Background
Acute otitis media (AOM) is the leading cause of bacterial infections in childhood, about 700 million cases each year, and the leading cause of antibiotic prescription [
1]. More than 80% of children have at least one episode of AOM before age 4 years and 40% will have 6 or more recurrences by age 7 years [
2,
3]. Otitis media is a multifactorial disease, from uncomplicated AOM to more complex recurrent and chronic cases. Most cases of AOM resolve spontaneously, but complications that can occur include suppurative ones and long-term effects such as hearing loss.
The major pathogens involved in AOM, also called “otopathogens”, are
Streptococcus pneumoniae and non-typable
Haemophilus influenzae (NT-
Hi) (Casey et al., 2004, Block et al., 2004) as well as
Moraxella catarrhalis [
4,
5]. NT-
Hi is frequently associated with AOM treatment failure, recurrence and otitis media with effusion [
6,
7].
The role of biofilm production has been suggested in several types of AOM. A biofilm is dynamic multimicrobial community adhering to a surface and enclosed in a matrix rich in exopolysaccharides, proteins, and nucleic acids. Bacteria living inside this structure are protected against external aggression such as the host immune system and antibiotic treatment [
8]. In vivo biofilm production may involve multiple bacteria species, and several studies involving dual-species biofilm experiments have suggested complex inter-species interactions [
9‐
11]. Some studies suggest that the persistence of bacterial species and notably NT-
Hi in a biofilm-structured community plays a role in the pathogenesis of chronic, recurrent or non-responsive otitis media. Indeed, because of inefficient clearance of bacteria from the middle ear, the biofilm acts as a pathogen reservoir [
12‐
16]. In situ hybridization or immunohistology techniques are probably the best methods to detect biofilm in these cases.
In the opposite spectrum of OM diseases, oto-pathogens are present in nasopharyngeal (NP) flora in biofilm, and some authors suggest that AOM episodes occur when bacteria escape from the biofilm surface to the surrounding space, in the region of adenoids, then Eustachian tube dysfunction promotes the penetration of the strains in the middle ear [
17]. In these cases, hybridization and/or immunohistology techniques cannot be used, and other methods, animal models or in vitro methods, are needed to assess the role of biofilm. The capacity for NT
-Hi and
S. pneumoniae to produce biofilm can be demonstrated in vitro or in vivo. In vivo studies require a Chinchilla model of otitis media or direct detection by confocal laser scanning microscopy [
18‐
22]. In vitro studies, such as microtiter plates or continuous-culture once-through flow cell, were described in several studies [
14,
23‐
25]. Methods based on PCR techniques have been described, but they cannot discriminate between planktonic and biofilm-growing bacteria [
26]. In our previous study, by using a modification of the microtiter plate assay with crystal violet (CV) stain, we found that 49% of
H. influenzae strains isolated from children’s nasopharynx produced biofilm [
27].
Because of the pain caused by tympanocentesis, most clinical-practice guidelines for AOM generally do not recommend taking bacteriological samples of middle ear fluid except for treatment-failure and recalcitrant cases [
28]. Kaur et al. used multilocus sequence typing to compare strains isolated from NP and middle-ear fluid samples in 34 children during an AOM episode and found the same sequence type of NT-
Hi in 31/34 children (84%), which highlighted the similarity of strains isolated from both sites [
28]. NP colonization with potential middle-ear pathogens is considered the initial event leading to AOM in humans, frequently preceded by or associated with viral infection [
29,
30]. Furthermore, some studies suggested that biofilm production ability differs by pneumococcal serotype [
24,
25,
31,
32].
Here, we evaluated biofilm production by NT-Hi and S. pneumoniae strains isolated alone or together in the nasopharynx of the same patient and analyze biofilm production according to serotypes.
Results
Methods for calculating the biofilm production
The cut-offs used to determine the intensity of biofilm production in isolates of S. pneumoniae and H. influenzae were as followed where AB represented the stained wells containing attached bacteria, CW the stained control wells containing bacteria-free medium only and G the bacterial growth control i) AB – CW: strong production > 0.30, moderate production 0.10–0.30, negative < 0.10 ii) AB / CW: strong production > 6, moderate production 2–6, negative < 2 iii)[(AB – CW) / G]: strong production > 1.10, moderate production 0.35–1.10, negative < 0.35.
