1. Dynamics of Nasopharyngeal Colonisation & Correlations with Lymphocyte Populations in the Adenoid & Peripheral Blood of Chronic Otitis Media Prone Children
Jessica J Browne1,3, Evan H Matthews2, Andrew W Taylor-Robinson3 & Jennelle M Kyd4
1Southern Cross University, School of Health & Human Sciences, Gold Coast, Australia, 2Otolaryngology Head & Neck Surgery, Mater Misericordiae Hospital, Rockhampton, Australia, 3CQUniversity, School of Medical & Applied Sciences, Rockhampton, Australia, 4Office of Senior DVC & Provost, Swinburne University of Technology, Melbourne, Australia.
This study was supported by the CQUniversity Health Collaborative Research Network grant from the Australian Government's CRN Program.

2. Bacterial Tolerance in OM
Increased bacterial nasopharyngeal colonisation.
Inflammation subsides in OM with effusion.
Why?
Mucosal colonisation of bacteria/virus maintained through Treg – mediated immune suppression:
Gut – Helicobacter pylori & probiotics
Urogenital – HPV16
Oropharynx – oral commensal flora
Nasopharynx - Neisseria meningitidis, Spn

3. Research Questions
Is nasopharyngeal carriage of Spn, Mcat & NTHi in children associated with Treg cells in the blood or adenoid?
What lymphocyte associations or changes occur with nasopharyngeal colonisation & are there differences between COM prone & non COM prone children?
How do these relationships contribute to a child’s susceptibility to OM?

4. Non-COM prone control group
Research Design
Non-COM prone control group
(enlarged adenoids, with or without URTI other than OM)
COM-prone group
(history of OM)
Samples
Lymphocyte Proportional Analysis
20 participants
Blood
Adenoid
Clinical Microbiology
NPA
Demographics
Questionnaire Clinical Records

5. Research Methods COM prone & non-COM prone children Lymphocytes
B cell / T cell subset distributions
8-colour
Flow Cytometry
Adenoid
Blood
NPA/Adenoid Biopsy
Bacterial culture
Correlate lymphocyte subset percentages with clinical bacteriology in COM and non-COM prone children to determine immunological factors associated with OM

6. Lymphocyte Phenotypic Analysis
Analysed B, T, TC, TH & Treg cell markers

7. Treg Cell Proportions are Similar in non-COM & COM Prone Children
Adenoid
PBMC
Non-COM prone
4.38 (1.19)
4.76 (2.09)
COM prone
3.82 (1.10) p = 0.15
3.85 (1.43) p = 0.12
Treg cell proportions in non-COM prone & COM prone groups analysed by independent student t-tests (n = at least 17 children per group; Treg cells presented as mean percentage of live TH cells with standard deviation).
No differences or correlations in other lymphocyte subset percentages between groups.

8. Circulating Treg Cells are Increased in Children with Positive Nasopharyngeal Culture
Adenoid
PBMC
NPA culture -ve
3.56 (0.86)
3.29 (0.59)
+ve
4.15 (1.25)
4.57** (1.93)
Adenoid culture -ve
3.94 (1.40)
3.21 (1.55)
4.04 (1.11)
4.52* (1.74)
Total Nasopharyngeal culture -ve
3.65 (0.53)
3.14 (0.49)
4.06 (1.23)
4.36** (1.84)
* p =<0.05, ** p =<0.005
Treg cell proportions in children with positive or negative otopathogen nasopharyngeal culture analysed by independent student t-tests (n = at least 36 children; Treg cells presented as mean percentage of live TH cells with standard deviation).

9. Otopathogen Trends – all children
Otopathogen positive culture
Adenoid
%(n)
NPA
Total Nasopharynx
Spn
27 (10)
28 (10)
38 (14)
S. aureus
36 (13)
NTHi
14 (5)
35 (13)
Mcat
19 (7)
22 (8)
24 (9)
Overall otopathogen culture
76 (28)
72 (26)
87 (32)
Multiple otopathogen culture
51 (19)
Frequencies of adenoid & NPA otopathogen culture. Values presented as % (n).
No differences in otopathogen cultures between non-COM & COM prone children.

10. Otopathogen Trends in COM Prone Children
Otopathogen positive culture
Adenoid
%(n)
NPA
Total Nasopharynx
Spn
39 (7)
28 (5)
44 (8)
S. aureus
33 (6)
NTHi
22 (4)
11 (2)
Mcat
17 (3)
Overall otopathogen culture
78 (14)
67 (12)
83 (15)
Multiple otopathogen culture
56 (10)
Frequencies of adenoid & NPA otopathogen culture. Values presented as % (n).
No differences in otopathogen cultures between non-COM & COM prone children.

