is a normal inhabitant of the skin and mucosa of healthy dogs and cats. Over the last decade, S. pseudintermedius
has emerged as a critically opportunistic animal pathogen causing primarily infections such as pyoderma and otitis [1
]. The spread of methicillin-resistant Staphylococcus pseudintermedius
(MRSP) worldwide represents a health problem for both companion animals and humans since zoonotic transmission has been documented [2
]. The MRSP strains possess a mobile genetic element, the staphylococcal cassette chromosome (SCCmec
), that include the mec
) and its regulatory genes mecR1
. Additionally, mecB
has recently been detected in methicillin-resistant Staphylococcus aureus
]. The mec
gene encodes a modified PBP, namely the PBP2A, that presents a decreased binding affinity to beta-lactams, leading to ineffectiveness of antibiotic [4
]. Since mec
gene is located on a mobile element of the chromosome it can be easily transferred between staphylococcal species. The ability to form biofilm is one of the main virulence factors studied in bacteria [5
]. Stefanetti et al. demonstrated that up to 96% of S. pseudintermedius
strains isolated from canine infections were able to produce biofilm [6
]. Biofilm formation can facilitate the colonization of different body sites in dogs and hinder the infection treatment. Walker et al. reported MIC values for biofilm-associated S. pseudintermedius
significantly higher than for planktonic bacteria [7
]. In another study, Meroni et al. reported that 68% of S. pseudintermedius
isolates were able to form biofilms, but 100% of the multidrug resistant strains were slime producers; therefore, it seems that biofilm production can facilitate bacteria horizontal gene transfer [8
]. Thus, discovery of novel agents for treatment of MRSP-associated and biofilm related infections is highly warranted.
To restore inefficacious critically important antibiotics, some research has been focused on the use of old drugs in synergic association with new antimicrobial, able to potentiate or restore their efficacy. The combination therapy is an important aspect of modern medicine in the design of new antimicrobials. The advantage relies in the possibility to repurpose existing, clinically-approved agents, accelerating the discovery and development of new therapies [9
]. In this context, phytomedicals in combination with commercially available antibiotics represent an alternative.
Natural products from plants, fungi and bacteria have been successfully used in the past for their antimicrobial activity, but this unlimited source has only been partially investigated in the search of new antimicrobial agents [10
]. Abietic acid is a diterpenoid with interesting antiviral, antibiotic and antifungal properties, whose main source is the resin of pine trees and other conifers [14
]. Such resin is produced to defend the plant from the attack of insects or microorganisms in presence of tissue injury. This can in part explain the intrinsic activity of abietic acid against bacteria (mainly Gram-positive) and fungi [17
Here, we first analyze the antimicrobial and antibiofilm properties of abietic acid against S. pseudintermedius. We also investigate the synergistic interaction between abietic acid and oxacillin against MRSP strains.
In recent years, the high prevalence of methicillin-resistant staphylococcal species in veterinary medicine has become increasingly evident [20
]. Among methicillin-resistant staphylococci (MRS), S. pseudintermedius
(MRSP) is a pathogen of great importance not only in veterinary, but also in human medicine. However, infections caused by S. pseudintermedius
in humans are often underreported due to inaccurate identification as S. aureus
. The frequency to develop infections associated with colonization of MRSP in people daily in contact with animals is considerably increased. Rodrigues et al. [21
] showed that 60% of participants to their study were found to be colonized by at least one MRS. Methicillin-resistance is caused by expression of modified penicillin-binding protein (PBP). S. pseudintermedius
gene is not only resistant to penicillins, cephalosporines and carbapenemes, but often also possess resistance to macrolides, lincosamides and streptogramins. For this reason, the treatment options for MRSP infections have become very limited and represent a new challenge in veterinary medicine.
As a response to the imminent lack of effective antibiotics in veterinary field and the increase of zoonotic transmission, the antimicrobial properties of abietic acid were here investigated. Abietic acid and its derived compounds are already known for their antibacterial activity. Helfenstein et al. [22
] reported an MIC value for abietic acid of 60 μg/mL against S. aureus
and 8 μg/mL against methicillin-resistant S. aureus, S. epidermidis
and Streptococcus mitis
; in another study, an MIC value of 800 μg/mL against S. epidermidis
was reported [23
]. A recent review discussed about the promising role of antibiotic resistance breakers (ARB) classes of compounds, among which abietane diterpenes are enumerated with the efflux pumps inhibitors [24
]. Among these, carnosic acid and carnosol, two diterpenes, at concentrations of 10 μg/mL induced two- and four-fold reductions in the MIC of tetracycline, respectively, against S. aureus
strains containing Msr(A) and Tet(k) pumps [25
Here, we demonstrated that abietic acid strongly reduced both MSSP and MRSP growth showing a bacteriostatic activity. Bacterial growth was inhibited already 1 h after, reaching the peak after 6 h of exposure. However, despite the growth resumption observed 24 h after, a slight decrease of cell growth was demonstrated, compared to control strain. Noteworthy, our results are strengthened by activity of abietic acid on the MRSP strains isolates from human samples. This result is encouraging, in consideration of high risk of zoonotic transmission of multiresistant strains.