Strains were classified according to agreement of at least 2 of the 3 methods used to calculate biofilm production by
S. pneumoniae (Additional file
1: Supplementary Data S1) and
H. influenzae (Additional file
2: Supplementary Data S2). Among 182 NT
-Hi strains, 62 (34%) were classified as strong biofilm producers, 55 (30.2%) moderate producers and 64 (35.1%) non-producers. One strain could not be evaluated. Overall, 117/181 (64.6%) NT-
Hi strains produced biofilm (Table
1). By using the same classification, among the 191
S. pneumoniae strains, 63 (33%) were classified as strong biofilm producers, 64 (33.5%) moderate producers and 63 (33%) non-producers. Overall, 128/191 (66.8%)
S. pneumoniae strains produced biofilm (Table
1).
Table 1
Biofilm production by H. influenzae and S. pneumoniae isolated alone or together by pneumococcal conjugate vaccine (PCV) period
H. influenzae
|
Isolated alone | 19/32 (59.4%) | 15/24 (62.5%) a | 11/30 (36.7%) a | 45/86 (52.3%) c |
Isolated with S. pneumoniae | 24/32 (75%) | 28/32 (87.5%) b | 20/32 (63.3%) b | 72/96 (75.5%) c |
Total | 43/64 (67.2%) | 43/56 (76.8%) | 31/61 (50.8%) | 117/181 (64.6%) |
S. pneumoniae
|
Isolated alone | 21/32 (65.6%) | 22/32 (68.8%) | 21/31 (67.7%) | 64/95 (67.4%) |
Isolated with H. influenzae | 23/32 (71.9%) | 21/32 (64.5%) | 20/32 (62.5%) | 64/96 (66.3%) |
Total | 44/64 (68.8%) | 43/64 (66.7%) | 41/63 (65.1%) | 128/191 (66.8%) |
Evolution of the proportion of biofilm-producing strains
Between the pre-PCV7 and post-PCV7/pre-PCV13 periods, the proportion of NT-
Hi biofilm-producing strains was stable (67.2 and 76.8%, respectively, p = ns), whether isolated alone or with
S. pneumoniae strains (Table
2). However, the proportion of NT-
Hi biofilm-producing strains was lower in the post-PCV13 than post-PCV7/pre-PCV13 period for strains isolated alone (62.5 and 36.7%, respectively,
p = 0.05) or with
S. pneumoniae strains (87.5 and 63.3%, respectively,
p = 0.02). Overall, the proportion of NT
-Hi biofilm-producing strains was greater when isolated with
S. pneumoniae (75.5% vs 52.3%,
p = 0.001) (Table
2).
Table 2
Serotypes of S. pneumoniae isolates producing biofilm, isolated alone or with H. influenzae
PCV7 |
6B (11) | 10 (91) | 7/8 (88) | 3/3 (100) | 1 | 2 | | | 1/3(33) | 3/3 (100) |
9 V (2) | 2 (100) | 1/1 (100) | 1/1 (100) | 1 | | | | 1/1 (100) | 1/1 (100) |
14 (10) | 6(60) | 1/2 (50) | 5/8 (63) | 5 | | 3 | | 8/8 (100) | 8/8 (100) |
18C (1) | 1 (100) | 0 | 1/1 (100) | 1 | | | | 1/1 (100) | 1/1 (100) |
19F (17) | 7 (41) | 2/6 (33) | 5/11 (45) | 4 | 1 | 4 | 2 | 8/11 (73) | 9/11 (82) |
23F (13) | 5 (38) | 3 /7(43) | 2/5 (40) | 1 | 1 | 2 | 1 | 3/5 (60) | 4/5 (80) |
Total | 31/54 (57.4) | 14/24 (58.3) | 17/29 (58.6) | 13 | 4 | 9 | 3 | 22/29 (75.9) | 26/29 (89.9) |
PCV13 additional serotypes |
1 (1) | 0 | 0 | 0/1 | | | 1 | | 1/1 (100) | 1/1(100) |
3 (1) | 0 | 0/1 | 0/0 | | | | | 0 | 0 |
5 (1) | 1 (100) | 0 | 1/1 (100) | | 1 | | | 0/1 | 1/1 (100) |
6A (6) | 3 (50) | 2/5 (40) | 1/1 (100) | 1 | | | | 1/1(100) | 1/1 (100) |
7F (2) | 2 (100) | 1/1 (100) | 1/1 (100) | 1 | | | | 1/1(100) | 1/1 (100) |
19A (17) | 16 (94) | 8/8 (100) | 8/9 (89) | 6 | 2 | 1 | | 7/9 (78) | 9/9 (100) |
