Co-occurrence of linear and cyclic pelgipeptins in broth cultures of Paenibacillus elgii AC13

Abstract

Paenibacillus elgii AC13 produces antimicrobial lipopeptides of agricultural and pharmaceutical importance. It secretes four cyclic lipopeptides named pelgipeptins, previously characterized in P. elgii B69. These lipopeptides result from the expression of a nonribosomal peptide gene cluster. P. elgii AC13 also produced two linear lipopeptides with ratios of [M + H] + 1105 and 1119 m/z. These compounds were previously observed in Paenibacillus sp. strain OSY-N, but due to purification difficulties, their characterization was executed using synthetically produced linear pelgipeptins. In the present study, purification was achieved from the supernatants of cultures from three complex media by high-performance liquid chromatography. The partial characterization of linear pelgipeptins revealed the similar antimicrobial activity and cytotoxicity of their synthetically produced counterparts, known as paenipeptins. Cyclic forms were highly stable to changes in pH, temperature, and organic extraction with n-butanol as shown by mass spectrometry (MALDI-TOF); therefore, these steps did not cause the hydrolysis of pelgipeptins. A low-activity thioesterase could also generate the linear isoforms observed; this enzyme catalyzes the cyclization process and is coded in the same gene cluster. Alternatively, the cyclic forms were hydrolyzed by an unknown protease produced during growth in the complex medium used in the present study. Although culture conditions are known to produce pelgipeptins with different yields and amino acid compositions, the occurrence of linear and cyclic forms simultaneously has not yet been reported. A mixture of cyclic and linear pelgipeptins presents a potential advantage of the higher antimicrobial activity of cyclic forms combined with the lower cytotoxicity of linear isoforms.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-021-00597-x.

Introduction

Pelgipeptins are lipopeptides first described in Paenibacillus elgii B69. Currently, they comprise a family of five homologous compounds, or isoforms, of cyclic cationic lipopeptides containing nine amino acids and a fatty acid chain. These lipopeptides are also known for their broad-spectrum antimicrobial activity against various bacteria and soil fungi [1, 2].

Pelgipeptins are synthesized by nonribosomal peptide synthases (NRPSs) coded by the plp gene cluster (40.8 kb) identified and characterized in P. elgii B69 [3]. The molecular weights of pelgipeptins A, B, C, D [1, 2], and E [4] are 1072, 1100, 1086, 1086, and 1072 Da, respectively. Variations among pelgipeptins A to D also include substitution at the second amino acid position, which may be either a valine or isoleucine. Moreover, pelgipeptins A and D exhibit a lipid tail with a methyl group linked at its penultimate (iso) carbon, whereas pelgipeptins B and C show this to the antepenultimate (anteiso) carbon. P. elgii BC34-6 isolated from red clay soil produces pelgipeptin E [4]. This compound exhibits the same stereochemistry as the amino acid and the ester bond of pelgipeptins A and C, but it is attached to a straight lipid chain.

A study has reported that Paenibacillus sp. strain OSY-N synthesizes three lipopeptides belonging to a new polypeptide family named paenipeptin A to C [5]. Paenipeptins A and B are linear lipopeptides, whereas paenipeptin C is a cyclic lipopeptide with a m/z ratio of 1133 [M + H]⁺. Paenipeptins A and B are, in fact, linear counterparts of pelgipeptins B and C, respectively [5]. However, the authors only detected these isoforms in Tryptic Soy Agar (TSA) solid culture medium and not on liquid culture. Due to the low yield of linear paenipeptins from cultures, the synthetic analogs of paenipeptins A and B were used to assess their antimicrobial activity showing the potential to eradicate biofilms. They were thus reported as an alternative strategy to fight multidrug-resistant bacteria or fungi [6].

P. elgii AC13 studied in the present work was isolated from soil samples; its draft genome was obtained previously [7]. Culture supernatants on Nutrient Broth (NB) revealed the presence of ions [M + H] + 1119 and 1105 m/z in addition to pelgipeptins A to D. These ions exhibited the same mass/charge ratio as the linear counterparts of pelgipeptins B and C, previously reported as paenipeptins A and B described in P. elgii OSY-N [5]. The current work aimed to investigate the influence of culture media on the occurrence of linear lipopeptides produced by P. elgii AC13. Culture conditions can influence the diversity of lipopeptides produced by bacteria, but it was not yet demonstrated that they could also influence the production of linear and cyclic lipopeptides. This study also investigated whether the linear pelgipeptins purified from culture supernatants presented the same characteristics and properties as their synthetic counterparts.