To get a better understanding of antimicrobial activity of abietic acid against MRSP, we examined the interaction between abietic acid and oxacillin via the checkerboard method and described it in terms of FIC. The FIC index of abietic acid in combination with oxacillin was 0.375, which according to Pillai et al. [26
] indicates a synergistic interaction. MIC of oxacillin for our MRSPs was 10 μg/mL, while if used in combination with abietic acid MIC value of oxacillin was reduced to 2.5 μg/mL. Although the value is still far from oxacillin breakpoint for S. pseudintermedius
], this result is of great interest since we demonstrated that abietic acid was able to increase oxacillin susceptibility of MRSP strains. This could result from the ability of abietic acid to modulate mec
operon. In line with the literature, here we showed that sub-inhibitory concentration of oxacillin strongly induced all the genes of the mec
operon in the MRSP strains, while abietic acid treatment had the opposite effect [28
]. However, we did not observe a strong inhibition of the genes included in the operon, but rather a mild repression. We can speculate that the reduced production of PBP2A enzyme in part abrogates the MRSP defense mechanism against beta-lactam antibiotics, thus partially restoring the activity of oxacillin. In fact, it has already been proposed that compounds having synergic effect with beta-lactams may inhibit the PBP2A activity [29
]. In addition, the abietic acid activity here observed might be attributable to the carboxylic group, which interacts with the lipid component of the bacterial cellular membrane and allows the penetration of the molecule inside the membrane, altering its functions [23
An important virulence factor of S. pseudintermedius
is the ability to produce biofilm. In a study on 710 clinical isolates from healthy and diseased dogs, Little et al. showed that the proportion of biofilm-producing isolates from diseased dogs was significantly greater when compared with isolates from healthy ones [32
]. Here we showed that at concentrations much lower than MIC values, abietic acid was able to inhibit by 73% MSSP biofilm formation, whereas it was unable to prevent biofilm formation by MRSP. At dose just above MIC, abietic acid induced a drastic reduction of preformed biofilm vitality of both MSSP and MRSP strains. Abietic acid efficacy on the viability of mature Streptococcus mutans
biofilm was described by Ito et al., but at higher concentrations [33
]. Although abietic acid had no effect on biofilm mass, we can hypothesize that due to its small size it penetrates biofilm matrix and kill the embedded bacteria. Further studies are needed to clarify the penetration and killing mechanisms. However, these findings might be of therapeutic interest because biofilm significantly affects antimicrobial susceptibility of both MSSP and MRSP from dogs [7
]. Of interest, human S. pseudintermedius
isolates also form structurally complex biofilm that are intrinsically resistant to antibiotics [34
], thus complicating zoonotic infections.
In our opinion, a future use of abietic acid to fight bacterial infections is also supported by its proven antioxidant activity [23
]. As known, the infectious process is associated to inflammatory response and the resulting oxidative stress is, undoubtedly, of importance in the mechanism defence of the host, but can cause tissue damage during the inflammation. In this context, the reported antioxidant properties of abietic acid might be protective during the infection for the host tissues. Worthy of note, abietic acid presented good biocompatibility as assessed by hemolytic assay, demonstrating a negligible lytic activity, and it was no toxic against human normal fibroblasts [23
4. Materials and Methods
4.1. Chemical Used
Abietic acid (75%) (Alfa Aesar, Ward Hill, MA, USA) was dissolved in ethanol and diluted in Mueller–Hinton broth to give a stock solution. XTT (Roche Diagnostics, Monza, Italy); PBS, Gram staining, crystal violet-staining (Sigma-Aldrich, Milan, Italy); Penicillin G, ampicillin, amoxicillin-clavulanic acid and oxacillin, ceftriaxone, ceftazidime, imipenem, chloramphenicol, ciprofloxacin, gentamicin, streptomycin, clindamycin, erythromycin, vancomycin, tetracycline, linezolid, sulfametoxazole-trimethoprim (Oxoid Ltd., Basingstoke, UK).
