Antibacterial and antibiofilm activity of 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H pyrazoles and thiazoles in multidrug-resistant pathogens

Abstract

To find novel antibiotic drugs, six 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H derivatives named 1b, 1d (pyrazoles), 2a, 2b, 2c, and 2d (thiazoles) were evaluated in silico and in vitro. The in silico analyses were based on ADME pharmacokinetic parameters (absorption, distribution, metabolism, and excretion). The in vitro antibacterial activity was evaluated in Gram-positive and Gram-negative species (Staphylococcus aureus ATCC® 25904, Staphylococcus epidermidis ATCC® 35984, Klebsiella pneumoniae ATCC® 700603, and Acinetobacter baumannii ATCC® 19606), by determination of minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), kinetics curve, and antibiofilm assays. As results, the azoles have activity against the Gram-negative species K. pneumoniae ATCC® 700603 and A. baumannii ATCC® 19606. No antibacterial activity was observed for the Gram-positive bacteria evaluated. Thus, the azoles were evaluated against clinical isolates of K. pneumoniae carbapenemase (KPC) and A. baumannii multidrug-resistant (Ab-MDR). All azoles have antibacterial activity against Ab-MDR isolates (Gram-negative) with MIC values between 512 μg/mL and 1,024 μg/mL. Against KPC isolates the azoles 1b, 1d, and 2d present antibacterial activity (MIC = 1,024 μg/mL). In the kinetics curve assay, the 1b and 1d pyrazoles reduced significantly viable cells of Ab-MDR isolates and additionally inhibited 86.6 to 95.8% of the biofilm formation. The in silico results indicate high possibility to permeate the blood–brain barrier (2b) and was predict human gastrointestinal absorption (all evaluated azoles). Considering that the research and development of new antibiotics is a priority for drug-resistant pathogens, our study revealed the antibacterial and antibiofilm activity of novel azoles against K. pneumoniae and A. baumannii pathogens.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-023-01110-2.

Introduction

Bacteria resistance is considered a major threat to public health since the therapeutic options available become increasingly limited [1]. Some opportunistic pathogens are frequent in cases of infections associated with the hospital environment such as Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, and Staphylococcus epidermidis [2]. These bacteria express different virulence factors that benefit the colonization of their host and are commonly associated with infections acquired in the community and can easily infect immunocompromised individuals or with an illness that increases the risk of infections [3].

Considering the estimative in the next three decades, approximately 300 million people will die due to cases of infections by multidrug-resistant (MDR) micro-organisms [1]. The indiscriminate use of antimicrobials directly affects public health, but they are also responsible for affecting the economy since there is a need for greater spending on medical care for the treatment of individuals affected by infections by these agents [4]. In addition to multidrug-resistance, these bacteria express different virulence factors, including biofilm formation. This three-dimensional structure helps cells to persist in the environment, also facilitating the transfer of resistance and virulence genes [5, 6]

The research of new antibiotics that present activity against different micro-organisms, especially against MDR bacteria, is extremely relevant since therapeutic alternatives are increasingly reduced. Therefore, some recent studies have proposed different approaches, such as changes in the structure of drugs already known or even the synthesis of new compounds from natural polymers [7, 8]. Thus, pyrazoles and thiazoles heterocyclic compounds belonging to the family of azoles have the potential for exploration due to their different biological properties as antitumor activity [9], antidepressant [10], antidiabetic [11], analgesic [12], antiparasitic [13, 14], antimycotic [15], and antibacterial [16, 17].

During the process of searching for and developing novel drugs, the in silico analysis possibility evaluates a large number of molecular structures according to diverse parameters to select compounds that have the best chance to become an effective medicine for patients. The estimative of absorption, distribution, metabolism, excretion, and toxicity (ADME-tox) parameters reduces drastically the fraction of pharmacokinetics failure, being a valid alternative to experimental procedures, especially at initial steps [18]. Furthermore, the use of in silico analysis can be a great alternative to understanding what structural changes may be necessary for the structure of synthetic compounds to improve their activity.