Methods

Bacteria strains

The antimicrobial compounds were isolated and purified from P. elgii AC13 (= CBMAI 2485) isolated from Cerrado soils, the Brazilian savanna [7]. The antimicrobial assays were performed using Escherichia coli ATCC11229 and Staphylococcus aureus ATCC14458.

Culture conditions

The occurrence of pelgipeptins in culture supernatants was evaluated on the following complex media: (i) Nutrient Broth (beef extract 0.3% and peptone 0.5%), this medium was from BD-DIFCO; (ii) Müller Hinton Broth (MH) (beef extract 0.2%, acid digest of casein 1.75%, and starch 0.15%) was from FLUKA-SIGMA; and (iii) Tryptic Soy Broth (TSB) (triptone 1.7%, bactosoytone 0.3%, glucose 0.25%, NaCl 0.5%, K₂HPO₄ 0.25%) was from KASVI. All cultures were executed in triplicates, and each experiment was repeated at least three times.

A spore stock from P. elgii AC13 was inoculated on 100 mL of each medium tested at a final concentration of 10³ spores/mL and incubated at 37 °C with shaking at 200 rpm. The lipopeptides were extracted and purified within 48 h.

Cultures starting from vegetative cells of AC13 were also obtained in NB. This procedure has been commonly utilized in other studies, including our previous work [8]. Briefly, a pre-inoculum was obtained from a single colony from P. elgii AC13 that was incubated on NB at 37 °C with shaking at 200 rpm. Growth was monitored by absorbance at 600 nm. Upon reaching an optical density of 0.6 to 0.8, a volume of 1 mL was transferred to 100 mL of sterile NB and incubated for 48 h at 37 °C with shaking at 200 rpm.

Purification and quantification of pelgipeptins

The cultures were centrifuged at 9000 × g for 10 min to remove the cells, and the lipopeptides were extracted from the supernatant with n-butanol and water (1:1; v/v). The organic layer was lyophilized, and the dry content was suspended in ultrapure water. This solution was then filtered (0.22 µm) and purified by reversed-phase high-performance liquid chromatography (RP-HPLC) using a Shim-pack Shimadzu C₁₈ column VP-ODS, 4.6 × 150 mm, and 4.6 µm particle size (Shimadzu, Japan). Mobile phases were HPLC-grade water and acetonitrile (ACN), both containing 0.1% trifluoroacetic acid (TFA). The solvent gradient was as follows: 0–10 min, 20% ACN, and 10–40 min, 20–60% ACN. The flow rate was kept at 1.0 mL/min, and the elution was monitored by measuring the absorbance at 216 nm using a UV detector (Prominence, Shimadzu, Japan). The fractions containing purified lipopeptides were collected and purified for a second time. Alternatively, a mixture of cyclic pelgipeptins A to D (pelgipeptin mix) was used; this mixture was produced and purified as described by Fulgencio et al. (2021). The purified lipopeptides were quantified by Murphy’s method [9].

Pelgipeptins were then identified using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF) (Autoflex Speed, Bruker Daltonics). The lipopeptides were mixed with an α-cyano-4-hydroxycinnamic acid solution (10 mg/mL) in a proportion of 1:1, and the spectra were acquired in reflected positive mode (RP 700–3500 m/z) with external calibration using peptide calibration standard II (Bruker Daltonics).

The quantification of the lipopeptide isoforms in the cell-free supernatant from cultures was determined by RP-HPLC. A standard curve was obtained from stock solutions with known concentrations of the lipopeptides. The quantitative chromatography of standards and supernatants after organic extraction was performed using the same column, gradient, and detection method. Purified solutions from several extractions from each culture medium were then concentrated as follows: NB 100 × , MH 25 × , and TSB 25 × to obtain a better resolution of the chromatographic profile allowing the quantification of the fractions corresponding to the linear and cyclic lipopeptide isoforms investigated in the present study.

