Abstract
The demand for plant-based cheese analogues has increased in recent years, driven both by the growing adoption of vegan and vegetarian diets and by consumers seeking plant-based alternatives to animal-derived products. Their production relies on diverse raw materials and often incorporates fermentation processes involving molds, yeasts, and lactic acid bacteria (LAB). Within this context, ensuring food safety, including the antimicrobial resistance (AMR) aspects, is essential due to the complex microbial communities involved in fermentation. Therefore, this study investigated the presence of bacterial species of potential public health relevance and assessed AMR profiles and genes in plant-based cheese analogues acquired in Brazil. None of the samples tested positive for Salmonella spp., coagulase-positive Staphylococcus, or Escherichia coli, while Bacillus cereus was detected in one sample. Two atypical colonies recovered on selective media for Salmonella were identified as Cronobacter malonaticus and Delftia acidovorans. Most samples exhibited high mold and yeast counts, likely associated with fermentation and maturation processes. Among 176 LAB isolates, 11 species were identified by MALDI-TOF, and twenty representative strains were selected for antimicrobial susceptibility testing. Resistance was detected in a limited number of isolates, mainly to ciprofloxacin, tetracycline, and gentamicin, whereas C. malonaticus exhibited resistance to penicillin. Whole-genome sequencing was performed on Lactiplantibacillus plantarum, Levilactobacillus brevis, Leuconostoc mesenteroides, Enterococcus faecium, and C. malonaticus. A total of eight antimicrobial resistance genes were identified, four of which were located on plasmids in C. malonaticus and L. brevis. These findings suggest that microorganisms associated with plant-based cheese analogues may act as reservoirs of antimicrobial resistance determinants, highlighting the importance of safety assessment and microbiological surveillance of strains intended for technological or probiotic applications.
Supplementary Information
The online version contains supplementary material available at https://doi.org/10.1007/s42770-026-02092-7.
Keywords: Microbiological safety, Lactic acid bacteria, Whole genome sequencing, Mobile genetic elements, Resistance genes
Introduction
Food safety remains a major public health concern worldwide, particularly in foods that rely on microbial fermentation [1–3]. Fermented foods contain complex microbial communities that contribute to product quality, stability, and sensory characteristics, but may also include undesirable microorganisms introduced through raw materials, processing environments, or post-production contamination [4–8].
In recent years, the market for plant-based cheese analogues has increased substantially due to the growing consumer demand for sustainable, ethical, and health-conscious alternatives to animal-derived products [9]. These products are commonly produced from nuts, legumes, seeds, starches, and vegetable oils, and frequently involve fermentation processes mediated by yeasts, molds, and LAB to improve flavour, texture, shelf life and overall product acceptance [10–14]. Among these microbes, LAB play a central technological role by contributing to acidification, flavour development and inhibition of spoilage microorganisms.
Despite the rapid growth of the plant-based cheese sector, information regarding the microbiological safety of these products remains limited. This is particularly relevant because many products are manufactured by small-scale producers and may not undergo heat treatment or fermentation [15, 16], potentially increasing their susceptibility to contamination by opportunistic or pathogenic microorganisms. Furthermore, the microbial populations present in fermented foods have attracted increasing attention as potential reservoirs of antimicrobial resistance (AMR) determinants [15, 17, 18]. Although LAB are generally recognized as safe and are widely used as starter cultures and probiotics, some strains have been reported to harbor acquired antimicrobial resistance genes (ARGs), especially those associated with mobile genetic elements that may facilitate horizontal gene transfer within microbial communities [5].
The presence of ARGs in food-associated microorganisms does not necessarily represent a direct health risk; however, monitoring these determinants contributes to a better understanding of the potential role of foods in the ecology and dissemination of AMR. Consequently, the assessment of both microbiological safety and AMR profiles has become increasingly relevant in fermented food systems [19].
Given this context, the present study evaluated the microbiological safety of Brazilian cashew-based plant-based cheese analogues and investigated the antimicrobial susceptibility profiles of lactic acid bacteria and bacterial contaminants isolated from these products. In addition, selected isolates were characterized by whole-genome sequencing to assess the occurrence of AMR genes.
