Characterization of Genetic and Phenotypic Diversity of Invasive Nontypeable Haemophilus influenzae

Abstract

The ability of unencapsulated (nontypeable) Haemophilus influenzae (NTHi) to cause systemic disease in healthy children has been recognized only in the past decade. To determine the extent of similarity among invasive nontypeable isolates, we compared strain R2866 with 16 additional NTHi isolates from blood and spinal fluid, 17 nasopharyngeal or throat isolates from healthy children, and 19 isolates from middle ear aspirates. The strains were evaluated for the presence of several genetic loci that affect bacterial surface structures and for biochemical reactions that are known to differ among H. influenzae strains. Eight strains, including four blood isolates, shared several properties with R2866: they were biotype V (indole and ornithine decarboxylase positive, urease negative), contained sequence from the adhesin gene hia, and lacked a genetic island flanked by the infA and ksgA genes. Multilocus sequence typing showed that most biotype V isolates belonged to the same phylogenetic cluster as strain R2866. When present, the infA-ksgA island contains lipopolysaccharide biosynthetic genes, either lic2B and lic2C or homologs of the losA and losB genes described for Haemophilus ducreyi. The island was found in most nasopharyngeal and otitis isolates but was absent from 40% of invasive isolates. Overall, the 33 hmw-negative isolates were much more likely than hmw-containing isolates to have tryptophanase, ornithine decarboxylase, or lysine decarboxylase activity or to contain the hif genes. We conclude (i) that invasive isolates are genetically and phenotypically diverse and (ii) that certain genetic loci of NTHi are frequently found in association among NTHi strains.

Adhesin genes and serum resistance in Haemophilus influenzae type f isolates

“…b-Lactamase activity was detected in 2/26 (7.7 %) Hif strains, R3788 and R3907, by an ampicillin hydrolysis assay. Consistent with the detected b-lactamase activity, strain R3907, a blood isolate, and R3788, a sinus isolate, were the only isolates determined to carry a large conjugative plasmid associated with blactamase activity, as they produced an~450 bp amplicon by PCR with primers topoF and topoR (Erwin et al, 2005).…”

“…b-Lactamase activity was detected in 2/26 (7.7 %) Hif strains, R3788 and R3907, by an ampicillin hydrolysis assay. Consistent with the detected b-lactamase activity, strain R3907, a blood isolate, and R3788, a sinus isolate, were the only isolates determined to carry a large conjugative plasmid associated with blactamase activity, as they produced an~450 bp amplicon by PCR with primers topoF and topoR (Erwin et al, 2005).Hif isolate typing by repPCR fingerprinting DNA fingerprinting for the confirmed Hif isolates was performed using repPCR with primers detecting ERIC sequences (Healy et al, 2005a), and amplicons were resolved using bioMérieux Diversilab Bacterial Barcodes software to generate a similarity index. Isolates with similarity indices .85 % were considered identical in this study (Healy et al, 2005a, b).…”

“…Genomic DNA was prepared using a DNeasy Tissue kit (Qiagen). Routine PCR conditions and DNA manipulations were performed as described previously (Erwin et al, 2005). For classification purposes, the absence of an amplicon, or an amplicon of ,100 bp, was considered negative for that locus.…”

“…Genomic DNA was prepared using a DNeasy Tissue kit (Qiagen). Routine PCR conditions and DNA manipulations were performed as described previously (Erwin et al, 2005). Initial screening of putative type f strains was with a multiplex PCR, using primers and methods reported previously (Nelson & Smith, 2010).…”

“…In R2846 (strain 12) and~20 % of other non-typable strains, the locus contains the glycosyltransferases losA and losB, whose expression is phase variable due to the presence of the octameric repeat 59-CGAGCATA-39, with the number of repeats varying between two and ten, modifying the translational reading frame; expression of these enzymes alters the structure of the surface LOS, affecting resistance to NHS (Erwin et al, 2006b). Both the lic2B-lic2C and the losA-losB regions are within the 2.2 kb amplicon found with the infA-ksgA primers; these regions can be differentiated by subsequent PCR and/or sequencing using allele-specific primers as reported previously (Erwin et al, 2005). Because of the heterogeneity of this island we examined the composition of 26 type f strains by PCR for the infA-ksgA locus using the infA-ksgA primer set (Table 2) and subsequent PCR using the lic2B-F and lic2BA primer pair to identify the lic2BC allele, or the infA-ksgA F and losB-R primer pair to identify the losAB allele.…”