Determination of the primary structure of the lipopeptides

The amino acid sequence was obtained using tandem mass spectrometry (MALDI-TOF MS/MS) LIFT method in positive ion mode. The primary sequence of the peptides was determined using FlexAnalysis software (Bruker Daltonics®).

Determination of minimal inhibitory concentration (MIC)

The minimal inhibitory concentration assay of purified lipopeptides was measured in 96-well microplates according to the Clinical and Laboratory Standards Institute [10]. The peptide solutions were prepared to final concentrations of 1.5–100 µM. Commercial polymyxin B (Sigma-Aldrich), penicillin G (Sigma-Aldrich), and chloramphenicol (Sigma-Aldrich) were employed as positive controls, and sterile ultrapure water was used as a negative control. The tested bacterial strains and peptide solution mixtures were incubated at 37 ºC for 16 h, and the absorbance (595 nm) was measured once per hour in a BioTek (Winooski, VT, USA) microplate reader. MICs were defined as the lowest peptide concentration that inhibited bacterial growth.

Stability assays

A mixture containing only cyclic isoforms was produced according to Fulgêncio et. al. (2021) and purified by solid-phase extraction [11]. This mixture was used to determine the stability of the pelgipeptins. The antimicrobial stability was evaluated using the agar diffusion method against E. coli ATCC ATCC11229 on the MH medium. Temperature stability assays were performed with a solution containing 100 µg/mL of the pelgipeptin mix. This solution was incubated for 12 h in the following temperatures: 40, 60, 80, and 100 °C. The pH assays were executed in a range of values between 2.0 and 12.0. The pelgipeptin mix solution was incubated for 12 h in each pH and then neutralized to pH 7.0.

The antimicrobial activity after stability tests was executed by applying 10 µg of the pelgipeptin mix (after each treatment) on a Whatman 6 mm filter paper on the MH plates pre-inoculated with E. coli. After each treatment, the inhibition halo was measured and compared with the control, which was the diameter of the halo observed for the pelgipeptins mix incubated at 37 °C and pH 7.0. In addition, the structural stability of the cyclic isoforms of pelgipeptins after each temperature and pH was investigated using RP-HPLC and MALDI-TOF. The presence of linear isoforms indicated that the treatment caused the cleavage of the cyclic lipopeptides. The structural stability was also evaluated before and after the n-butanol purification step.

Cytotoxic activities of pelgipeptins A, B, C, and D and isoform B’

The cytotoxicity of pelgipeptins A, B, C, D, and isoform B’ was evaluated using human fibroblast cells. Initially, fibroblasts were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and incubated at 5% CO₂ and 95% humidity at 37 ºC until the cytotoxicity assays were conducted.

The cell viability tests were performed on technical replicates and were assessed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) (Sigma-Aldrich, USA) colorimetric assay [12]. Briefly, the cells were seeded onto 96-well plates (10³ cells/well; 100 µL) and allowed to adhere and grow for 24 h. The cells were then incubated with fresh prepared medium and lipopeptide or polymyxin B to final concentrations of 3–50 µmol l⁻¹. After the treatment, 90 µL was added to 10 µL of MTT (50 µg), and then to each well, the cells were incubated for 4 h. Then, 100 µL of dimethyl sulfoxide (DMSO) was added to all wells and mixed to dissolve the dark blue crystals (formazan). Finally, the absorbance was measured on a microplate spectrophotometer at 570 nm. Cells treated with 50 µL of the lysis solution of sodium hypochlorite (2.5%) were used as negative control, and the untreated cells were used as the positive control group.

Statistical analysis

The data for cytotoxicity and pelgipeptin yield were expressed as the mean ± standard deviation (SD) of three independent experiments. Multiple comparisons were performed by one-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test using GraphPad Prism 9 (GraphPad Software, San Diego, CA). The comparisons between two samples were performed using the t test for unpaired data.

