Proteomic analysis revealed the biofilm-degradation abilities of the bacteriophage UPMK_1 and UPMK_2 against Methicillin-resistant Staphylococcus aureus.

OBJECTIVE: The degradation activity of two bacteriophages UPMK_1 and UPMK_2 against methicillin-resistant Staphylococcus aureus phages were examined using gel zymography. METHODS: The analysis was done using BLASTP to detect peptides catalytic domains. Many peptides that are related to several phage proteins were revealed. RESULTS: UPMK_1 and UPMK_2 custom sequence database were used for peptide identification. The biofilm-degrading proteins in the bacteriophage UPMK_2 revealed the same lytic activity towards polysaccharide intercellular adhesin-dependent and independent of Methicillin-resistant Staphylococcus aureus (MRSA) biofilm producers in comparison to UPMK_1, which had lytic activity restricted solely to its host. CONCLUSION: Both bacteriophage enzymes were involved in MRSA biofilm degradation during phage infection and they have promising enzybiotics properties against MRSA biofilm formation.

Genomic analyses of two novel biofilm-degrading methicillin-resistant Staphylococcus aureus phages.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) biofilm producers represent an important etiological agent of many chronic human infections. Antibiotics and host immune responses are largely ineffective against bacteria within biofilms. Alternative actions and novel antimicrobials should be considered. In this context, the use of phages to destroy MRSA biofilms presents an innovative alternative mechanism. RESULTS: Twenty-five MRSA biofilm producers were used as substrates to isolate MRSA-specific phages. Despite the difficulties in obtaining an isolate of this phage, two phages (UPMK_1 and UPMK_2) were isolated. Both phages varied in their ability to produce halos around their plaques, host infectivity, one-step growth curves, and electron microscopy features. Furthermore, both phages demonstrated antagonistic infectivity on planktonic cultures. This was validated in an in vitro static biofilm assay (in microtiter-plates), followed by the visualization of the biofilm architecture in situ via confocal laser scanning microscopy before and after phage infection, and further supported by phages genome analysis. The UPMK_1 genome comprised 152,788 bp coding for 155 putative open reading frames (ORFs), and its genome characteristics were between the Myoviridae and Siphoviridae family, though the morphological features confined it more to the Siphoviridae family. The UPMK_2 has 40,955 bp with 62 putative ORFs; morphologically, it presented the features of the Podoviridae though its genome did not show similarity with any of the S. aureus in the Podoviridae family. Both phages possess lytic enzymes that were associated with a high ability to degrade biofilms as shown in the microtiter plate and CLSM analyses. CONCLUSIONS: The present work addressed the possibility of using phages as potential biocontrol agents for biofilm-producing MRSA.

Methicillin-Resistant Staphylococcus aureus Biofilms and Their Influence on Bacterial Adhesion and Cohesion.

Twenty-five methicillin-resistant Staphylococcus aureus (MRSA) isolates were characterized by staphylococcal protein A gene typing and the ability to form biofilms. The presence of exopolysaccharides, proteins, and extracellular DNA and RNA in biofilms was assessed by a dispersal assay. In addition, cell adhesion to surfaces and cell cohesion were evaluated using the packed-bead method and mechanical disruption, respectively. The predominant genotype was spa type t127 (22 out of 25 isolates); the majority of isolates were categorized as moderate biofilm producers. Twelve isolates displayed PIA-independent biofilm formation, while the remaining 13 isolates were PIA-dependent. Both groups showed strong dispersal in response to RNase and DNase digestion followed by proteinase K treatment. PIA-dependent biofilms showed variable dispersal after sodium metaperiodate treatment, whereas PIA-independent biofilms showed enhanced biofilm formation. There was no correlation between the extent of biofilm formation or biofilm components and the adhesion or cohesion abilities of the bacteria, but the efficiency of adherence to glass beads increased after biofilm depletion. In conclusion, nucleic acids and proteins formed the main components of the MRSA clone t127 biofilm matrix, and there seems to be an association between adhesion and cohesion in the biofilms tested.