A chemically defined medium that supports the growth of both encapsulated and nontypeable Haemophilus influenzae strains in broth to densities that are > 10 9 CFU/ml or on agar plates is described. The mean generation time of a panel of clinical isolates was comparable to that in rich, chemically undefined media (brain-heart infusion broth supplemented with heme and ␤-NAD).Haemophilus influenzae is a fastidious, gram-negative coccus that inhabits the upper respiratory system of humans and has an obligate requirement for heme and ␤-NAD for aerobic growth. Prior investigators studying Haemophilus influenzae sought a chemically defined medium to facilitate genetic and metabolic studies. Multiple defined media were devised for use with Rd derivatives of H. influenzae (2,3,4,5,6,8,13,15), but these media would not support the growth of many nontypeable clinical isolates or encapsulated strains.During the course of experiments studying the invasion of human cell lines by pathogenic H. influenzae, we found that RPMI medium-based tissue culture media appeared to support bacterial growth. Further studies led to the development of the chemically defined media described herein. Table 1 describes the strains used in this study, which were stored at Ϫ70°C in 10% skim milk and were subcultured onto chocolate agar. One liter of chocolate agar was prepared by adding 36 g of GC base (catalog no. 228920; Difco, Detroit, Mich.) and 10 g of hemoglobin (catalog no. ZIZ392; BD Biosciences, Sparks, Md.) and autoclaving at 121°C at 15 lb/in 2 for 20 min; after cooling to 55°C, 5,000 U of bacitracin and 10 ml of GCHI Rehydrating Solution (catalog no. 450411; Remel, Lenexa, Kans.) were added and the plates were poured. Supplemented brain-heart infusion (sBHI) agar was prepared by adding 37 g of BHI media (catalog no. 211059; BD Biosciences) and 15 g of BactoAgar (catalog no. 214530; BD Biosciences) to 1 liter of deionized water, autoclaving, cooling to 55°C, and adding 10 ml of ␤-NAD and 10 ml of heme and L-histidine stock. The heme-histidine stock was prepared by dissolving 0.2 g of L-histidine (freebase; Sigma catalog no. H 8000) in 200 ml of deionized water and then adding 0.2 g of hemin HCl (catalog no. H 5533, bovine; Sigma) and 4 ml of 1 N NaOH and steaming over a boiling water bath for 5 to 10 min to solubilize the mixture. The solution was then cooled to room temperature, filter sterilized (0.2-m pore size), and placed in a foil-covered bottle at 4°C. We found that the histidine decreased the rate at which the hemin precipitated from solution. ␤-NAD ϩ stock was prepared by dissolving 100 mg of ␤-NAD ϩ (catalog no. N 7004; Sigma) in 100 ml of deionized water, filter sterilizing (0.2-m pore size), and storing at 4°C. sBHI broth was prepared without the agar.Defined liquid medium was made with the following: 191 ml of RPMI 1640 with L-glutamine and 25 mM HEPES, pH 7.26 (catalog no. 61870036; InVitrogen), 2 ml of a 100 mM MEM sodium pyruvate solution (catalog no. 11360070; InVitrogen), 2 ml of ␤-NAD ϩ stock, 4 ml of heme-L-histidine ...

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We are investigating a nontypeable Haemophilus influenzae (NTHI) strain, R2866, isolated from a child with meningitis. R2866 is unusually resistant to killing by normal human serum. The serum 50% inhibitory concentration (IC 50 ) for this strain is 18%, approaching that of encapsulated H. influenzae. R3392 is a derivative of R2866 that was found to have increased sensitivity to human serum (IC 50 , 1.5%). Analysis of tetrameric repeat regions within lipooligosaccharide (LOS) biosynthetic genes in both strains indicated that the glycosyltransferase gene lgtC was out of frame ("off") in most colonies of R3392 but in frame with its start codon ("on") in most colonies of the parent. We sought antigenic and biochemical evidence for modification of the LOS structure. In a whole-cell enzyme-linked immunosorbent assay, strain R3392 displayed reduced binding of the Gal␣1,4Gal-specific monoclonal antibody 4C4. Mass spectrometry analysis of LOS from strain R2866 indicated that the primary oligosaccharide glycoform contained four heptose and four hexose residues, while that of R3392 contained four heptose and three hexose residues. We conclude that the R2866 lgtC gene encodes a galactosyltransferase involved in synthesis of the 4C4 epitope, as in other strains, and that expression of lgtC is associated with the high-level serum resistance that has been observed for this strain. This is the first description of the genetic basis of high-level serum resistance in NTHI, as well as the first description of LOS composition in an NTHI strain for which the complete genome sequence has been determined.The gram-negative bacterium Haemophilus influenzae is a common commensal and occasional pathogen of the upper respiratory tract. Prevalence studies have shown that H. influenzae strains isolated from the upper airway are predominately unencapsulated (nontypeable H. influenzae [NTHI]). While most disease associated with NTHI is limited to mucosal sites, a small proportion of NTHI infections are systemic. Many of these are in elderly adults, occurring as a complication of pneumonia. In the past decade, there have been several reports of NTHI strains or strains with capsules of types other than b isolated from the blood or cerebrospinal fluid (CSF) of apparently normal children who had been immunized with an H. influenzae type b (Hib) vaccine (7,8,10,38). To facilitate development of vaccines able to prevent all H. influenzae infections, an understanding of the virulence of invasive NTHI is critical.A 1996 case report was one of the first to describe NTHI meningitis in a child with normal immunological and anatomical status (36). We found that R2866, the strain isolated from this child, is nearly as resistant as a virulent type b strain, E1a, to the bactericidal effects of normal adult human serum. We demonstrated that R2866 lacks the genes for capsule synthesis and secretion. Using flow cytometry, we showed that when R2866 is incubated in 40% normal human serum, the classical pathway of complement activation is initiated normally, but t...

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