We previously demonstrated that mutation of the staphylococcal accessory regulator (sarA) in a clinical isolate of Staphylococcus aureus (UAMS-1) results in an impaired capacity to form a biofilm in vitro (K. E. Beenken, J. S. Blevins, and M. S. Smeltzer, Infect. Immun. 71:4206-4211, 2003). In this report, we used a murine model of catheter-based biofilm formation to demonstrate that a UAMS-1 sarA mutant also has a reduced capacity to form a biofilm in vivo. Surprisingly, mutation of the UAMS-1 ica locus had little impact on biofilm formation in vitro or in vivo. In an effort to identify additional loci that might be relevant to biofilm formation and/or the adaptive response required for persistence of S. aureus within a biofilm, we isolated total cellular RNA from UAMS-1 harvested from a biofilm grown in a flow cell and compared the transcriptional profile of this RNA to RNA isolated from both exponential- and stationary-phase planktonic cultures. Comparisons were done using a custom-made Affymetrix GeneChip representing the genomic complement of six strains of S. aureus (COL, N315, Mu50, NCTC 8325, EMRSA-16 [strain 252], and MSSA-476). The results confirm that the sessile lifestyle associated with persistence within a biofilm is distinct by comparison to the lifestyles of both the exponential and postexponential phases of planktonic culture. Indeed, we identified 48 genes in which expression was induced at least twofold in biofilms over expression under both planktonic conditions. Similarly, we identified 84 genes in which expression was repressed by a factor of at least 2 compared to expression under both planktonic conditions. A primary theme that emerged from the analysis of these genes is that persistence within a biofilm requires an adaptive response that limits the deleterious effects of the reduced pH associated with anaerobic growth conditions.

Global Gene Expression in Staphylococcus aureus Biofilms

A crucial role for exopolysaccharide modification in bacterial biofilm formation, immune evasion, and virulence.

In vitro and in vivo model systems to study microbial biofilm formation

The influence of microorganisms growing as sessile communities in a large number of human infections has been extensively studied and recognized for 30-40 years, therefore warranting intense scientific and medical research. Nonetheless, mimicking the biofilm-life style of bacteria and biofilm-related infections has been an arduous task. Models used to study biofilms range from simple in vitro to complex in vivo models of tissues or device-related infections. These different models have progressively contributed to the current knowledge of biofilm physiology within the host context. While far from a complete understanding of the multiple elements controlling the dynamic interactions between the host and biofilms, we are nowadays witnessing the emergence of promising preventive or curative strategies to fight biofilm-related infections. This review undertakes a comprehensive analysis of the literature from a historic perspective commenting on the contribution of the different models and discussing future venues and new approaches that can be merged with more traditional techniques in order to model biofilm-infections and efficiently fight them.

https://www.mdpi.com/2076-0817/2/2/288/pdf

From in vitro to in vivo Models of Bacterial Biofilm-Related Infections

Molecular basis of in-vivo biofilm formation by bacterial pathogens

Microbial adhesion and biofilm formation on medical implants is a common occurrence and represents a serious medical problem. Since biofilm microorganisms are difficult to eradicate with antibiotic therapy, chronic, recurrent infections often develop. With the increased use of prosthetic biomedical implants, chronic nosocomial infections have become prevalent in recent years (9, 41). Bacterial colonization of indwelling devices can be a prelude to both systemic infection and malfunction of the device.

A variety of techniques, such as direct microscopic enumeration, total viable count, metabolically active dyes, radiochemistry, and fluorescence, have been used to investigate microbial biofilms (1, 4, 8, 14, 17, 18, 23, 29, 33, 38). While some of these methods are useful for in vitro studies, they have not proved ideal for the investigation of biofilms in experimental infection models. The difficulty in analyzing biofilms in vivo lies in the lack of tools that allow noninvasive longitudinal study design. Assays developed to date, both direct and indirect, are time-consuming and laborious and involve the extraction of bacteria from support surfaces. To better understand and control biofilms on medical devices, rapid, direct, nondestructive, real-time quantitative monitoring methods that are adaptable to the clinical situation are needed. These assays may be used to develop new preventive and therapeutic methods to combat biofilm related infections.

To this end, bioluminescent reporters offer a method of labeling pathogens that is innocuous and allows the sensitive detection of only live, metabolically active cells by optical biophotonic imaging. Since bacterial luciferase requires reduced flavin mononucleotide for the generation of bioluminescence within the cell, it is a good indicator of the metabolic state of cells (22). Many biological applications of bioluminescent and fluorescent reporter gene systems have been developed during the last few years (1, 3, 10, 14, 19-23, 26-28, 33, 35-38). Monitoring a disease process in a living animal by using bioluminescent-tagged pathogens was first demonstrated in 1995 by Contag et al. (5). This noninvasive, rapid, real-time monitoring approach has been applied to a myriad of animal infection models and has proven to have significant advantages over conventional methods for studying disease and treatment in animals (6, 7, 11, 12, 31).

Monitoring bioluminescence as a measure of metabolic activity provides a rapid, quantitative in situ measure of biofilm development and physiological activities of bacteria within biofilms. In this study, we describe the detection of bioluminescent strains of Staphylococcus aureus and Pseudomonas aeruginosa, two of the most predominant biofilm-forming pathogens, using real-time monitoring to nondestructively, image catheter-based infections in an experimental animal model.

(Parts of the present study were presented at the 102nd General Meeting of the American Society for Microbiology, abstr. J6, 2002.)

https://iai.asm.org/content/iai/71/2/882.full.pdf

Michael Rubin

Papers

Direct continuous method for monitoring biofilm infection in a mouse model