11. Otopathogens Co-culture
Nasopharyngeal culture
Spn within co-culture
Mcat within co-culture
NTHi within co-culture
S. aureus within co-culture
-ve
+ve
Spn -ve
62 (23)
38 (14)
87 (20)*
57 (8)
 13 (3)
43 (6)
 78 (18)*
 22 (5)
44 (10)
93 (13)**
55 (13)
7 (1)
Mcat -ve
 71 (20)*
33 (3)
29 (8)
67 (6)
76 (28)
24 (9)
 71 (20)
44 (4)
 29 (8)
56 (5)
57 (16)
78 (7)
 43 (12)
22 (9)
NTHi -ve
75 (18)*
39 (5)
25 (6)
61 (8)
 83 (20)
62 (8)
 17 (4)
38 (5)
65 (24)
35 (13)
42 (10)
100 (13)
 58 (14)
0 (0)***
S. aureus -ve
56 (13)
70 (16)
86 (12)
30 (7)
14 (2)
100 (14)
Co-colonisation was analysed between species by chi-square analyses (Fisher’s exact test where cell counts were less than 5).
The results displayed here show the colonisation of otopathogens relative to the co-colonised otopathogen. These results support finding in murine models of S. pneumoniae pre-existing colonisation enhancing NTHi in nasopharyngeal co-colonisation (Margolis et al. 2010), & synergistic mechanisms among co-colonising bacterial species that are yet to be elucidated in children (Krishnamurthy et al. 2009).
* p = < 0.05, **p = < 0.005, ***p = < 0.000
Associations of otopathogen culture analysed by chi-square analyses using Fisher’s exact test where cell counts were less than 5. Values presented as % (n).
Spn positive correlations with Mcat & NTHi, while in the absence of Spn culture, the proportions of NTHi & Mcat cultures were also reduced.

12. B Cells were Increased in Children with Positive Mcat Culture
Total Nasopharyngeal Culture
% B cells
Adenoid
PBMC
Mcat culture -ve
51.18 (8.23)
8.59 (3.08)
+ve
55.97 (5.44)
12.37* (4.04)
* p =<0.01
Lymphocyte subsets in positive & negative otopathogen nasopharyngeal culture analysed by independent student t-tests (n = at least 36 children; Treg cells presented as mean percentage of live TH cells with standard deviation).
Previously, it has been shown that M. catarrhalis induces proliferation of B lymphocytes from the blood in a dose-dependent manner with the MID protein (Wingren et al. 2002).
In a study that included children & adults, M. catarrhalis induced proliferation of mixed tonsillar lymphocytes and isolated B lymphocytes ex vivo. With MID deficient M. catarrhalis stimulation isolated lymphocyte proliferation was reduced, & this was significant for isolated B cells (Jendholm et al. 2008). This suggested that MID might have strong mitogenic effects & that T lymphocytes enhance B lymphocyte responses to MID.
No differences or correlations in other lymphocyte subset percentages with otopathogen positive & negative culture.

13. Spn Culture Predicts NTHi Culture
Independent Determinant
Spn +ve culture
Mcat +ve culture
NTHi +ve culture
S. aureus +ve culture
Univariate
Multivariate
5.00 (1.00–25.02)*
 ns
 4.80 (1.13–20.46)*
 7.32 (1.09–49.43)*
0.06 (0.01–0.53)*
0.07 (0.01–0.70)* ns
* p = < 0.05
Otopathogen culture determinants of other otopathogen culture, analysed by univariate & multivariate logistic regression analyses, controlling for the independent determinants of different species-specific colonisation, including environmental tobacco smoke exposure, the youngest child among siblings, & antibiotics within the previous 6 months. Values presented as OR (95% CI), n = 37.
The results displayed here demonstrate the nature of the relationships between colonising species. These results supports finding in murine models of S. pneumoniae pre-existing colonisation enhancing NTHi in nasopharyngeal co-colonisation (Margolis et al. 2010), & synergistic mechanisms among co-colonising bacterial species that are yet to be elucidated in children (Krishnamurthy et al. 2009).
Spn was an independent determinant for NTHi colonisation. For Spn & S. aureus colonisation, the relationship was inverse; children with +ve culture of either species were 93% less likely to have nasopharyngeal culture of the other species.

14. Outcomes
Treg cells in the adenoid & blood were similar in COM prone & non-COM prone children.
Circulating Treg cells were increased in children with increased nasopharyngeal bacterial carriage.
Spn presence may enhance NTHi colonisation in children.
No immune deficiencies or differences were detected between the groups
Increased B cells associated with Mcat colonisation, but not COM specific
The correlations and strong predictive abilities found with NPA cultures for adenoid nasopharyngeal culture will be of direct benefit to clinicians in making diagnoses.

15. Participants & their families
Many Thanks to…
Participants & their families
Prof. Jennelle Kyd
Primary Supervisor, PI
Dr. Evan Matthews
Assoc. Supervisor, ENT Specialist
Prof. Andrew Taylor-Robinson
Assoc. Supervisor, Laboratory Manager
Dr. Ajay Krishnamurthy
Post-Doc. Research Fellow
Clinic & theatre staff
This study was supported by the CQUniversity Health CRN grant  the Australian Government's Collaborative Research Networks Program.