Materials and methods
Sample collection
Thirteen samples (S1–S13) of Brazilian cashew-based plant-based cheese analogues were acquired from local supermarkets, fairs, and e-commerce platforms (Supplementary Table 1). The sample selection was carried out through searches on networks such as Google, considering products available between August 2023 and January 2024. The samples were stored at 4 °C, and microbiological analyses were performed before the expiry date indicated on the product label.
Identification of indicator and pathogenic microorganisms
The selection of microorganisms investigated was based on Annex I of Normative Instruction (IN) No. 161, dated July 1, 2022, issued by the Brazilian Health Regulatory Agency (ANVISA), specifically in group 4 (other plant-based products), category (c): Refrigerated tofu, sufu, and similar products [20]. Microbiological analyses included the detection of Salmonella spp. and the enumeration of coagulase-positive Staphylococcus, Escherichia coli, presumptive Bacillus cereus, and molds and yeasts. The methods were based on the International Standards Organization (ISO) 6579:2002, ISO 6888-1:1999, ISO 16649-2:2001, ISO 7932:2004 e ISO 21527-1:2008, with adaptations [21].
For each assay, 25 g of the sample was homogenized in 225 mL of buffered peptone water (BPW; Oxoid, Basingstoke, UK). Coagulase-positive Staphylococcus, presumptive B. cereus, and molds and yeasts were enumerated by surface plating 0.1 mL of the diluted sample in selective media. Baird-Parker Agar (BP; Oxoid) was used for coagulase-positive Staphylococcus, Mannitol Egg Yolk Polymyxin Agar (MYP, Oxoid) for presumptive B. cereus and Yeast Glucose Chloramphenicol Agar (YGC; Merck, Germany) for molds and yeasts. Plates were spread using a Drigalski spatula and incubated under aerobic conditions at 35 ± 1 °C for 48 ± 2 h (BP), 30 ± 1 °C for 18 to 24 h (MYP), and 25 ± 1 °C for 5 days (YGC).
Escherichia coli was enumerated using the pour plate method, where 1 mL of the diluted sample was transferred to a Petri dish, followed by the addition of Tryptone Bile X-glucuronide Agar (TBX; Neogen, USA). After homogenization, plates were incubated aerobically at 44 ± 1 °C for 18 to 24 h.
For the detection of Salmonella spp., BPW suspensions were pre-enriched at 37 ± 1 °C for 18 to 24 h. Subsequently, 0.1 mL and 1 mL aliquots were transferred to Rappaport-Vassiliadis Soy (RVS; Oxoid, Basingstoke, UK) and Muller-Kauffmann Tetrathionate Novobiocin (MKTTn; Oxoid) and incubated at 45 ± 1 °C and 37 ± 1 °C for 24 ± 3 h, respectively. For subsequent selective enrichment, 10 µL of the pre-enriched sample was streaked onto Xylose Lysine Deoxycholate Agar (XLD) and Brilliant Green Agar (BGA; Oxoid).
For mold and yeast enumeration, serial decimal dilutions were prepared in BPW and 0.1 mL of each dilution were surface plated onto YGC agar. All plates were analyzed for colonies displaying typical morphology of the target microorganism. Additionally, morphologically distinct colonies cultured on Nutrient Agar (NA; Neogen, USA) were selected and identified using Matrix-assisted Laser Desorption/ionization-Time of Flight mass spectrometry (MALDI-TOF MS).
Isolation and identification of lactic acid bacteria (LAB)
LAB were isolated following a method adapted from ISO 15214:1998 [20]. Serial decimal dilutions of the samples were prepared in BPW and 0.1 mL were surface plated onto Man, Rogosa & Sharpe (MRS) agar using a Drigalski spatula. Plates were incubated anaerobically at 30 ± 2 °C for 48 ± 3 h.
Following incubation, colonies were selected based on morphological characteristics such as size, shape, color, texture, and margin, with up to 24 colonies recovered per sample. Isolates were purified in MRS broth and incubated under the same conditions prior to identification by MALDI-TOF MS.