In addition, the synthesis of these compounds has the advantage of using clean chemistry, using renewable solvents, presenting high yields ad purity, and the reactions can be carried out in a short time [19, 20]. Among the pyrazoles and thiazoles synthesized and described in the literature to date, the antibacterial activity of compounds derived from 1-thiocarbamoyl-3.5-diaryl-4,5-dihydro-1H has not been reported. Thus, here we evaluate for the first time the in silico and in vitro antibacterial and antibiofilm activity of six novel pyrazoles and thiazoles, against pathogens listed as a priority to the World Health Organization (WHO) for research and development (R&D) of new antibiotics [1].

Material and methods

Chemicals

In a 50-mL rounded flask, the pyrazoles 1a–d [19] (2.0 mmol) and 2-bromoacetophenone (2.0 mmol) were mixed with ethanol (10 mL) without a catalyst (Fig. 1). The reaction mixtures were then in a microwave CEM, model Discover System, with power 100 W, 250 psi at 50 °C for 5 min. The obtained precipitates were filtered and washed with cold ethanol to give pure 2-(3,5-diaryl-4,5-dihydro-1H- pyrazol-1-yl)-4-phenyl thiazoles 2a–d. The ¹H and ¹³C NMR spectra were recorded on a Bruker DRX500 Spectrometer in CDCl₃ (500 MHz for ¹H and 126 MHz for ¹³C) using TMS as an internal standard. Melting points were recorded in open capillary on an electrothermal apparatus. All solvents and chemicals were of research grade and were used as obtained from Sigma-Aldrich (St. Louis, USA).

Fig. 1.

Synthesis of 2-(3,5-diaryl-4,5-dihydro-1H-pyrazol-1-yl)-4-phenyl thiazoles

Reference strains and clinical isolates

The antibacterial activity of the six pyrazoles and thiazoles was evaluated against bacterial species commonly associated with human infection. The reference strains were obtained from American Type Culture Collection (ATCC) provided by Oswaldo Cruz Foundation (FIOCRUZ, Rio de Janeiro, Brazil), which are Gram-positive Staphylococcus aureus ATCC® 25904, Staphylococcus epidermidis ATCC® 35984 and Gram-negative Klebsiella pneumoniae ATCC® 700603 and Acinetobacter baumannii ATCC® 19606. Four clinical isolates of A. baumannii multidrug-resistant (Ab-MDR) and four of K. pneumomiae carbapenemase (KPC) previously characterized [21, 22] were included in this study. The isolates belonging to the collection of the Laboratory of Bacteriology and Bioassays (LaBBio), Federal University of Pelotas (UFPel, Pelotas, RS, Brazil) were provided by the Microbiology Laboratory of School Hospital, Federal University of Pelotas (UFPel, Pelotas, RS, Brazil).

Minimal inhibitory and bactericidal concentration (MIC and MBC)

The pyrazoles (1b and 1d) and thiazoles (2a–d) were evaluated for their antibacterial activity by minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). The MIC and MBC were determined by broth microdilution on polystyrene plates as established by Clinical and Laboratory Standards Institute standards [23]. For the tests, stoke solutions of azoles were prepared in dimethyl sulfoxide 5% (DMSO) (Sigma®), and they were added to Mueller–Hinton broth (MH) (Kasvi®). The concentration of azoles in the culture medium ranged from 1 to 1,024 μg/mL. As controls, only the culture medium, culture medium plus the bacterial inoculum, and culture medium plus azoles were used. The tests were performed in triplicate, with three replicates for each bacterial strain, and incubated at 36 °C for 24 h. After the incubation period, 10 μL of resazurin 0.2% (Sigma ®) was added to wells, and the plates were incubated at 36 °C for 2 h. The formation of pink staining indicated possible antibacterial activity, making it possible to determine MIC. Following, MBC was determined from the results obtained by MIC. Aliquots were collected from each well that did not show a color change after adding the resazurin, being sown in plates containing BHI agar (Kasvi®), which were incubated for 24 h at 36 °C. After the incubation period, it was observed which azole present bactericidal activity, indicated by the absence of bacterial growth. The tests were performed in triplicate with three replicates for each strain.

