Microbiome Analysis of Sugarcane Juices and Biofilms from Louisiana Raw Sugar Factories

ABSTRACT During postharvest processing of sugarcane for raw sugar, microbial activity results in sucrose loss and undesirable exopolysaccharide (EPS) production. Historically, culture-based approaches have focused on the bacterium Leuconostoc mesenteroides as the main contributor to both processes. However, recent studies have shown that diverse microbes are present in sugarcane factories and may also contribute to sugarcane juice deterioration. In the present study, high-throughput amplicon-based sequence profiling was applied to gain a more comprehensive view of the microbial community in Louisiana raw sugar factories. Microbial profiling of the bacterial and fungal microbiomes by 16S V4 and ITS1 sequences, respectively, identified 417 bacterial amplicon sequence variants (ASVs) and 793 fungal ASVs. While Leuconostoc was indeed the most abundant bacterial genus overall (40.9% of 16S sequences), multiple samples were dominated by other taxa such as Weissella and Lactobacillus, underscoring the microbial diversity present in sugarcane factories. Furthermore, flask cultures inoculated with the same samples demonstrated differences in the rate of sucrose consumption, as well as the production of exopolysaccharides and other organic acids, which may result from the observed differences in microbial composition. IMPORTANCE Amplicon-based sequencing was utilized to address long-ignored gaps in microbiological knowledge about the diversity of microbes present in processing streams at Louisiana sugarcane raw sugar factories. These results support an emerging model where diverse organisms contribute to sugarcane juice degradation, help to contextualize microbial contamination problems faced by raw sugar factories, and will guide future studies on biocontrol measures to mitigate sucrose losses and operational challenges due to exopolysaccharide production.


RESULTS
Microbial community composition. Samples were collected during the October 2021 to January 2022 sugarcane harvest season in Louisiana (see Materials and Methods). DNA was extracted and amplicon-based sequencing was performed to identify both bacteria by the V4 region of the 16S rRNA gene (16S V4), and fungi by the rRNA Intergenic Transcribed Spacer 1 (ITS1) region. This yielded 1,522,114 paired-end reads for 16S V4 and 2,867,360 reads for ITS1. After processing (see Materials and Methods), 417 bacterial unique amplicon sequence variants (ASVs) and 793 fungal ASVs were identified.
Three genera of lactic acid bacteria (LAB), Leuconostoc, Weissella, and Lactobacillus, were the most abundant in the 16S data and comprised 40.9%, 14.2%, and 12.6%, respectively, of all sequences (Fig. 1A). In total, the 12 most abundant bacterial genera represented 86.0% of all bacterial sequences. Fungal communities in juices were much more evenly distributed, with the most abundant genus Saccharomyces representing 15.4% of all sequences in the juice samples (Fig. 1B). In total, the 12 most abundant genera represented 76.0% of all fungal sequences. Notably, Saccharomyces represented 91.0% of ITS1 sequences in the biofilm samples. Principal Coordinate Analysis (PCoA) plots were generated using unweighted UniFrac distances to graphically represent the structure of microbial communities. In general, biofilm samples were well-separated from juice samples, but crusher juice and mixed juice samples were not easily distinguishable ( Fig. 1C and D). Two mixed juice samples (MJ-Fact1-Oct and MJ-Fact3-Jan) clustered less closely with crusher juice samples in the 16S data set. Alpha diversity metrics were calculated for the 16S and ITS1 data using rarefied data ( Fig. 1E and F). In Permutational multivariate analysis of variance (PERMANOVA) was performed on unweighted UniFrac distances between samples using sample type as the variable (crusher juicer, mixed juice, and biofilm), and showed that overall, both bacterial and fungal community compositions significantly differed between sample types (16S: P = 0.012; ITS: P = 0.005). Pairwise PERMANOVA showed significant differences between bacterial communities in crusher juice and biofilm samples (P = 0.031), fungal communities in crusher juice and biofilms (P = 0.026), and fungal communities in mixed juices and biofilm samples (P = 0.024). There were no significant differences in the examined alpha diversity metrics or unweighted UniFrac distances when comparing samples by factory or month of sampling (P . 0.05).
