We sought to determine whether commensal staphylococci collected from the skin, nasal, oral and perineal sites of companion dogs and cats exhibit antimicrobial activity against methicillin-resistant S. pseudintermedius (MRSP) ST71 (Figure 1AMa et al., 2020; Worthing et al., 2018b). Fifty-eight staphylococcus isolates across the coagulase-positive (CoPS) and coagulase-negative (CoNS) groups were screened, including validated antimicrobial strains of human origin, S. hominis A9 (Nakatsuji et al., 2017) and S. capitis E12 (O’Neill et al., 2020) and a non-active S. aureus 113 negative control strain (Figure 1B). The animal test isolates were screened for antimicrobial activity by live co-culture on agar plates or in the presence of sterile conditioned supernatant, as illustrated in Figure 1A. Amongst all test isolates, five strains demonstrated greater than 80 % inhibition of S. pseudintermedius growth (dashed line) across all three different dilutions of supernatant (1:1, 1:4, 1:8) (Figure 1C). Surprisingly, these strains exhibited greater potency compared to the positive control S. hominis A9 supernatant (indicated by black circle), which inhibited growth of S. pseudintermedius in a 1:1 dilution, but not at 1:4 or lower. Amongst the five positive hits, three were identified as S. felis and two S. pseudintermedius. In the second independent antimicrobial assay, all five isolates including positive control S. hominis A9, produced an observable zone of inhibition against S. pseudintermedius during live co-culture on agar (Figure 1D). The two feline S. felis species (C4, N26 labelled with white arrows) produced the largest inhibitory zones, extending 3.0–3.3 mm outward from the edge of the growing colony. The S. felis C4 strain was chosen for further analysis, as it demonstrated potent activity and was isolated from healthy skin. To investigate the significance and selectivity of the S. felis antimicrobial supernatant, we tested its capacity to inhibit the growth of other clinically relevant, gram-positive and gram-negative pathogens (of which several belong to the clinically-relevant ESKAPE group). Of the four gram-negative strains tested, only moderate inhibition was demonstrated after 18 hr incubation with 80–100% of the S. felis C4 supernatant (Figure 1E). In contrast, bacterial culture in the presence of just 1–5% of S. felis C4 supernatant was sufficient to inhibit >80% growth of all four gram-positive organisms, including S. pseudintermedius, E. faecium, B. subtilis, and S. aureus.

Of the three antimicrobial S. felis isolates, only the C4 supernatant retained activity after precipitation with 75 % ammonium sulfate (AS) (Figure 1—figure supplement 1A). Moreover, 75 % AS was highly effective in precipitating the antimicrobial factor(s) from the C4 supernatant since no activity could be visualized in the non-precipitate fraction (Figure 1—figure supplement 1B). This effect was also achieved with a simpler extraction by n-butanol. The antimicrobial butanol extract remained active up to 1 week at room temperature (RT) and was stable after boiling (Figure 1—figure supplement 1C). As expected, the butanol extraction provided a partially purified and enriched antimicrobial fraction compared to that of the crude supernatant and AS precipitation (Figure 1—figure supplement 1D). Therefore, this S. felis C4 extract obtained via n-butanol extraction was then adopted for further experiments. The S. felis extract was found to be effective against multiple clinical isolates of S. aureus and S. pseudintermedius with a MIC range of 0.4–6.25 μg/ml (Figure 1—figure supplement 1E). In contrast, some CoNS strains, including S. hominis and S. lugdunensis were less sensitive and had a higher MIC of 12.5 μg/ml.

We next evaluated the activity of S. felis C4 extract to disrupt bacterial biofilms. Biofilm formation is considered an important determinant of staphylococci virulence and is associated with increased skin colonization and severity of disease (Di Domenico et al., 2018; Kwiecinski et al., 2015). Reports have shown that most clinically-derived S. pseudintermedius strains are biofilm producers (Singh et al., 2013). A 4 hr preformed biofilm of pseudintermedius ST71 showed a significant decrease in crystal violet (CV) staining over time, when incubated with 100 % conditioned supernatant of S. felis C4, indicating biofilm disruption and degradation (Figure 1—figure supplement 2A). The S. felis C4 extract had anti-biofilm activity similar to crude conditioned supernatant, with biofilm mass reduced by 48 % at 250 μg/ml and 58 % at 500 μg/ml (Figure 1—figure supplement 2B).

