Pathogens and Disease. 2016 Feb;74(1):ftv106. doi: 10.1093/femspd/ftv106.
Persistence of non-typeable Haemophilus influenzae in the pharynx of children with adenotonsillar hypertrophy after treatment with azithromycin.
Olszewska-Sosińska O1, Zielnik-Jurkiewicz B1, Stępińska M2,3, Antos-Bielska M2, Lau-Dworak M4, Kozłowska K2, Trafny EA2,3
1Department of Otolaryngology Children’s Hospital, Niekłańska 4/24, 03-924 Warsaw, Poland.
2Department of Microbiology, Military Institute of Hygiene and Epidemiology, Kozielska 4, 01-163 Warsaw, Poland.
3Biomedical Engineering Center, Institute of Optoelectronics, Military Institute of Technology, 2 Gen. S. Kaliskiego, 00-908 Warsaw, Poland,
4Department of Laboratory Diagnostics, Children’s Hospital, Niekłańska 4/24, 03-924 Warsaw, Poland.
The study was performed in children with adenotonsillar hypertrophy to evaluate the effect of azithromycin (AZT) on the presence of NTHi in monocyte/macrophages (CD14+ cells) of adenoids/tonsils and the persistence of NTHi after adenotonsillectomy. A total of 36 pediatric patients participated in the study: 20 children were treated with AZT before adenotonsillectomy and 16 children did not receive the antibiotic prior to surgery. NTHi were identified by culture and PCR in swabs and tissue samples. NTHi was detected in the lysates of CD14+ cells by fluorescence in situ hybridization (FISH) and by culture. The molecular typing was used to cluster NTHi isolates from each child. The intracellular NTHi was found in 10 (62.5%) untreated patients and was identified in three (15%) azithromycin-treated patients (p=0.003). The proportion of the persistent NTHi strains was similar in both groups. AZT treatment followed by adenotosillectomy did not completely eliminate NTHi from pharynges; however, it significantly reduced the risk of carriage of H. influenzae inside the CD14+ cells.
KEYWORDS: NTHi; adenoid; azithromycin; children; macrophages
Nontypable Haemophilus influenzae (NTHi) is a commensal of human nasopharynx and predominant pathogen isolated from the nasopharynx in children. Only periodic detection of NTHi in nasopharyngeal swabs does not indicate the absence of the pathogen because NTHi may be present in upper respiratory tract in biofilms and intracellulary.
The potential of NTHi to persist as the intracellular pathogen may contribute to its chronic existence in lymphoid tissue and result in the lengthening of the disease period. Moreover, NTHi can hide inside host cells thereby avoiding contact with the most commonly used antibiotics. Bearing in mind that NTHi may be present in the children throat in the form of a biofilm and as the intracellular bacteria, the clinicians should be aware of the potential lack of antibiotics effectiveness for treatment of the NTHi chronic respiratory tract infections. The long-term existence of NTHi (in a biofilm and the intracellular compartments of host cells) inaccessible to the commonly used β-lactams is related to the risk of recurrent and life-threatening infections. Macrolides are not the first-line antibiotics in NTHi acute infections but their use in chronic upper respiratory tract diseases is beneficial [1, 2 ,3]. NTHi strains are clinically sensitive to azithromycin.
Therefore, this study was performed to verify whether the administration of macrolides followed by adenotonillectomy is clinically justified in chronic adenoid hypertrophy in children and leads to eradication of NTHi from the upper respiratory tract of young children. Accordingly, azithromycin as representative of the macrolide class with high activity against biofilms and the intracellular bacteria was chosen for treatment of the patients. The antibiotic was administered at a dose of 20 mg/kg body weight for three days to 20 children with adenotonsills hypertrophy. The presence of NTHi on the surface of adenoids and tonsils, in the lymphoid tissue homogenates, and in the lysates of CD14+ cells was evaluated. NTHi were identified by culture and multiplex PCR in swabs and tissue samples of adenoids and tonsils. The CD14+ cells (monocyte/macrophages) were isolated from the tissue homogenates by the immunomagnetic separation according to the described methods by Forsgen et al.  and Stępińska et al. . The intracellular H. influenzae were detected by FISH in the lysates of CD14+ cells from the lymphoid tissues (Figure 1). The persistence of NTHi strains in each individual child’s throat was monitored during 13 weeks, starting from a week before the surgery, by using PFGE genotyping of at least 10 NTHi isolates per one sampling occasion.
We discovered that the administration of azithromycin before adenotosillectomy did not completely eliminate NTHi from the patient’s throat. The treatment with azithromycin followed by the surgery had also no significant effect on the presence of persistent (present in the swabs collected before or during surgery and after surgery), transient (present before or during surgery and absent after surgery), and the acquisition of the new NTHi strains (after surgery)  in the throat of patients. However, there were a statistically significant lower number of children that carried the intracellular NTHi in monocytes/macrophages (CD14+ cells) after treatment with azythromycin followed by adenotonsillectomy when compared to the children, which were not treated with azythromycin but underwent the surgery. These results suggested that azithromycin strongly reduced the risk of carrying the intracellular NTHi. Only few previous studies have found that in vitro azithromycin had the bactericidal effect against viable NTHi and this was observed for human bronchial epithelial cells and HEp-2 cells .
Many other antibiotics have higher intracellular bactericidal activity against NTHi than azithromycin. The advantage of azithromycin is a simultaneous inhibition of biofilm formation and a reduction of already established H. influenzae biofilms by subinhibitory concentrations of the antibiotic . Therefore, the inhibitory effect of azithromycin on the growth of biofilm and its bactericidal effects towards the intracellular NTHi could encourage its wider use in infections of the upper respiratory tract in children.
Figure 1. The intracellular H. influenzae after lysis of CD14+ cells isolated from adenotonsilar tissues. The presence of the bacteria was detected with blue fluorescence DAPI (A, C), and with red fluorescence Cy3 (B, D) using FISH procedure for staining. The conglomerates of bacteria visible on micrographs of the same field of microscopic view (A, B) and (C, D) are marked by white arrows.
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We want to acknowledge the excellent technical assistance of Agnieszka Wielechowska. The study was financed from the funds of the National Science Centre of Poland (Project No NN404139838).
Elżbieta Anna Trafny
Biomedical Engineering Center, Institute of Optoelectronics,
Military Institute of Technology,
2 Gen. S. Kaliskiego, 00-908 Warsaw, Poland,
Phone: +48 22 6839544,
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