Common and unique LAB. A significant consequence of microbial activity in the deterioration of sugarcane juice is the formation of dextran polysaccharides. Most reports of dextrans have been linked to LAB, including Leuconostoc, Weissella, Lactobacillus, and Streptococcus (25). As such, the ASVs belonging to the order Lactobacillales were specifically examined for commonalities and differences between samples (Fig. 2). There was FIG 1 Legend (Continued) of unweighted UniFrac distances between (C) bacterial and (D) fungal communities. Alpha diversity metrics of (E) bacterial and (F) fungal communities. CJ, crusher juice; MJ, mixed juice. Different letters represent fractions that were significantly different (P , 0.05) in pairwise Kruskal-Wallis tests. Alpha and beta diversity metrics were calculated based on rarefied data (see Materials and Methods). Comparisons were made between (A) sample groups and between individual samples of (B) crusher juice, (C) mixed juice, and (D) biofilm samples. The numbers in each region represent the shared or unique ASVs, and the numbers in the parentheses represent the relative abundance of those ASVs out of all Lactobacillales reads. significant overlap between the Lactobacillales ASVs present in crusher juice and mixed juice samples (25 out of 34), while biofilms had the highest number of unique ASVs between the three sample categories ( Fig. 2A). Nevertheless, the 15 ASVs common to all three sample categories (crusher juice, mixed juice, and biofilms) were the most abundant by read count (91% of all Lactobacillales reads). Similarly, the LAB population in individual samples were compared within each sample group, to explore whether a sample contained a unique population of LAB, which may affect dextran production ( Fig. 2B-D). In general, there was a larger number of "unique" ASVs (present in one sample) and "accessory" (present in multiple, but not all samples) ASVs than "core" (present in all samples) ASVs. However, the core ASVs made up the majority of the LAB population by the relative abundance of reads. A notable exception to this trend were the biofilm samples, which had very few unique ASVs (Fig. 2D).
Expected frequency of sucrose-dependent EPS production by sugarcane factory bacteria. The amplicon sequencing data revealed many taxa that were previously unreported from sugarcane factories. To explore whether these other taxa are likely to produce dextrans or levans like other previously reported bacteria, BLAST searches were used to query genomes deposited in the NCBI RefSeq database for the 12 most abundant bacterial genera and any other genera found in recent culture-based studies (11)(12)(13)(14) (Fig. 3). Out of 15 genera that were surveyed, Leuconostoc had the highest average copy number of dextransucrases per genome (1.90). Interestingly, Oenococcus, a previously uncultured genus, had the second highest average copy number of dextransucrases per genome (1.46). Levansucrases were present in more genera, with Gluconobacter and Zymomonas having the highest average copy number per genome (1.10 and 1.00, respectively). Fungi have not been reported to encode either dextransucrases or levansucrases (26).
Experimental analysis of sugarcane factory microbial communities. To investigate how microbial composition affects sucrose consumption and production of EPS during postharvest degradation, frozen samples were used to inoculate flask cultures. While these experimental conditions differed greatly from the factory environment, they allowed for various measurements to be taken, such as the optical density at 600 nm (OD 600 ) for the rate of growth, consumption of sucrose, and the production of organic acids and EPS, over the course of the experiment (Fig. 4-6). The OD 600 of the inoculum varied greatly and may have reflected differences in microbial load, but these Names are marked with an asterisk (*) for genera that were abundant in the 16S V4 data set and/or a caret (^) for genera of bacteria reported in recent culture-based studies. The average copy number of dextransucrases (orange; left axis) and levansucrases (blue; left axis) were calculated as the ratio of tBLASTn hits against the number of genomes deposited in the NCBI RefSeq database (gray bars; right axis) for each genus. Superscript letters a and b indicate taxonomic groups consisting of multiple genera: a Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium and b Methylobacterium-Methylorubrum.