To determine the nature of the antimicrobial product produced by S. felis C4, sterile culture supernatant was purified by HPLC. This yielded two major peaks that eluted at 44% and 47% acetonitrile (Figure 2A). Anti-S. pseudintermedius activity was predominantly associated with fraction 32 which eluted at 47 % acetonitrile (Figure 2B). SDS PAGE and protein silver staining of the active and inactive fractions revealed a unique band of roughly 5 kDa in size in the active fraction 32 (Figure 2C). To determine if this small protein was responsible for antimicrobial activity, gel slices of the fraction 32 lane corresponding to small, medium and larger proteins ( ≤ 5 kDa, 5–20 kDa and 20–50 kDa, respectively) were excised and extracted by acetone precipitation as previously described (Botelho et al., 2010; Zhang et al., 2015). Only the ≤5 kDa band demonstrated antimicrobial activity after incubation with S. pseudintermedius (Figure 2D), thereby suggesting the likely candidate to be a small peptide. Mass spectrometry (MS) analysis of the top eight hits in the active and non-active HPLC fractions identified several putative small antimicrobial peptides (AMP), representing the phenol soluble modulin beta (PSMβ1–3) and gamma (PSMγ, aka delta-hemolysin) families and a peptide of unknown function containing the EF-HAND domain, common amongst some antimicrobial Ca²⁺ binding proteins, such as S100A8/S100A9 (Chazin, 2011; Figure 2E). Whole genome analysis of the S. felis C4 strain confirmed the presence of three PSMβ-encoding genes (Figure 2F). Based on sequence similarities to mammalian cationic AMPs such as cathelicidin LL-37 the PSMβ and EF-HAND domain peptides were predicted to have α-helical amphipathic structure (Figure 2G). Synthetic N-formylated versions of all three S. felis C4 PSMβ1–3 and the EF-hand domain containing peptide were then generated and added to primary cell cultures of normal human keratinocytes (NHEK) for 24 hr to determine the level of cytotoxicity as measured by lactate dehydrogenase (LDH) release. The positive control cytotoxic PSMγ induced maximal cell death with 100 % LDH release at 62.5 ug/ml, yet at this concentration none of the PSMβ peptides nor the crude S. felis extract exceeded 20 % LDH release (Figure 2H), suggesting that S. felis C4 PSMβs are not cytotoxic.

Unlike the well-characterized cytolytic and inflammatory activities of PSMα, a defined role for PSMβ in mediating host interactions has been largely unexplored (Da et al., 2017). To investigate the potential effects of S. felis C4 on the host immune response, we stimulated cells with various TLR agonists in the presence or absence of the extract or the individual PSMs and measured inflammatory gene expression. NHEKs were treated with S. felis PSMβ2, PSMβ3, extract, DMSO control alone, or each in combination with the TLR2 agonist MALP-2 (200 ng/μl) or the TLR3 agonist Poly I:C (0.4 μg/ml) for 4 hr. Neither PSMβ nor the extract resulted in any detectable increase in gene expression, whereas MALP-2 significantly increased the expression of hBD-2, and Poly I:C significantly increased CXCL10 and IL-6 expression (Figure 3A–C). Interestingly, these TLR-mediated responses were significantly reduced during co-treatment with S. felis C4 PSMβ or extract. This result was confirmed by ELISA, showing that PSMβ2 had a significant effect on suppressing CXCL10 secretion in NHEKs after 24 hr co-treatment (Figure 3D). To determine if this interaction is specific for epithelial cells, we also stimulated human THP-1 macrophage-like cells with MALP-2, or the TLR4 agonist LPS and found that IL-6 and TNFα expression was decreased during co-treatment with S. felis C4 PSMβ2 (Figure 3—figure supplement 1). The addition of S. felis C4 PSMβ2 to NHEK activated by poly I:C demonstrated that PSMβ2 inhibited phosphorylation of TBK1 and IRF3 at 15 min post-stimulation with the peptide (Figure 3E). This inhibition of inflammatory target gene and kinase activity was further evaluated by the analysis of changes in global gene expression using RNA-Seq analysis of NHEKs stimulated with Poly I:C, with and without PSMβ2 at 4 hr and 24 h. Gene ontology (GO) analyses revealed the significant down-regulation of several gene clusters associated with ‘immune effector process’ and ‘type I IFN signaling’ at 4 h during co-treatment of PSMβ2 and Poly I:C (Figure 3F). A heatmap of selected genes within the ‘Immune response’ GO term at 4 h post-treatment further highlighted the suppressive effect, and also importantly showed that PSMβ2 treatment alone did not induce an immunological response in NHEKs (Figure 3G). However, when NHEKs were treated with PSMβ2 in the absence of an inflammatory stimulus, we identified the downregulation of genes within GO terms such as “cytokine-mediated signaling” and “pathogenic E. coli infection” (Figure 3—figure supplement 2), suggesting that exposure to PSMβ2 primes cells to dampen potential inflammatory mediators in response to TLR ligands. This contrasted with PSMβ2-upregulated genes which were mostly associated with biosynthetic pathways including lipid and amino acid metabolism (data not shown).