Antimicrobial susceptibility testing
Antimicrobial susceptibility test was performed using the disk diffusion method (Kirby-Bauer) and BrCAST reference. Prior to testing, LAB isolates and distinct colonies of C. malonaticus and D. acidovorans were cultured in MRS and BHI broths. The following antibiotics were tested: vancomycin (30 µg), tetracycline (30 µg), chloramphenicol (30 µg), amoxicillin (10 µg), gentamicin (120 µg), ampicillin (10 µg), streptomycin (300 µg), erythromycin (15 µg), ciprofloxacin (5 µg), and penicillin (10 µg).
Bacterial suspensions were adjusted to a 0.5 McFarland standard in 0.85% saline solution and spread onto the appropriate agar medium using a sterile swab. Antibiotic disks were placed to the agar surface, and plates were incubated either anaerobically at 30 ± 2 °C (MRS) or aerobically at 36 ± 2 °C for 24 h (BHI), according to the growth requirements of each isolate. Inhibition zone diameters were measured and interpreted as susceptible or resistant according to BrCAST criteria.
Whole genome sequencing and genomic analysis
Five isolates were selected for whole-genome sequencing based on their antimicrobial susceptibility profiles: Lactiplantibacilus plantarum, Levilactobacillus brevis, Leuconostoc mesenteroides, Enterococcus faecium and Cronobacter malonaticus. Genomic DNA was extracted using the PureLink™ Genomic DNA Mini Kit and quantified using the Qubit™ dsDNA Quantification Assay Kit. Samples with DNA concentration ≥ 10 ng/µL were submitted to shotgun sequencing by IPEC Guarapuava (Brazil) using the Illumina NovaSeq 6000 platform with paired-end 150 bp reads.
Data analysis included an initial quality control step for the reads using the fastp (v0.23.2) [22], with the flags -q 30 -w 30 --cut_front --cut_tail --n_base_limit 0 --length_required 50. Genome assembly de novo was carried out using SPAdes software (v3.15.5) [23], applying the “--isolate” flag due to the high coverage obtained.
AMR genes were identified using the RGI (Resistance Gene Identifier, v6.0.0) tool against the CARD (Comprehensive Antibiotic Resistance Database, v3.2.6) [24], with main function and strict alignment thresholds and ≥ 85% identity of matching region. Mobile genetic elements and associated resistance genes were investigated using geNomad [25] with default parameters.
Sequence types (STs) were determined using the Multi-Locus Sequence Typing (MLST) database. The pathogenic potential of each genome was subsequently assessed using the PathogenFinder 1.1 web server, which predicts the probability of a bacterium being a human pathogen based on genomic features. Genomes with a predicted pathogenicity probability ≥ 0.5 were classified as potentially pathogenic.
Results and discussion
Identification of indicator and pathogenic microorganisms
None of the analyzed samples tested positive for Salmonella spp., E. coli or coagulase-positive Staphylococcus (Table 1). No presumptive B. cereus colonies were confirmed according to the ISO-based enumeration method. However, one isolate recovered during LAB isolation was classified as B. cereus by MALDI-TOF MS.
Table 1.
