Antimicrobial activity of harzianic acid against Staphylococcus pseudintermedius

Abstract The emerging concern about the increase of antibiotic resistance has encouraged research efforts to develop effective alternatives to counteract bacterial infections. Herein, we studied a new perspective to therapeutic treatment against Staphylococcus pseudintermedius, an opportunistic pathogen documented as the major cause of skin, ear, and post-operative bacterial infections in dogs and cats. Antimicrobial activity of secondary metabolites produced by selected microbial strains belonging to Trichoderma, Talaromyces, Clonostachys and Coniothyrium fungal genera has been tested against S. pseudintermedius. Several extracts, particularly those obtained from Trichoderma harzianum E45 and ET45, showed a significant antimicrobial activity towards S. pseudintermedius methicillin-resistant (MRSP) and methicillin-susceptible (MSSP) strains. Bioassay-guided fractionation of E45 and ET45 extracts allowed to isolate harzianic acid as the major compound responsible for biological activities (e.g. antimicrobial, antibiofilm formation and biofilm disaggregation). Graphical Abstract


Introduction
In the last ten years, fungi have received increasing consideration for their widespread ability to develop symbiotic associations with other microorganisms and plants (Harman et al. 2004). Filamentous fungi are able to produce different types of secondary metabolites, and some of them are known for their antitumor and/or antibiotic properties. In particular, many bioactive compounds are produced by the fungi belonging to the genera Aspergillus, Penicillium, Talaromyces and Trichoderma (Nicoletti and Vinale 2018).
Genome sequencing revealed that many microorganisms have much greater ability to produce specialised metabolites than those obtained using classical bioactivity screening. This finding is supported by several studies reporting that many specialised metabolite biosynthetic gene clusters (BGCs) are not expressed in laboratory cultures, but can be activated using specific elicitors (biotic or abiotic agents capable of inducing, in the plant, the biosynthesis of metabolites involved in defensive responses) (Vinale, Nicoletti, Borrelli, et al. 2017). These fungi have been widely studied for their ability to protect plants and contain pathogen populations under different soil conditions. Moreover, secondary bioactive metabolites showed a crucial role during interactions within microbial communities, such as competition for space/nutrients, parasitism and antagonism, as well as the induction of plant defense responses (Harman et al. 2004).
In agriculture, the use of biopesticides and biofertilizers based on beneficial organisms, such as Trichoderma fungi, is widespread worldwide. Trichoderma spp. can act as bioagent improving plant growth and resistance to pathogens, increasing plant nutrient assimilation and tolerance to abiotic stress, etc. The efficacy of beneficial strains of Trichoderma has been often related to the production of secondary metabolites (Harman et al. 2004). Beneficial microorganisms also include endophytic fungi that colonise plants, resulting in numerous benefits for the host, such as protection against pathogens, the promotion of growth, the induction of disease resistance and the production of bioactive compounds with antibiotic and antitumor properties. Talaromyces pinophilus is another fungal species that can produce a variety of bioactive metabolites, including alkaloids, peptides, lactones, polichetides and compounds of multiform structure, with different chemical and biological activities . Clonostachys rosea is a mycoparasite that showed inhibiting effects on nematodes (Rodr ıguez-Mart ınez et al. 2018), but whose activity against bacteria of animal or human origin has not been reported yet. Coniothyrium aleuritis represents a small part of beneficial endophytic fungal communities of switchgrass plants and have a good aptitude to improve the growth of these plants (Xia et al. 2018). The use of natural bioactive compounds from environmental sources could be important to manage multidrug-resistant bacteria of human or animal origin.
Staphylococcus pseudintermedius represents the most common opportunistic bacterial pathogen of skin and ear infections in dogs and cats, frequently isolated from canine pyoderma and otitis externa (De Martino et al. 2016). It has been sporadically isolated also from humans, suggesting a possible zoonotic potential (Pompilio et al. 2015).
Antibiotic resistance is an important and emerging alarm for veterinary medicine and public health. In a previous study, the clonal spread of methicillin-resistant S. pseudintermedius (MRSP), which is causing growing concern in small animal practice, was investigated (Perreten et al. 2010). Moreover, several bacterial pathogens produce biofilms, that are virulence factors causing persistent chronic infections (Grant and Hung 2013). Biofilms formed by MRSP are resistant to many antimicrobial active ingredients, including b-lactams (Davies, 2003). In this study, we evaluated the in vitro antimicrobial and antibiofilm capability of bioactive compounds extracted from Trichoderma, Talaromyces, Clonostachys and Coniothyrium spp., against both methicillin-resistant (MRSP) and methicillin-susceptible (MSSP) S. pseudintermedius strains.
LC-MS qTOF analysis detected different compounds in ESI positive ion mode directly from crude fungal extracts (Tafuri et al. 2019). Table S2 reports the metabolites identified by comparing raw data with an in-house database. In particular, several metabolites, belonging to different classes of natural compounds, have been identified: i) the tetramic acid derivatives harzianic acid and iso-harzianic acid (both from T harzianum E45 and ET45); ii) the polyketides monocillin III (from T. pinophilus K9 and T. flavus K5), as well as harzianolide, deydro-harzianolide and T39 butenolide (from T. harzianum E36); iii) the diketopiperazine cyclo-(L-Phe-L-Pro) (from T. saturnisporum K14); iv) the funicone 3-O-methylfunicone (from T. pinophilus E2). From C. aleuritis K6 no compounds included in the database have been identified.
Harzianic acid (HA; Figure 1), isolated from both T harzianum E45 and ET45, showed the same chromatographic and spectroscopic properties of a standard sample. HA is the main compound isolated in this work in term of amount per litre of culture filtrate (190 mg per litre of culture filtrate).