differences also could have been affected by particulates like soil and bagasse fibers, so the measurements were not used to make any conclusions regarding the microbial load in the inoculum (Fig. 4). The first OD 600 measurement taken was at approximately 5.5 h and showed that several samples had already entered logarithmic growth while others still appeared to be experiencing a lag phase. At 24 h, some flasks had already reached stationary phase, and the remaining flasks had all reached stationary phase by around 48 h. This matches closely with the high-pressure liquid chromatography (HPLC) measurements of sucrose, where various amounts of sucrose had been consumed at 24 h, but most of the sucrose was consumed in all flasks by 48 h (Fig. 5). There was significant correlation between the amount of sucrose consumed in the first 24 h and the change in OD 600 in the first 24 h (Spearman's rho = 0.605; P = 0.00212). However, the consumption of sucrose and change in OD 600 between 24 and 48 h were not significantly correlated. It is possible that some of the OD 600 measurements at 48 h were inaccurate, as several flasks had visible formation of large clumps, which were likely aggregates of cells and EPS. Additionally, flask cultures inoculated with two of the samples, CJ-Fact2-Oct and MJ-Fact3-Dec, became very noticeably viscous likely due to production of water-insoluble EPS (Fig. 7). Water-soluble EPS was measured from the final time point by modified Haze method (Fig. 7). Measured EPS values ranged from about 1 g/L in biofilm-inoculated cultures to up to 19 g/L in CJ-Fact2-Nov. Viscosity was also measured after 96 h and only the CJ-Fact2-Oct and MJ-Fact3-Dec flasks had any measurable viscosity. Finally, the viscosity did not correlate with the soluble EPS measured by Haze method. Several methods were used to try and identify any taxa that might be associated with higher EPS or viscosity measurements, including analysis of compositions of microbiomes with bias correction (ANCOM-BC), random forest regression, and Spearman's correlation. However, possibly due to the limited data set or changes in the microbial community during the experiment, no taxa could be positively linked to higher EPS and viscosity.
HPLC was also used to characterize the presence of ethanol and several organic acids known to be by-products of some of the more abundant microbes present in the  (27)(28)(29)(30). All flask cultures produced high levels of ethanol during the first 48 h. However, ethanol levels from CJ-Fact2-Oct and MJ-Fact3-Dec could not be accurately measured because their high viscosity (Fig. 7) required the addition of ethanol to precipitate EPS so the samples could be analyzed by HPLC. Unexpectedly, acetic acid levels increased over each time point for most cultures, even after total sugars were consumed. The acetic acid was likely produced by some microbes that oxidized ethanol, which decreased as acetic acid increased.

DISCUSSION
In summary, we present the first use of amplicon-based sequencing to profile the microbial communities present in crusher juice, mixed juice, and biofilm samples collected from three Louisiana raw sugar mills. In general, crusher juices, which are produced from the first set of mills from sugarcane as they enter the factory, were generally similar to each other ( Fig. 1C and D). On the other hand, the bacterial community in the mixed juice tanks, which collect the crusher juice and imbibition water before heating and clarification, showed remarkable range in both community structure (Fig. 1C) and alpha diversity (Fig. 1E). This may indicate that differences in factory practices (sanitation, biocide application, temperature of imbibition water) may affect the bacteria present in juices.
Lactobacillales, which are the most abundant bacteria in isolation-based studies (11)(12)(13)(14), were similarly the most abundant in the 16S V4 data set in this study. However, Lactobacillales do not appear as an abundant taxon in amplicon-sequencing results of the sugarcane rhizosphere (19)(20)(21), which suggests that they may become enriched during the delay between harvesting and milling. Our finding that Weissella or Lactobacillus were more abundant than Leuconostoc in certain samples, such as MJ-Fact1-Oct and Biofilm1-Fact3-Dec support recent commentary that these bacteria may play a larger role in EPS production than previously thought (12). A survey of the fungi present using the ITS1 marker region showed a large diversity of taxa in juices, but notably revealed that biofilms are predominantly Saccharomyces. This was interesting as yeasts produce invertases to hydrolyze sucrose (31) and are known to have mutualistic interactions with lactic acid bacteria, which were also abundant in biofilms (32)(33)(34). Due to the limitations from the COVID-19 pandemic, a limited number of samples were collected. A more expanded study would be required to examine microbiome variation resulting from the time in harvest season (relating to effects from weather) or differences in factories (relating to effects from factory practices or differences in sugarcane varieties grown in localities).
