European Review for Medical and Pharmacological Sciences 2019; 23(1 Suppl.): 60-66 Modulation of opportunistic species Corynebacterium diphtheriae, Haemophilus parainfluenzae, Moraxella catarrhalis, Prevotella denticola, Prevotella melaninogenica, Rothia dentocariosa, and pseudopneumoniae by intranasal administration of Streptococcus salivarius 24SMBc and Streptococcus oralis 89a combination in healthy subjects

R. DE GRANDI1, M. BOTTAGISIO1, S. DI GIROLAMO2, A. BIDOSSI1, E. DE VECCHI1, L. DRAGO3

1Laboratory of Clinical Chemistry and Microbiology, IRCCS Galeazzi Orthopaedic Institute, Milan, Italy 2Department of Clinical Sciences and Translational Medicine, Tor Vergata University, Rome, Italy 3Clinical Microbiology, Department of Biomedical Sciences for Health, University of Milan, Milan, Italy

Abstract. – OBJECTIVE: Probiotics S. sali- be interesting to evaluate also this positive im- varius 24SMBc and S. oralis 89a comprised in pact of longer administration of this probiotic the nasal spray Rinogermina are known to exert formulation. inhibition of harmful pathogens and ameliorate the outcome of patients with chronic upper air- Key Words ways infections. In this study, for the first time, Microbiota, Upper respiratory tract infections, Pro- the effect of this formulation on the modulation biotics, Streptococcus salivarius, Streptococcus oralis, of the microflora of healthy subjects was eval- Pathobionts. uated, with particular interest on pathobionts and pathogens present. PATIENTS AND METHODS: Metagenomic List of Abbreviations identification and quantification of bacterial PEG-PPG: poly(ethylene glycol)–poly(propylene glycol); abundances in healthy subjects were carried DNA: Deoxyribonucleic acid; rRNA: Ribosomal Ribonu- out by means of Ion Torrent Personal Machine. cleic Acid; OTU: operational taxonomic unit. In particular, nasal swabs were sampled one, two and four weeks after seven days of treat- ment with Rinogermina. RESULTS: The modulation of the abundance Introduction of pathobionts and pathogenic species (i.e., Co- rynebacterium diphtheriae, Haemophilus parain- fluenzae, Moraxella catarrhalis, Prevotella den- The human nasal cavity harbors a rich bacte- ticola, Prevotella melaninogenica, Rothia den- rial ecosystem characterized and influenced both tocariosa, Staphylococcus aureus and Strepto- by the host itself and the environmental conditions coccus pseudopneumoniae) was characterized to which it is exposed1,2. When the non-sterile air and a significant temporary decrease in their is inhaled, potential pathogens together with dust presence was identified. CONCLUSIONS: and environmental particles are firstly filtered The beneficial effects of S. and trapped by vibrissae placed in the nostrils. salivarius 24SMBc and S. oralis 89a nasal in- take was assessed but seemed to be restrict- Subsequently, the local microbial flora plays a ed in specific temporal windows. Thus it would crucial role in protecting the host by preventing

