into Detroit562 cell, (B) penetrations of NTHi through Detroit562 cell.
Fig. 5. Accumulation of antibiotics in Detroti562 cells. Values represent the concentrations of antibiotics in cytoplasm after incubation with antibiotics. Concentrations of azithromycin in cytoplasm after incubation at 1 mg/ml (closed triangle) and at 10 mg/ml (closed circle). Concentration of ampicillin in cytoplasm after incubation at 10 mg/ml (open square).
Fig. 6. Bacteriological effects of azithromycin on NTHi interacted with Detroti562 cells. Values represent the mean S.D. log 10 CFUs/ml of triplicate measurements. The MIC of NTHi strain I-270 to azithromycin was 1.0 mg/ml. Bacteriological effects of azithromycin against NTHi are shown by solid bars. Controls without treatments are shown in open bar. (A) Bacteriological effects of azithromycin against NTHi internalized into Detroti562 cells. (B) Bacteriological effects of azithromycin against NTHi penetrated through Detroit562 cells. *p < 0.05 by ANOVA test with the Dunnet method.
reagent could not kill the NTHi associated with HEp-2 cells. After treatments with ceftriaxone, the NTHi strain persisted in the cytoplasm of HEp-2 cells and actually exhibited regrowth. Even though gentamicin is frequently used for studies of bacteria internalized into eukaryotic cells, small numbers of NTHi were still alive even after treatment with gentamicin [16]. Recent observations suggest that NTHi can enter a variety of eukaryotic cells [8,9,17–19]. Persistent colonization of NTHi is identified in the lower respiratory tract of 33% of clinically stable patients who were previously diagnosed with respiratory infections and 87% of patients with acute infections, whereas healthy volunteers have no NTHi in
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their lower respiratory tracts [20]. Molecular studies of culture-negative sputum have revealed persistent colonization by NTHi in patients with chronic obstructive pulmonary disease [21]. Histopathological studies demonstrated the presence of H. influenzae in the epithelium and submucosa of bronchi and respiratory tissues [22]. Intracellular localization of NTHi in the epithelial cells seems to play a role in the persistent existence of this pathogen which may result in the pathogenesis of intractable AOM. In this report, we applied a trans-well chamber assay to evaluate NTHi internalized into the epithelial cells separating from planktonic NTHi. NTHi clinical isolates showed various degree of internalization into Detroit562 cells and penetration through the cell monolayer. Holmes and Bakaletz showed that NTHi cells adhered to epithelial cells induced cytoskeletal changes [23]. Other studies also suggested that Hap protein or gag-dependent binding of the high molecular weight proteins on bacterial surfaces was associated with the internalization of NTHi into epithelial cells [13,24]. The present study showed a wide spectrum of internalization and penetration of NTHi clinical isolates. A number of NTHi were capable of entering into epithelial cells by the process of micropinocytosis. The possibility that NTHi enter mammalian cells and thereby escape the local immune system suggests that the host cells act as a reservoir for NTHi. Although we did not evaluate the internalization and subsequent penetration of NTHi via cultured human epithelial cells of NTHi isolated from simple AOM, the intracellular existence of NTHi might be a feasible explanation for the recurrent or prolonged clinical course of infections. For the treatment of infections caused by facultative intracellular pathogens, both the accumulation of antibiotics and their magnitude become important. b-Lactams do not accumulate in the cytoplasm and cannot kill the NTHi internalized into the epithelial cells although most of the antimicrobial reagent has been shown to exhibit bactericidal efficacy against NTHi in vitro [14]. We further evaluated the bactericidal efficacies of azithromycin against intracellular NTHi. Azithromycin can kill both cellassociated and planktonic NTHi. Although the b-lactams do not accumulate in the cytoplasm, azithromycin can accumulate intracellularly [25]. The unique pharmacokinetics of AZM will bring the advanced efficacies of AZM against intracellular NTHi. The administration of azithromycin, which is effective over an excellent magnitude of intracellular concentrations, results in enhanced phagocytic killing [26]. Azithromycin may indeed be of high therapeutic significance in the treatment of prolonged respiratory tract infections due to NTHi internalized in to the epithelial cells. In conclusion, the present study showed a wide spectrum in the level of NTHi internalization and penetration. The possibility that NTHi could enter mammalian cells and thereby escape the local immune system suggests that the host cells act as a reservoir for NTHi.
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Azithromycin showed a marked bacteriological efficacy against cell-internalized NTHi.
Conflict of interest All of the authors have no conflict of interest.
Acknowledgements We greatly thank Miss Yuki Tatsumi (Department of Otolaryngology-Head and Neck Surgery, Wakayama Medical University, Wakayama, Japan), Sekie Saito and Tsutomu Tomita (Cine Science Ltd., Tokyo, Japan) for their technical assistance. This work was supported by national grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology (Grant Nos. 19791224 and 19791226).