4.2. Bacterial Strains and Culture
Five S. pseudintermedius strains were used in the study. Two strains were isolated from dogs with otitis externa at the Microbiology Laboratory of the Department of Veterinary Medicine and Animal Production, University of Naples Federico II (Italy). Three S. pseudintermedius strains were isolated by human wounds at the Bacteriology and Mycology Section of the Department of Molecular Medicine and Medical Biotechnology, School of Medicine and Surgery, University of Naples Federico II (Italy). The samples were plated on blood agar base supplemented with 5% sheep blood and on mannitol-salt agar and incubated aerobically at 37 °C for 24–48 h. Bacteria were firstly examined by macroscopic observation of the colonies, Gram staining, standard laboratory methodologies (catalase and staphylocoagulase tube test), and, finally, identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonics, Macerata, Italy).
4.3. Molecular Analysis
Genomic DNA extraction was performed by using GenUp Bacteria gDNA kit (BiotechRabbit, Berlin, Germany) according to the manufacturer’s instructions. All the isolates were tested for S. pseudintermedius
-specific thermonuclease nuc
gene and mec
operon using the polymerase chain reaction (PCR) [36
]. One µL of DNA was amplified in a reaction mixture containing 10 mM Tris–HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 10 μM dNTP, 10 μM forward and reverse primers (MecA
F-5′-TCCACCCTCAAACAGGTGAA-3′, R-5′-TGGAACTTGTTGAGCAGAGGT; Mec1
F-5′-TCATCTGCAGAATGGGAAGTT, R-5′-TTGGACTCCAGTCCTTTTGC; MecR1
F-5′-AGCACCGTTACTATCTGCACA, R-5′- AGAATAAGCTTGCTCCCGTTCA; rRNA16S F-5′- CGGTCCAGACTCCTACGGGAGGCAGCA, R-5′-GCGTGGACTACCAGGGTATCTAATCC) and 2.5 U of Taq DNA polymerase (BiotechRabbit, Berlin, Germany) in a final volume of 25 µL. The cycling conditions were as follows: preheating for 5 min at 95 °C, followed by 32 cycles of denaturation at 95 °C/30 s, annealing at 55 °C/40 s, extension at 72 °C/30 s and final extension for 5 min at 72 °C. The expected PCR products were 139 bp for MecA
, 103 bp for Mec1
and 142 bp for MecR1
primers. The reaction was carried out in a DNA thermal cycler (MyCycler, Bio-Rad, Milan, Italy). The PCR products were analyzed by electrophoresis on 1.8% agarose gel in TBE and analyzed on a Gel Doc EZ System (Bio-Rad, Milan, Italy)
4.4. Antimicrobial Susceptibility Testing
The antimicrobial susceptibility patterns of isolated strains were determined by disk diffusion test on Mueller–Hinton agar (Oxoid Ltd., Basingstoke, UK). The inhibitory zone diameters obtained around the antibiotic disks were measured after incubation for 24 h at 37 °C and evaluated according to the Clinical and Laboratory Standards Institute (2013). Twenty-two commercial antibiotic discs (Oxoid Ltd., Basingstoke, UK) were tested. MIC of abietic acid was determined in Mueller–Hinton medium by the broth microdilution assay, according to the European Committee on Antimicrobial Susceptibility Testing. The compound was added to bacterial suspension in each well yielding a final cell concentration of 5 × 105 CFU/mL and a final compound concentration ranging from 0.125 to 128 μg/mL. Negative control wells were set to contain bacteria in Mueller–Hinton broth plus the amount of ethanol used to dilute each compound. Positive controls included oxacillin (2 μg/mL) and vancomycin (2 μg/mL). Medium turbidity was measured by a microtiter plate reader (Tecan, Milan, Italy) at 595 nm. Minimum bactericidal concentration (MBC) was defined as the concentration that caused ≥3log10 reduction in colony count from the starting inoculum plated on TSA, incubated for 24 h at 37 °C.
4.5. Killing Rate
Time kill assay was carried out as previously described by Olajuyigbe et al. [37
] with minor modifications. Bacterial suspension (105
CFU/mL) was added to microplates along with abietic acid at the MIC value. Plates were incubated at 37 °C on an orbital shaker at 120 rpm. Viability assessments were performed at 0, 2, 4, 6 and 24 h by plating 0.1 mL undiluted and 10-fold serially diluted samples onto Mueller–Hinton plates in triplicate. After the overnight incubation at 37 °C, bacterial colonies were counted and compared with counts from control cultures.