Total | 22/28 (78.5) | 11/15 (73.3) | 11/13(84) | 8 | 3 | 2 | | 10/13 (77) | 13/13 (100) |
Other serotypes |
15B/C (15) | 14 (93) | 8/8 (100) | 6/7 (86) | 4 | 2 | | 1 | 4/7 (57) | 6/7 (86) |
15A (12) | 6 (50) | 3/5 (60) | 3/7 (43) | 3 | | 4 | | 7/7 (100) | 7/7 (100) |
11A (9) | 6 (67) | 3/3 (100) | 3/6 (50) | 3 | | 2 | 1 | 5/6 (83) | 5/6 (83) |
23B (9) | 3 (33) | 0/4 | 3/5 (60) | 3 | | 2 | | 5/5 (100) | 5/5 (100) |
23A (12) | 8 (67) | 6/8 (75) | 2/4 (50) | 1 | 1 | 2 | | 2/3 (67) | 4/4 (100) |
10A (6) | 3 (50) | 0/2 | 3/4 (75) | 1 | 2 | 1 | | 2/3 (67) | 4/4 (100) |
21 (5) | 2 (40) | 1/2 (50) | 1/3 (33) | | 1 | 2 | | 2/3(67) | 3/3 (100) |
35F (4) | 4 (100) | 1/1 (100) | 3/3 (100) | 2 | 1 | | | 2/3 (67) | 3/3 (100) |
35B (6) | 5 (83) | 3/4 (75) | 2/2 (100) | | 2 | | | 0/2 | 2/2 (100) |
6C (4) | 2 (50) | 1/2 (50) | 1/2 (50) | 1 | | 1 | | 2/2 (100) | 2/2 (100) |
17F (4) | 4 (100) | 3/3 (100) | 1/1 (100) | 1 | | | | 1/1(100) | 1/1 (100) |
22F(2), 29(2) | 2 (50) | 0/2 | 2/2 (100) | 1 | | | | 2/2 (100) | 2/2(100) |
24F (2) | 1 (50) | 1/1 (100) | 0/1 | | | | 1 | 0/1 | 0 |
15F (1) | 0 (0) | 0/1 (0) | 0/1 | | | 1 | | 1/1 (100) | 1/1 (100) |
16(1) | 1 (100) | 0 | 1/1 (100) | 1 | | | | 1/1 (100) | 1/1 (100) |
38 (1) | 1 (100) | 0 | 1/1 (100) | | 1 | | | 0/1 | 1/ 1(100) |
8(2), 12F (1), 25A (1) 31 (1), 33F (1) | 5 (83.3) | 5/6 (83.3) | 0/0 | | | | | 0 | 0 |
Total | 67/101 (69.3) | 43/62 (69.3) | 36/57 (63.1) | 24 | 11 | 17 | 4 | 36/48 (75) | 46/48 (94.6) |
NT (7) | 7 (100) | 3/3(100) | 4/4 (100) | 3 | 1 | | | 3/4 (75) | 4/4 (100) |
Total | 127/190 (66.8%) | 64/95 (67.4%) | 64/97 (66%) | 46/65 (71%) | 18/65 (27.6%) | 26/32 (81.2%) | 6/32 (18.7%) | 71/94 (75.5%) |
89/94 (94.6%)
|
Conversely, the proportion of
S. pneumoniae biofilm-producing strains did not change over the study periods, whether isolated alone (65.6 and 67.7% in pre-PCV7 and post-PCV13 periods) or with NT
-Hi strains (71.9 and 62.5% in pre-PCV7 and post-PCV13 periods). Overall, the proportion of
S. pneumoniae biofilm-producing strains did not differ whether isolated alone or with NT-
Hi strains [67.4% vs. 66.3%,
p = 0.92] (Table
2).
Characteristics of S. pneumoniae biofilm-producing strains
We found no differences in biofilm-producing ability by pneumococcal serotype (Table
2). The proportion of
S. pneumoniae serotypes included in PCV7 (54/191) that produced biofilm was similar to that for other serotypes: 57.4% (31/54) versus 70.6% (96/137),
p = 0.09 (data not shown). Moreover, the proportion did not differ between the 6 additional
S. pneumoniae serotypes included in PCV13 and other serotypes [78.5% (22/28) vs 64.4% (105/163),
p = 0.14)]. Strains of serotypes 6B, 15B/C, 19A, 35F, and 35B produced biofilm in more than 80% of the cases. In contrast, strains of serotypes 23B, 23F,19F were the lowest producers (40% of strains) (Table
2).
The S. pneumoniae serotypes frequently isolated with NT-Hi strains were 11A, 14, 15A, 15B/C, 19A, 19F, 23F and 23B, representing 60.4% (58/96) of the combination. H. influenzae biofilm-producing strains were isolated more often with serotypes 11A, 14, 15A, 19F and 19A. This association did not affect biofilm production by S. pneumoniae.
