Nonencapsulated Streptococcus pneumoniae causes otitis media during single-species infection and during polymicrobial infection with nontypeable Haemophilus influenzae

Abstract

Streptococcus pneumoniae strains lacking capsular polysaccharide have been increasingly reported in carriage and disease contexts. Since most cases of otitis media involve more than one bacterial species, we aimed to determine the capacity of a nonencapsulated S. pneumoniae clinical isolate to induce disease in the context of a single-species infection and as a polymicrobial infection with nontypeable Haemophilus influenzae. Using the chinchilla model of otitis media, we found that nonencapsulated S. pneumoniae colonizes the nasopharynx following intranasal inoculation, but does not readily ascend into the middle ear. However, when we inoculated nonencapsulated S. pneumoniae directly into the middle ear, the bacteria persisted for two weeks post-inoculation and induced symptoms consistent with chronic otitis media. During coinfection with nontypeable H. influenzae, both species persisted for one week and induced polymicrobial otitis media. We also observed that nontypeable H. influenzae conferred passive protection from killing by amoxicillin upon S. pneumoniae from within polymicrobial biofilms in vitro. Therefore, based on these results, we conclude that nonencapsulated pneumococci are a potential causative agent of chronic/recurrent otitis media, and can also cause mutualistic infection with other opportunists, which could complicate treatment outcomes.

Keywords: Haemophilus; coinfection; nonencapsulated; pneumococcus.

Graphical Abstract Figure.

Pneumococcal variants lacking capsular polysaccharide are increasingly common in carriage and in some disease states; in this study we show that one such clone can persist and cause chronic otitis media.

Figure 1.

NTSp colonizes the chinchilla nasopharynx with minimal ascension into the middle ear. Chinchillas were inoculated intranasally with NTSp strain MNZ1113. Pneumococcal bacterial loads were quantitated at one, four and seven days post-inoculation in the nasopharynx (A), Eustachian tubes (B) and middle ear bullae (C). Dotted line indicates limit of detection.

Figure 2.

NTSp colonizes the chinchilla middle ear and induces otitis media. Chinchillas were inoculated via transbullar injection with a high- or low-dose S. pneumoniae strain MNZ1113. At three and seven days post-inoculation, pneumococcal bacterial loads were quantitated in homogenized middle ear bullae (A) and middle ear fluid (B). The dashed line indicates the bacterial limit of detection.

Figure 3.

NTSp persists through two weeks in the chinchilla middle ear. Chinchilla middle ears were inoculated via transbullar injection of NTSp strain MNZ1113. Total pneumococcal cfu were quantitated at 1, 7 and 14 days post-inoculation in homogenized middle ear bullae (A) and middle ear fluid (B). Disease was assessed by via otoscopic examination (C). The solid horizontal lines indicate geometric mean (A) or mean (C). The dashed lines indicate the bacterial limit of detection.

Figure 4.

NTSp and NTHi persist together in the chinchilla middle ear. Chinchillas were inoculated via transbullar injection with a high or low dose of NTSp, alone or in combination with NTHi (see the section ‘Materials and Methods’). At four and seven days post-inoculation, animals were euthanized and bacterial loads were assessed for S. pneumoniae (A and C) and NTHi (B and D) in homogenized middle ear tissue (A and B) and middle ear fluid (C and D). The geometric mean (solid horizontal line) was calculated for each group and did not include values below the bacterial limit of detection (dashed horizontal lines). Inflammation was evaluated by otoscopic examination at one, four and six days post-inoculation (E); graph shows mean otoscopy scores, with error bars denoting the range of scores in each group. *P < 0.05; **P < 0.01.

Figure 5.

NTHi protects NTSp against beta-lactam antibiotics in vitro. NTSp and NTHi were grown as static biofilm cultures, alone or in co-culture. At 24 h, cultures were treated with 125 μg ml⁻¹ of amoxicillin and cultures were incubated for an additional 24 h. Surface-attached bacteria were removed and plated on selective media to quantitate NTSp (A) and NTHi (B). Graphs indicate mean bacterial cfu; error bars denote 95% confidence interval. The dashed lines denote the bacterial limit of detection.