Pelgipeptin gene cluster

The putative sequence for the pelgipeptin gene cluster was identified by gene similarly to the P. elgii B69 plp gene cluster [3] (accession number, JQ745271.1). BLASTn tool [13] was utilized to identify the contigs containing the plp genes from the previously obtained draft genome of P. elgii AC13 [7]. Genome annotation revealed forty putative NRPS gene clusters also identified in P. elgii strain B69. The genes from the plp biosynthetic gene cluster of P. elgii B69 were aligned against the contigs from the P. elgii AC13 draft genome using Geneious 11.1.4 [14]. The missing base pairs between contigs were obtained by PCR amplification followed by Sanger sequencing of the PCR products. The NRP domains were identified using antiSMASH 5.0 [15]. The complete pelgipeptin gene cluster was obtained, and it is deposited under accession number MN577593.

Results and discussion

Paenibacillus elgii AC13 isolated from tropical soils produced four cyclic lipopeptides corresponding to pelgipeptins A to D, described previously from cultures of P. elgii B69 [1, 2]. During the maintenance of P. elgii AC13 cultures, two other compounds were detected in the supernatant of NB, and these compounds had ions of masses [M + H]⁺ 1105.7 and 1119.7 m/z. The present study identified these two ions as the linear isoforms of pelgipeptins C and B, named herein pelgipeptins C’ and B’ (Table S1). Additionally, they occurred in all complex media tested in the study: MH, NB, and TSB, but there were a few differences in their supernatants.

The RP-HPLC of supernatants obtained from MH and NB resulted in six fractions with a retention time of 24 to 30 min (Fig. 1A and B; Table S1); supernatants in TSB presented a retention time of 30 to 35 min and lower resolution in fractions (Fig. 1C). Fractions C’ and B’ corresponded to compounds with monoisotopic masses of [M + H]⁺ 1105.7 and 1119.7, respectively. Moreover, MS/MS predicted sequence of fraction C’ was the same as that found for pelgipeptin C (Fig. S1, Table S1), and fraction B’ was the same as pelgipeptin B (Fig. S2, Table S1). Thus, the purified lipopeptides obtained from P. elgii AC13 cultures in complex media were identified as follows: fractions B’ and C’ were the linear counterparts of pelgipeptins B and C, respectively.

Fig. 1.

Chromatographic profile of P. elgii AC13 supernatant after organic extraction with butanol, cultivated in A Nutrient Broth, B Müller Hinton Broth, and C Tryptic Soy Broth. C’, linear pelgipeptin C; D’, putative linear pelgipeptin D; B’, linear pelgipeptin B; A, B, C, and D indicate pelgipeptin cyclic isoforms

The remaining fractions contained the antimicrobial cyclic lipopeptides, namely, pelgipeptins A to D (Fig. 1). Fraction D’ is likely to be the linear counterpart of pelgipeptin D, but its sequencing was not performed due to difficulties in obtaining the pure fraction. Cyclic and linear forms of pelgipeptin A were also not obtained in sufficient quantities to allow their characterization.

The linear isoforms of pelgipeptins were previously detected in the cultures of Paenibacillus sp. OSY-N, and they were named paenipeptins A and B [5]. The linear lipopeptides of OYS-N were only detected in cells harvested from Tryptic Soy Agar plates. Differently from OYS-N, P. elgii AC13 produced linear and cyclic pelgipeptins in all the complex media tested. This result may be due to species or strain genetic differences, but the classification of OSY-N is not yet clear, impairing further genomic comparisons. Alternatively, the occurrence of linear pelgipeptins could result from differences in culture growth conditions, as observed in the present study.

P. elgii AC13 cultures in NB derived from vegetative cells or spores did not show significant differences (P < 0.01) in the concentration of cyclic or linear lipopeptides. Nonetheless, cultures originating from spores presented higher repeatability and were easier to set up; hence, inoculation using spores was used in subsequent experiments.

Cultivation in MH medium yielded the highest concentration of total isoforms (Fig. 2), which may be due to the abundance and nutrient variety this medium offers. Alternatively, the chemical components of the media could interact with other reagents employed in the purification procedures [16], hindering the recovery of linear forms in some complex media. This result was observed in the supernatant of TSB medium that presented a variety of other undesired compounds in the detriment of linear isoforms (Fig. 1B). Cultures in TSB produced 5.6 times fewer linear isoforms than cyclic pelgipeptins, and it was the lowest yield of linear pelgipeptins obtained from all tested media (Fig. 2). These results are in accordance with the data reported for Paenibacillus sp. OSY-N, because linear pelgipeptins (paenipeptins) were not obtained from TSB cultures but only from its solid medium counterpart, TSA [5]. Although it is true that some peptides are only produced on solid media, this may not be the case for the linear forms of pelgipeptins, as demonstrated in the present study.