Kinetics curve assay

The pyrazoles 1b and 1d were evaluated when their inhibitory activity on the bacterial growth curve for A. baumannii ATCC® 19606 and Ab-MDR 15 followed the protocol described by Daniel Janbun et al. [24], with modifications. The 2 × MIC of pyrazoles was prepared in BHI broth, and a bacterial suspension was added (1 × 10⁶ CFU/mL). At times 0, 1, 2, 6, and 24 h, 100 μL was removed and diluted (1:10) in saline solution. From the dilutions, drop counts were made using MacConkey Agar (Kasvi®), and plates were incubated at 37 °C for 24 h. The number of viable colonies was enumerated and expressed as a log.CFU/mL.

Biofilm inhibition

In this assay, the biofilm formed by A. baumannii ATCC® 19606 and Ab-MDR 15 was treated with the pyrazoles 1b and 1d before induction of biofilm formation [25]. For this, 20 μL of bacterial suspension (1 × 10⁶ CFU/mL) was added to 180 μL of MH Broth (Kasvi®) with MIC and 2 × MIC of pyrazoles. Controls were included: MH with the concentration of pyrazoles tested, biofilm-forming strain (C +) and MH broth without bacteria (C −). The microplates were incubated for 24 h at 37 °C. After, the contents of the plates were removed and washed with a sterile saline solution (200 μL) to removed not added cells. The added cells were fixed with 99.8% methanol (200 μL) and stained with 0.5% of violet crystal (200 μL). Next, the wells were washed with sterile saline, and the dye was dissolved with 100% ethanol (200 μL). The optical density of biofilms was measured at 540 nm using POLARIS EE Spectrophotometer (Celer Biotecnologia S.A., Brazil). The results were expressed as a percentage of inhibition through the formula: 100 × (1 − (OD_treatment − OD_{negative control})/(OD_{positive control} − OD_{negative control})), where OD_treatment is the OD of bacterial biofilm exposed to different concentrations of Bio-AgNP, OD_{negative control} is the OD of wells containing MH broth only, and OD_{positive control} is the OD of wells containing the biofilm untreated.

In silico assays

The in silico prediction of ADME pharmacokinetic parameters (absorption, distribution, metabolism, and excretion) was performed using free software: Swiss ADME (available at: http://www.swissadme.ch/) and ADMETlab (available at: https://admet.scbdd.com/) [26]. With the chemical notation SMILES (Simplified Molecular Input Line Entry System) for the molecular structures, each of the azoles was evaluated according to lipophilicity (XLOGP3 _o/w), water solubility (Log S), blood–brain barrier permeator, P-glycoprotein substrate, human gastrointestinal absorption, and mutagenicity. Also, the azoles were screened for their physicochemical properties using the Lipinski rule of five (molecular weight, logarithms of partial coefficient, hydrogen bond donor, and hydrogen bond acceptor) [27].

Statistical analysis

The results were analyzed through two-way analysis of variance (ANOVA) and Dunnett’s post-test to detect significant differences between the treatments. For the antibiofilm, one-way ANOVA and Tukey post-test were used. For the analysis, the GraphPad Prism 8.2.0 software was used, where values of p<0.05 were considered statistically significant.

Results

1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H pyrazoles and thiazoles

A sequence of 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H-pyrazoles was synthesized by thiosemicarbazide and the different substituted chalcones [19], as shown in Fig. 1. The reaction was performed in microwave irradiation to avoid excessive reaction times and high temperatures. All compounds exhibited physical and spectrometric properties consistent with the proposed structures and following the literature [20]. Table 1 presents the chemical structure and molecular weight of pyrazoles 1b and 1d and thiazoles 2a–d obtained from 1-thiocarbamoyl-3,5–diaryl-4,5–di–hydro-1H.

Table 1.

Chemical structure and molecular weight of pyrazoles and thiazoles obtained from 1-thiocarbamoyl-3,5–diaryl-4,5–di–hydro-1H–pyrazole

In vitro antibacterial activity

The in vitro antibacterial activity of azoles against the standard strains and isolates is described in Table 2. The azoles 1a and 1c have not been evaluated for their antibacterial activity because were not soluble in the culture medium. According to the results, the pyrazoles and thiazoles derivates demonstrated activity against the Gram-negative species K. pneumoniae ATCC® 700603 and A. baumannii ATCC® 19606 with MIC of 1,024 μg/mL. The azoles evaluated did not demonstrate activity against the Gram-positive species S. aureus ATCC® 25904 and S. epidermidis ATCC® 35984 at the concentrations tested. It was not possible to determine MBC among the concentrations evaluated for all azoles.

