Characterizing antimicrobial activity of environmental Streptomyces spp. and oral bacterial and fungal isolates from Canis familiaris and Felis catus

Abstract

Antimicrobials are a pillar of modern medicine, yet our limited arsenal of antibiotics and antifungals is currently threatened by widespread drug resistance. Ongoing efforts are focused on developing strategies to identify compounds that enhance the efficacy of current antimicrobials and develop novel, resistance-evasive therapeutic strategies. In this study, we characterized microbial isolates from two distinct environments to identify those that exhibit antimicrobial activity alone and in combination with current antimicrobials: (i) oral isolates from domesticated animals and (ii) environmental Streptomyces spp. First, conditioned media prepared from bacterial and fungal oral isolates that were collected from Canis familiaris and Felis catus were screened for antibacterial and antifungal activity. Three supernatants from bacterial isolates exhibited antifungal activity against the human fungal pathogen Candida albicans in the presence of subinhibitory concentrations of fluconazole, the most widely deployed antifungal. Additionally, two bacterial isolates displayed antibacterial activity against Escherichia coli alone and in combination with the antibacterial ampicillin. Furthermore, 32 environmental isolates of confirmed and predicted Streptomyces spp. were screened for activity against C. albicans and E. coli. Cell-free media harvested from isolates WAC5038 and WAC5287 exhibited antifungal activity against Candida spp., while only the WAC5038-conditioned medium displayed antibacterial activity. Bioactivity-guided fractionation, coupled with UV/Vis absorbance spectra, suggested that the bioactive compound in WAC5287 has a similar absorbance spectrum to the antifungal class of polyenes, while the bioactive component of WAC5038 remains unknown. Overall, this work highlights a strategy to collect and screen environmental isolates for the identification of novel antimicrobials.

Importance: The emergence and spread of antimicrobial resistance presents a global health challenge. As such, researchers are focused on developing pipelines to discover novel antimicrobials. In this study, we screened two distinct collections of microbes for antimicrobial activity. First, we collected bacterial and fungal isolates from the oral cavities of domesticated dogs and cats and identified these isolates using 16S (bacteria) and ITS (fungi) sequencing. Follow-up analyses confirmed that some conditioned media from bacterial isolates had antibacterial activity against Escherichia coli and antifungal activity against Candida albicans both alone and in combination with the current antimicrobial drugs. Additionally, screening 32 predicted or confirmed Streptomyces environmental isolates for antifungal and antibacterial activity identified two isolates with antifungal activity (WAC5038 and WAC5287), with only one isolate demonstrating antibacterial activity (WAC5038). Overall, this study provides a framework to identify and characterize environmental microbes with antimicrobial activity.

Keywords: Candida albicans; Canis familiaris; Escherichia coli; Felis catus; Streptomyces; antimicrobials; drug combinations; resistance.

Fig 1

Isolates from F. catus and C. familiaris exhibit antimicrobial activity. (A) Schematic depicting the workflow of swabbing C. familiaris and F. catus for microbial samples and assessing for antimicrobial activity. Schematic created in BioRender. (B) Growth inhibitory assays were conducted using 50% (vol/vol) conditioned media in a 96-well plate with 200 µL total volume per well. Bacterial inoculum was diluted to an OD₆₀₀ of 0.01 in LB, and 100 µL of inoculum was added to 100 µL conditioned medium per well in a 96-well plate, in the absence and presence of subinhibitory concentrations of ampicillin (0.5 µg/mL). Relative growth was measured by absorbance at 600 nm (OD₆₀₀) after a 24-hour incubation at 37°C (see color bar). Fungal inoculum was diluted to 1 × 10⁴ cells/mL in YPD, and 100 µL of inoculum was added to 100 µL conditioned medium per well in a 96-well plate, in the absence and presence of subinhibitory concentrations of fluconazole (1.5 µg/mL). Relative growth was measured by absorbance at 600 nm (OD₆₀₀) after 48 hours at 30°C (see color bar). Microbial isolates belonging to the same genera are color-coded when multiple species were recovered from that genus. Blue: Staphylococcus. Green: Rothia. (C) Dose-response assays were conducted using a titration of 10, 20, 30, 40, or 50% (vl/vol) conditioned media in LB alone or in combination with subinhibitory concentrations of ampicillin (0.5 µg/mL). Experiments were conducted as described in panel B. (D) Conditioned media were incubated at 37°C for 1  hour with 100  mg/L DNase I, RNase A, or proteinase K. Enzymes were inactivated by heating the treated conditioned media at 100°C for 10  minutes. The activity of the conditioned media (20% vol/vol) was assessed against DH5α as described in panel B. (E) Dose-response assays were conducted as described in panel B, without or with subinhibitory concentrations of fluconazole (1.5 µg/mL). Grey box: bioactivity could not be determined due to conditioned media precipitating out of solution. (FC) Conditioned medium was incubated at 37°C for 1  hour with 100  mg/L DNase I, RNase A, or proteinase K. The activity of the conditioned media was assessed by incubating CaCi-2 at 30°C for 48  hours in the presence (+) or absence (−) of 1.5 µg/mL of fluconazole. Fungal growth was quantified by measuring OD₆₀₀. All data represent technical duplicates in two biological replicates.

Fig 2

Conditioned media from distinct Streptomyces spp. have antimicrobial activities. (A) Schematic depicting the workflow for testing conditioned media from Streptomyces spp. for bioactivity. Schematic created in BioRender. (B) Conditioned media from two prioritized Streptomyces spp., WAC5038 and WAC5287, show bioactivity against C. albicans (CaCi-2) and C. auris (Ci6684). Twelve subfractions from conditioned media (10 mL) were generated by butanol extraction followed by separation by HPLC, eluting from a C18 column with a step gradient from 5% to 97% acetonitrile with 0.1% formic acid. Dose-response assays were conducted as described in Fig. 1C against C. albicans or C. auris in the presence or absence of fluconazole (see color bar). (C) Dose-response assay with subfractions from two prioritized Streptomyces spp., WAC5038 and WAC5287, against E. coli (DH5ɑ) were performed, as described in Fig. 1C. WAC5038 displayed antibacterial activity as low as 10% (vol/vol), with a slight decrease in bioactivity observed with the addition of ampicillin. Conditioned medium from WAC5287 displayed no antibacterial activity. (D) Subfractions of butanol-extracted conditioned media were generated by HPLC. Dried subfractions were dissolved in 20 µL of DMSO. C. albicans cells (1 × 10³) in YPD were seeded per well in a 96-well plate (200 µL total volume), and 2 µL of each subfraction was added. Relative growth was measured by OD₆₀₀ after a 48-hour incubation at 30°C. (E) Subfractions were tested as described above. E. coli inoculum was diluted to an OD₆₀₀ of 0.01 before being adding to the 96-well plate. Relative growth was measured by OD₆₀₀ after a 24-hour incubation at 37°C. (F) Absorbance spectra of WAC5287 fraction 5 (rt 6.0 min) reveal the triple absorption peaks (at 362, 382, and 482 nm) highly characteristic of a polyene. (G) C. albicans amphotericin B-resistant isolate is resistant to WAC5287 conditioned medium and is hypersensitive to WAC5038 conditioned medium. Dose-response assays were conducted, as described in Fig. 1C, and incubated for 24 hours at 30°C. All dose-response assays were conducted in technical duplicates and are representative of biological replicates.