Virulence phenotypes of low-passage clinical isolates of nontypeable Haemophilus influenzae assessed using the chinchilla laniger model of otitis media

Abstract

Background: The nontypeable Haemophilus influenzae (NTHi) are associated with a spectrum of respiratory mucosal infections including: acute otitis media (AOM); chronic otitis media with effusion (COME); otorrhea; locally invasive diseases such as mastoiditis; as well as a range of systemic disease states, suggesting a wide range of virulence phenotypes. Genomic studies have demonstrated that each clinical strain contains a unique genic distribution from a population-based supragenome, the distributed genome hypothesis. These diverse clinical and genotypic findings suggest that each NTHi strain possesses a unique set of virulence factors that contributes to the course of the disease.

Results: The local and systemic virulence patterns of ten genomically characterized low-passage clinical NTHi strains (PittAA - PittJJ) obtained from children with COME or otorrhea were stratified using the chinchilla model of otitis media (OM). Each isolate was used to bilaterally inoculate six animals and thereafter clinical assessments were carried out daily for 8 days by blinded observers. There was no statistical difference in the time it took for any of the 10 NTHi strains to induce otologic (local) disease with respect to any or all of the other strains, however the differences in time to maximal local disease and the severity of local disease were both significant between the strains. Parameters of systemic disease indicated that the strains were not all equivalent: time to development of the systemic disease, maximal systemic scores and mortality were all statistically different among the strains. PittGG induced 100% mortality while PittBB, PittCC, and PittEE produced no mortality. Overall Pitt GG, PittII, and Pitt FF produced the most rapid and most severe local and systemic disease. A post hoc determination of the clinical origins of the 10 NTHi strains revealed that these three strains were of otorrheic origin, whereas the other 7 were from patients with COME.

Conclusion: Collectively these data suggest that the chinchilla OM model is useful for discriminating between otorrheic and COME NTHi strains as to their disease-producing potential in humans, and combined with whole genome analyses, point the way towards identifying classes of virulence genes.

Figure 1

Rapidity of local disease onset. Scatterplot showing the number of days it took for chinchillas inoculated with the 10 clinical NTHi strains to develop moderate or worse (score =/> 2) local (otologic) disease. X-axis = the clinical NTHi strains (PittAA-PittJJ, left to right); Y-axis = the days post inoculation that moderate or worse local disease developed.

Figure 2

Rapidity of most severe local disease. Scatterplot showing the number of days it took for chinchillas inoculated with the 10 clinical NTHi strains to develop their maximum (most severe) otologic score – regardless of what that score was. X-axis = the clinical NTHi strains (PittAA-PittJJ, left to right); Y-axis = the days post inoculation that moderate or worse local disease developed.

Figure 3

Maximum otologic score per animal. Scatterplot showing the maximum otologic severity – regardless of time – recorded for each of the chinchillas inoculated with the 10 clinical NTHi strains. X-axis = the clinical NTHi strains (PittAA-PittJJ, left to right); Y-axis = the otologic clinical score based upon the criteria in Table 1.

Figure 4

Rapidity of systemic disease onset. Scatterplot showing the number of days it took for chinchillas inoculated with the 10 clinical NTHi strains to first develop their maximum (most severe)significant systemic signs (systemic score ≥1 – regardless of what their eventual maximum severity was. X-axis = the clinical NTHi strains (PittAA-PittJJ, left to right); Y-axis = the days post inoculation that the first signs of systemic disease developed.

Figure 5

Maximum systemic severity. Scatterplot showing the maximum systemic severity – regardless of time – recorded for each of the chinchillas inoculated with the 10 clinical NTHi strains. X-axis = the clinical NTHi strains (PittAA-PittJJ, left to right); Y-axis = the systemic clinical score based upon the criteria in Table

Figure 6

Differences in mortality. Kaplan-Meyer plots showing the differences in mortality induced by the 10 NTHi strains, PittAA-PittJJ. X-axis = time in days following inoculation; Y-axis percentage of surviving animals.

Figure 7

Degree of genic sharing of distributed or non-core genes. Dendrogram developed using the unweighted pair group method algorithm demonstrating the degree of genic sharing of distributed or non-core genes [26,28] which are defined as the set of genes not universally present among all strains of the species. The figure compares 15 NTHi strains, which include 9 of the strains phenotyped in the current study (PittDD was omitted due to incomplete genomic data and PittFF and PittGG collapse to a single strain using this method) and the laboratory strain Rd. The sequence for the 86028NP strain has been previously published [18], and the unannotated sequences for the R2866 and R2846 NTHi strains were obtained from Genbank (accession #s NZ_AADP00000000, and NZ_AADO00000000, respectively) and used with permission of the depositing authors. The X-axis lists the number of genic differences between strains; y-axis lists the H. influenzae strains. Strain 86028NP is a nasopharyngeal (NP) isolate obtained from a patient suffering from OM; R2866 is an invasive strain; CHSHi22121 is an NP isolate from a well child; R2846 is an COME isolate; CGSHiR3021 and CGSHi22421 are NP isolates from healthy children; and CHSHi3655 is an OME strain.