Biochemical and physiological characterization
Biochemical characterization was carried out for the isolated bacterial strains L1, L2, L3, L4, L8 and L12. The strains were tested positive for gram staining and chalk agar test while they were tested negative for catalase test, oxidase test, indole test, citrate test, urease test, H2S production and pigment production as shown in Table 1. By finding out the ideal growth temperature (Fig. 1), pH (Fig. 2) and sodium chloride concentrations (Fig. 3), physiological characterisation was carried out.
Resistance to pH variation of strains L1, L2, L3, L4, L8 and L12.
Examination of optimal growth rate at different NaCl concentrations (%) of strains L1, L2, L3, L4, L8 and L12.
Identification of CrR resistance strains
In order to assess the isolated probiotic bacteria resistance to potassium dichromate (K2Cr2O7), their minimum inhibitory concentrations (MICs) were calculated shown in Table 2. Furthermore, the diphenylcarbazide (DPC) assay was performed to evaluate isolated probiotic strains ability to reduce chromate. Reduction potential (%) of the isolated strains was measured mentioned in Table 3.
Antibiotic resistance
Antibiotic resistance in Lacticaseibacillus rhamnosus strains L1, L2, L3, L4, L8, and L12 determined as shown in the Table 4. Ampicillin resistance (R) was present in all strains. The susceptibility to clindamycin and erythromycin was highest in strains L1 and L12, while several strains exhibited resistance to gentamicin, streptomycin, and tetracycline.
Molecular characterization
“WizPure™ PCR 2X Master Mix” Cat number W1401-5 was used for PCR. The thermal profile for the PCR reaction.
Agarose gel electrophoresis
Gel electrophoresis on a 2% agarose gel was performed as shown in Fig. 4.
2% Agarose gel electrophoresis: Lane M represents Ladder (Thermo Scientific GeneRuler 100 bp Plus DNA Ladder) L1 to L12 represents putative flavin reductase gene.
Bioinformatics analysis
BioEdit
Sequencing chromatogram was obtained from bioedit software (BioEdit 7.2 download (Free) – bioedit.exe (informer.com).
Phylogenetic analysis
Phylogenetic analysis of bacterial species of the Lactobacillus on the basis of 16 S rRNA gene.
Phylogenetic analysis revealed that the putative flavin reductase proteinbelongs to the chrR and yieF clade, a specific branch of chromate reductases. Interestingly, this protein shows substantial sequence and structural divergence from other characterized chromate reductases. Furthermore, the amino acid sequence of flavin reductase and REF was paired with other characterized protein sequences of Bacteroides eggerthii (PDB:4ICI), Bacteroides uniformis (PDB:4J8P), Bacteroides fragilis (PDB:3KLB) and Bacteroides fragilis (NCBI ID: CAH09789.1). Notable residues (of flavin reductase/REF) 42Ala and 90Leu and 112Asp is highly conserved in all proteins, while some residues like 44Phe, 46Thr and 156Ile exhibit absolute conservation among all proteins.
Protein structure prediction
Physiochemical characterization and domain analysis
Protein family, domain, and motif identification were performed using the InterPro online portal available at (InterPro (ebi.ac.uk)), which combines predictions from multiple signature databases. Amino acid sequence positioned the protein within the FMN-binding split-barrel superfamily and flavin reductase-like domain. This domain organization implies FMN-dependent catalytic activity.
The Expasy ProtParam tool (Expasy – ProtParam) was utilized to determine the protein’s physiochemical parameters, including molecular weight (MW), isoelectric point (pI), grand average of hydropathicity (GRAVY), and instability index as shown in Table 5. These calculations facilitated the prediction of its functional behaviour and stability.
Molecular docking
Structural comparison of flavin reductase and 3hmz was conducted.
Simulation of FMN to flavin reductase and REF protein
Molecular docking simulation of FMN bound to flavin reductase protein, generated using AutoDock Vina. The predicted binding energy is -8.8 kcal/mol, indicating strong affinity (Fig. 9).
Molecular docking simulation of FMN bound to REF protein, generated using AutoDock Vina. The predicted binding energy is -9.1 kcal/mol, indicating strong affinity (Fig. 10).
Animal studies
Histopathology (H&E) results for hexavalent chromium induced allergic contact dermatitis22.
Histopathological analysis of skin samples shows a progression of chromate heavy metal-induced damage from normal tissue to severe damage. With its intact epidermis, sebaceous glands, and hair follicles, as well as the absence of any granulomas, burns, or infections, the control sample exhibits normal skin architecture. The remodeled skin, on the other hand, shows epidermal growth along with regions of epithelial thinning, destruction, and irregular growth of hair follicles and sebaceous glands. Active remodeling and inflammation are indicated by the dermis’s fibrocollagenous tissue, edema, mild chronic inflammatory cell infiltrates, and inflamed granulation tissue.