Susceptibility or resistance to penicillin did not differ with and without biofilm production: 64.2% (61/96) versus 69.5% (66/95), p = 0.44.
Overall, 75.5% (72/96) of NT-Hi strains produced biofilm when isolated with S. pneumoniae strains. Serotypes of S. pneumoniae were not associated with biofilm production by NT-Hi strains.
When isolated from the same nasopharynx,
S. pneumoniae or
H. influenzae or both produced biofilm in 94.6% (89/94) of cases (Table
3).
Table 3
Demographic characteristics and clinical signs of children according to the biofilm production
Day-care center | 47/72 (65.3) | 0.66 | 42/63 (66.7) | 0.99 |
Recurrent acute otitis media | 22/33 (66.7) | 0.77 | 19/29 (65.5) | 0.87 |
Conjunctivitis | 59/97 (60.8) | 0.27 | 42/66 (63.6) | 0.52 |
Otalgia | 96/145 (66.2) | 0.26 | 105/156 (67.3) | 0.68 |
Temperature > 38.5 °C | 42/56 (75.0) |
0.047
| 45/71 (63.4) | 0.46 |
Fever + otalgia | 50/67 (74.6) |
0.025
| 58/86 (67.4) | 0.87 |
Antibiotics 3 months before enrollment | 53/87 (60.9) | 0.34 | 55/82 (67.1) | 0.95 |
Relation between clinical signs and biofilm production
Table
3 presents the demographic characteristics and clinical signs by biofilm production. Biofilm was produced significantly more often by NT-
Hi strains isolated from children with than without fever (temperature > 38.5 °C) or fever + otalgia [75% (42/56) vs 59.7% (74/124),
p = 0.047 and 74.6% (50/67) vs 56.5% (65/112),
p = 0.025]. These relations were not observed for
S. pneumoniae (
p = 0.46 and
p = 0.87, respectively).
Antimicrobial susceptibility and serotyping of the strains
The proportion of resistant strains significantly decreased from the pre-PCV7 to post-PCV13 periods for both NT-Hi and S. pneumoniae. Among NT-Hi strains, the proportion of β-lactamase–producing strains decreased from 51.5% (33/64) in the pre-PCV7 period to 17.8% (10/56) and 17.7% (11/62) in the post-PCV7/pre-PCV13 and post-PCV13 periods, respectively (p < 0.001). As well, the proportion of S. pneumoniae strains with decreased susceptibility to penicillin decreased from 65.7% in the pre-PCV7 period to 45.4% (29/64) and 39.7% (25/63) in the post-PCV7/pre-PCV13 and post-PCV13 periods (p = 0.003). Among S. pneumoniae strains, the proportion of those with erythromycin susceptibility increased from 46.8% (30/64) to 81% (51/63) between the post-PCV7/pre-PCV13 and post-PCV13 periods, respectively (p < 0.001).
After PCV implementation, the distribution of the S
. pneumoniae serotypes changed according to the study period (Additional file
3: Supplementary Data S3). In the pre-PCV7 period, the serotypes of 47/64
S. pneumoniae strains were included in PCV7, 10/64 involved the 6 additional serotypes included in PCV13, and 3 were serotype 15B/C. In the post-PCV7/pre-PCV13 period, the serotypes of 6/64
S. pneumoniae strains were included in PCV7, 19/64 involved the 6 additional serotypes included in PCV13, and 38/62 were of various serotypes. In the post-PCV13 period, only 1/63 serotypes was included in PCV13.
Discussion
To our knowledge, it is the first study exploring in a large cohort the production of biofilms of
H. influenzae and
S. pneumoniae isolated alone or together from NP flora of children with AOM. Previously, the role of biofilm production in AOM was mainly explored for
S. pneumoniae [
25,
31,
40] or
H. influenzae [
22,
27,
41] or both in Chinchilla models [
42].
We used here three methods of calculation because there is no reference method for biofilm production. We found high biofilm production for both
S. pneumoniae and
H. influenzae (66.8 and 64.4%, respectively) with no variation in proportion for
S. pneumoniae over PCV implementation periods. Of note, the proportion of NT-
Hi biofilm-producing strains was greater when
H. influenzae strains were isolated with
S. pneumoniae, which agreed with the results of Hong et al. [
10].
