In vitro Multi-Species Biofilms of Methicillin-Resistant Staphylococcus aureus and Pseudomonas aeruginosa and Their Host Interaction during In vivo Colonization of an Otitis Media Rat Model

Abstract

Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) are known to cause biofilm-related infections. MRSA and PA have been frequently isolated from chronically infected wounds, cystic fibrosis, chronic suppurative otitis media (CSOM), and from indwelling medical devices, and these bacteria co-exist; however, their interaction with each-other or with the host is not well known. In this study, we investigated MRSA and PA multi-species biofilm communities in vitro and their interaction with the host during in vivo colonization using an OM rat-model. In-vitro biofilm formation and in-vivo colonization were studied using CV-microtiter plate assay and OM rat-model respectively. The biofilms were viewed under scanning electron microscope and bacteria were enumerated using cfu counts. The differential gene expressions of rat mucosa colonized with single or multi-species of MRSA or PA were studied using RNA-sequencing of total transcriptome. In multi-species in-vitro biofilms PA partially inhibited SA growth. However, no significant inhibition of MRSA was detected during in-vivo colonization of multi-species in rat bullae. A total of 1,797 genes were significantly (p < 0.05) differentially expressed in MRSA or PA or MRSA + PA colonized rat middle ear mucosa with respect to the control. The poly-microbial colonization of MRSA and PA induced the differential expression of a significant number of genes that are involved in immune response, inflammation, signaling, development, and defense; these were not expressed with single species colonization by either MRSA or PA. Genes involved in defense, immune response, inflammatory response, and developmental process were exclusively up-regulated, and genes that are involved in nervous system signaling, development and transmission, regulation of cell growth and development, anatomical and system development, and cell differentiation were down-regulated after multi-species inoculation. These results indicate that poly-microbial colonization induces a host response that is different from that induced by single species infection.

Options and Limitations in Clinical Investigation of Bacterial Biofilms

“…RNA sequencing (RNA-seq) analyses based on next-generation sequencing (NGS) are now replacing conventional microarrays and provide higher sensitivity and information pertaining to small noncoding RNAs actively participating in oral pathogen dysbiotic processes (180). Additionally, RNA-seq-based transcriptomic analyses have provided a multitude of genes associated with biofilm formation by both Gram-positive and Gram-negative bacterial species (such as MRSA, Enterococcus faecalis, and P. aeruginosa) in clinical entities, including otitis media, CF, chronic wounds, and endodontic and indwelling device infections (181,182). NGS platforms provide massive parallel sequencing of DNA segments physically or artificially produced from a single sample.…”

Asian Sand Dust Particles Increased Pneumococcal Biofilm Formation in vitro and Colonization in Human Middle Ear Epithelial Cells and Rat Middle Ear Mucosa

“…The in vitro biofilms of Pseudomonas aeruginosa, MRSA, and E. coli were grown as described above and quantified using resazurin staining (Yadav et al, 2017). Resazurin stain is a blue colored, non-fluorescent dye that is reduced and then emits pink fluorescence (resorufin) in the presence of actively growing bacteria.…”

Subversion of host immune responses by otopathogens during otitis media

“…This influences the quorum signaling pathway and can result in the development as well as the establishment of biofilms . Biofilms can be particularly pathogenic in the case of OM, as the infections are polymicrobial and can hence create synergistic reactions, increased growth of organisms, antimicrobial tolerance, increased virulence and persistence, as well as exaggerated levels of exopolysaccharide (EPS) . Biofilms have been found to be associated with adenoid hypertrophy and middle ear effusion .…”