60 Corresponding Author: Lorenzo Drago, MD; e-mail: [email protected] Intranasal administration of bacteriotherapy the colonization of the nasal vestibule by airborne Patients and Methods pathogens. Indeed, the nasal flora discourages the pathogens attachment by means of a mechanism Patients Recruitment, Probiotics called “competitive exclusion”, in which prokary- Administration, and Sample Collection otic cells must compete with their neighbors for Twenty-three healthy volunteers (19-64 years space and resources3. Furthermore, the commen- old; 9 males and 11 females) were recruited to this sal harbored in the nasal cavity stimulate study between January and February 2017. the consistent secretion of a mucus layer or snot The exclusion criteria were (1) the antibiotic containing enzymes and immunoglobulins apt to consumption within two months before the exper- stimulate the host immune system and impeding imental treatment and (2) the use of any medical the spread of a disease4. devices or treatment for nasal congestion. Fur- Different aerobic and anaerobic bacterial thermore, (3) subjects suffering from allergies to communities physiologically constitute the nasal pollen, grasses, poplars and dust and also (4) in- microbiota. Staphylococcus spp., Haemophilus dividuals with sinusitis or rhinosinusitis were ex- spp., Streptococcus spp., Moraxella spp., Neis- cluded. The volunteers had no pets in their homes, seria spp., and Corynebacterium spp. are some and they lived in the same geographical area, in of the commensal aerobic bacteria that inhabit a limit of 10 km from the Milan urban center in the healthy human nasal cavity5; whereas were order to ensure that all the subject were exposed Prevotella spp., Porphyromonas spp., Fusobac- to similar climatic and environmental conditions terium spp., and spp. are (e.g., humidity, temperature, pollution, etc.). the most common anaerobic isolates6. However, All the enrolled volunteers were treated with when external events such as alterations of the a mixture of S. salivarius 24SMBc, and S. oralis environmental conditions (e.g., humidity, oxygen 89a suspended in a 98:2 ration in buffered isotonic concentration, climate change, pollution, pollen, solution with pH 7.0 composed by poly(ethylene exposure to chemicals, etc.) or trauma alter the glycol)–poly(propylene glycol) (PEG-PPG) copo- homeostasis, a progressive change in the phys- lymer and PEG-14 Dimethicone (Rinogermina, iologic flora might occurs leading to a microbi- DMG, Rome, Italy). The probiotic solution was al imbalance, called dysbiosis. This occurrence administrated with two bilateral spray injections might increase the host susceptibility favoring the into each anterior nostril for a week, in the morn- establishment of inflammatory, infectious or al- ing after personal care/washing. lergic diseases7,8. In particular, pathobionts might Samples were collected by means of sterile switch from carriage to a pathological state com- pernasal swabs in polypropylene tubes (Thermo promising the individual welfare. Thus, there is Fisher, Waltham, MA, USA). In particular, the the need to restore the levels of a healthy nasal surface of the mucosa of the left anterior nos- microflora. A recent study investigated the use of tril was sampled in a 1 cm depth from the outer a nasal spray containing a mixture of probiotics edge. Per subject, four nasal swabs were collect- (Streptococcus salivarius 24SMBc and Strepto- ed according to the following experimental time coccus oralis 89a, 98:2 ratio) to evaluate the safe- points: (1) before the treatment, (2) 1 week, (3) 2 ty of the treatment on the nasal flora of healthy weeks and (4) 1 month after the end of the treat- subjects9. Indeed, the use of α-hemolytic strepto- ment with probiotics. Samples were collected at cocci as prophylaxis is now a promising emerging the Laboratory of Clinical Microbiology (Uni- strategy being S. salivarius and S. oralis biosafe versity of Milan, Milan, Italy) and store at -80°C colonizers characterizing a healthy and balanced until the analyses performed 48 h after sampling. nasopharyngeal microbiota9,10. Even if it was described the valuable role of the DNA Extraction, 16S rRNA Amplification, analyzed treatment in the reduction of the harmful and Sequencing pathogens and the increase of beneficial one, there Bacterial genomic deoxyribonucleic acid is still the need to characterize these populations (DNA) was extracted by means of the QIAamp to have a deeper insight into the modulation of DNA Mini Kit (Qiagen, Milan, Italy). Then, part the described bacterial hub. With this aim, in the of the 16S Ribosomal Ribonucleic Acid (rRNA) present study, the ability of S. salivarius 24SMBc gene sequences were amplified using the 16S and S. oralis 89a to modulate the nasal microbiota Metagenomics Kit (Life Technologies, Monza, It- composition was evaluated through the analysis aly) to analyses of the microbial populations char- of abundance of the identified species. acterizing the samples through the Ion Torrent