When
S. pneumoniae and
H. influenzae were isolated together, 93.7% of cases showed biofilm production by
S. pneumoniae and/or
H. influenzae. Residence of a bacterium within a biofilm allows for global changes in gene and protein expression profiles, which has many effects on cell physiology, promoting adhesion and cohesion properties of biofilm cells, thereby increasing its persistence [
43]. Recent studies demonstrated that
H. influenzae and
S. pneumoniae modulate the expression of each other’s virulence genes, which results in persistent biofilm, mainly by upregulati type IV pilus structural protein (
pilA) by
H. influenzae, thereby playing an important role in adhesion and biofilm stability [
9,
12]. Hong et al. reported significantly downregulated expression of pneumococcal genes regulating autolysis and fratricide,
lytA and
cbpD, on co-culture with NT
-Hi, which suggests that pneumococcal survival and biofilm production can be enhanced in the presence of NT-
Hi [
10] .
We found that S
. pneumoniae serotypes 6B, 15B/C, 19A, 35F and 35B were the best biofilm producers. Previously, serotypes 14, 6B, 15B/C, and 11A were found efficient in producing biofilm [
25,
40], whereas Domenech et al., reported serotypes 35B and 11A, 19A as efficient [
24]. Therefore, these results support the validity of our method because the 5 serotypes we found as the best biofilm producers were those previously described. Analysis of a large database of AOM serotypes revealed that serotype 19A has the highest disease potential for AOM [
44]. The high production of biofilm may be one explanation for this phenomenon.
Our study has several limitations. The first is the lack of direct evaluation of biofilm in biological samples in middle ear fluid (MEF) with alternative methods such as confocal microscopy after live/dead staining [
45,
46]. However, we assumed that this method could not be used for a large population with AOM. Second, one can argue that the studied strains were isolated from the nasopharynx and not from MEF. We postulate, as do other authors, that the reservoir of bacterial species implicated in AOM is the nasopharynx, and their carriage precedes AOM [
29,
30]. Previously we demonstrated no significant difference in biofilm production between NT-
Hi strains from MEF and NP samples [
27]. In a previous prospective study, Bingen et al., using pulsed-field gel electrophoresis in conjunctivitis-otitis syndrome
, revealed identical NT
-Hi strains isolated from MEF and conjunctivitis tissue [
47] . Van Dongen et al., in a systematic review found NT-
Hi strains isolated from both MEF and NP samples in 80% of cases [
48]. More recently, Van Hoecke et al., investigating the presence of otopathogenic bacteria in middle ear effusion and adenoids of children with chronic otitis, found
NT-Hi and
S. pneumoniae, isolated from both locations, genetically identical in 13/14 cases [
22]. The third limitation is that we could have used knock-out mutants as a control. However, many genes appear to be involved, although the role of each appears to vary when biofilm is produced in batch or continuous culture. Proteomic studies have revealed an increase in number of proteins synthesized de novo and differences in protein production patterns during
S. pneumoniae biofilm development [
23]. In these conditions, in vivo studies are difficult. Another limitation is that these isolates came only from children with AOM and not from healthy children or children with chronic AOM. However, these results support the hypothesis that multispecies biofilm is the basis for the chronicity of otitis media as previously suggested [
13]. Finally, even if a larger number of strains would have allowed us to have more statistical power, our sample allowed us to detect some statistical differences.
The last limitation of our study is that biofilm production was studied in monoculture because of the different culture requirements of the 2 strains. One reason was the difficulty in co-cultivating the 2 bacteria because of the rapid lysis of S. pneumoniae in a liquid medium. The incubation time required for the experiment was not the same for both bacteria.
The role of biofilm in otitis media is not fully understood, and we lack a universally accepted or feasible method to study biofilm formation in vitro and in vivo in humans. We are aware that we are simply bringing some modest information to a complex puzzle. However, if our study has several limitations, it has also several strengths as follows. 1) To our knowledge, it is the first study exploring in a large cohort the production of biofilm of H. influenzae and S. pneumoniae isolated from NP flora of children with AOM. 2) We were able to demonstrate H. influenzae and/or S. pneumoniae biofilm production in all the clinical situations and more particularly by NT-Hi strains isolated from children with fever or fever associated with otalgia; this seems an important point reinforcing biofilm production as a ubiquitous phenomenon in carriage. 3) More than 60% of S. pneumoniae and H. influenzae strains produced biofilm, and this proportion increased significantly for H. influenzae when the 2 bacterial species were isolated in the same sample. Furthermore, biofilm production globally did not differ by period or vaccine and non-vaccine type. 4) Our results agree with those of previous studies regarding the ability of some S. pneumoniae serotypes to produce biofilm.
Acknowledgements
We are grateful to the following paediatricians from the Association Clinique et Thérapeutique Infantile du Val de Marne (ACTIV) who provided the samples: E. Sobaral, M. Boucherat, I. Ramay, M. Fernandes, C. Prieur, D. Kern and A. Prieur.