Genetic requirement for pneumococcal ear infection

Abstract

Background: Ear infection or otitis media (OM) accounts for most bacterial respiratory infections in children in both developed and developing nations. Streptococcus pneumoniae, nontypeable Haemophilus influenzae, and Moraxella catarrhalis are the major OM pathogens. However, little is known about the genetic basis of bacterial OM largely due to practical difficulties in conducting research in ear infection models and genetically manipulating clinical isolates. Here, we report the first genome-scale in vivo screen for bacterial genes required for ear infection in a chinchilla model by signature tagged mutagenesis (STM), a high throughput mutant screen technique.

Methodology/principal findings: STM strains were constructed with a multi-drug resistant OM isolate ST556 (serotype 19F) and screened in a chinchilla OM model. Out of 5,280 mutants tested, 248 mutants were substantially underrepresented in the mutant pools recovered from the middle ear fluids of the infected chinchillas, indicating the impaired ability to survive and replicate in the middle ears due to genetic disruptions in the chromosome of strain ST556. Further DNA sequencing analysis mapped the mutations to 169 pneumococcal genes. Surprisingly, only 52 of these genes were required for pneumococcal nasopharyngeal colonization in a murine model. This infection site-specific gene requirement was verified by targeted mutagenesis in the selected genes.

Conclusions/significance: These findings suggest that there are a subset of pneumococcal genes required for ear infection and that these may be distinct from those required for nasal colonization. Our data thus provide comprehensive gene targets for mechanistic understanding of pneumococcal ear infection. Finally, this study has also developed a model for future genome-scale search for virulence determinants in other pathogens associated with ear infections.

Figure 1. Middle ear infection by pneumococcal strain ST556.

(A) Detection of ST556 encapsulation by transmission electron microscopy. (B) Detection of ST556 infection in the middle ears. Chinchillas were infected with bioluminescent pneumococci via tympanotomy and monitored daily for bacterial burden by bioluminescence. The scales on the right indicate the levels of photon counts.

Figure 2. Construction of STM strains in ST556.

(A) Sequence diversity of the STM constructs. The pneumococcal DNA inserts from 7 E. coli transformants (1–7) were released from the pID701t plasmid by restriction digestion with XhoI and XbaI, which flanked the original SmaI cloning site. DNA standards are indicated in kilobase (kb). (B) Random plasmid insertions in ST556 chromosome. Genomic DNA preparations from seven ST556 STM strains (1–7) were digested by HindIII, separated by agarose gel electrophoresis, and hybridized with plasmid-specific probe.

Figure 3. Negative selection of the STM strains.

(A) Detection of the attenuated STM strains. The same input pool #3 was used to infect the middle ears of chinchillas or nasopharynx of mice in the 2^nd round screen. The animals were sacrificed 3 days post infection to recover the bacteria (output pool) from the middle ears (chinchillas) or nasopharynx (mouse). The input and output pools were compared by PCR amplification and agarose gel electrophoresis. Pound signs (#) and asterisks (*) indicate the mutants that were missing in the output pools from the middle ear and nasopharynx, respectively. (B) Schematic representation of the STM strains identified in the 2^nd round screen. The red and yellow colors indicate the total numbers of the mutants that were attenuated only in the middle ear infection or nasal colonization, respectively. The mutants attenuated in both models are represented by blue color. (C) Schematic representation of the genes identified in the 2^nd round screen as in (B).

Figure 4. Quantification of the attenuation with ST556 deletion mutants.

(A) Middle ear co-infection. Both the left and right ears of chinchillas (n = 3) were infected by tympanotomy with a 1∶1 mixture of the wild type and isogenic deletion mutants ST1594 (SP0308), ST1595 (tetM), ST1601 (SP1732, stkP) or ST1603 (SP1448). Each symbol represents the CI value from a single ear of infected chinchillas. CI is defined as the output CFU ratio (mutant/wild type) divided by the input CFU ratio (mutant/wild type). Bar indicates the geometric mean CI value for each group of chinchillas. Statistical significance was determined by Student's t test. *, P<0.05, **, P<0.01. (B) Nasal co-infection. BALB/c mice (n = 5) were intranasally infected as in (A). The data were analyzed and presented as in (A).