Table 2.

In vitro antibacterial activity of six derivates of 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H- pyrazoles and thiazoles against reference strains and clinical isolates

MIC (μg/mL)
Bacteria strains	2a	2b	2c	2d	1b	1d
S. aureus ATCC® 25904	> 1,.024	> 1,.024	> 1,024	> 1,024	> 1,024	> 1,.024
S. epidermidis ATCC® 35984	> 1,.024	> 1,024	> 1,24	> 1,024	> 1,024	> 1,.024
A. baumannii ATCC® 19606	1,.024	1,024	1,.024	1,024	1,024	1,.024
K. pneumoniae ATCC® 700603	1,024	1,024	1,024	1,024	1,024	1,.024
Ab-MDR 2	> 1,024	1,.024	1,024	> 1,024	1,024	1,024
Ab-MDR 13	1,024	1,024	1,024	1,.024	512	1,024
Ab-MDR 15	1,024	1,024	1.,024	1,024	512	512
Ab-MDR 47	1,024	1,024	1,024	1,024	1,024	1,024
KPC 1	> 1,024	> 1,024	> 1,024	1,024	> 1,024	1,024
KPC 2	> 1,024	> 1,024	> 1,024	1,024	1,024	1,024
KPC 3	> 1,024	> 1,024	> 1,024	1,024	> 1,024	1,024
KPC 4	> 1,024	> 1,024	> 1,024	1,024	1,024	1,024

Ab-MDR multidrug-resistant clinical isolate of A. baumannii, KPC K. pneumoniae carbapenemase producers, MIC minimal inhibitory concentration

The azoles showed inhibitory activity against four Ab-MDR isolates (2, 13, 15, and 47), except the thiazoles 2a and 2d which did not present antibacterial activity against Ab-MDR 2. The 1b pyrazole was the more active against Ab-MDR 13 and 15 (MIC = 512 μg/mL) and 1d against Ab-MDR 15 (MIC = 512 μg/mL). For the other azoles, the MIC was observed in the highest concentration tested (MIC = 1,024 μg/mL). For the KPC isolates, 2d and 1d azoles showed activity against all KPC isolates (MIC = 1,024 μg/mL). The 1b azole presented inhibitory activity (MIC = 1,.024 μg/mL) against two KPC isolates (KPC 2 and KPC 4), and other azoles were not active against KPC isolates.

Kinetic of activity

The pyrazoles 1b and 1d demonstrated increased antibacterial activity against Ab-MDR isolates, thus being evaluated in the growth curve and antibiofilm assay at 2 × MIC. In the kinetic curve assay of A. baumannii ATCC® 19606 and Ab-MDR 15, it is possible to observe in Fig. 2 that both pyrazoles inhibited cell multiplication after 6 h of treatment presenting a significant difference when compared to the control (p < 0.05). In 24 h, the pyrazoles were able to reduce 5.8 to 10.8 logs.CFU for both ATCC® 19606 and Ab-MDR 15. No significant difference was observed between the pyrazoles.

Fig. 2.

Kinetic curve assay of pyrazoles 1b and 1d against Acinetobacter baumannii ATCC® 19606 and A. baumannii multi-drug resistant isolate (Ab-MDR 15) Control: standard curve (untreated bacteria); Different letters (a and b) means the statistical difference between treatments (p < 0.05)

Antibiofilm activity

The inhibition of biofilm formation was evaluated for both pyrazoles 1b and 1d at MIC and 2 × MIC against the standard strain and Ab-MDR 15 isolate (Fig. 3). These pyrazoles showed percentages of biofilm-forming inhibition varying between 86.6% and 95.2% for ATCC® 19606 and between 94.4% and 95.6% for the Ab-MDR 15 isolate. There was no statistical difference between treatments, which can be justified because pyrazoles belonging to the same class and has a similar chemical structure.

Fig. 3.