The antimicrobial activity of all three synthetic S. felis C4 PSMβ (PSMβ1–3) peptides in inhibiting S. pseudintermedius was observed to occur at a concentration of 50 μg/ml and the synthetic EF-HAND domain-containing peptide did not inhibit bacterial growth at concentrations up to 200 μg/ml (Figure 3H). In contrast, the native extract prepared from S. felis supernatant was found to be more potent than the synthetic PSM when added individually (Figure 3H) or in combination (Figure 3—figure supplement 3). A non-N-formylated version of PSMB2 exhibited similar activity to the N-formylated PSMβ2 version (Figure 3H). This disparity in the potency of the synthetic peptides compared to the native extract suggested that another antimicrobial molecule is produced by S. felis C4 that was not detected in the prior analyses.

To investigate this, we conducted further genome analysis using the web server anti-SMASH to identify the potential presence of secondary metabolites encoded from biosynthetic gene clusters (BCG) (Weber et al., 2015). Interrogation of the S. felis C4 genome revealed a BCG with similarity to micrococcin P1 from Macrococcus caseolyticus (Figure 4A). Micrococcin P1 is a macrocyclic antibiotic that inhibits ribosome translation in gram-positive bacteria (Figure 4B Carnio et al., 2000; Ciufolini and Lefranc, 2010). To determine if micrococcin P1 could be present within the S. felis C4 extract, we compared the C8 HPLC elution profile of synthetic micrococcin P1 to the native S. felis C4 extract and identified that the synthetic peptide eluted as a single major peak at 59 % acetonitrile in fractions 27 and 28 (Figure 4C). These fractions were active against S. pseudintermedius ST71 by agar radial diffusion (Figure 4C inset). Purification by HPLC of the S. felis C4 extract revealed a similar major peak that eluted at 59 % acetonitrile in fractions 27 and 28 (Figure 4D) and this also was active against S. pseudintermedius ST71 (Figure 4D inset). Mass spectrometry analysis of this elution fraction from S. felis C4 extract showed a peptide with mass of 1,144 daltons which was identical to synthetic micrococcin P1 (Figure 4E and Figure 4—figure supplement 1). Furthermore, both synthetic micrococcin P1 and the S. felis C4 antimicrobial activity could be extracted with butanol (Figure 4F). These observations strongly suggested that S. felis produces a peptide similar to micrococcin P1 and this peptide together with the previously identified PSMs contributed to the highly potent antimicrobial activity of S. felis C4.

Next, we sought to better understand how the antimicrobial action of S. felis C4 negatively affects bacterial physiology. Given the selective nature of the S. felis C4 supernatant against gram-positives (Figure 1E), we speculated that S. felis C4 activity may target and compromise the bacterial membrane and/or cell wall. To address this question, we utilized bacterial cytological profiling (BCP) to distinguish between the different cellular pathways targeted by antimicrobials (Nonejuie et al., 2013). B. subtilis cells exposed to 1 X MIC of the S. felis C4 extract and micrococcin P1 showed greater elongation compared to DMSO control and nucleoids that were highly condensed into toroidal structures, a characteristic of compounds that block translation, such as tetracycline (Figure 5A). Nucleoids were also highly condensed in micrococcin P1-treated cells and to a lesser extent in PSMβ2-treated cells. To examine the ultrastructural profile of staphylococci, we conducted transmission electron microscopy (TEM) imaging on sectioned S. pseudintermedius ST71 cells that were exposed to sub-MIC (0.8 μg/ml), MIC (1.5 μg/ml), or 5 X MIC (25 μg/ml) amounts of S. felis C4 extract or DMSO control (Figure 5B). TEM observations upon control DMSO treatment showed normal, uniform spherical cocci physiology and septum formation indicating active replication. In contrast, short exposure to the extract showed evidence of cell wall thickening, membrane invagination, and major perturbations in the structure and rigidity of the cell membrane (Figure 5B, lower zoom inset panels). Moreover, treatment with the extract showed evidence of greater chromosomal compaction compared to control, evidenced by the increased (electron) density of the nucleoid (highlighted yellow arrows). This observation is consistent with the cytological profile of toroidal structures in B. subtilis by BCP, indicating a similar mechanism of action in staphylococci. Additionally, exposure of S. pseudintermedius ST71 to increasing concentrations of the S. felis extract, resulted in a dose-dependent increase in reactive oxygen species (ROS) (Figure 5C) together with a concomitant decrease in the intracellular ATP levels (Figure 5D). Furthermore, evidence to support an additional membrane-active component was provided by flow cytometry analysis of bacterial cells positively stained with the membrane impermeable dye propidium iodide (PI). Greater than 20 % of S. pseudintermedius cells were PI-positive after 4 h exposure to 1 X-4X MIC of S. felis extract (Figure 5E). Lastly, S. felis C4 was shown to potentiate the effects of antibiotics against S. pseudintermedius ST71. The MIC values of gentamicin, rifampicin, and chloramphenicol were decreased in the presence of sub inhibitory 0.25 X MIC of S. felis extract compared to TSB control alone (Figure 5F). Remarkably, growth of erythromycin-resistant S. pseudintermedius ST71 in the presence of subMIC S. felis C4 extract restored sensitivity to erythromycin from MIC >100 μg/ml – MIC = 0.2 μg/ml.

