Characterization of nasopharyngeal isolates of type b Haemophilus influenzae from Delhi

Abstract

Background & objectives: Haemophilus influenzae is an important cause of mortality and morbidity among young children in developing countries. Increasing incidence of antibiotic resistance especially production of extended spectrum beta lactamase (ESBL) has made treatment and management of H. influenzae infection more difficult. Nasopharyngeal H. influenzae isolates are excellent surrogate for determination of antibiotic resistance prevalent among invasive H. influenzae isolates. In this study, we characterized nasopharyngeal H. influenzae isolates obtained from healthy school going children in Delhi.

Methods: Nasopharyngeal H. influenzae isolates were collected from healthy school going children and subjected to serotyping, fimbrial typing and antibiogram profiling. ESBL production was recorded using phenotypic as well as molecular methods. Multi locus sequence typing (MLST) of 13 representative nasopharyngeal H. influenzae isolates was performed as per guidelines.

Results: A significant proportion (26 of 80, 32.5%) of nasopharyngeal isolates of H. influenzae were identified as serotype b. Fimbrial gene (hifA) was detected in 23 (28.75%) isolates. Resistance against commonly prescribed antibiotics (Amp, Tet, Chloro, Septran, Cephalexin) were observed to be high among the nasopharyngeal commensal H. influenzae. Extended spectrum beta lactamase (ESBL) production was observed in a five (6.25%) isolates by both double disk diffusion and molecular typing. MLST identified several novel alleles as well as novel sequence types.

Interpretation & conclusions: Our findings showed high resistance against common antibiotics and detection of ESBL in nasopharyngeal H. influenzae isolates collected from normal healthy school going children in Delhi. Detection of H. influenzae type b capsular gene and the presence of fimbrial gene (hif A) suggest virulence potential of these isolates. Discovery of novel alleles and presence of new sequence types (STs) among nasopharyngeal H. influenzae isolates may suggest wider genetic diversity.

Fig. 1

PCR amplified product (capB) in 1.5% agarose gel. Lane M: 100 bp Ladder, Lane 1: Positive control, Lanes 2-9: amplification of 480 bp from H. influenzae isolates, Lane 10: Negative control.

Fig. 2

PCR amplified product (hifA) electrophoresed in 1.5% agarose gel. Lane M: 50 bp Ladder, Lane1: Positive control, Lanes 2-15: amplified 800 bp product H. influenzae isolates, Lane 16: Negative control.

Fig. 3

PCR amplified product (TEM) electrophoresed in 1.5% agarose gel. Lane M: 100 bp Ladder, Lanes 1-2: amplification of 717 bp from H. influenzae isolates, Lanes 3: Klebsiella pneumoniae ATCC 700603 and Lane 4: Negative control.

Fig. 4

H. influenzae isolates UPGMA dendrogram based on MLST data for 13 type b.