Poster: 49Sublethal doses of the microbicidal compound hypothiocyanite convert methicillin-resistant Staphylococcus aureus to small colony variants

Abstract

Background

Staphylococcus aureus lung infection is a hallmark of cystic fibrosis (CF), affecting up to 80% of all people with CF (PwCF) by their mid-teens. Additionally, antibiotic resistance also represents an increasing concern, with methicillin-resistant S. aureus (MRSA) infecting up to 20% of all PwCF. One way S. aureus can survive in the lungs of PwCF is through the formation of small colony variants (SCVs) which are characterized by colonies that are up to 10 times as small as normal colonies, are non-pigmented, and slower growing. SCVs arise due to alterations in metabolism and may increase resistance to antibiotics and environmental stressors. One way to treat antibiotic resistant infections is with hypothiocyanite (OSCN^-). OSCN^- is produced through the action of peroxidases such as lactoperoxidase (LPO) and myeloperoxidase (MPO) reacting with hydrogen peroxide (H₂O₂) and thiocyanate (SCN^-). OSCN^- has known antimicrobial effects against a broad range of viral and bacterial pathogens. Our goal was to test the ability of OSCN^- to kill CF clinical isolates of MRSA.

Staphylococcus aureus lung infection is a hallmark of cystic fibrosis (CF), affecting up to 80% of all people with CF (PwCF) by their mid-teens. Additionally, antibiotic resistance also represents an increasing concern, with methicillin-resistant S. aureus (MRSA) infecting up to 20% of all PwCF. One way S. aureus can survive in the lungs of PwCF is through the formation of small colony variants (SCVs) which are characterized by colonies that are up to 10 times as small as normal colonies, are non-pigmented, and slower growing. SCVs arise due to alterations in metabolism and may increase resistance to antibiotics and environmental stressors. One way to treat antibiotic resistant infections is with hypothiocyanite (OSCN^-). OSCN^- is produced through the action of peroxidases such as lactoperoxidase (LPO) and myeloperoxidase (MPO) reacting with hydrogen peroxide (H₂O₂) and thiocyanate (SCN^-). OSCN^- has known antimicrobial effects against a broad range of viral and bacterial pathogens. Our goal was to test the ability of OSCN^- to kill CF clinical isolates of MRSA.

Methods

OSCN^- was produced in vitro by combining LPO (175 µg/ml) with NaSCN (7.5 mM) and H₂0₂ (5.6 mM) at 4°C. Any remaining H₂O₂ was removed by adding catalase (13.1 mg/ml) at 4°C. Catalase and LPO were removed through filtration with 10 kDa amicon spin filters and OSCN^- concentration was measured using a DTNB assay. OSCN^- was then incubated with 1 × 10⁵ CFU/ml of four CF clinical isolates of MRSA (MRSA24, MRSA25, MRSA47, and MRSA74) and two MRSA reference strains (USA300 and JE2) at concentrations ranging from 0 µM to 500 µM for 2.5 hours at 37°C. The MRSA isolates were then plated on blood agar plates and grown over night at 37°C. Colonies were counted and killing was measured in relation to a time zero control which received no OSCN^-. SCVs were determined as colonies without pigmentation nor hemolysis and smaller than those produced by the time zero control.

OSCN^- was produced in vitro by combining LPO (175 µg/ml) with NaSCN (7.5 mM) and H₂0₂ (5.6 mM) at 4°C. Any remaining H₂O₂ was removed by adding catalase (13.1 mg/ml) at 4°C. Catalase and LPO were removed through filtration with 10 kDa amicon spin filters and OSCN^- concentration was measured using a DTNB assay. OSCN^- was then incubated with 1 × 10⁵ CFU/ml of four CF clinical isolates of MRSA (MRSA24, MRSA25, MRSA47, and MRSA74) and two MRSA reference strains (USA300 and JE2) at concentrations ranging from 0 µM to 500 µM for 2.5 hours at 37°C. The MRSA isolates were then plated on blood agar plates and grown over night at 37°C. Colonies were counted and killing was measured in relation to a time zero control which received no OSCN^-. SCVs were determined as colonies without pigmentation nor hemolysis and smaller than those produced by the time zero control.

Results

OSCN^- slowed the growth of all MRSA isolates in a dose-dependent manner. Additionally, at the highest concentration of 500 µM, OSCN^- killed 58% (n=5) of MRSA24, 55% (n=6) of MRSA25, 58% (n=4) of MRSA47, 76% (n=5) of MRSA74, 34% (n=6) of USA300, and 21% (n=6) of JE2. Those bacteria that were not killed were converted to SCVs. 92% of MRSA24 colonies, 100% of MRSA25 colonies, 96% of MRSA47 colonies, 99% of MRSA74 colonies, 44% of USA300 colonies, and 51% of JE2 colonies converted to SCVs following incubation with 500 µM OSCN^-.

OSCN^- slowed the growth of all MRSA isolates in a dose-dependent manner. Additionally, at the highest concentration of 500 µM, OSCN^- killed 58% (n=5) of MRSA24, 55% (n=6) of MRSA25, 58% (n=4) of MRSA47, 76% (n=5) of MRSA74, 34% (n=6) of USA300, and 21% (n=6) of JE2. Those bacteria that were not killed were converted to SCVs. 92% of MRSA24 colonies, 100% of MRSA25 colonies, 96% of MRSA47 colonies, 99% of MRSA74 colonies, 44% of USA300 colonies, and 51% of JE2 colonies converted to SCVs following incubation with 500 µM OSCN^-.

Conclusions

Our results indicate that OSCN^- can effectively kill both CF clinical isolates and laboratory reference strains of MRSA. However, killing is not complete at the tested dose range, and at sub-lethal concentrations, OSCN^- exposure converts MRSA to SCVs. The CF clinical isolates were killed more efficiently by OSCN^- than the reference strains but were also more susceptible to conversion to SCVs. This represents an interesting dynamic in the treatment of MRSA infections in CF as bacterial killing is beneficial, but conversion to SCVs may lead to increased bacterial persistence and enhanced antibiotic resistance. More work is needed to map the complete microbicidal range of OSCN^- against MRSA and to understand the mechanism by which sublethal OSCN^- doses induce its transition to SCVs. This work will strengthen the opportunity to use OSCN^- as a future therapeutic in CF.

Our results indicate that OSCN^- can effectively kill both CF clinical isolates and laboratory reference strains of MRSA. However, killing is not complete at the tested dose range, and at sub-lethal concentrations, OSCN^- exposure converts MRSA to SCVs. The CF clinical isolates were killed more efficiently by OSCN^- than the reference strains but were also more susceptible to conversion to SCVs. This represents an interesting dynamic in the treatment of MRSA infections in CF as bacterial killing is beneficial, but conversion to SCVs may lead to increased bacterial persistence and enhanced antibiotic resistance. More work is needed to map the complete microbicidal range of OSCN^- against MRSA and to understand the mechanism by which sublethal OSCN^- doses induce its transition to SCVs. This work will strengthen the opportunity to use OSCN^- as a future therapeutic in CF.