Methods for the Diagnosis and Treatment of Biofilm-Related Infections

Abstract

Disclosed herein are methods and systems for rapid diagnosis and treatment of biofilm-related infections in a subject having a medical implant. A reporter cocktail composition is disclosed herein and may be used to detect a microorganism of interest and determine the presence of an infection. A therapeutic cocktail composition is also disclosed herein and may be used to treat a subject diagnosed with a biofilm-related infection.

Claims

1. A method for the diagnosis and treatment of a biofilm-related infection in a subject comprising the steps of: (i) providing a biological sample taken from the subject; (ii) diagnosing the subject with a biofilm-related infection by detecting the presence of at least one microorganism of interest comprising the steps of: (a) contacting at least one aliquot of the biological sample with an amount of a reporter cocktail composition comprising at least one recombinant bacteriophage; (b) detecting a signal produced following replication of the recombinant bacteriophage, wherein detection of the signal indicates the microorganism of interest is present in the sample; and (iii) treating the subject diagnosed with a biofilm-related infection comprising the steps of: (a) selecting a therapeutic cocktail composition based on the diagnosis of step (ii); (b) administering a therapeutically-effective amount of a therapeutic cocktail composition comprising at least one bacteriophage, wherein the bacteriophage is specific for the detected microorganism of interest; and (c) optionally administering at least one additional therapeutic agent.

2. The method of claim 1, wherein the biofilm is on or around the surface of an implant in a subject suspected of having an infection.

3. The method of claim 1, wherein the step of diagnosing the subject comprises contacting a plurality of aliquots with a plurality of reporter cocktail compositions.

4. The method of claim 1 wherein the step of diagnosing the subject further comprises determining the antibiotic resistance of the detected microorganism of interest.

5. The method of claim 4, wherein determining the antibiotic resistance of the detected microorganism of interest further comprises the step of contacting the biological sample with an antibiotic prior to contacting the biological sample with the reporter cocktail composition.

6. The method of claim 1, wherein the recombinant bacteriophage of the reporter cocktail composition comprises a genetic construct inserted into a bacteriophage genome, wherein the genetic construct comprises an indicator gene and an additional bacteriophage late promoter.

7. The method of claim 6, wherein the indicator gene does not encode a fusion protein and transcription of the indicator gene is controlled by the additional bacteriophage late promoter.

8. The method of claim 7, wherein expression of the indicator gene during bacteriophage replication following infection of a host bacterium results in the indicator protein product.

9. The method of claim 8, wherein the indicator gene encodes a luciferase enzyme.

10. The method of claim 1, wherein the bacteriophage of the therapeutic cocktail composition is a recombinant bacteriophage.

11. The method of claim 10, wherein the recombinant bacteriophage of the therapeutic cocktail composition comprises a genetic construct inserted into a bacteriophage genome, wherein the genetic construct comprises an enzyme.

12. The method of claim 11, wherein the enzyme is a glycosidase, amidase, or an endopeptidase.

13. The method of claim 1, wherein the microorganism of interest is Staphylococcus species, Klebsiella species, Pseudomonas species, Cutibacterium acnes, and Shigella species.

14. The method of claim 1, wherein at least one type of recombinant bacteriophage is constructed from Phage K, MP115, or ISP.

15. The method of claim 1, wherein the biological sample is first incubated in conditions favoring growth for an enrichment period of 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or 2 hours.

16. The method of claim 1, wherein the therapeutic agent is an antibiotic.

17. A method of preventing or inhibiting infection in a subject comprising applying a cocktail composition comprising at least one recombinant bacteriophage to a surgical implant, dressing, or suture.

18. A surgical implant, dressing, or suture coated in a cocktail composition comprising at least one recombinant bacteriophage.

Description

EXAMPLES

[0164] The following examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.

Example 1. Staphylococcus aureus Biofilm and Irrigation Wash Testing Protocol

[0165] Overnight cultures of S. aureus were diluted into either 200 μL TSB (100% Tryptone Soya Broth), TSBg (66% TSB+0.2% glucose), or TSB-HS (90% TSB+10% human serum). The initial inoculum was a 200-fold dilution of overnight culture and prepared in 96-well plates. Plates were covered and incubated statically at 37° C. for at least 16 hours. Non-biofilm planktonic cells were removed by discarding the media and washing gently with 200 μL saline. Saline irrigation wash was performed by pipetting 200 μL of saline forcibly onto the biofilm. This direct wash is expected to mechanically release portions of the adherent biofilm. 150 μL of each saline irrigation wash sample was transferred to a separate 96-well plate containing dried-down concentrated BHI (Brain Heart Infusion). The final concentration of BHI in each well was 1× (37 g/L). In order to assess the residual adherent biomass, 150 μL of BHI was added to each well containing the post-irrigation washed biofilm. All samples (residual biofilm+saline irrigation wash) were covered and incubated statically at 37° C. to facilitate enrichment over a four-hour period. After enrichment, 10 μL of a recombinant phage cocktail was added to each well and mixed by pipetting. Plates were once again covered and incubated statically at 37° C. for two hours. After infection, 65 μL of detection master mix containing NANO-GLO® buffer, NANO-GLO® substrate and Renilla lysis buffer was added to each well and mixed again by pipetting. Samples were read on a GLOMAX® luminometer after a 3-minute wait time and utilizing a 1 second integration. Data is presented as relative light unit (RLUs) in Table 1.

TABLE-US-00001 TABLE 1 Phage Detection of S. aureus Saline Irrigation RLU Signal (Positive Wash of Biofilm cutoff is 3x Background) ATCC TSB TSBg TSB-HS S. aureus Strain Type Biofilm Biofilm Biofilm BAA-1763 MRSA 387000 3336000 984800 BAA-1768 MRSA 611900 9260000 2628000 BAA-42 MRSA 35890 120500 18010 BAA-1720 MRSA 7652000 11030000 27120000 33592 MRSA 401700 1041000 4530000 12600 MSSA 998000 9317000 14800000 Background Saline 335 395 371 Control Post-Wash RLU Signal (Positive Residual Biofilm cutoff is 3x Background) ATCC TSB TSBg TSB-HS S. aureus Strain Type Biofilm Biofilm Biofilm BAA-1763 MRSA 171600 2772000 114300 BAA-1768 MRSA 262400 2304000 117300 BAA-42 MRSA 18990 78880 8318 BAA-1720 MRSA 2854000 9420000 13000000 33592 MRSA 581200 919900 1010000 12600 MSSA 211500 35220000 308700 Background BHI 150 173 152 Control TSB-100% TSB TSBg-66% TSB + 0.2% glucose TSB-HS-90% TSB + 10% Human serum