Fig. 2.

Yields of linear and cyclic isoforms of pelgipeptins in different culture media. Significance level between samples obtained by Bonferroni’s post hoc test after ANOVA. * p < 0.001. NB, Nutrient Broth; MH, Muller Hinton; TSB, Tryptic Soy Broth

The reasons for the presence of linear pelgipeptins in our culture conditions are still unclear. The genome of P. elgii AC13 revealed the presence of a gene cluster highly similar to the gene cluster responsible for the synthesis of these lipopeptides in P. elgii B69 (Table 1) containing a thioesterase domain (plpB) [3].

Table 1.

Identification and similarity scores of the deduced amino acid composition of Plp proteins from Paenibacillus elgii AC13 and Paenibacillus elgii B69¹

Protein	Similarity	Function
PlpA	100%	Diaminobutyrate-2-oxoglutarate aminotransferase
PlpB	97.2%	Esterase and lipase
PlpC	98.5%	4′-phosphopantetheinyl transferase
PlpD	97.2%	Nonribosomal peptide synthetase
PlpE	96.5%	Nonribosomal peptide synthetase
PlpF	97.7%	Nonribosomal peptide synthetase
PlpG	97.9%	ABC transporter
PlpH	96.9%	ABC transporter

¹Qian et al., 2012

Thioesterases guide the lactone ring formation characteristic of pelgipeptins and other cyclic lipopeptides such as surfactin and iturin [17]; thus, linear forms could result from a low thioesterase activity. Another hypothesis is that the culture conditions or the purification process caused the hydrolysis of cyclic pelgipeptins. However, the cyclic forms of pelgipeptins were highly stable. Hydrolysis was not observed in high temperatures (Fig. S3), and it was only obtained from pH 12 (Fig. S4). In addition, the n-butanol extraction did not cause major structural changes in the purified mix of pelgipeptins (Fig. S5), indicating that this critical purification step did not cause the hydrolysis of this lipopeptide. Finally, an enzymatic activity could be responsible for the linear isoforms of pelgipeptins, but cyclic lipopeptides are generally highly resistant to protease activity. Pelgipeptins were previously reported to be resistant to degradation by pepsin, proteinase K, and trypsin (Wu et al. 2010). The hydrolysis of surfactin was only observed for Staphylococcus aureus V8 endoprotease and a novel enzyme from Streptomyces sp. MG1 [18, 19].

Antimicrobial activity of purified pelgipeptins

The antimicrobial activity of synthetically produced paenipeptins A and B was tested against 19 bacterial strains [5]. Purified pelgipeptins B’ and C’ tested here also exhibited a higher MIC than its cyclic counterpart against E. coli ATCC11229 and S. aureus ATCC14458 (Table 2). As previously reported, pelgipeptin cyclization improved the antimicrobial activity against gram-negative bacteria, but it was not a required step for its activity [5].

Table 2.

Minimum inhibitory concentration values (µM) against tested strains exhibited by pelgipeptins cyclic isoforms (A, B, C, and D), linear isoforms (B’ and C’), pelgipeptin mix, and reference antibiotics

Strains	Pelgipeptins							Controls
	A 1073 m/z	B 1101 m/z	C 1087 m/z	D 1087 m/z	B’ 1119 m/z	C’ 1105 m/z	Pelgipeptin mix	Ampicillin	Chloramphenicol	Polymyxin B
E. coli ATCC11229	6.25	12.5	6.25	6.25	50	> 100	12.5	25	NT	3.1
S. aureus ATCC14458	12.5	12.5	12.5	12.5	> 100	> 100	6.25	NT	12.5	25

