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Evaluation of phage-based treatments for combating Staphylococcus aureus biofilms on dairy surfaces

ABSTRACT: The ability of Staphylococcus aureus to establish strong biofilms in milk poses a significant threat to the consumer’s health and a challenge for the dairy industry. Here, we have evaluated the efficacy of phage-based treatments (phage Kayvirus rodi and lytic protein LysRODI∆Ami) for combating biofilm formation on polystyrene and stainless steel surfaces submerged in UHT skim milk compared to growth medium. An adaptive transcriptional response was observed in cells intended to metabolize lactose and to increase biofilm formation. Also, biofilms grown in milk exhibited a denser and more complex matrix, mainly composed of proteins, presumably casein. These changes in matrix composition and structure offered a high resistance to both LysRODI∆Ami (4 µM) and phage Kayvirus rodi (108 PFU/ml) individual treatments, although combination of the two led to some reduction in the number of attached cells. Both antimicrobials were more successful at biofilm inhibition, exhibiting a synergistic interaction when used together. Unexpectedly, the bacteriophage treatment exhibited enhanced efficacy to prevent biofilm development in milk compared to fresh medium. The shift in matrix composition and pH modulation might be behind these differences. This research contributes to the development of innovative strategies for ensuring milk quality and safety. Moreover, our results demonstrate the importance of testing antimicrobials under conditions that reflect the real-life application as much as possible.

ORGANISM(S): Staphylococcus aureus

PROVIDER: GSE283972 | GEO | 2025/06/15

REPOSITORIES: GEO

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Project description:Environmental cues sometimes have a direct impact on phage particle stability, as well as bacterial physiology and metabolism, having a profound effect on phage infection outcome. Here, we explore the impact of temperature on the interplay between phage Kayvirus rodi (phiIPLA-RODI) and its host, Staphylococcus aureus. Our results show that phiIPLA-RODI is a more effective predator at room (25 °C) compared to body temperature (37 °C) against planktonic cultures of several strains with varying degrees of phage susceptibility. This result differs from most known examples of temperature-dependent phage infection, in which optimum infection is correlated with the host growth rate. Further characterization of this phenomenon was carried out with strains IPLA15 and IPLA16, whose respective MICs were 7 log units and a 1-log unit higher at 37 °C than at 25 °C. Our results demonstrated that the phage also had a greater impact at room temperature during biofilm development and for the treatment of preformed biofilms. There was no difference in phage adsorption between the two temperatures for strain IPLA16. Conversely, adsorption of phiIPLA-RODI to IPLA15 was reduced at 37 °C compared to 25 °C. Moreover, confocal microscopy analysis indicated that the biofilm matrix of both strains has a greater content of PIA/PNAG at 37 °C than at 25 °C. Regarding infection parameters, we observed longer duration of the lytic cycle at 25 °C for both strains, and infection of IPLA15 by phiIPLA-RODI resulted in a smaller burst size at 37 °C than at 25 °C. Finally, we also found that the rate of phage resistant mutant selection was higher at 37 °C for both strains. Altogether, this information highlights the impact that bacterial responses to environmental factors have on phage-host interactions. Moreover, phage phiIPLA-RODI appears to be a highly effective candidate for biofilm disinfection at room temperature, while its efficacy in biofilm-related infections will require combination with other antimicrobials.

Project description:For Streptococcus pneumoniae, biofilms have been suggested to promote long-term colonization of the nasopharynx and contribute to the pathology of recurrent middle ear infections. To date numerous studies have investigated the contribution of specific genetic determinants for the development of pneumococcal biofilms, however, studies examining the global changes that occur during biofilm development and how they contribute to disease are lacking. Using Scanning and Transmission electron microscopy we examined development of a mature pneumococcal biofilm in a continuous flow through reactor. We determined that a mature biofilm is formed in discrete stages, is marked by the formation of complex 3-dimensional structures, and is primarily composed of dead pneumococci. Using genomic microarrays we determined that pneumococci in mature biofilms down regulate genes involved in protein synthesis, energy production, metabolism, capsular polysaccharide production, and virulence. We confirmed these changes by testing bacterial resistance to antimicrobials, measuring capsule production by ELSIA, and immunoblotting for pneumolysin production. We determined that biofilm pneumococci are hyper-adhesive, binding to cell lines at levels 9 to 11-fold greater than planktonic counterparts. Using Western blot and ELISA, we determined that biofilm bacteria produce greater amounts of the adhesins PsrP, CbpA, and surface exposed phosphorylcholine. We subsequently determined that the hyper-adhesive phenotype was in part due to selection of the transparent phase variant during biofilm growth. Intranasal, intratracheal and intraperitoneal challenge of mice with biofilm and planktonic pneumococci determined that biofilm bacteria were highly attenuated for invasive disease but not nasopharyngeal colonization. Immunization of mice with ethanol-killed biofilm pneumococci of serotype 4 conferred protection against challenge with same isolate but not a serotype 3. ELISA for reactive IgG levels subsequently determined that biofilm pneumococci do not provide high levels of cross-reactive protein antigens. Together these studies suggest that biofilms do not directly contribute to disease but instead confer a protected mode of growth for the pneumococcus.

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Evaluation of phage-based treatments for combating Staphylococcus aureus biofilms on dairy surfaces

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