Microbiological counts of artisanal cashew-based plant-based cheese analogues
| Samples | Microorganisms | ||||
|---|---|---|---|---|---|
| Salmonella spp. | Escherichia coli | coagulase positive Staphylococcus | Bacillus cereus | Molds and yeasts | |
| Detection in25 g | CFU/g | ||||
| S1 | ND | < 10 | < 100 | < 100 | > 300 × 10³ |
| S2 | ND | < 10 | < 100 | < 100 | 100 |
| S3 | ND | < 10 | < 100 | < 100 | 9.7 × 10⁴ |
| S4 | ND | < 10 | < 100 | < 100 | 4.6 × 10³ |
| S5 | ND | < 10 | < 100 | < 100 | 1.9 × 10⁴ |
| S6 | ND | < 10 | < 100 | < 100 | < 100 |
| S7 | ND | < 10 | < 100 | < 100 | < 100 |
| S8 | ND | < 10 | < 100 | < 100 | < 100 |
| S9 | ND | < 10 | < 100 | < 100 | > 300 × 10³ |
| S10 | ND | < 10 | < 100 | < 100 | 210 × 10³ |
| S11 | ND | < 10 | < 100 | < 100 | 8.4 × 10⁴ |
| S12 | ND | < 10 | < 100 | < 100 | < 100 |
| S13 | ND | < 10 | < 100 | < 100 | 7.9 × 10³ |
ND Not detected
The absence of the main foodborne pathogens and hygiene indicators investigated suggests that the analyzed products were manufactured under satisfactory hygienic conditions. Similar findings were reported by Correia et al. (2026) [26], who found no evidence of Salmonella spp. or Listeria monocytogenes in commercial plant-based cheese analogues from Brazil, but detected B. cereus spores in 27% of the products evaluated. In the present study, although only one B. cereus isolate was detected and was not recovered through the selective enumeration procedure, its occurrence deserves attention because some strains can produce toxins associated with foodborne illness [27]. As this microorganism is commonly associated with plant-derived ingredients and environmental sources, its presence may reflect contamination originating from raw materials or processing environments [27], reinforcing the importance of monitoring spore-forming bacteria throughout the production chain of plant-based cheese analogues.
Most samples exhibited high mold and yeast counts, with predominance of yeasts (Table 1). According to Brazilian legislation (IN No. 313/2024), microbiological limits for molds and yeasts do not apply to foods intentionally produced with viable microorganisms. Therefore, this high counts in fermented plant-based cheese analogues should not necessarily be interpreted as indicators of poor microbiological quality. Several samples analyzed in this study declared the use of probiotic, mesophilic, or vegan cultures on their labels, which may partially explain the observed counts. In addition, undeclared microorganisms associated with spontaneous fermentation may also contribute to these microbial populations [5].
From a technological perspective, yeasts and molds may be desirable in fermented plant-based cheese analogues due to their contribution to flavour development during maturation. Species such as Geotrichum candidum are commonly used as ripening cultures because of their lipolytic and proteolytic activities, which can enhance sensory properties [10, 28]. However, in products not intended to fermentation or maturation processes, elevated yeast counts may indicate spoilage potential or insufficient process control.
Isolation and identification of lactic acid bacteria (LAB)
Presumptive LAB were detected in 10 of the 13 analyzed samples, yielding a total of 176 isolates, while no LAB were recovered from the other three samples. The distribution of bacterial species varied among products (Fig. 1A). Marked variation in bacterial community structure was observed across samples. Whereas some products were dominated by a single species, others contained a broader range of bacterial taxa. These patterns may reflect differences in raw materials, processing conditions, starter culture application, and the occurrence of spontaneous fermentation.
Fig. 1.
Distribution of bacterial isolates among the analyzed samples (a) and the total number of isolates identified for each species (b)
The LAB community was dominated by E. faecium (39 isolates), L. plantarum (32 isolates), Pediococcus acidilactici (24 isolates), L. mesenteroides (23 isolates), Pediococcus pentosaceus (17 isolates), and Lacticaseibacillus paracasei (15 isolates) (Fig. 1B). Additionally, a few non-LAB species were identified, namely Bacillus subtilis, B. cereus, Cronobacter malonaticus, and Delftia acidovorans.
Several of the predominant species identified in this study have previously been reported in fermented plant-based cheese analogues [16]. In particular, L. plantarum, Leuconostoc spp., and Pediococcus spp. are well adapted to plant-based substrates and play important roles in acidification, flavour development, and texture formation during fermentation [12, 29]. However, the predominance of E. faecium observed here differs from previous studies, in which Lactococcus lactis and L. mesenteroides were reported as the most abundant species [16]. Although Enterococcus spp. are commonly found in fermented foods and may contribute to fermentation processes [8], some strains have been associated with AMR and opportunistic infections [30]. Consequently, their high prevalence in the samples analyzed highlights the need for further strain-level characterization to assess their safety and technological relevance.