We next sought to investigate the potential of S. felis C4 and its antimicrobial products as a therapy against S. pseudintermedius colonization and infection on an animal model. Since S. felis C4 is a commensal bacterium that was isolated from healthy feline skin, we speculated it should be safe and well tolerated on mammalian skin. S. felis C4 was sensitive to several common antibiotics (Figure 6A) and as such represents a suitable strain for further investigation as a bacteriotherapy. We therefore assessed the skin tolerability of a 3 day topical application of S. felis C4 on SKH1 hairless mice. Whereas S. pseudintermedius and the non-antimicrobial S. felis ATCC 49168 showed evidence of disease as observed by scaling and redness on back skin, S. felis C4 did not promote any adverse reaction (Figure 6B).

To test the antimicrobial activity of S. felis C4 on skin, we first applied 5 × 10⁷ CFU/cm² S. pseudintermedius directly onto mouse back skin, 48 hr later we then applied an equal density of S. felis C4 or 1 mg of the S. felis C4 extract to the same site. Both treatments were repeated daily for three days. The skin showed a reduction in scaling and redness post-treatment with S. felis C4 or extract compared to control (Figure 6C). Enumeration of S. pseudintermedius ST71 CFUs after plating onto selective agar revealed a significant 2.9 log decrease in CFU after extract application and a 3.3 log decrease after live S. felis C4 application (Figure 6D). Enumeration of total staphylococci CFUs after plating onto selective agar confirmed that the extract treatment significantly reduced bacterial colonization (Figure 6E). In contrast, total staphylococci CFU counts after the S. felis C4 application were more similar to the control group, suggesting that the S. felis bacteria successfully colonized and outcompeted S. pseudintermedius ST71 on the skin (Figure 6E). To further test the S. felis C4 extract as an anti-MRSP intervention, we also evaluated its efficacy in limiting deep tissue infection by S. pseudintermedius injected into the dermis. One hour after an intradermal injection of 1 × 10⁷ CFU S. pseudintermedius ST71, two intradermal inoculations of 250 μg of S. felis C4 extract were administered adjacent to the infection site. Infection was monitored by observations of skin necrotic lesion size over a 14-day period. Compared to controls, the extract-treated mice exhibited slower lesion progression from day 1 to day 2, and significantly better protection from S. pseudintermedius skin disease from day four onwards (Figure 6F and G). These results demonstrate the in vivo efficacy and clinical potential for S. felis C4 as a bacteriotherapy against S. pseudintermedius skin colonization and infection.

The bacterial strains used in this study were all grown overnight, with the exception of E. faecium which was grown for 48 hr, in 3 % Tryptic Soy Broth (TSB) with shaking at 250 rpm in a 37 °C incubator or otherwise grown on agar at 37 °C under static conditions.

Animal-derived staphylococci samples came from two previously described collections: the first collection consisted of clinical isolates of skin and soft tissue infection from Australian dogs and cats (Worthing et al., 2018a), and the second collection was comprised of staphylococci isolated from the nose, mouth and perineum of healthy dogs and cats in Australia (Ma et al., 2020). All samples had previously been identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF), as previously described (Worthing et al., 2018b), and the ST71 MRSP isolate had been characterized by whole genome sequencing (Worthing et al., 2018a). Two human derived antimicrobial skin commensal isolates were used as positive controls: S. hominis A9 (Nakatsuji et al., 2017) and S. capitis E12 (O’Neill et al., 2020) and a non-antimicrobial S. aureus 113 isolate served as a negative control.

For the initial staphylococci screen, single clone-derived cultures of animal-derived staphylococci were used as competitor isolates against the growth of methicillin-resistant S. pseudintermedius ST71. Each pure culture, including positive and negative control strains, were first streaked onto 3 % TSB agar plates and a single colony was transferred to 1 ml of TSB in a deep 96 well plate (Thermo). The CoNS plate was sealed with sterile Aeraseal film (Sigma, St. Louis, MO) and cultured at 37 °C overnight with shaking at 250 rpm. Bacterial growth was evaluated by measuring OD600 with only bacteria that grew to a density (OD600 >6.0) used for subsequent analysis. To measure antimicrobial activity in the secreted supernatant, the animal-derived staphylococci supernatant from overnight cultures were harvested and centrifuged through a 96-well 0.2 μm sterile filter plate (Corning). Next, 1 × 10⁵ CFU of S. pseudintermedius ST71 was inoculated into 150 μl of TSB media containing 50 %, 20% or 12.5 % sterile supernatant and grown on a plate shaker for 18 h at 30 °C. To measure antimicrobial activity from the live agar co-culture assays (radial diffusion), 20 μl of overnight S. pseudintermedius ST71 culture was first inoculated into 45 °C molten TSB and immobilized after pouring and cooling into square petri dishes with grids. Overnight cultures of animal-derived staphylococci were centrifuged to pellet the bacteria, washed 2 X with PBS and resuspended in fresh TSB. The culture (10 μl) was inoculated onto a 13 mm grid of the S. pseudintermedius agar plates and cultured overnight at 30 °C. The resulting zones of inhibition from antimicrobial isolates were imaged using the camera feature on an iPhone 12.