Antibiofilm activity of pyrazoles 1b and 1d in Acinetobacter baumannii ATCC® 19606 and A. baumannii multi-drug resistant isolate (Ab-MDR 15). Control: standard curve (untreated bacteria); *statistical difference when compared to control (p < 0.05)

ADMET-tox analysis

The results of the pharmacokinetics properties of the azoles are presented in Table 3. Only two compounds (2a and 1d) violate Lipinski’s rule. The partition coefficient between n-octanol and water (XLOGP3_o/w) is the descriptor for lipophilicity used in this work (threshold: − 0.7 and + 5.0), and only 2a, 2b, and 1d were considered lipophilic. When water solubility was predicted we observed that these three compounds also have low LogS values and are considered P-glycoprotein substrates. All compounds have high levels of possibility to permeate the blood–brain barrier, with the lowest value in compound 2b (74%). Also, all the compounds showed high levels of human gastrointestinal absorption prediction, and 2b demonstrates the highest mutagenicity percentage of all the compounds.

Table 3.

In silico analysis of six compounds derivates of 1-thiocarbamoyl-3,5-diaryl-4,5-dihydri-1H-pyrazoles and thiazoles

Compounds	Violates Lipinski*	HGA (%)**	LogS *	Permeates BBB (%)**	P-gp substrate*	Mutagenicity (%)**	Lipophilicity*
2a	Yes	82.5	L	83	Yes	54	6.05
2b	No	73.8	L	74.1	Yes	54.8	6.02
2c	No	87	M	99.2	No	32.8	2.83
2d	No	86.3	M	97.9	No	30.8	3.36
1b	No	74.7	M	96.1	No	40.6	2.7
1d	Yes	84.8	L	85.4	Yes	37.2	6.68

HGA human gastrointestinal absorption, LogS water solubility, L lower, M moderate

^*Swiss ADME

^**ADMETlab

Discussion

The increase in rates of nosocomial infections caused by MDR bacteria in recent decades has become a major public health problem due to high rates of morbidity and mortality [1, 27, 28]. For the WHO [1] the research and development of new antibacterial against A. baumannii and K. pneumoniae resistant to carbapenems, such as isolates evaluated in this study, are considered a critical priority. Thus, research that evaluates new compounds with antibacterial potential is extremely necessary to arrive at an efficient alternative in the control of these bacteria. Our results demonstrated no activity of compounds against Gram-positive bacteria, although indicate a potential application against important Gram-negative MDR pathogens.

Several studies demonstrate the activity of pyrazoles against micro-organisms such as that carried by Hafez, El-Gazzar, and Al-Hussain [29] evaluating 20 compounds derived from ethyl 4-amino-3-(4-chlorophenyl)-pyrazole-5-carboxylate described in vitro activity against Escherichia coli, Pseudomonas aeruginosa, Streptococcus lactis, S. aureus, Candida albicans, and Aspergillus flavus in concentrations ranged from 10 to 50 μg/mL. Anush et al. [8] report that pyrazoles obtained from chitosan present MIC = 100 μg/mL against standard strains of S. aureus, Bacillus subtilis, E. coli, and K. pneumoniae. However, after changes to the Schiff bases of these compounds, they began to exert inhibitory activity in lower concentrations, ranging from 6 to 50 μg/mL; thus, some changes in the chemical structure of the evaluated compounds may be interesting to improve the results.

So far, a few studies have evaluated the antibiofilm activity of azoles [30]; however, they did not investigate the antibiofilm potential against A. baumannii. Considering the ability to form biofilms in A. baumannii made it even more difficult to control in the hospital environment and clinical treatment. In this sense, these virulence factors become a potential target for new drugs [27, 29]. Our study evaluated for the first time the potential antibiofilm of azoles against this important pathogen, including a clinical isolate MDR.

The antibacterial and antibiofilm activity observed against A. baumannii in our study can be attributed to the similarity in the chemical structure of the evaluated azoles (Table 1) since the antibacterial activity is directly linked to the grafted chains in the azoles, and it is a degree of hydrophobicity [30]. The pyrazolic nucleus present in these compounds is responsible for their biological activity, such as antimicrobial, anti-tumor, anti-inflammatory, and anti-thermal activity [31]. Compounds derivatives from 1-thiocarbamoyl-3,5-diaryl-4,5-dihydri-1H- pyrazoles (1b and 1d) and thiazoles (2a–d), like those evaluated in our study, have been described as active against bladder cancer [9] and had a synergistic effect when combined with doxorubicin against breast cancer [32].