Identification of other bacteria with potential pathogenicity
In addition to the target microorganisms investigated, atypical colonies recovered on BGA medium were identified as C. malonaticus and Delftia acidovorans (Fig. 1) in two samples (S4 and S5). Among these species, C. malonaticus warrants particular attention due to its opportunistic pathogenic potential and association with human infections. Members of the genus Cronobacter have been isolated from a wide variety of food products, including cereals, herbs, vegetables, dairy products, and powdered infant formula [31, 32]. Although the primary reservoirs of Cronobacter spp. remain poorly defined, plant-derived materials and food-processing environments are considered important sources of contamination. Furthermore, the capacity of these bacteria to form biofilms may enhance their persistence in processing facilities, thereby increasing the risk of contamination during food production [33, 34].
Delftia acidovorans is an environmental bacterium frequently detected in water systems and biofilms [35]. Although generally regarded as a low-virulence microorganism, it has occasionally been associated with opportunistic infections, particularly among immunocompromised individuals [36]. Its detection in the present study most likely reflects environmental contamination originating from raw materials or processing environments rather than a direct role in the fermentation process.
Antimicrobial resistance
From 69 representative isolates obtained, 20 strains were selected for antimicrobial susceptibility testing. Resistance was detected in 11 isolates (55%), representing four bacterial species (Table 2). The resistance phenotypes were primarily associated with ciprofloxacin, tetracycline, gentamicin, and penicillin. Two E. faecium isolates exhibited resistance to gentamicin and ciprofloxacin, consistent with previous reports describing AMR among food-associated Enterococcus spp [30]. In contrast, D. acidovorans was susceptible to all antimicrobials tested, whereas C. malonaticus displayed resistance to penicillin.
Table 2.
Antimicrobial susceptibility profiles of selected representative bacterial isolates
| Isolate | Sample | Number of isolates | Number of isolates analyzed (%) | Antimicrobial resistance | Number of resistant isolates (%) |
|---|---|---|---|---|---|
| Pediococcus pentosaceus | S2 | 6 | 2 (33) | Ciprofloxacin | 2 (100) |
| S10 | 11 | 3 (27) | NI | 0 (0) | |
| Pediococcus acidilactici | S6 | 1 | 1 (100) | Tetracycline | 1 (100) |
| Ciprofloxacin | 1 (100) | ||||
| S9 | 23 | 3 (13) | NI | 0 (0) | |
| Enteroccocus faecium | S7 | 3 | 2 (67) | Gentamicin | 1 (50) |
| 2 (67) | Ciprofloxacin | 1 (50) | |||
| S8 | 1 | 1 (100) | NI | 0 (0) | |
| S9 | 1 | 1 (100) | NI | 0 (0) | |
| S13 | 20 | 3 (15) | Ciprofloxacin | 3 (100) | |
| Cronobacter malonaticus | S4 | 3 | 2 (67) | Penicillin | 2 (100) |
| Total | 69 | 20 (29) | 11 (55) |
NI Not identified
Previous studies have also documented resistance among LAB isolated from fermented foods, particularly to vancomycin, aminoglycosides, and quinolones [37]. These findings reinforce the importance of monitoring AMR in microorganisms associated with fermented food products.
Genomic sequencing and resistome analysis
Whole-genome sequencing was performed on five representative isolates selected based on their antimicrobial susceptibility profiles. Among the sequenced strains, AMR genes were detected only in C. malonaticus genome. The identified resistance determinants, their associated antimicrobial classes, and the corresponding resistance mechanisms are presented in Table 3. C. malonaticus is a Gram-negative opportunistic pathogen known for its virulence potential and its ability to acquire and maintain AMR determinants [38, 39]. Most of the resistance genes detected in this species were associated with multidrug efflux systems and regulatory pathways, which may contribute to reduced susceptibility to multiple antimicrobial classes, including fluoroquinolones, cephalosporins, and tetracyclines, as well as disinfectants and antiseptic compounds.
Table 3.