Supernatant from overnight cultures of selected human and animal-derived staphylococci were first sterilized by filtration through a low-protein binding 0.22 μm Millipore filter. Activity was precipitated by ammonium sulfate (75 % saturation) for 1 hr, under constant rotation followed by centrifugation at 4,000 x g for 45 min and re-suspension of the pellet in dH₂O. To test stability, the precipitate was boiled at 95 °C for 30 m or stored in a sterile eppendorf tube at room temperate for 1 week. Antimicrobial activity was measured by radial diffusion assay. Sterile supernatant of S. felis strains were also subject to n-butanol extraction and purification as previously described (Joo and Otto, 2014). Briefly, in each tube 10 ml of butanol was added to 30 ml of supernatant and incubated at 37 °C for 2 h under constant rotation. The tubes were then set aside for several mins until the butanol phase settled. After centrifugation at 2000 x g for 5 min, the upper butanol phase was collected and lyophilized in a SpeedVac vacuum concentrator. The lyophilized extract was resuspended and concentrated to 10 mg/ml in DMSO. Protein concentration was determined by Pierce BCA Protein Assay Kit.

MIC values were determined using a broth micro dilution method. Bacterial cells were grown to mid-late log phase, to an OD600 nm value of roughly 1.0 for each bacterial strain and then normalized to 1 × 10⁷ CFU/mL. The PSM peptides or butanol extracts were dissolved in DMSO to a stock concentration of 10 mg/ml. The stock concentrations of antibiotics that were water-soluble were prepared with H₂O or 100 % ethanol if water-insoluble. The 1 × 10⁷ CFU/ml bacterial cultures (10 μl) were aliquoted into 96-well microtiter plates and mixed with 95 μL of media with or without twofold dilutions of the conditioned supernatant, PSM peptides, butanol extracts or antibiotics and incubated for 16–18 hr at 30 °C with shaking at 250 rpm. Growth inhibition was determined by measuring the OD600 nm readings of each well using a microplate reader (SpectraMax iD3, Molecular Devices). The MIC of each bacterial strain was determined by the lowest peptide concentration that inhibited more than 80 % bacterial growth.

Overnight culture of S. pseudintermedius ST71 was diluted in fresh TSB to 1 × 10⁷ CFU/ml by OD600 reading. A total of 100 μl of bacteria was transferred to a flat-bottom 96 well plate and incubated at 37 °C without shaking for 4 hr to initiate biofilm formation. Next, the supernatants were removed by washing the plates three times with 200 μl of dH₂O. Subsequently, 150 μl of the S. felis C4 supernatant, sterile extract or TSB control was added at various concentrations to the biofilm for periods between 2 and 24 hr. After incubation, the supernatant was gently removed, and the biofilm was gently washed three times with dH₂O followed by air drying. Next, 150 μl of 0.1 % crystal violet (CV) solution was added to all wells containing biofilm. After 20 min of incubation with CV dye, the excess CV was removed and each well was gently washed twice with dH₂O. Fixed CV dye was released from the biofilm by 70 % ethanol, and absorbance was measured at 595 nm.

Prior to BCP, MIC of all the antimicrobial compounds to be tested were generated first. MICs for B. subtilis PY79 were conducted in 96-well plates. Cultures were taken from glycerol stocks and plated on LB plates for 24 hr at 30 °C. On the day of the experiment, single colonies were transferred into 3 mL liquid LB media and rolled until they reached early exponential phase (OD600 0.12–0.15). In 96-well plates, antibiotics were serially diluted down twofold across the plate. One μl of cells was added to 100 μL of LB+ antibiotic. Plates were incubated for 24 hr in a 30 °C plate shaker. Plates were then read in plate reader using an OD 600 nm spectrophotometer at T0 and T24. MIC was determined by the concentration of antibiotic at which the T24 OD600 value was 10 % or less than the control cell density. BCP was performed as described previously (Lamsa et al., 2012; Nonejuie et al., 2013; Peters et al., 2018). Briefly, early exponential phase B. subtilis PY79 was incubated with 0.5 x, 1 x, or 5 x MIC concentrations of antibiotics and rolled at 30 °C for 2 hr. Samples were taken at 30 m and 2 hr, and then dye mix was added to each for each strain. The dye mix contained 30 μg mL−1 FM 4–64, 20 μg mL−1 DAPI, and 2.5 μM Sytox Green in 1× Tbase. Six μL of cells were mixed with 1.5 μL of dye mix, and then 6 μL of that mixture was added to agarose pads (1 % agarose, 20 % LB). Cells were then imaged on an Applied Precision DV Elite optical sectioning microscope with a Photometrics Cool-SNAP-HQ2 camera, and images were deconvolved using SoftWoRx v5.5.1. Deconvolved images were then converted into TIFFs using Fiji, and then adjusted for clarity in Photoshop, producing the final images.