As a culture-independent method, amplicon-based sequencing was able to reveal a much greater diversity of bacteria present in raw sugar factories in comparison to isolation-based studies carried out from raw sugar factory samples, including an isolation study that was carried out with some of the same samples that were extracted for amplicon-based sequencing (11)(12)(13)(14) (Table 1). One limitation of amplicon-based sequencing is the inability to reveal phenotypic differences arising from species-or strain-level diversity. In the context of raw sugar processing, this may include traits such as rates of sucrose consumption, EPS production, and biocide susceptibility, which must be studied using isolated microbes (11,14). Relating to this, bacterial taxa identified in prior isolation-based studies at sugar factories comprised 70.6% of the 16S reads, which demonstrated that most bacteria present in sugarcane factory juice and biofilm samples are culturable and can be studied in further laboratory experiments. However, the 16S V4 sequencing data paired with BLAST queries against genomes in public databases (Fig. 3) also highlighted some previously uncultured bacteria (notably Oenococcus and Zymomonas) that should be studied for potential contributions to the EPS problem in factories.
Finally, the same samples were used to inoculate laboratory cultures for examination of how differences in microbial composition affect rates of sucrose consumption, organic acid production, and EPS formation. A key result from this experiment was the difference in soluble EPS production and viscosity (caused by insoluble EPS) between flasks. To date, various bacteria have been reported to produce EPS with differing monomeric compositions and linkage structures than typical a-1,6-linked dextran, which have differences in solubility and viscosity (35)(36)(37). These results highlighted the need for method development to comprehensively characterize and quantify microbial production of both soluble and insoluble EPS, which can pose different challenges during sugarcane processing. Examining the Lactobacillales (high dextran producers) revealed some ASVs that were specific to different samples or sample groups, but the core ASVs comprised most of the LAB reads (Fig. 2). It seems more likely that high EPS results from increased abundance of certain organisms rather than their presence or absence. However, several factors, including the limited sample size and possible changes in the microbial community during the experiment, likely contributed to our inability to positively link any microbial taxa to higher EPS or viscosity using methods such as ANCOM-BC, random forest regression, and Spearman's correlation. Additionally, more useful measurements might have come from the juice samples used for DNA extraction or the raw sugar factories. However, due to operational limitations imposed during the COVID-19 pandemic, we were limited in the experiments that could be performed during the sugarcane harvest season. In summary, we report the first use of amplicon-based sequencing to study the microbiomes present in processing streams at raw sugar factories. These methods will enable researchers to gain a more comprehensive understanding of microbial biodiversity than previously available and lay the foundations for development of more effective methods to inhibit microbial degradation of sugarcane juice during processing.

MATERIALS AND METHODS
Collection of sugarcane juice and biofilm samples. Samples were collected from three raw sugar factories in southern Louisiana during the 2021 to 2022 harvest season, which ran from October to January. The factories are referred to anonymously at the request of factory operators. Due to operational limitations that were in place in response to the COVID-19 pandemic, only four sampling trips could be arranged: Factory no. 1 in October 2021 (Fact1-Oct), Factory no. 2 in November 2021 (Fact2-Nov), and Factory no. 3 in December 2021 (Fact3-Dec) and January 2022 (Fact3-Jan). Long-handled dippers were used to collect juice into clean Nalgene bottles from the juice flow exiting the first rolling mill (crusher juice, CJ) and a tank that was fed by the crusher juice and a countercurrent stream of imbibition water that extracted juice from the cane as it was pressed through the later mills (mixed juice [MJ]). A list of samples is presented in Table S1 in the supplemental material. Additionally, samples were collected from two biofilms found near the processing streams at Factory no. 3. These were thick, sludge-like materials that were scraped from the factory surfaces with a clean gloved hand into Ziploc bags. All samples were chilled on ice and transported to the laboratory within 3 h. Most studies on sugarcane juice spoilage will allow the juice to spoil at ambient temperature for several days (2, 3), so additional microbial growth and spoilage during transport should have been minimal. Juice samples were stored at 220°C, while biofilm samples were stored at 280°C until further analysis. For flask experiments, aliquots of the juice samples were also mixed with an equal volume of sterile 40% glycerol and stored at 280°C. Due to their viscous nature, samples of the biofilms were scooped into 50-mL tubes and mixed with minimal amounts of sterile water by vortexing until they could be pipetted into a vial with the 40% glycerol solution.