61 R. De Grandi, M. Bottagisio, S. Di Girolamo, A. Bidossi, E. De Vecchi, L. Drago sequencing technology (Life Technologies, Mon- Results za, Italy). The hypervariable regions of the 16S rRNA were amplified using the primer set V2-4-8 During the experimental time course, no se- and V3-6, 7-9 at the conditions reported in Table vere side effects were recorded in any enrolled I using the SimpliAmp Thermal Cycler (Thermo patients after the treatment with S. salivarius Fisher, Waltham, MA, USA). 24SMBc and S. oralis 89a. In particular, only The quality of the obtained amplicon was as- 10% of the subject suffered from allergic cold, sessed by electrophoresis on a 2% agarose gel. and 80% did not show any problems breathing. Finally, the sequencing of the bacterial DNA was However, 90% of people reported nasal dripping performed through the Personal Genome Ma- immediately after the treatment administration. chine, as previously described 11. A total amount of 4,901,713 high-quality fil- tered reads were obtained (126,674±552,453 per Ethic Statement patients), clustered in OTUs and consequently clas- All the performed procedures were conformed sified into taxonomic ranks (phyla, classes, orders, to the Helsinki of 1975, as revised in 2000 and families, genera, and species). The changes detect- 2008. The experimental protocols were approved ed in the presence of corynebacteria species in the by the internal review board of the IRCCS Galeazzi nasal microbiota composition were represented in Orthopaedic Institute and by the Ethics Committee Figure 1A. In particular, after an initial decrease of the IRCCS Ospedale San Raffaele (MicroSP no. one week after the treatment, Corynebacterium 31/INT/2017, Milan, Italy). The study was conduct- diphtheriae showed a progressive increase at T3 ed according to the International Council for Har- with a peak registered one month after the treat- monisation of Technical Requirements for Phar- ment. Even if the initial drop was also detected for maceuticals for Human Use guidelines for Good Corynebacterium accolens, Corynebacterium du- Clinical Practice. Finally, all the enrolled patients rum, Corynebacterium macginleyi, Corynebacte- were extensively informed about the purpose of rium tuberculostearicum, these microorganisms the study and a written consent was obtained from had a progressive decrease starting from T3, es- each participant. At the beginning and at the end pecially concerning C. durum, C. macginleyi. A of the trial, volunteers were asked to fill a ques- similar trend was observed in Staphylococcus tionnaire reporting any adverse effects and nasal spp. (Figure 1B) where the effects of Rinogermina disorders related to the treatment with probiotics. seemed to affect the abundance of these bacteria, especially one week after the administration. How- Statistical Analysis ever, the increase at T3 highlighted the positive The raw abundances of the species of each op- effects of the probiotic intake on Staphylococcus erational taxonomic unit (OTU) were firstly nor- warneri and Staphylococcus hominis, two benefi- malized to 100,000 reads per sample. The signifi- cial commensal strains. Although S. aureus was cant differences in microbial species were assessed widely present in nares of enrolled subjects, a re- through the nonparametric tests based on the Kru- duction in the abundance of this ubiquitous species skal-Wallis and Wilcoxon rank-sum test, coupled was appreciable during all the experimental time with Dunn’s post-hoc test (R software v 3.3.1, USA). points. More importantly, the synergistic action of All data are expressed as means, and only the signif- S. salivarius 24SMBc and S. oralis 89a resulted in icant differences were reported in graphs; p-values a reduction of S. aureus up to one month after the < 0.05 were considered statistically significant. treatment. Concomitantly, after an initial decrease at T2, the treatment helped Staphylococcus epider- midis to colonize nasal mucosae with a growing Table I. Amplification condition. trend also a month after the probiotic intake. Step Temperature Time Due to the different abundance of streptococ- ci, these bacteria were depicted in two graphs to Initial denaturation 95°C 10 min appreciate also the differences in underrepresent- Denaturation 95°C 30 sec ed species (Figure 2). Annealing x 30 cycles 58°C 20 sec displayed a consistent increase in the colonization Extension 72°C 20 sec of the nostrils one week after the probiotic in- Final extension 72°C 7 min take followed by a sudden drop at T3. In contrast, Amplification conditions for the hyper variable regions of the Streptococcus sanguinis, Streptococcus thermo- 16S rRNA using the primer set V2-4-8 and V3-6, 7-9. philus, Streptococcus sinensis and Streptococcus