4.6. Checkerboard Method
The interaction between abietic acid and oxacillin against MRSP strains was evaluated by the checkerboard method in 96-well microtiter plates containing Mueller–Hinton broth. Briefly, abietic acid and oxacillin were serially diluted along the y and x axes, respectively. The final concentration ranged from 1/16 to 1 × MIC for oxacillin and from 1/64 to 1 × MIC for abietic acid. The checkerboard plates were inoculated with bacteria at an approximate concentration of 105
× CFU/mL and incubated at 37 °C for 24 h, following which bacterial growth was assessed visually and the turbidity measured by microplate reader at 595 nm. To evaluate the effect of the combination treatment, the FIC index for each combination was calculated as follows: FIC index = FIC of abietic acid + FIC of oxacillin, where FIC of abietic acid (or oxacillin) was defined as the ratio of MIC of abietic acid (or oxacillin) in combination and MIC of abietic acid (or oxacillin) alone. The FIC index values were interpreted as follows: ≤0.5, synergistic; >0.5 to ≤1.0, additive; >1.0 to ≤2.0, indifferent; and >2.0, antagonistic effects [26
4.7. RNA Isolation and RT-PCR
MRSP strain was treated with sub-MIC concentration (16 μg/mL) of abietic acid for 30 min. Total RNA was isolated by using GenUp Total RNA kit (BiotechRabbit, Berlin, Germany) according to the manufacturer’s instructions. DNA contamination from the total RNA was removed by incubation with DNase I (RNase-free DNase Set, Qiagen, Hilden, Germany). Measuring A260/A280 nm ratio assessed the nucleic acid purity. To generate cDNA, total RNA was reverse transcribed by using (RevertUP II Reverse Transcriptase, BiotechRabbit, Berlin, Germany) into cDNA using random hexamer primers (Random hexamer, Roche Diagnostics, Monza, Italy) at 48 °C for 60 min according to the manufacturer’s instructions. RT-PCR was carried out using 2μl of cDNA. Quantification data were normalized to the reference gene for 16S rRNA gene and analyzed by Image Lab software 5.2.1 (Bio-Rad, Milan, Italy).
4.8. Effect of Abietic Acid on Biofilm Formation
Abietic acid was added at concentrations ranging from 0.25 μg/mL to 2 μg/mL to an equal volume of medium containing 106 CFU/mL of MSSP and MRSP1. Control cells were grown in medium broth alone. After culturing for 24 h at 37 °C, the supernatant was gently removed, and the wells were rinsed with 200 μL of PBS. The biofilm biomass was measured by staining with 0.1% crystal violet-staining and the absorbance measured at 595 nm using a microtiter plate reader (Bio-Rad, Milan, Italy).
4.9. Effect of Abietic Acid against Preformed Biofilm
Biofilm was formed in 96-well microtiter plates. After 24 h planktonic cells were removed, and the wells were rinsed with PBS. Abietic acid was then added at the final concentration ranging from 10 μg/mL to 128 μg/mL and the plates were incubated for 24 h at 37 °C. Control biofilms were grown without abietic acid. At the end of the experiment crystal violet-staining was performed to assess biofilm biomass, as above described.
4.10. Quantitation of Metabolic Activity of Mature Biofilm
The XTT [2,3-bis(2-methyloxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay was used to quantify the metabolic activity of preformed biofilms of MSSP and MRSP1. The assay was conducted as described by Vollaro et al. with some modifications [38
]. After the treatment, XTT was added to each well and the plate incubated at 37 °C for 40 min in the dark. Changes in the absorbance of XTT, due to the reduction of the tetrazolium salt, were measured spectrophotometrically at 490 nm.
4.11. Confocal Laser Scanning Microscopy
Confocal laser scanning microscopy (CLSM) was used to illustrate the effect of abietic acid on viability and architecture of mature biofilms of MSSP and MRSP1. Cells were grown in chambered cover glass (μ Slide 4 well; ibidi GmbH, Gräfelfing, Germany) in a static condition. Abietic acid was added on a 1-day-old biofilm at 2 × MIC value for each strain. After 24 h, biofilms were rinsed and stained by using a LIVE/DEAD® BacLight Bacteria Viability stains (Life Technologies, Monza, Italy). The images were observed using an LSM 700 inverted confocal laser-scanning microscope (Zeiss, Arese, Milano, Italy), using a 10× objective lens with the signal recorded in the green channel for Syto9 (excitation 488 nm, emission 500–525 nm) and in red channel for PI (excitation 500–550 nm, emission 610–650 nm).
4.12. Statistical Analysis
Each experiment was performed in triplicate and repeated three times on different days. Arithmetic means and standard deviations were calculated. Student’s t-test was used to determine statistical differences between group means.