DNA extraction. Juice samples were centrifuged at 9,000 g for 15 min and the supernatants were discarded. Approximately 0.3 g of pellet material containing microorganisms and soil were used for total DNA extraction with the DNeasy Powersoil DNA extraction kit (Qiagen, Hilden, Germany). For DNA extraction from biofilm samples, the material was thawed and approximately 0.3 g of biofilm material was also extracted with the DNeasy Powersoil DNA extraction kit. Due to uncertainties about the quality of DNA extracted from the sugarcane factory samples, each DNA extraction was performed in triplicate, which were sequenced as technical replicates. DNA samples with 260/280 ratios below 1.8 as measured by a Nanodrop OneC (Thermoscientific, Waltham, MA) were further purified using a DNA clean and concentrator-5 kit (Zymo Research, Irvine, CA).
Amplicon-based sequencing. DNA samples were sent to Argonne National Laboratory (Lemont, IL) for amplicon-based sequencing of rRNA gene. The V4 region of the 16S rRNA gene was amplified with primers 515f/806r for bacteria and the internal transcribed spacer 1 (ITS1) was amplified with primers ITS1f/ITS2r for fungi (38)(39)(40)(41). Further details on the library preparation provided by Argonne National Laboratory can be found in the supplemental material. Additionally, a sample of a fungal mock community DNA control (42) (kindly provided by Briana Whitaker, USDA-ARS, Peoria, IL) was included to evaluate taxonomic resolution (Table S2). We were able to identify 16 of 17 expected genera. The ZymoBIOMICS Microbial Community DNA Standard (Zymo Research, Irvine, CA) was also included as a control sample for the 16S data set, but these libraries failed and produced zero reads. The barcoded libraries were sequenced on an Illumina MiSeq platform with 2 Â 251 bp reads.
Data analysis. The raw sequences were imported into QIIME2 version 2022.2 for analysis (43). Raw sequences were demultiplexed and filtered based on quality with the q2-demux plugin. The adapter and primer sequences were removed from both ends of the read using the q2-cutadapt plugin before reads were denoised with DADA2 (44). For the ITS1 data, too many read pairs were unable to be assembled due to low quality, so only the forward reads were used for denoising and subsequent analysis. Taxonomy was assigned using the q2-feature-classifier plugin, with a Naive Bayes classifier that was trained on the Silva version 138 99% ASVs database containing the 515f-806r region for bacteria (45,46) and the UNITE version 8.2 (release date, 4 February 2020) ITS database for fungi (47). The 16S V4 data were also filtered to remove sequences longer than 260 nucleotides or shorter than 100 nucleotides using the q2-rescript plugin. Additionally, the 16S data were filtered for ASVs that were not assigned to a phylum, as well as chloroplast and mitochondrial sequences. The ITS1 data were only filtered for ASVs that were unassigned to a phylum, as no nonfungal sequences were found. Next, each technical triplicate was filtered to select only the replicate with the highest sequencing depth for downstream analysis. Finally, the ASVs were filtered to omit those that were only present in 1 sample.
For phylogenetic diversity metrics, fragment insertion using the SEPP algorithm was used to insert bacterial ASVs into a reference tree based on the SILVA version 128 database (48). For fungal ASVs, mafft was used to perform multiple sequence alignment, and the unrooted tree generated by FastTree was used for subsequent analysis. Alpha and beta diversity were calculated using data sets rarefied to the lowest feature counts in each sample set (16S V4 = 9,099, ITS1 = 16,672). The sequencing depths for each sample and rarefaction curves are presented in the supplemental material (Table S1, Fig. S1). The significance of differences in alpha diversity were evaluated using the Kruskal-Wallis test (a = 0.05). Differences in community structure were visualized by applying principal coordinate analysis (PCoA) to the unweighted UniFrac distance matrices. PERMANOVA was used to calculate significance in comparisons of unweighted UniFrac distances between sample groups with 999 permutations. QIIME2 artifacts produced from the steps described above were imported into R using the package qiime2R, and key packages used in producing the figures depicting the microbiome data include phyloseq (49), microViz (50), and ggvenn.
Expected frequency of dextransucrases and levansucrases. A representative dextransucrase from Leuconostoc citreum strain NRRL B-1299, DsrA (51), and levansucrase from Bacillus subtilis strain 168, SacB (52), were used to query the NCBI refseq_genomes database (24) with tBLASTn. Due to sequence similarity to other carbohydrate-active enzymes, E-value thresholds of 1E-10 and 1E-5 were used to filter out likely false-positive hits for dextransucrases and levansucrases, respectively. These values were empirically chosen after examining the annotations and query coverage values for hits with higher E values.