Antimicrobial resistance genes, antimicrobial classes, associated antibiotics, and resistance mechanisms detected in Cronobacter malonaticus
| ARG | Antibiotic class | Antibiotic | AMR mechanisms |
|---|---|---|---|
| CMA-2 | cephalosporin | benzalkonium chloride | antibiotic inactivation |
| marA | fluoroquinolone; monobactam; carbapenem; cephalosporin; glycylcycline; penicillin beta-lactam; tetracycline; rifamycin; phenicol; disinfecting agents and antiseptics | tigecycline; tetracycline; rifampin; chloramphenicol; ampicillin; cefalotin; triclosan | antibiotic efflux; reduced permeability to antibiotic |
| emrR | fluoroquinolone | nalidixic acid | antibiotic efflux |
| emrB | fluoroquinolone | nalidixic acid | antibiotic efflux |
| rsmA | fluoroquinolone; diaminopyrimidine; phenicol | trimethoprim; chloramphenicol | antibiotic efflux |
| CRP | macrolide antibiotic; fluoroquinolone; penicillin beta-lactam | erythromycin; cloxacillin; oxacillin; norfloxacin | antibiotic efflux |
| Shigella flexneri acrA | fluoroquinolone; cephalosporin; glycylcycline; penicillin beta-lactam; tetracycline; rifamycin; phenicol; disinfecting agents and antiseptics | tigecycline; ciprofloxacin; tetracycline; rifampin; chloramphenicol; ampicillin; cefalotin; triclosan | antibiotic efflux |
| E. coli AcrAB-TolC with MarR mutations conferring resistance to ciprofloxacin and tetracycline | fluoroquinolone; cephalosporin; glycylcycline; penicillin beta-lactam; tetracycline; rifamycin antibiotic; phenicol; disinfecting agents and antiseptics | tigecycline; ciprofloxacin; tetracycline; rifampin; chloramphenicol; ampicillin; cefalotin; triclosan | antibiotic target alteration; antibiotic efflux |
Regarding resistance determinants located in plasmid-associated regions, genes related to vancomycin, chloramphenicol, fosfomycin, and tetracycline resistance were detected in C. malonaticus and L. brevis (Table 4). In C. malonaticus, most of the identified genes were associated with multidrug efflux systems and regulatory mechanisms commonly found in Gram-negative bacteria. In contrast, L. brevis harbored genes encoding tetracycline efflux transporters belonging to the Major Facilitator Superfamily (MFS).
Table 4.
Plasmid-associated antimicrobial resistance genes identified in the sequenced isolates
| Bacteria | ||||
|---|---|---|---|---|
| Cronobacter malonaticus | Levilactobacillus brevis | |||
| Contig | NODE_20 | NODE_11 | NODE_48 | |
| Length | 55,793 | 131,011 | 11,767 | |
| Coverage | 370.357707 | 381.537737 | 520.296920 | |
| Topology | DTR | DTR | No terminal repeats | |
| No. genes | 59 | 114 | 12 | |
| Genetic code | 11 | 11 | 11 | |
| Plasmid score | 1 | 1 | 9,999 | |
| FDR | NA | NA | NA | |
| n hallmarks | 3 | 4 | 0 | |
| Marker enrichment | 262,956 | 378,914 | 20,608 | |
| Conjugation genes | NA | NA | NA | |
| AMR genes | NF000402 | NF033134 | NF000496 | NF012174 |
| Annotation accessions | PF16968; K18941; TIGR02915; COG4567 | K08162; COG2211 |
PF14506; COG3565; TIGR00068; K11210 |
PF00083; TIGR00903; K08221; COG2807 |
| Annotation description | PEP-CTERM-box response regulator transcription factor | Na+/melibiose symporter or related transporter |
CppA N-terminal |
Major facilitator 4 family protein |
| NIH | Vancomycin resistance response regulator transcription factor VanR-B | CmlA/FloR family chloramphenicol efflux MFS transporter | Fosfomycin resistance glutathione transferase | Tet(A)/Tet(B)/Tet(C) family tetracycline efflux MFS transporter |
FDR False discovery rate
Although intrinsic resistance is a recognized characteristic of several LAB species, the presence of AMR genes carried by mobile genetic elements in food-associated bacteria remains a concern due to their potential for horizontal dissemination [14, 17, 37, 40]. In this context, the detection of resistant microorganisms and resistance determinants in fermented plant-based foods reinforces the importance of integrating microbiological and genomic monitoring strategies, particularly for strains with potential technological or probiotic applications. Although the occurrence of resistance was limited among the isolates evaluated, the detection of these genes highlights the potential role of food-associated microorganisms as reservoirs of AMR determinants and underscores the value of combining phenotypic and genomic approaches for surveillance purposes.