NT, not tested. Pelgipeptin mix contains cyclic pelgipeptins A to D

The disk diffusion tests revealed that pH and temperature treatments did not suppress the antimicrobial activity of the pelgipeptin mix (containing cyclic pelgipeptins A to D). The inhibition halos of E. coli growth were observed in all treatments, except for the treatment at pH 12. The structural analysis of cyclic pelgipeptins submitted to different temperatures and pH conditions also revealed the presence of linear isoforms only for the assays at pH 11 and 12 (Fig. S4). Antimicrobial activity stability of cyclic lipopeptides is already known; for instance, surfactin maintains its surfactant and antimicrobial activity in pHs 5 to 13 and stability after autoclaving [20]. The cell-free extract containing cyclic pelgipeptins was previously reported to only reduce its antimicrobial activity after 2 h at 80 or 100 °C (Wu et al. 2010). Differently from the cell-free extract (Wu et al. 2010), the purified pelgipeptins studied here lost their antimicrobial activity exclusively after high pH treatments. The results presented here showed that this treatment also caused the hydrolysis of cyclic pelgipeptins, thereby producing the less active linear isoforms.

The linear isoforms isolated from P. elgii AC13 cultures presented higher MICs and were less toxic to the primary culture of human fibroblasts than the cyclic isoforms (Fig. 3). This result is in accordance with the cytotoxicity of paenipeptins (the synthetically produced linear pelgipeptins) which was tested on defibrinated rabbit blood cells [5]. Therefore, the culture-obtained linear pelgipeptins present similar characteristics as their synthetic counterparts.

Fig. 3.

Cell viability assay performed after the 24-h incubation period with different concentrations of pelgipeptins (PGP) and polymyxin B. Each bar represents the mean ± SD of cellular absorbance (abs), n = 3. *p < 0.05, **p < 0.01, ****p < 0.0001

The results obtained in the present study verified that the linear isoforms of pelgipeptins C and B occur in the liquid cultures of P. elgii AC13 along with their cyclic counterparts. It is common to observe a preferential synthesis of certain molecules to the detriment of others caused by changes in the culture medium composition or incubation conditions [16, 21]. However, this effect was not yet reported to produce the linear forms of cyclic lipopeptides.

In conclusion, the culture medium composition, incubation conditions, and extraction methods can modify the yield and the repertoire of the lipopeptides produced by Paenibacillus species. The present study demonstrated that culture media combined with the method of extraction allows the purification of the linear counterparts of the cyclic pelgipeptins produced by P. elgii AC13. This knowledge may be useful in further modulation of the quantitative and qualitative production of pelgipeptins, providing a mix of cyclic and linear isoforms from one single culture. Finally, studies on the mechanism of action and the cell toxicity of the linear pelgipeptins were executed using the synthetic linear lipopeptides C or B, named paenipeptins [6]. It would be appropriate to keep the name paenipeptins for these synthetic molecules, making it easier to differentiate them from the linear compounds that co-occur with cyclic isoforms and can be easily obtained from cultures.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contribution

All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Thiago Fellipe de Araújo, Daniel Barros Ortega, Rosiane Andrade da Costa, Isadora Emanoela Pereira Costa de Andrade, Débora Luiza Albano Fulgêncio, Marise Leite Mendonça, Flávio Silva Costa, and Michel Lopes Leite. The first draft of the manuscript was written by Cristine Chaves Barreto and Thiago Fellipe de Araújo. The final critical analysis of data was executed by Cristine Chaves Barreto, Marcelo Henrique Soller Ramada, and Rosiane Costa. All authors commented on previous versions of the manuscript.

All authors read and approved the final manuscript.

Funding

This study was financed in part by the Brazilian National Council for Scientific and Technological Development/Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Grant, 560915/2010–1). Graduate scholarships were provided by the Coordination for the Improvement of Higher Education Personnel/Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES).

Data Availability

Pelgipeptin gene cluster full sequence was deposited in the GenBank under number MN577593. Paenibacillus elgii AC13 is deposited in the Brazilian Collection of Environmental and Industrial Microorganisms – CBMAI under the number CBMAI 2485. This culture collection is part of the World Federation for Culture Collections. Paenibacillus elgii AC13 studies are registered at the SisGen System of the Brazilian government under number A356834.

Code availability

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Declarations

Ethics approval

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Conflict of interest

The authors declare no competing interests.