Flask culture experiments. Glycerol stocks were thawed, and 0.5 mL were used to inoculate 50 mL of TSY medium (0.5% tryptone, 0.5% yeast extract, 0.1% K 2 HPO 4 , and 12% sucrose, pH 7.2 adapted from TGY medium as described by Haynes et al [53]) in Erlenmeyer flasks and incubated at 28°C for 4 days with 225 rpm shaking. The OD 600 was measured at 0, 5.5, 24, 48, 72, and 96 h. Samples were collected at 24, 48, 72, and 96 h to measure sucrose consumption. Additional samples were collected at 96 h to measure EPS formation. The results were plotted in Excel.
Viscosity measurement. Viscosity was measured using a Brookfield DV II1 viscometer (AMETEK Brookfield, Middleboro, MA) with a T-bar spindle. The spindle and shear rate were selected to obtain a torque between 10 and 100% for viscosity measurements. Measurements were taken at room temperature.
HPLC. Analysis of sugars and products were carried out by high-pressure liquid chromatography (HPLC; Series 1100 Hewlett Packard/Agilent, Santa Clara, CA) with a refractive index detector. The mobile phase was 5 mM H 2 SO 4 , which was pumped at 0.6 mL/min through an Aminex HPX-87H column with a Cation H guard column (Bio-Rad, Hercules, CA). Samples were injected (2 to 5 mL) with the column maintained at 20°C to prevent hydrolysis of sucrose (54). One standard stock solution was made with (25 g/L) of each of the sugars (sucrose, glucose, and fructose). The other standard stock solutions contained succinic acid (10 g/L), acetic acid (7 g/L), lactic acid (11 g/L), and ethanol (15 g/L). Stock solutions were portioned out in glass vials and frozen. When used, vials were thawed and 2, 4, 6, 8, and 10 mL were injected onto the column to generate calibration curves. All peak-area-based calibrations were found to be linear, stable over time, and with excellent correlations (R 2 . 0.99). The results were plotted in Excel and Tukey's honestly significant difference (HSD) test was used in R to determine significant differences (P , 0.05) in measured sugars and by-products between samples at each time point.
Measurement of total exopolysaccharide in flask cultures. Total soluble EPS in culture samples were measured by a modified Amstar contract method for dextran, which is a Haze-based method (55). A small amount of filter aid (;25 mg, Celatom EP-905, EP Minerals, Reno, NV) was mixed with 300 mL 10% (wt/vol) trichloroacetic acid and 1,500 mL sample (containing , 1,000 mg/L EPS) in a 2-mL Eppendorf microcentrifuge tube. The content was mixed and centrifuged for 5 min at 9,500 g. Then, 900 mL was transferred to a Costar Spin-X HPLC microcentrifuge 0.45-mm Nylon Filter (Corning Inc., Corning NY) and centrifuged again for 5 min at 9,500 g. The filtered sample was diluted (300 mL:300 mL) with deionized water (for blank) and (300 mL:300 mL) with 100% pure ethanol. After 30 min, the absorbance was read at 700 nm (10 mm path length) using a JENWAY Genova Bio spectrophotometer (Cole-Parmer, Vernon Hills, IL). Calibration was performed using Dextran 2000 (Amerham Biosciences, Uppsala, Sweden) in deionized water, as well as sterile media samples, with the concentration ranging from 0 to 1,000 mg/L. The calibration was linear. The results were plotted in Excel and Tukey's HSD test was used in R to determine significant differences (P , 0.05) in measured dextran haze.
Data availability. Raw sequence reads were deposited on the Sequence Read Archive with accession number PRJNA892996.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, DOCX file, 0.44 MB.

ACKNOWLEDGMENTS
Bretlyn Pancio with the Oak Ridge Institute for Science and Education is acknowledged for her technical assistance. Evan Terrell and Emily Heck at USDA-ARS SRRC are acknowledged for assistance with viscosity measurements. Brian Mack is acknowledged for discussions on data analysis. The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by USDA. USDA is an equal opportunity provider and employer. The authors are employed by the funding organization. However, the funders had no role in the design of the study; in the collection, analyses, or interpretation of data or in the writing of the manuscript; but approved the decision to publish the results.
We declare no conflict of interest.