First step HPLC purification was carried out with 1 mg of S. felis C4 supernatant loaded onto a Capcell Pak C8 column (5 mm, 300 A°, 4.6 mm 250 mm) (Shiseido, Tokyo, Japan) using a linear acetonitrile gradient from 10% to 60% in 0.1 % (v/v) trifluoroacetic acid (TFA) at a flow rate of 1.0 ml/min. The resulting fractions were lyophilized, then resuspended in water, and antimicrobial activity assessed by liquid culture assay. Up to five sequential purifications were carried out with each antimicrobial fraction pooled together for the second HPLC purification. A linear gradient of acetonitrile from 25% to 50% was used for the second purification. The eluted fractions were tested for antimicrobial activity by liquid culture and agar radial diffusion assays and the active and nonactive fractions were submitted for mass spectrometry analysis. For the purification of micrococcin P1 peptide and S. felis extract by HPLC, 200 μg of micrococcin P1 or 1 mg of S. felis C4 extract were loaded onto the C18 column using a linear acetonitrile gradient from 5% to 80% in 0.1 % TFA. The active fractions were pooled and submitted for mass spectrometry analysis to identify the presence of micrococcin P1 in the S. felis C4 extract. Synthetic peptides were synthesized with or without N-terminal formulation to at least 80 % purity by commercial vendors (LifeTein LLC, Somerset, NJ) and (Biomatik LLC, Delaware (PSMβ1)). Sequences of the peptides are as follows:

Twenty μg of protein from sources including the antimicrobial HPLC fractions, butanol extracts and crude supernatant were loaded onto a Novex 16 % Tricine gel and subjected to SDS-PAGE. Silver staining and de-staining of the protein gels were performed according to the manufacturer’s instructions (Thermo Pierce Silver Stain Kit). A previously published protocol for acetone extraction of AMPs from SDS gels was used (Burgess, 2009). Briefly, a sterile razor blade was used to excise gel slices according to protein size. The gel slices were cut into small pieces and immersed in dH₂0 for 4 hr, with regular vortexing to elute proteins. The eluted protein was mixed with four volumes of ice-cold acetone for 1 hr at –20 °C. The samples were centrifuged at 16,000 x g for 15 min at 4 °C. The supernatant was removed and lyophilized (acetone-soluble fraction) and the resulting pellet air dried briefly and resuspended in dH₂0 (acetone-insoluble fraction). The antimicrobial activity of both fractions was tested by radial diffusion agar assay against S. pseudintermedius ST71.

NHEKs (ThermoFisher) were cultured in EpiLife medium containing 60 μM CaCl₂ (ThermoFisher) supplemented with 1 X human keratinocyte growth supplement (ThermoFisher) and 1 X Antibiotic Antimycotic (Millipore Sigma) at 37 °C, 5 % CO₂. All experiments performed on NHEKs were between passages 3 and 5 with cells at 70–80% confluency. For synthetic PSM treatments, the peptides (10–1000 μg/ml) were added to the NHEKs for 4 hr or 24 hr in dimethyl sulfoxide (DMSO).

NHEK cells were treated with Poly I:C (0.4 μg/ml), PSMβ2 (10 μg/ml) or Poly I:C and PSMβ2 combined for 15 min, 30 min, or 45 min and cells lysed in complete RIPA buffer supplemented with 1 X protease and phosphatase inhibitor cocktail (ThermoFisher). The lysate was centrifuged at 4 °C, at 13,000 rpm for 20 min and the total cytoplasmic supernatant fraction was kept at –80 °C, until future use. The total protein amount was quantified for each treatment using the Pierce BCA Protein Assay Kit according to manufacturer’s instructions. Fifteen µg of total protein was reduced with β-mercaptoethanol, boiled for 5 min loaded onto a 4–20% Mini-PROTEAN TGX gel (Bio-Rad). After electrophoresis, the gel was transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked for 1 hr in Intercept (PBS) Blocking Buffer (Licor) then incubated and probed overnight at 4 °C with the following primary antibodies: P-TBK1/NAK (D52C2), TBK1/NAK (D1B4), P-IRF-3 (S396), IRF-3 (D83B9), COX IV (3E11) in Intercept Blocking Buffer supplemented with 0.05 % Tween 20. The membranes were washed for 3 X for 10 min in PBS/Tween and incubated with IRDye conjugated anti-rabbit and anti-mouse secondary antibodies (IRDye800CW and IRDye680RD; Licor, USA) in Intercept Blocking Buffer/Tween for 1 hr at room temperature, followed by 4 X washing in PBS/Tween. Images of the membranes were acquired on an Odyssey DLx Imaging System (Licor, USA).

