Two Phages, phiIPLA-RODI and phiIPLA-C1C, Lyse Mono- and Dual-Species Staphylococcal Biofilms

Abstract

Phage therapy is a promising option for fighting against staphylococcal infections. Two lytic phages, vB_SauM_phiIPLA-RODI (phiIPLA-RODI) and vB_SepM_phiIPLA-C1C (phiIPLA-C1C), belonging to the Myoviridae family and exhibiting wide host ranges, were characterized in this study. The complete genome sequences comprised 142,348 bp and 140,961 bp and contained 213 and 203 open reading frames, respectively. The gene organization was typical of Spounavirinae members, with long direct terminal repeats (LTRs), genes grouped into modules not clearly separated from each other, and several group I introns. In addition, four genes encoding tRNAs were identified in phiIPLA-RODI. Comparative DNA sequence analysis showed high similarities with two phages, GH15 and 676Z, belonging to the Twort-like virus genus (nucleotide identities of >84%); for phiIPLA-C1C, a high similarity with phage phiIBB-SEP1 was observed (identity of 80%). Challenge assays of phages phiIPLA-RODI and phiIPLA-C1C against planktonic staphylococcal cells confirmed their lytic ability, as they were able to remove 5 log units in 8 h. Exposure of biofilms to phages phiIPLA-RODI and phiIPLA-C1C reduced the amount of adhered bacteria to about 2 log units in both monospecies and dual-species biofilms, but phiIPLA-RODI turned out to be as effective as the mixture of both phages. Moreover, the frequencies of bacteriophage-insensitive mutants (BIMs) of Staphylococcus aureus and S. epidermidis with resistance to phiIPLA-RODI and phiIPLA-C1C were low, at 4.05 ؋ 10 ؊7 ؎ 2.34 ؋ 10 ؊9 and 1.1 ؋ 10 ؊7 ؎ 2.08 ؋ 10 ؊9 , respectively. Overall, a generally reduced fitness in the absence of phages was observed for BIMs, which showed a restored phage-sensitive phenotype in a few generations. These results confirm that lytic bacteriophages can be efficient biofilm-disrupting agents, supporting their potential as antimicrobials against staphylococcal infections.T wo staphylococcal species, Staphylococcus aureus and Staphylococcus epidermidis, are the main causes of nosocomial infections due to their ability to adhere to, colonize, and develop biofilms in medical devices and human organs (1). Staphylococcal biofilms are complex structures in which bacterial cells are surrounded by an extracellular material (polysaccharides, teichoic acids, proteins, and extracellular DNA) which confers protection against antibacterial drugs and the host immune system. In addition, growth of bacteria in a biofilm facilitates the development of antibiotic-resistant organisms (2). S. epidermidis is one of the most abundant species in the human skin microbiota, from which it easily reaches catheters, heart valves, and contact lenses. Despite being regarded as an innocuous bacterium, it is now accepted as an opportunistic pathogen and one of the most common causes of bacteremia in immunocompromised patients (3), preterm infants (4), and biofilm-related infections (5). In addition, resistance to methicillin due to the presence of the mecA gene is widely spread in hospital isolates (6). Similarly, virule...

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

“…In a previous study performed in our laboratory, we described that treatment with phiIPLA-RODI reduced the number S. aureus IPLA16 cells by 2.4 log units in monospecies biofilms and by 4.2 log units in dual-species biofilms formed with the phagesensitive strain S. epidermidis LO5081 (15). Thus, the presence of a second species, also susceptible to the phage, seemed to enhance the efficacy of phage treatment.…”

“…For selective growth of the different species, the following media were used: Baird-Parker agar (BP) for S. aureus, Kenner Fecal (KF) agar for E. faecium, and MRS agar plates for the other three strains. Bacteriophage phiIPLA-RODI was propagated on S. aureus IPLA1 as previously described (15).…”

“…Notably, this lytic phage has a wide host range among staphylococcal species, including S. aureus and S. epidermidis. Thus, exposure of 24-h biofilms to phage phiIPLA-RODI resulted in a 2-log decrease in the number of S. aureus cells in single-species biofilms and 4 log units in S. aureus-S. epidermidis dual-species biofilms (15).…”

“…Phages, due to their antimicrobial activity, have been proposed as natural weapons for the control of pathogenic bacteria ("phage therapy") in clinical, veterinary, food safety, and environmental settings (13,14). Moreover, numerous studies have already shown the ability of phages to remove biofilms formed by a variety of bacteria, including staphylococcal species (15)(16)(17)(18). Additionally, phage-encoded proteins, such as endolysins and exopolysaccharide depolymerases, can also be used for biofilm removal (19)(20)(21).…”

Current Research Approaches to Target Biofilm Infections.

“…They have also been shown to clear biofilm infections caused by P. aeruginosa (41). Two lytic phages were recently described that were found to reduce Staphylococcal biofilms by 2 logs and the frequency of bacteriophage resistance developing in the bacteria was sufficiently low to merit these bacteriophages as potential candidates for therapy (42). Another recent study found a bacteriophage, EFDG1, to have effective lytic activity against planktonic and biofilm cultures of Enterococcus faecalis and E. faecium isolates, regardless of their antibiotic resistance profile (43).…”