The occurrence of resistance genes in plasmid-associated regions is particularly relevant because mobile genetic elements may facilitate the horizontal dissemination of AMR determinants within bacterial communities. Although transferability assays were not conducted in the present study, the genomic context of these genes highlights the need for further investigations into their mobility and dissemination potential in food-associated microbiota.
Pathogenicity prediction analyses revealed marked differences among the sequenced isolates. C. malonaticus exhibited a high predicted pathogenicity score (0.9559), whereas L. brevis showed a substantially lower probability (0.0374). These results are consistent with the recognized opportunistic pathogenic potential of Cronobacter species [31].
The detection of tetracycline resistance determinants, including tet(A), tet(B), and tet(C), in L. brevis underscores the importance of evaluating AMR in microorganisms associated with fermented foods. Although this species is commonly found in fermented products and is generally regarded as safe [36], the presence of acquired resistance genes warrants attention when selecting strains for technological or probiotic applications. Collectively, these findings support previous reports indicating that food-associated bacteria, including LAB, may serve as reservoirs of AMR determinants and reinforce the importance of continued surveillance from a food safety perspective.
Conclusions
This study is one of the firsts to investigate the microbiological safety and antimicrobial resistance of plant-based cheese analogues produced in Brazil, contributing to a better understanding of this emerging and relatively underexplored area. Overall, the analyzed products showed a low occurrence of classical foodborne pathogens and hygiene indicators, suggesting satisfactory microbiological quality. However, opportunistic bacteria of potential public health relevance, including B. cereus and C. malonaticus, were detected in a limited number of samples. AMR phenotypes were identified in a subset of isolates, and whole-genome sequencing revealed resistance-associated genes, including determinants located in plasmid-associated regions. Although the transferability of these genes was not evaluated, their occurrence highlights the importance of monitoring AMR in microorganisms associated with fermented plant-based foods. These findings reinforce the need for continued microbiological monitoring in fermented plant-based foods, particularly regarding AMR surveillance and the safety evaluation of microorganisms used for technological purposes. Further studies should investigate the transferability of resistance genes and the ecology of microbial communities associated with these emerging fermented foods.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
This study was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grants 303574/2024-0 and 383394/2024-4; ResGen - Grant number 408717/2022-0, funded by CNPq, FNDCT, and MCTI) and by the Fundação de Amparo à Pesquisa do Rio Grande do Sul (FAPERGS; grant 24/2551-0000616-8). NZD received a fellowship from CAPES. FQM is a CNPq Level C Research Fellow (305705/2025-3), and VK received a CNPq fellowship. The study was also supported by BRIGHT, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark (NNF24SA0100980).
Author contributions
Writing – original draft: NZD; Writing – review & editing: FQM, JF, RFM, APGF, CIK, VK, LK, HM. Conceptualization: JF, FQM; Methodology: NZD, FQM, JF, VK, HM; Formal analysis: NZD, VK and HM; Investigation: ALT, KRKK, NZD, VK, HM, JF; Data curation: NZD, TS; Resources: JF, FQM, LK, APGF; Supervision: FQM, JF.
Funding
The Article Processing Charge (APC) for the publication of this research was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) (ROR identifier: 00x0ma614).
Data availability
Sequence data that support the findings of this study have been deposited in the National Center for Biotechnology Information - NCBI GenBak under the BioProject ID PRJNA1406699.
Declarations
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
Sequence data that support the findings of this study have been deposited in the National Center for Biotechnology Information - NCBI GenBak under the BioProject ID PRJNA1406699.