Four fractions of interest (ranging from 10 to 20 μg/mL) were dried under vacuum and resuspended in 15 μL of 5 % acetonitrile with 5 % formic acid. Next, individual LC-MS experiments were conducted on 6 μL of each sample through 85 min of data acquisition on an Orbitrap Fusion (Thermo Fisher Scientific) mass spectrometer with an in-line Easy-nLC 1000 (Thermo Fisher Scientific). A home-pulled and packed 30 cm column was triple-packed with 0.5 cm, 0.5 cm and 30 cm of 5 μm C4, 3 μm C18, and 1.8 μm C18 respectively and heated to 60 °C for use as the analytical column. Peptides were first loaded at 500 bar which was followed by a chromatography gradient ranging from 6% to 25% acetonitrile over 70 min followed by a 5 min gradient to 100 % acetonitrile, which was held for 10 min. Electrospray ionization was performed by applying 2000 V through a stainless-steel T-junction connecting the analytical column and Easy-nLC system. Each sample was followed by four washes starting with a gradient from 3% to 100% acetonitrile over 15 min with an additional 10 min at 100 % acetonitrile. An m/z range of 375–1500 was scanned for peptides with charge states between 2 and 6. Centroided data was used for quantitation of peaks. Acquisition was run in a data-dependent positive ion mode. Raw spectra were searched in Proteome Discoverer Version 2.1 and PEAKS Studio X (Ma et al., 2003) against 6-frame translated databases based of a uniprot reference database for Staphylococcus felis ATCC 49168 (Uniprot proteome UP000243559, accessed 06/26/2019) as well as in-house sequencing of S. felis C4 and de-novo algorithm of PEAKS Studio X. Data were searched using the Sequest algorithm (Eng et al., 1994) using a reverse database approach to control peptide and protein false discoveries to 1 % (Elias and Gygi, 2007). No enzyme was specified in the search and a minimum peptide length was set to six amino acids. Search parameters included a precursor mass tolerance of 50 ppm and fragment mass tolerance of 0.6 Da and variable oxidation for modifications.

DNA was extracted from S. felis C4 using the UltraClean Microbial DNA Isolation Kit (MoBio) according to the manufacturer’s instructions. The library was prepared using Nextera DNA Flex library preparation kit according to the manufacturer’s instructions (Illumina, San Diego, CA). The library was diluted to 1.0 nM, then sequenced for 300 cycles using the Illumina NovaSeq system to generate 150 bp paired-end reads with 794 x coverage that was reduced to 100 x coverage for read mapping. Fastq files from S. felis C4 were trimmed using Trimmomatic (Bolger et al., 2014), then assembled using SPAdes Genome Assembler v.3.14.1. The S. felis C4 genome was annotated using the RAST tool kit via the Pathosystems Resource Integration Center (PATRIC) database (Wattam et al., 2014). Genes encoding PSMβ were identified by BLASTn and secondary metabolite biosynthesis gene clusters by anti-SMASH bacterial version (Weber et al., 2015).

The intracellular ATP levels of S. pseudintermedius ST71 treated with S. felis C4 butanol extract were measured following the manufacturer’s instructions (ReadiUse Rapid Luminometric ATP Assay Kit). Briefly, an overnight S. pseudintermedius ST71 culture was sub-cultured to an OD of 0.5 at 37 °C. The bacteria were pelleted, washed with fresh TSB and incubated with various concentrations (0–320 μg/ml) of S. felis C4 butanol extract for 1 hr. The bacterial cultures were centrifuged at 10,000 x g for 5 min at 4 °C. The bacterial pellet was lysed by lysozyme and centrifuged. The bacterial supernatant was mixed with an equal volume of detecting solution in a 96-well plate and incubated at room temperature for 20 min. ATP luminescence was read using a SpectraMax iD3 (Molecular Devices).

The levels of reactive oxygen species (ROS) in S. pseudintermedius ST71 that was treated with S. felis C4 butanol extract were measured with 2′,7′-dichlorofluorescein diacetate (DCFDA) following the manufacturer’s instructions (DCFDA/H2DCFDA Abcam cellular ROS assay kit). Briefly, an overnight culture of S. pseudintermedius ST71 was sub-cultured to an OD of 0.5 at 37 °C. The bacteria were pelleted and re-suspended in fresh TSB. DCFDA was added to a final concentration of 20 μM to the bacterial culture incubated with various concentrations of extract (0–320 μg/ml) at 37 °C for 1 hr. Fluorescence intensity was immediately measured at an excitation wavelength of 488  nm and an emission wavelength of 525  nm using a SpectraMax iD3 (Molecular Devices).