62 Intranasal administration of bacteriotherapy

A B

Figure 1. Corynebacteria e staphylococci modulation over time. A, The graph shows the changes in corynebacteria abun- dance before Rinogermina intake (T1) and 1 week (T2), 2 weeks (T3) and 4 weeks (T4) after the treatment. B, Changes in staphylococci abundance before Rinogermina intake (T1) and 1 week (T2), 2 weeks (T3) and 4 weeks (T4) after the treatment. mitis had an opposite behavior, with a prompt oralis 89a seemed not to affect the Haemophilus reduction in relative abundance at T2. While the parainfluenzae and Rothia dentocariosa abundanc- presence of S. mitis and S. thermophilus progres- es, which remained stable overtime. Differently, a sively increased over time, the levels of S. sangui- discontinuous trend in the M. catarrhalis abundance nis had a drastic decreased (Figure 2A). Among was found during the experimental time points. In- the most abundant streptococci, the presence of deed, after a drop at T3, a gradual recovery was Streptococcus australis and S. salivarius enor- observed up to the one month after the end of the mously increased after seven days of treatment treatment. A similar increase at T4 was also detected and returned to baseline values from day 14 (T3). in Neisseria perflava (Figure 3A). Conversely, even if Streptococcus pseudopneu- Finally, among the anaerobic bacteria, moniae was detected at basal values at one month Prevotella melaninogenica was the most abun- after the end of the probiotic consumption, a peak dant species detected. The probiotic administra- in its abundance was observed at T3 (Figure 2B). tion affected this pathobiont in which a decrease The remaining identified microorganisms were was found up to T3 and a return towards basal grouped in accordance with their aerobic or anaer- valued at T4. A similar trend as that described for obic metabolism. Concerning the aerobic bacteria, corynebacteria was also observed for the remain- the administration of S. salivarius 24SMBc and S. ing representatives of Prevotella ssp. (Figure 3B).

A B

Figure 2. Streptococci modulation over time. A, The graph shows the changes in less abundant streptococci before Rinoger- mina intake (T1) and 1 week (T2), 2 weeks (T3) and 4 weeks (T4) after the treatment. B, Changes in more abundant strepto- cocci before Rinogermina intake (T1) and 1 week (T2), 2 weeks (T3) and 4 weeks (T4) after the treatment.

63 R. De Grandi, M. Bottagisio, S. Di Girolamo, A. Bidossi, E. De Vecchi, L. Drago

A B

Figure 3. Other aerobes and anaerobes modulation over time. A, The graph shows the changes in Moraxella catarrhalis, Haemophilus parainfluenzae, Dolosigranulum pigrum, Rothia spp., and Neisseria spp. before Rinogermina intake (T1) and 1 week (T2), 2 weeks (T3) and 4 weeks (T4) after the treatment. B, Changes in Prevotella spp. and Veillonella spp. before Rino- germina intake (T1) and 1 week (T2), 2 weeks (T3) and 4 weeks (T4) after the treatment.

