Beta- Lactam Antibiotics Stimulate Biofilm Formation in Non-Typeable Haemophilus influenzae by Up-Regulating Carbohydrate Metabolism

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.

Sub-inhibitory concentrations of penicillin G induce biofilm formation by field isolates of Actinobacillus pleuropneumoniae

“…Numerous studies have shown that b-lactams below their MIC (e.g. 1/4 and 1/2 MIC) could induce biofilm formation of different bacterial species (Ahmed et al, 2009;Davies et al, 2006;Frank et al, 2007;Gomes et al, 2013;Kaplan, 2011;Mirani and Jamil, 2011;Nucleo et al, 2009;Wu et al, 2014). Our findings demonstrated that 1/2 MIC of penicillin G induced biofilm formation in A. pleuropneumoniae.…”

“…Low concentrations of antibiotics including b-lactams can potentially function as signalling molecules and induce virulence, quorum sensing and biofilm formation (Andersson and Hughes, 2014;Kaplan, 2011;Wu et al, 2014;Yim et al, 2007). The mechanisms associated with these effects differ for each species, isolates and antibiotics.…”

“…The biofilm matrix of A. pleuropneumoniae is mainly composed of an extracellular polymer of N-acetyl-D-glucosamine (PGA) (Kaplan et al, 2004). Numerous studies have shown that sub-minimum inhibitory concentrations (sub-MIC) of some antibiotics can affect growth, morphology, surface properties, pathogenicity and biofilm formation (Andersson and Hughes, 2014;Davies et al, 2006;Fonseca and Sousa, 2007;Gomes et al, 2013;Kaplan, 2011;Wu et al, 2014). Specifically, antibiotics at sub-MIC may induce bacterial biofilm.…”

Cephalosporin-3′-Diazeniumdiolate NO Donor Prodrug PYRRO-C3D Enhances Azithromycin Susceptibility of Nontypeable Haemophilus influenzae Biofilms

“…Biofilm formation by NTHi has been identified as playing a key role in both colonization and disease, and it contributes to ineffective antibiotic treatment (28). NO has been shown to signal a dispersal response in several biofilm-forming species, rendering the released bacteria susceptible to antibiotics (19)(20)(21).…”

Development of Broad-Spectrum Antibiofilm Drugs: Strategies and Challenges

“…Addition of exogenous indole or indole derivatives have a significant effect in the biofilm formation of both indoleproducing and nonindole producing bacteria and both Gram-negative and Gram-positive Escherichia coli Trancriptome Microarray Gene expression omnibus GSE3905 [84] Haemophilus influenzae Trancriptome Microarray Gene expression omnibus GSE58213 [85] Pseudomonas aeruginosa Trancriptome Microarray Gene expression omnibus GSE10030 [86] Staphylococcus epidermidis Trancriptome RNA-seq Gene expression omnibus GSE60834 [87] Vibrio cholerae Trancriptome Microarray Gene expression omnibus GSE42674 [88] Candida albicans, Streptococcus mutans Trancriptome Microarray Gene expression omnibus GSE52543 [89] Fusobacterium nucleatum, Streptococcus mutans, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Streptococcus sanguinis Trancriptome Microarray Gene expression omnibus GSE28841 [90] Aggregatibacter actinomycetemcomitans Proteome LC-MS/MS analysis PRIDE archive PXD000411 [91] Candida albicans Proteome LC-MS/MS analysis PRIDE archive PRD000640 [92] Pseudomonas bacteria are reported to involve TCS system in regulation of biofilm formation. Apart from the aforementioned bacterial regulatory pathways, the EPS matrix of various bacteria are found to share similar components and further studies in EPS may provide broad-spectrum drug targets for combating mature polymicrobial biofilm [113,114].…”