Histidine auxotrophy in commensal and disease-causing nontypeable Haemophilus influenzae

Abstract

Histidine biosynthesis is one of the best studied metabolic pathways in bacteria. Although this pathway is thought to be highly conserved within and between bacterial species, a previous study identified a genetic region within the histidine operon (his) of nontypeable strains of Haemophilus influenzae (NTHI) that was more prevalent among otitis media strains than among throat commensal NTHI strains. In the present study, we further characterized this region and showed that genes in the complete his operon (hisG, -D, -C, -NB, -H, -A, -F, and -IE) are >99% conserved among four fully sequenced NTHI strains, are present in the same location in these four genomes, and are situated in the same gene order. Using PCR and dot blot hybridization, we determined that the his operon was significantly more prevalent in otitis media NTHI strains (106/121; 87.7%) than in throat strains (74/137; 54%) (prevalence ratio, 1.62; P<0.0001), suggesting a possible role in middle ear survival and/or acute otitis media. NTHI strains lacking the his operon showed attenuated growth in histidine-restricted media, confirming them as his-negative auxotrophs. Our results suggest that the ability to make histidine is an important factor in bacterial growth and survival in the middle ear, where nutrients such as histidine may be found in limited amounts. Those isolates lacking the histidine pathway were still able to survive well in the throat, which suggests that histidine is readily available in the throat environment.

FIG. 1.

Genomic map of the his operon in strain Rd. □, location of sJPX132;▒, genes deleted in 23221 (throat strain) but present in G622 (middle ear strain) and Rd; ▪, genes present in 23221 (throat strain), G622 (middle ear strain), and Rd. (a) PCR protocol for each individual his gene and the flanking genes to determine size of insertion/deletion; (b) PCR protocol spanning the his region, used to verify size and location of the his indel. If an isolate is missing the complete his operon, a 2-kb product is amplified. If an isolate has the complete his operon, then a negative PCR will result, because the genomic region is too large to amplify (10.2 kb) using traditional PCR techniques.

FIG. 2.

Growth curves of selected NTHI isolates grown in specific media for 12 h. ⧫, growth in histidine-restricted medium; ▪, growth in histidine-supplemented medium, ▴, growth in Levinthal control medium. (a) Growth curves of isolates possessing the complete his operon; (b) growth curves of isolates missing the his operon. Isolate 23221 (throat isolate) gave inconsistent results in the histidine medium as well as the Levinthal control medium.