General characteristics of S. pseudintermedius strains used in this study
In this study, two strains of S. pseudintermedius, one methicillin-resistant (MRSP) and another methicillin-susceptible (MSSP), both of canine origin, have been used to evaluate the activity of fungal extracts. Oxacillin resistance, confirmed by the detection of mecA gene by PCR, allowed us to identify the strain as MRSP.
The antibiotic resistance pattern of MRSP and MSSP evidenced several resistances (Table S3). Both strains showed resistance to amoxycillin/clavulanic acid, ampicillin, penicillin, erythromycin, clindamycin, tetracyclin and sulfamethoxazole/trimethoprim. Furthermore, both bacterial isolates showed biofilm formation activity, with MRSP strain resulted to be a strong biofilm producer compared with MSSP, which was classified as moderate biofilm producer (data not shown).

Antimicrobial and antibiofilm activity of fungal extracts
Fungal extracts demonstrated antimicrobial activity against MRSP and MSSP, with minimum inhibitory concentration (MIC) values of 32 or 64 mg ml À1 , except when strains K6 or E43 were used (MIC >128 mg ml À1 ). Furthermore, the extracts obtained by T. harzianum E45 and ET45, as well as T. pinophilus K9, showed the highest antimicrobial activity against both MRSP and MSSP (Table S4).
The crystal violet staining assay demonstrated that only T. harzianum ET45 and E45 significantly inhibited the biofilm biosynthesis in both MRSP and MSSP. In particular, both ET45 and E45 extracts showed a maximum of about 80% biofilm inhibition at 128 lg ml À1 ( Figure S1(A and B)). Significant antibiofilm activity was already observed at 16 lg ml À1 (p < 0.05) for ET45 extract on both MRSP and MSSP, whereas E45 extract reduced biofilm formation at concentrations ! 32 mg ml À1 ( Figure S1(A and B)). No disaggregation of pre-formed biofilms produced by both MRSP and MSSP after 24 h exposure of fungal extracts at concentrations ranging from 8 to 128 lg ml À1 was observed (data not shown).

Harzianic acid activities
Harzianic acid (HA, Figure 1) was the major compound isolated by E45 and ET45 extracts using bioassay-guided fractionation. HA showed significant antimicrobial activity against MRSP and MSSP, with MIC values of 32 mg ml À1 and 16 mg ml À1 , respectively ( Figure S2(A and B)). The antibiofilm effects of HA, assessed by microtiter plate method, resulted in a significant inhibition of biofilm formation in both MRSP and MSSP already at 16 mg ml À1 , and lasting in a dose-dependent manner ( Figure S3(A)). Interestingly, in the presence of HA at 32 and 64 mg ml À1 , statistically significant (p < 0.05) disaggregation of pre-formed biofilm produced by MSSP was also observed ( Figure S3(B)).
Harzianic acid is a tetramic acid derivative defined by the presence of an unnatural 4,4-disubstituted glutamic acid unit. This natural product was prepared in a six steps synthesis with an over-all yield of 22% (Healy et al. 2015). HA biosynthesis has not been studied yet, but it could possibly derive from a pentaketide bounded with an amino acid (Sivasithamparam and Ghisalberti 1998). Vinale et al. (2013) demonstrated the capability of this tetramic acid derivative to bind Fe 3þ with a good affinity, which may represent a significant mechanism altering nutrient availability in S. pseudintermedius. Moreover, bacteria need iron acquisition for biofilm formation (Kang and Kirienko 2018); harzianic acid may possibly interfere with biofilm formation by limiting bacterial iron availability, but further experiments are necessary in order to demonstrate this hypothesis.

Effect of fungal extracts on HaCaT cells viability
Cytotoxic effects of fungal extracts were tested on HaCaT cells. Cells were treated at the concentrations of 32 and 64 mg ml À1 and the viability was recorded after 24 h. Eight out of the ten tested fungal extracts did not display a statistically significant variation of the cell viability, whereas K6 and E43 extracts reduced HaCaT cells viability of 36% and 25%, respectively, compared to the untreated control ( Figure S4). In addition, all fungal extracts neither displayed cytotoxic activity, nor induced modification of cell morphology or evident signs of cell death at any tested concentrations. Moreover, pure harzianic acid did not significantly alter HaCaT cells viability at concentrations ranging from 8 to 128 mg ml À1 ( Figure S5).

Conclusions
This study focuses on fungal compounds/extracts to be used against pathogenic strains of animal origin such as S. pseudintermedius, whose infections are difficult to manage due to their multidrug resistance profiles (Frank and Loeffler 2012). Moreover, harzianic acid, the main metabolite produced by strains ET45 and E45 of T. harzianum, demonstrated the capability to act as a natural antimicrobial and antibiofilm agent, highlighting its potential use in S. pseudintermedius-associated canine skin diseases. Finally, considering the closeness between companion animals and humans, our results could have also a public health outcome.