In addition to in vitro and in vivo analysis the use of computational tools can be a complementary and important step to select new antimicrobials [18]. The drug-likeness of a compound is ranged with some properties, which are important to become a bioavailable oral drug. The Lipinski rule of five is the pioneer rule that implemented a method to distinguish between drug-like and no drug-like molecules [33, 34]. Only two compounds violate the Lipinski rule (2a and 1d) because both are too lipophilic molecules.

Lipophilicity plays a vital role in the therapeutic suitability of a molecule candidate for drug development. One study demonstrates that lipophilic substances are more quickly metabolized but are also toxicologically worrisome [35]. The increase of lipophilicity always leads to enhanced blood–brain barrier (BBB) diffusion, so lipophilicity of the drugs correlates significantly with the tendency to permeate the BBB and the central nervous system [36]. All the compounds showed up to 74% predicted in silico levels to permeate BBB and mutagenicity levels down to 54%, which is very crucial to a new antibacterial approach.

The compounds 2c, 2d, and 1b showed water solubility (log S), which facilitate the drug development activities. Compounds with very high hydrophilic values are not able to diffuse passively through membranes because of the hydrophobic interior of the lipophilic bilayer. One property that contributes to water solubility is molecular complexity, and this measure accounts for the number of rings and aromatic rings for example [37]. The most complex molecules with more aromatic rings analyzed in this study are the ones with less predicted solubility in water.

One crucial point in drug development is to know the metabolic routes of the studied molecule. The knowledge about compounds being substrate or non-substrate of the permeability P-glycoprotein is needed to understand the efflux through biological membranes [33]. P-glycoprotein is the most important member among ATP-binding cassette transporters, and compounds that are P-gp substrates can modulate the physiological functions of this protein by regulating the distribution of drugs [35]. Our study shows that three compounds (2a, 2b, and 1d) are predicted as P-gp substrate.

Currently, there are no studies on the in silico parameters and antimicrobial activity of 1-thiocarbamoyl-3,5-diaryl-4,5-dihydri-1H-pyrazoles and thiazoles; only research related to it is biological activity against cancer cells [9, 32] and antidepressant activity [38]. The other research describes the synthesis and characterization of their chemical structure [19, 26]. Considering the extreme necessity for studies on new potential drugs for application against MDR bacteria, our study demonstrated for the first time the activity of 1-thiocarbamoyl-3,5-diaryl-4,5-dihydri-1H- pyrazoles and thiazoles against reference bacteria and MDR human clinical isolates and their potential antibiofilm activity.

Conclusion

According to the results, we conclude that compounds derived from 1-thiocarbamoyl-3,5-diaryl-4,5-dihydri-1H-pyrazoles and thiazoles demonstrated activity in vitro against Gram-negative reference strains and clinical isolates of A. baumannii MDR and K. pneumoniae KPC pathogens, important agents of nosocomial infections. Besides, the azoles also demonstrated inhibition of biofilm formation in vitro against A. baumannii MDR and reduce cell viability. In silico analysis indicates a high possibility to permeate the blood–brain barrier and present human gastrointestinal absorption. To the best of our knowledge, this is the first study that evaluates the antibacterial and antibiofilm activity of these azoles.

Author contribution

DDH and CMPP conceived and designed the study; KFC, BNR, DTFA, MOG, SOA, and ILP carried out experimental work; DDH, CMPP, KFC, and BNR carried out the analysis and interpretation of data, analysis, and technical support; KFC, BNR, and ILP co-wrote the manuscript; DDH and CMPP supervised the work and reviewed the manuscript.

Funding

This work was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES, http://www.capes.gov.br/)—Finance Code 001 and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, http://www.cnpq.br/) which provided research (DDH and CMPP) and scholarship (KFC, MOG, SOA, BNR, and ILP).

Data Availability

All data generated or analysed during this study are included in this published article and it supplementary information file.

Declarations

Competing interests

The authors KFC, MOG, SOA, DTFA, BNR, CMPP, and DDH are inventors of a patent protecting the use of azoles as antibacterials (Instituto Nacional de Propriedade Industrial—BR1020210261064).