Dead or damaged bacteria induced by S. felis C4 extract were evaluated using the LIVE/DEAD BacLight Bacterial Viability Kit, according to manufacturer’s instructions (Invitrogen, catalogue no. L7012). An overnight S. pseudintermedius ST71 culture was washed with fresh TSB and OD adjusted to 0.5 under treatment with increasing concentrations of extract (0, 0.8, 1.0, 1.6, 3.1, 12.5  μg/ml). After incubation for 4  hr, the bacteria were harvested, washed and resuspended in 1 X PBS. Equal volumes of SYTO9 and propidium iodide (PI) were mixed and 3 μl was added to each sample to a final volume of 1  ml and incubated at room temperature in the dark for 15  min. Flow cytometry measurements were taken on a BioRad ZE5 Cell Analyzer with forward and side scatter parameters for detecting bacteria that were non-stained. Total bacteria were gated by dual stained but SYTO9-positive only population. The percentage dead or membrane-compromised bacteria were detected and recorded by the population that were SYTO9- and PI-positive. Analysis was performed using FlowJo V10 software (BD Biosciences).

S. pseudintermedius ST71 cell pellets were immersed in modified Karnovsky’s fixative (2 % glutaraldehyde and 2 % paraformaldehyde in 0.10 M sodium cacodylate buffer, pH 7.4) for at least 4 hr and further postfixed in 1 % osmium tetroxide in 0.1 M cacodylate buffer for 1 hr on ice. The cells were stained all at once with 2 % uranyl acetate for 1 hr on ice, then dehydrated in a graded series of ethanol (50–100%) while remaining on ice. The cells were washed with 100 % ethanol and washed twice with acetone (10 min each) and embedded with Durcupan. Sections were cut at 60 nm on a Leica UCT ultramicrotome and picked up on 300 mesh copper grids. Sections were post-stained with 2 % uranyl acetate for 5 min and Sato’s lead stain for 1 min. Grids were viewed using a JEOL JEM-1400Plus (JEOL, Peabody, MA) transmission electron microscope and photographed using a Gatan OneView 4 K digital camera (Gatan, Pleasanton, CA).

The day prior to bacterial infection, the dorsal skin of age-matched 8–10 week old C57BL/6 mice (n = 5, per treatment) was shaved and depilated by using Nair cream followed by removal with alcohol wipes. A 50 μl inoculum suspension containing 1 × 10⁷ CFU of late log phase S. pseudintermedius ST71 in PBS was intradermally injected into the dorsal skin using 0.3 mL/31-gauge insulin syringe (BD, Franklin Lakes, NJ). At 1 hr post-infection, a 50 μl suspension of the S. felis C4 extract (250 μg, at a concentration of 5 mg/ml in 25 % DMSO) or a control suspension of 1 X PBS in 25 % DMSO was injected twice in two separate skin sites directly adjacent to the bacterial injection site. Body weights of the mice were measured before and after infection every day for 14 days. To determine lesion size, a ruler was positioned adjacent to the mouse skin lesions and digital photos were taken daily with a Kodak PIXPRO Astro Zoom AZ421 and analyzed via ImageJ software (National Institutes of Health Research Services Branch, Bethesda, MD, USA). Lesion size in mm² was measured by calculating the length x width.

mRNA transcript abundance was measured by qPCR in NHEKs stimulated with or without TLR2/6 agonist MALP-2 (200 ng/ml) or TLR3 agonist Poly I:C (0.4 μg/ml) in the presence or absence of S. felis C4 extract, PSMβ2 or PSMβ3 (10 μg/ml) or DMSO control (0.1%) at 4 hr post-treatment. RNA was extracted from NHEK cells using Pure Link RNA isolation kit (Thermo Fisher) according to manufacturer’s instructions. RNA was quantified on a Nanodrop 2000/200c spectrophotometer (Thermo Fisher, USA). Purified RNA (1 μg) was used to synthesize cDNA using the iScript cDNA Synthesis Kit (Bio-Rad, USA). Pre-Developed SYBR-Green gene expression assays (Integrated DNA Technologies, USA) were used to evaluate mRNA transcript levels.

NHEK cells were treated with DMSO (0.1%) control, PSMβ2 (10 μg/ml), Poly I:C (0.4 μg/ml) or PSMβ2 and Poly I:C combined, all in triplicate wells, for 4 hr or 24 hr in and RNA was extracted using the PureLink RNA mini kit and triplicate samples were pooled together for each treatment. Isolated RNA was submitted to the UCSD IGM Genomics Center for RNA-sequencing performed on a high-output run V4 platform (Illumina, USA) with a single read 100 cycle runs. Data alignment was performed using Partek Flow genomic analysis software (Partek, USA) with Tophat2 (version 2.0.8) Gene ontology (GO) enrichment analysis was performed on differentially regulated genes ( ≥ 1.5-fold) using DAVID 6.8 or Metascape (Zhou et al., 2019).

Significant differences between the means of the different treatments were evaluated using GraphPad Prism version 7.03 (GraphPad Software, Inc, La Jolla, CA). Either unpaired, two-tailed Student’s t test or one-way analysis of variance (ANOVA) followed by Dunnett’s or Tukey’s multiple comparisons test were used for statistical analysis and indicated in the respective figure legends. Differences were considered statistically significant with a p value of < 0.05.