References [1] Berman S. Otitis media in children. N Eng J Med 1995;332:1560–5. [2] Bluestone CD, Stephenson JS, Martin LM. Ten-year review of otitis media pathogens. Pediatr Infect Dis J 1992;11(Suppl.8):7–11. [3] Hotomi M, Yamanaka N, Samukawa T, et al. Treatment and outcome of severe and non-severe acute otitis media. Eur J Pediatr 2005;164: 3–8. [4] Hotomi M, Yamanaka N, Shimada J, Ikeda Y, Faden H. Factors associated with clinical outcomes in acute otitis media. Ann Otol Rhinol Laryngol 2004;113:846–52. [5] Hotomi M, Fujihara K, Billal DS, et al. Genetic characteristics and clonal dissemination of b-lactamase non-producing ampicillin resistant (BLNAR) Haemophilus influenzae isolated from the upper respiratory tract in Japan. Antimicrob Agents Chemother 2007;51: 3969–76. [6] West-Barnette S, Rockel A, Swords WE. Biofilm growth increases phosphorylcholine content and decreases potency of nontypeable Haemophilus influenzae endotoxins. Infect Immun 2006;74:1828–36. [7] Jurcisek JA, Bookwalter JE, Baker BD, et al. The PilA protein of nontypeable Haemophilus influenzae plays a role in biofilm formation, adherence to epithelial cells and colonization of the mammalian upper respiratory tract. Mol Microbiol 2007;65:1288–99. [8] St Geme III JW, Falkow S. Haemophilus influenzae adhered to and enters cultured human epithelial cells. Infect Immun 1990;58: 4036–44. [9] van Schilfgaarde M, Eijk P, Regelink A, et al. Haemophilus influenzae localized in epithelial cell layers is shilded from antibiotics and antibody-mediated bactericidal activity. Microb Pathog 1999;26: 249–62. [10] Murphy TF, Apicella MA. Nontypeable Haemophilus influenzae: a review of clinical aspects, surface antigens and the human response to infection. Rev Infect Dis 1987;9:1–15. [11] Murphy TF. Respiratory infections caused by nontypeable Haemophilus influenzae. Curr Opin Infect Dis 2003;16:129–34. [12] Foxwell AR, Kyd JM, Cripps AW. Nontypeable Haemophilus influenzae: pathogenesis and prevention. Microbiol Mol Biol Rev 1998;62:294–308. [13] Ahren IL, Williams DL, Rice PJ, Forsgren A, Riesbeck K. The importance of a b-glucan receptor in the nonopsonic entry of nontypeable Haemophilus influenzae into human monocytic and epithelial cells. J Infect Dis 2006;184:150–8.
144
M. Hotomi et al. / Auris Nasus Larynx 37 (2010) 137–144
[14] Ahren IL, Karlsson E, Forsgren A, Riesbeck K. Comparison of the antibacterial activities of ampicillin, ciprofloxacin, clarithromycin, telithromycin and quinupristin/dalfopristin against intracellular nontypeable Haemophilus influenzae. J Antimicrob Chemother 2002;50: 903–6. [15] Kratzer C, Graninger W, Macfelda K, Buxbaum A, Georgopoulos A. Comparative activities of antibiotics against intracellular non-typeable Haemophilus influenzae. Wien Klin Wochenschr 2007;119:297–302. [16] Kihlstrom E, Andaker L. Inability of gentamicin and fosfomycin to liminate intracellular Enterobacteriaceae. J Antimicrob Chemother 1985;15:723–8. [17] Ketterer MR, Shao JQ, Hornick DB, Buscher B, Bandi VK, Apicella MA. Infection of primary human bronchial epithelial cells by Haemophilus influenzae: macropinocytosis as a mechanism of airway epithelial cell entry. Infect Immun 1999;67:4161–70. [18] Jonhson PDR, Oppedisano F, Bennett-Wood V, Gilbert GL, RobinsBrowne R. Sporadic invasion of cultured epithelial cells by Haemophilus influenzae type b. Infect Immun 1996;64:1051–3. [19] Ahren IL, Janson H, Forsgren A, Riesbeck K. Protein D expression promotes the adherence and internalization of non-typeable Haemophilus influenzae into human monocytic cells. Microb Pathog 2001;31: 151–8.
[20] Bandi V, Apicella MA, Mason E, et al. Nontypeable Haemophilus influenzae in the lower respiratory tract patients with chronic bronchitis. Am J Respir Cirt Care Med 2001;164:2114–9. [21] Murphy TF, Brauer AL, Schiffmacher AT, Sethi S. Persistent colonization by Haemophilus influenzae in chronic obstructive pulmonary disease. Am J Respir Cirt Care Med 2004;170:266–72. [22] Forsgren J, Samuelson A, Ahlin A, Jonasson J, Rynnel-Dagoo B, Lindberg A. Haemophilus influenzae resides and multiplies intracellularly in human adenoid tissue as demonstrated by in situ hybridization and bacterial viability assay. Infect Immun 1994;62:673–9. [23] Holmes KA, Bakaletz LO. Adherence of non-typeable Haemophilus influenzae promotes recognization of the actin cytoskelton in human or chinchilla epithelial cells in vitro. Microb Pathog 1997;23:157–66. [24] Fink DL, Green BA, St Geme III JW. The Haemophilus influenzae Hap autotransporter binds to fibronectin, laminin, and collagen IV. Infect Immun 2002;70:4902–7. [25] Gladue RP, Sinder ME. Intracellular accumulation of azithromycin by cultured human fibroblast. Antimicrob Agents Chemother 1990;34: 1056–60. [26] Wildfeuer A, Laufen H, Zimmermann T. Uptake of azithromycin by various cells and its intracellular activity under in vitro conditions. Antimicrob Agents Chemother 1996;40:75–9.