Comparison of bacterial biomass and PRP production between different isolation of Haemophilus influenza type-b (Hib) under different culture conditions

Heamophilus influenzae type-b (Hib) is a gram - negative pleomorphic bacterium that causes meningitis infections in children with the age of less than ۵ years particularly in two years old infants. In the present study various isolates of Heamophilus influenzae from infants suspected to meningitis were collected, identified, characterized and were used throughout our experiments. Different culture media namely Brain Heart Infusion Broth (BHIB), Tryptic Soy Broth (TSB) and GC medium Base (GCB) which this medium was modified and prepared in our own laboratory, were compared to determine the highest bacterial yield. All media were added supplements ۱۰mg/ml hemin & ۰.۰۱/ml IsovitaleX containing V factor. The bacterial yield for all Hib strains present in our laboratory were measured with an initial inoculums of ۱۰۴ cfu per ml. The result showed very closed amount of biomass for all isolates however, GCB had slightly higher yield and ultimately we chose this medium for cultivation and extraction of capsular polysaccharide (CPS-b). In our laboratory we have adapted the PRP production according to our technical and instrumental availabilities which exists in our laboratories replacing ultra centrifugation to phenol chloroform to remove contaminants like endotoxin and proteins to the minimum level and also decreased the number of some chemical treatments while some steps were added in purification process. Our study showed although there were not significant differences between the PRP extract of the three isolates with average amount of ۱۰۸ mg/lit, however, isolate ATCC۱۰۲۱۰ (ATF۲) showed the highest amount with ۱۹۲mg/lit and the least PRP was produced by isolate H.inf.۱, with ۱۶ mg/lit. It seems that the data can be categorized to a normal distribution with the mean of ۱۰۶.۴ and standard deviation of ۶.۲۵. This result was confirmed by one sample kolmogorov-Smirnov test, hence the PRP ≥۱۹۲ mg/lit is statistically significant at a significant level of α =۰.۱۰ (P=۰.۰۸۵). The amount of contaminants e.g protein along with nucleic acid present in PRP was estimated at optical density ۲۸۰ and ۲۶۰nm, were under ۱۰% and ۵% respectively.

Keywords:

Haemophoilus influenzae type b (Hb) , Isolation , identification , polyribosylribitol phosphate (PRP) , bacterial biomass