Beta- lactam antibiotics stimulate biofilm formation in non-typeable haemophilus influenzae by up-regulating carbohydrate metabolism.

Siva Wu,Xiaojin Li,Charles Wang,Debbie Guerrero-Given,Kathleen Maguire,Manjula Gunawardana,Paul Webster,Marc M Baum,Christoph Schaudinn,Tom Coenye

doi:10.1371/journal.pone.0099204

Abstract

Non-typeable Haemophilus influenzae (NTHi) is a common acute otitis media pathogen, with an incidence that is increased by previous antibiotic treatment. NTHi is also an emerging causative agent of other chronic infections in humans, some linked to morbidity, and all of which impose substantial treatment costs. In this study we explore the possibility that antibiotic exposure may stimulate biofilm formation by NTHi bacteria. We discovered that sub-inhibitory concentrations of beta-lactam antibiotic (i.e., amounts that partially inhibit bacterial growth) stimulated the biofilm-forming ability of NTHi strains, an effect that was strain and antibiotic dependent. When exposed to sub-inhibitory concentrations of beta-lactam antibiotics NTHi strains produced tightly packed biofilms with decreased numbers of culturable bacteria but increased biomass. The ratio of protein per unit weight of biofilm decreased as a result of antibiotic exposure. Antibiotic-stimulated biofilms had altered ultrastructure, and genes involved in glycogen production and transporter function were up regulated in response to antibiotic exposure. Down-regulated genes were linked to multiple metabolic processes but not those involved in stress response. Antibiotic-stimulated biofilm bacteria were more resistant to a lethal dose (10 µg/mL) of cefuroxime. Our results suggest that beta-lactam antibiotic exposure may act as a signaling molecule that promotes transformation into the biofilm phenotype. Loss of viable bacteria, increase in biofilm biomass and decreased protein production coupled with a concomitant up-regulation of genes involved with glycogen production might result in a biofilm of sessile, metabolically inactive bacteria sustained by stored glycogen. These biofilms may protect surviving bacteria from subsequent antibiotic challenges, and act as a reservoir of viable bacteria once antibiotic exposure has ended.

Highlights

Non-typeable Haemophilus influenzae (NTHi), a Gram-negative, rod-shaped bacterium is a common commensal organism in the human upper respiratory tract
Biofilm formation is stimulated by beta-lactam antibiotics A total of six strains of NTHi bacteria were chosen for this study based on their origin and history
The anomalous measurement obtained from the untreated PittGG biofilm was most probably due to the inability of the protein assay to detect the low level of protein present in the biofilm

Summary

Introduction

Non-typeable Haemophilus influenzae (NTHi), a Gram-negative, rod-shaped bacterium is a common commensal organism in the human upper respiratory tract. NTHi is a major causative agent of acute otitis media (AOM, middle ear infection) [4,5,6]. Tympanostomy tube insertion, a common treatment for chronic serous otitis media [8,9], has a treatment cost of $2,174 per patient [10]. NTHi has been linked to community-acquired pneumonia in children [11], infections in cystic fibrosis patients [12], and upper respiratory tract infections [13,14,15,16,17]. NTHi can cause sinus infections [3,18] and has been linked to morbidity in patients with chronic obstructive pulmonary disease (COPD) [14,15,16]

Methods

Results

Discussion

Conclusion