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

Abstract

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.

Keywords: Bacteriophage; Confocal laser scanning microscopy (CLSM); MRSA biofilm; Microtiter plates; Virus.

Fig. 1

Examples of MRSA isolated phage plaques. a, b, c, d and e represent phage UPMK_1 infected bacterial lawn host MRSA t127/4, while f, g, h and i represent phage UPMK_2 infected bacterial lawn host MRSA t223/20. Using 0.6% agar overlay method plaques morphology of UPMK_1 (B and E) and UPMK_2 (G) appeared as lysis center surrounded by turbid halos. The phage lysate in plates C, D, H, I was applied in 10-fold serial dilutions to lawn of the host bacteria by spotting 10 μl from the phage dilution. The turbid halo around the lysis zone appeared clearer and its size increased as shown in D and I after longer incubation

Fig. 2

Determination of phage lytic efficiency at an optical density of 600 nm. The quantification of the culture cell density before and after infection with the bacteriophage as at MOI = 1; (a) = UPMK_1 with its host MRSAt127/4, while (b) = UPMK_2 with its host MRSAt223/20. Error bars represent mean values ± standard deviation of three independent experiments

Fig. 3

Spot test method; ph 4 represents UPMK_1 and ph 20 represents UPMK_2 (a) MRSAt2249/9 after 24 h of adding 10 μL of UPMK_1 and UPMK_2; halo was observed around UPMK_2 spot when incubated at room temperature for a week (b); (c) Small plaques formed by other MRSA isolates during EOP assay; (d) Clear lysis spot as a result of adding 10 μL of UPMK_2 on MRSA ST239 compared to the addition of UPMK_1

Fig. 4

Electron micrographs of UPMK_1 (left) and UPMK_2 (right) infected MRSA-negatively stained with 1% uranyl acetate

Fig. 5

One-step phage growth was performed for UPMK_1/ MRSA t127/4 (a) and UPMK_2/ MRSA t223/20 (b) at 37 °C. The phage growth parameters are indicated in the figure and correspond to: L-latent period and B-burst size. Data points represent the mean of three independent experiments while the error bars are the mean ± standard deviations of the data sets

Fig. 6

Genome map of UPMK_1 generated using GenomeVx available at http://wolfe.gen.tcd.ie/GenomeVx. Genes are shown as colored arcs and are labeled with the annotation proteins. Genes on outside are forward strand genes and genes on the inside are reverse strand genes

Fig. 7

Genome map of UPMK_2 generated using GenomeVx available at http://wolfe.gen.tcd.ie/GenomeVx. Genes are shown as colored arcs and are labeled with the annotation proteins. Genes on outside are forward strand genes and genes inside are reverse strand genes

Fig. 8

Phylogenic analysis showing the evolutionary history of the intact prophage that detected in the UPMK_2 genome by PHASTER analysis. The phylogenetic analyses were performed by using the maximum likelihood (ML) method based on the Tamura-Nei model. The tree with the highest log likelihood (− 3,039,991.6946) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. All positions containing gaps and missing data were eliminated. The analyses were conducted in MEGA7

Fig. 9

Phylogenic analysis showing the evolutionary history of the intact prophage (on the left) and incomplete prophage (on the right) that detected in the UPMK_1 genome by PHASTER analysis. The phylogenetic analyses were performed by using the maximum likelihood (ML) method based on the Tamura-Nei model. The tree with the highest log likelihood (− 2,768,365.1605) for intact prophage and (− 1,752,398.3348) for the incomplete prophage are shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. All positions containing gaps and missing data were eliminated. The analyses were conducted in MEGA7

Fig. 10

Molecular phylogenetic analysis of phage integrase gene sequence by Maximum Likelihood method based on the Tamura-Nei model. The tree with the highest log likelihood (− 15,349.6750) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 24 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA7

Fig. 11

MRSA t127/4 and MRSA t223/20 biofilms treated with UPMK_1 and UPMK_2 for 2, 4, 6,8,12 and 24 h. A1 and B1 are biofilm biomass treated with the phages at MOI ~ 1 (OD570 reading after CV staining). The estimated number of viable bacterial cells is shown in A2 and B2, while A3 and B3 represent phage particles in the biofilms treated with UPMK_1 for 6 h and with UPMK_2 for 8 h, respectively. The assays were performed severally to assess the ability of the phages to degrade the biofilm, and thrice to estimate the other values. The values are shown as the mean ± standard deviation of the values; statistical significance of biofilm reduction was reported at p < 0.05

Fig. 12

Tissue culture plates (TCP) of of MRS t127/4 and MRSA t223/20 biofilms which achieved a maximum biofilm removel after treatment with UPMK_1 and UPMK_2 FOR 6 h (A1) and 8 h (B1), respectively. (A1) and (B1) represent the bottom of the TCP, (A2) and (B2) represent the side view of the wells after treatment with the phages, and (A3) and (B3) represent the side view of the wells before treatment with the phages (control)

Fig. 13

CLSM micrographs of the biofilms formed by MRSA before and after treated with bacteriophage. A1 (scale bar; 50 μm), A2 (scale bar; 20 μm), A3 (scale bar; 10 μm) and A4 (scale bar; 5 μm) were represented the biofilm of MRSAt127/4 that treated with SM buffer as control. B1 (scale bar; 50 μm), B2 (scale bar; 20 μm), B3 (scale bar; 10 μm) and B4 (scale bar; 5 μm) were represented the biofilm of MRSAt127/4 that treated with phage UPMK_1 for 6 h. C1 (scale bar; 50 μm), C2 (scale bar; 20 μm), C3 (scale bar 10 μm) and C4 (scale bar; 5 μm) were represented the biofilm of MRSAt223/20 that treated with SM buffer as control. D1 (scale bar; 50 μm), D2 (scale bar 20 μm), D3 (scale bar 10 μm) and D4 (scale bar; 5 μm) represents the biofilm of MRSAt223/20 that treated with phage UPMK_2 for 8 h. Three dimensional projections of biofilm structure were reconstructed using Lecia LAX software

Fig. 14

CsCl purification of the phages. The phages are in the bluish-white band