Bacteriophages against Proteus mirabilis Biofilms

Rice, C., Ganacias, C. J., Chai, Z., Flynn, P. B., Gilmore, B. F., Jones, B. V., Irwin, N., Kulakov, L. A., &Skvortsov, T. (2019). Bacteriophages against Proteus mirabilis Biofilms. Poster session presented atEurobiofilms 2019, Glasgow, United Kingdom.

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Download date:20. Oct. 2020

Bacteriophages For Eradication Of Clinically Relevant Biofilms

Cormac J. Rice1, Chris. J Ganacias1, Zheng Hua Chai1, Padrig B. Flynn1, Brendan F. Gilmore1, Brian V. Jones3, Nicola Irwin1

Leonid A. Kulakov2, Timofey Skvortsov1

Fig. 3. Calcium of PC3 cells following canonical activation by a. thrombin and b. trypsin, or synthetic peptide activation by c. TFFLR or d. SLIGKV. Fig. 3. Calcium of PC3 cells following canonical activation by a. thrombin and b.trypsin, or synthetic peptide activation by c. TFFLR or d. SLIGKV.

Introduction

Proteus mirabilis is a Gram-negative, rod-shaped, opportunisticallypathogenic bacterium, often found in the urinary tract. P. mirabilisstrains are characterised by their ability to produce urease, whichcatalyses the hydrolysis of the urine’s urea into ammonia and carbondioxide. The released ammonia increases the pH of the urine,promoting the formation and accumulation of struvite deposits whichcan in turn result in catheter blockages.

Bacteriophages are a unique type of virus that recognize a specifictype of bacteria and then infect and kill the host via cell lysis. Theapplication of phages and their enzymes for treating bacterial biofilmshas recently gained significant interest due to a number of significantadvantages compared to traditional antibiotics, including highspecificity and efficacy, low immunogenicity and production costs.

Aims

• To isolate novel bacteriophages which act against Proteus spp• Obtain high and pure phage titre of isolated phages• Characterize each phage to determine their novelty• Sequence bacteriophage genomes• Screen the phages for potential enzyme/depolymerase activity• Assess the ability of phages to degrade bacterial biofilms

Fig. 1. (Left) Spot tests of phages isolated from various animal faecal sources and wastewater effluent. (Right) Plaque assay confirming the presence and activity of one of thenewly isolated phages (SS) against P. mirabilis (BB2000).

• Environmental enrichment of phages• Top agar overlay spot test assay• Top agar plaque assay• Phenol:Chloroform DNA extraction• Genomic restriction analysis • RAPD PCR of Phage DNA• Genomic sequencing

Methodology

Summary of findings

Since the beginning of the project (Jan 2019), the following results have beenobtained:• 50 phages active against Proteus spp. isolated• 3 phage isolates with depolymerase activity identified• 12 Broad host range phages found• Several different plaque morphologies observed

Screening for unique plaque morphologies

Fig. 2. (Left) Spot tests of phages isolated from various animal faecal sources, environmental regions andwaste water effluent. (Right) Plaque assay confirming the presence and activity of one of the newlyisolated phages (PM) against P. mirabilis (Strain BB200).

Potential Depolymerase activity of PM PhageAn early indicator for the presence of a potential depolymerase associated withbacteriophage activity is the presence of an additional zone of clearance aroundthe initial lysis plaque. A secondary means of confirmation can be concluded uponobserving expansion of this halo zone.

Overnight Incubation

Fig. 3. (Left) shows the initial plaque assay and the additional Halo zones associated with the PM phage.(Right) Plaque assay showing the presence of the expanding halo zone indicating that this phage may beassociated with depolymerase activity against P. mirabilis (Strain BB200).

Future Work

• Assess the potential for a phage-cold plasma synergistic technique to facilitate total biofilm eradication

• Assess the effect of a cocktail of phages in a biofilm model to see if they enhance the degradation of the biofilm

• Sequence novel phages and identify genes of potential biofilm degradation enzymes

• Obtain phage enzymes and assess their potential as biofilm biocontrol agents

• Obtain and characterise phages against other biofilm-associated pathogenic bacteria

1School of Pharmacy, Queen’s University Belfast, Belfast, Northern Ireland, BT9 7BL, UNITED KINGDOM 2 School of Biological Sciences, Queen’s University Belfast, Belfast, Northern Ireland, BT9 5AJ, UNITED KINGDOM

3School of Biology and Biochemistry, The University Of Bath, Bath BA2 7AX, UNITED KINGDOM

Host PM PM2 CJR TS1 BGM1 SL21 PFLN SK19 LK19 ZHC SLRY S.PIT

Clinical P. mirabilis - - - - + + + - + + + -P. mirabilis BB2000 + + + + + + + + + + + +P. mirabilis 35508 + + + + + + + + + + + +P. mirabilis 51286 + + + + + + + + + + + +P. vulgaris UM266 + + + + + + + + + + + +P. penneri 12737 + + + + + + + + + + + +P. mirabilis B2 + + + + + + + + + + + +P. mirabilis B4 + + + + + + + + + + + +P. mirabilis RS1 - - + + + + + + + + - +P. mirabilis RS6 - - - - - - - - - - - -P. mirabilis RS17 - - + + + + + + + - - +P. mirabilis RS18 - - + + + + - + - + + +P. mirabilis RS28 + + + + + + - + - - + +P. mirabilis RS40 + + + + + + + + + + + +P. mirabilis RS47 + + - - - - - - - + + -P. mirabilis RS50A + + + + + + + + + + + +

Table 1. Shows the host range associated with each bacteriophage with + meaninglysis of the bacteria and – meaning there was little to no lysis. The host range ofeach phage is still currently being assessed and expanded