The Behavior of Staphylococcus aureus Dual-Species Biofilms Treated with Bacteriophage phiIPLA-RODI Depends on the Accompanying Microorganism

Abstract

The use of bacteriophages as antimicrobials against pathogenic bacteria offers a promising alternative to traditional antibiotics and disinfectants. Significantly, phages may help to remove biofilms, which are notoriously resistant to commonly used eradication methods. However, the successful development of novel antibiofilm strategies must take into account that real-life biofilms usually consist of mixed-species populations. Within this context, this study aimed to explore the effectiveness of bacteriophage-based sanitation procedures for removing polymicrobial biofilms from food industry surfaces. We treated dual-species biofilms formed by the food pathogenic bacterium Staphylococcus aureus in combination with Lactobacillus plantarum, Enterococcus faecium, or Lactobacillus pentosus with the staphylococcal phage phiIPLA-RODI. Our results suggest that the impact of bacteriophage treatment on S. aureus mixed-species biofilms varies depending on the accompanying species and the infection conditions. For instance, short treatments (4 h) with a phage suspension under nutrient-limiting conditions reduced the number of S. aureus cells in 5-h biofilms by ϳ1 log unit without releasing the nonsusceptible species. In contrast, longer infection periods (18 h) with no nutrient limitation increased the killing of S. aureus cells by the phage (decrease of up to 2.9 log units). However, in some cases, these conditions promoted the growth of the accompanying species. For example, the L. plantarum cell count in the treated sample was up to 2.3 log units higher than that in the untreated control. Furthermore, phage propagation inside dual-species biofilms also depended greatly on the accompanying species, with the highest rate detected in biofilms formed by S. aureus-L. pentosus. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) also showed changes in the three-dimensional structures of the mixed-species biofilms after phage treatment. Altogether, the results presented here highlight the need to study the impact of phage therapy on microbial communities that reflect a more realistic setting.IMPORTANCE Biofilms represent a major source of contamination in industrial and hospital settings. Therefore, developing efficient strategies to combat bacterial biofilms is of the utmost importance from medical and economic perspectives. Bacteriophages have shown potential as novel antibiofilm agents, but further research is still required to fully understand the interactions between phages and biofilm-embedded bacteria. The results presented in this study contribute to achieving a better understanding of such interactions in a more realistic context, considering that most biofilms in the environment consist of mixed-species populations. KEYWORDS Staphylococcus aureus, biofilms, phage

Using Bacteriophages as a Trojan Horse to the Killing of Dual-Species Biofilm Formed by Pseudomonas aeruginosa and Methicillin Resistant Staphylococcus aureus

“…In this scenario, bacteriophages appear to be an alternative strategy to treat biofilm-forming infections. Over the past few years, numerous studies have been carried out investigating the effectiveness of phages against mono-and dualspecies biofilm (Sillankorva et al, 2010;Chhibber et al, 2015;Gutierrez et al, 2015;Gonzalez et al, 2017;Melo et al, 2019). Many of these studies pointed to a notable dependency between the phages and the bacterial species involved on the biofilm for the efficacy of the phage treatment.…”

Bacteriophages inhibit biofilms formed by multi-drug resistant bacteria isolated from septic wounds

“…The lytic activity of bacteriophages was examined and confirmed by the CLSM. Disruption of the cell membrane due to lysis leads to penetration of propidium iodide stain, whereas live bacterial cells that are not lysed stain with SYTO ® 9 appear in green color (Ansari et al, 2015; Hu et al, 2015; González et al, 2017). The application of phages on MDR-bacterial pathogens present in the single species biofilms showed decreased biomass to almost 80%.…”

“…After 15 min incubation at room temperature in the dark, samples were washed with sterile saline for removing the excess dye and rinsed with water from the base of the support material. In order to minimize the air contact and maintain constant sample moisture conditions, a coverslip was used on the specimen (González et al, 2017). Due to the bacteriolytic activity of phages leads to the disruption of bacterial biofilm integrity and allows the propidium dye and appears in red color and live bacteria present in the biofilm appears in green color.…”

“…MDR-bacterial biofilms and respective phage treated slides were subjected to CLSM to detect the effect of bacteriophages on the MDR-bacterial biofilms. The staining with FilmTracer™ LIVE/DEAD ® Biofilm viability kit (Molecular Probes, Life Technologies Ltd) was performed according to the instructions provided by the manufacturer (Ansari et al, 2015; Hu et al, 2015; González et al, 2017).…”

Analysis of Different Parameters Affecting Diffusion, Propagation and Survival of Staphylophages in Bacterial Biofilms

“…Indeed, several studies have shown that staphylophages alone or in combination with other compounds can be effective for biofilm removal ( Son et al, 2010 ; Kelly et al, 2012 ). Quite recently, Gutiérrez et al (2015) isolated two virulent staphylophages, vB_SauM_ phiIPLA-RODI (phiIPLA-RODI) and vB_SepM_ phiIPLA-C1C (phiIPLA-C1C) that exhibited antibiofilm activity against both single-species and mixed-species biofilms formed by S. aureus ( Gutiérrez et al, 2015 ; González et al, 2017 ). Here, we set out to determine the ability of the aforementioned phages to penetrate biofilms formed by different species and strains, both sensitive and insensitive to the viruses.…”