Discussion for the evaluation of S. salivarius 24SMBc and S. oralis 89a on the microbial species composition of The human nares physiologically harbor dif- healthy nostrils was used in this study. The identi- ferent aerobic and anaerobic commensal microor- fication of species can be very challenging and de- ganisms that compose the nasal microbiota. This manding because of the high degree of 16S rRNA dynamic ecosystem is strongly influenced by en- similarities in members of the same genera, but at vironmental conditions and by the host itself. The the same time can be an extremely important tool bacterial communities and their metabolic prod- to study the microbe-microbe interaction deter- ucts influence the health of their habitat and that of mined by a peculiar condition14. the organism in which they live. Indeed, the micro- The transient - but not stable - peak in the abun- biota possesses the capability to reflect the health dance of S. salivarius after one week is correlated status of the host, and a dysbiosis caused by the to the high percentage of this streptococcal strain homeostasis alteration lead to changes that might in Rinogermina. Interestingly, even if it was not no- be used as potential diagnostic tools12. For this rea- ticed a similar trend in the S. oralis abundance, this son, numerous bacterial isolated from healthy sub- increase might be reflected in other members of the jects have been proposed as probiotics to restore S. mitis group, which include S. infantis, S. tiguri- this imbalance. The local administration of fully nus and S. australis. It is well-known that mem- characterized commensal bacteria which presence bers of the S. mitis group are able to influence each is associated to a healthy state might be beneficial other by means of the production of competence for both preventing and treating pathological con- stimulating peptides (CSP) pheromones, which ditions. Two streptococcal strains, S. salivarius also directly control the secretion of streptocine15. 24SMBc and S. oralis 89a, have been proposed This effect might contribute to the modulation in for this purpose as they were isolated from healthy the number of some observed pathogenic species individuals during pathological outbreaks and be- often involved in chronic upper airways infections cause they possess desirable characteristics ob- (C. diphtheriae, H. parainfluenzae, M. catarrhalis, served in in vitro experiments, such as the ability to Prevotella denticola, P. melaninogenica, R. dento- colonize the respiratory tract and to counteract the cariosa, S. aureus and S. pseudopneumoniae)6,16-19. presence of airborne pathogens13. Previous studies Contrary to other members of the mitis group, S. underlined the safety and tolerability of the combi- pseudopneumoniae had a temporary growth arrest nation of these two probiotic strains on healthy and probably related to a putative beneficial influence pathological subjects by evaluating the clinical out- of the probiotic administration and the potential in- comes after the administration of the nasal spray hibitory effected exerted by S. oralis, as similarly formulation9,10. However, the impact of Rinoger- observed in S. pneumoniae20. mina on the normal nasal microbiota has not been Notably, at the end of the follow-up, the action investigated yet. Hence, a metagenomic approach of S. salivarius 24SMBc and S. oralis 89a combi-

64 Intranasal administration of bacteriotherapy nation was appreciable in an important reduction late the nasal microbiota composition with partic- of pathogens. In particular, the reduction of the ular regards to pathobionts and pathogenic species. abundance of S. aureus during the follow up cor- The positive effects in most of the analyzed species relates with the concomitant increase in S. epider- seemed to be restricted in a specific temporal win- midis presence. Thus, it can be speculated that the dow. Hence it will be important also to elucidate nasal colonization of S. aureus was limited by the the impact of longer administration of Rinogermi- secretion of the extracellular serine protease (Esp) na, which might result in a more consistent modu- by S. epidermidis, which is known to negatively lation of the nasal microbiota during time. affect the mechanisms of adhesion and biofilm formation of the above-mentioned pathogen21. Interestingly, the use of S. salivarius 24SMBc Sources of Funding and S. oralis 89a resulted in the modulation of the This work was supported by the Italian Society of Rhi- presence of Corynebacterium spp. These microor- nology. The sponsors provided financial support for costs ganisms commonly colonize juvenile and adult nasal related to the publication of this article. The sponsor was not involved in the study design, in the collection and in- passage, thus its role in the establishment of several terpretation of data, in the writing of the study and in the commensal-pathobiont interactions is not surprising decision to submit the article for publication. 14. However, little is known about the molecular inter- actions of these genera with neighbor bacteria com- posing the human nasal microbiota. The temporary Conflict of Interests scarceness of the pathogen C. diphtheriae could be The authors declare that they have no conflict of interest. related to the concomitant predominance of S. sali- varius, which might exert by a direct inhibitory effect on this pathogen, as previously described22. References Our data might also indicate a positive impact of the Rinogermina administration by limiting the 1) Haug RH. Microorganisms of the nose and pa- presence of potential pathogenic anaerobe micro- ranasal sinuses. Oral Maxillofac Surg Clin North organisms such as P. denticola and P. melanino- Am 2012; 24: 191-196, vii-viii. 2) Mahdavinia M, Keshavarzian A, Tobin MC, Landay AL, genica previously largely recovered in infections Schleimer RP. 23,24 A comprehensive review of the nasal of upper and lower airways . In fact, of all the microbiome in chronic rhinosinusitis (CRS). 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