The chromate-induced samples exhibit the most drastic changes, with the dermis exhibiting severe damage, inflammation, and bleeding, and the epidermis displaying widespread necrosis and desquamation. The results demonstrate how chromate heavy metals harm skin tissue, causing necrosis, inflammation, and serious structural damage23.
A common occupational hazard for construction workers exposed to wet cement is cement allergy, a type of allergic contact dermatitis. The presence of heavy metal dichromates in cement, specifically hexavalent chromium (Cr(VI)), is the main cause of this condition. One well-known environmental contaminant that has serious health effects is Cr(VI) as stated by Kridin et al., 201623. Through anion transporters, Cr(VI) enters host cell membranes and causes a series of detrimental effects inside the cells. Hossini et al., 2022 reported similar toxic effects of chromate these include DNA damage, which plays a major role in the development of acute inflammatory responses, allergic reactions, and carcinoma, similar affects of chromate were reported by24. Interestingly, a number of investigations have shown that some bacterial species can convert Cr(VI) to its less hazardous trivalent form (Cr(III)). Plestenjak et al., 2022 studies different bacterial isolates and their bioremediation potential against hexavalent chromium. It has been suggested that this microbial reduction process could be used to lessen the negative effects of Cr(VI), including the way it can cause irritant dermatitis. These results are consistent with the goal of our study, which was to investigate the potential of probiotic bacterial strains to treat chromate-induced dermatitis Because of their resistance qualities, probiotic bacteria present a viable facultative approach to reducing the harmful effects of heavy metals like chromium. By leveraging their endurance potential, these bacteria can facilitate the transformation of chromium from its more toxic hexavalent form (Cr(VI)) to its less toxic trivalent form (Cr(III)), thereby protecting against cellular deterioration25.
Abdel-Megeed et al., 2021 stated about probiotics as promising agent for heavy metal detoxification. Among these, Lactobacillus strains are widely recognized for their safe application in the dairy and food industries. They are especially effective because of their strong oxidative stress-reducing properties and high capacity to detox heavy metals like chromium. By blocking its absorption and actively keeping it out of cells via particular operons, these strains can regulate the accumulation of chromium. In our study, we selected six strains of Lacticaseibacillus rhamnosus to evaluate their efficacy in treating heavy metal-induced dermatitis. These strains were picked because of their proven safety record and ability to reduce oxidative stress26.
In vivo tests using mice with chromium-induced dermatitis were conducted to assess the ability of these probiotic strains to mitigate allergic reactions and reduce chromium levels. The results underscore the therapeutic potential of L. rhamnosus strains in managing chromium-induced cellular damage and allergic conditions, highlighting their role as a safe and effective intervention27.
This study focused on the potential of probiotics—live microorganisms that provide health benefits when given in sufficient amounts—to reduce chromium toxicity. Quadrant streaking was used to find chromium-resistant strains, and the colonies were then purified and compared. Only six of the eight tested Lactobacillus strains—designated L1, L2, L3, L4, L8, and L12—showed resistance to media supplemented with hexavalent chromium as potassium dichromate28. These six strains were then chosen for in-depth examination. Biochemical characterization of the isolates was performed using standard test procedures. All strains were confirmed as gram-positive based on gram staining, with no gram-negative strains detected. This observation aligns with prior findings that probiotics are predominantly gram-positive bacteria. Additional biochemical tests, including catalase and oxidase tests, were conducted to further characterize the isolates, confirming their identity as lactic acid bacteria29. The capacity of these isolates to produce lactic acid—a characteristic that distinguishes Lactobacillus species—was demonstrated by particular tests, such as the use of chalk agar as reported by Salem et al., 2023 where the study discussed about the production of lactic acid on chalk agar confirming that the bacterial isolate under study is a lactic acid bacteria30. These strains biochemical traits and resistance to Cr (VI) highlight their potential use in treating illnesses brought on by heavy metal exposure. These results support the hypothesis that Lactobacillus strains, particularly those resistant to Cr(VI), are robust candidates for therapeutic interventions aimed at reducing chromium toxicity and its associated health impacts31. The presence of clear zones surrounding the growth of the isolated colonies indicated that all six strains tested positive in the chalk agar test. This result validates the strains capacity to generate lactic acid, which is a crucial trait of lactic acid bacteria. Significantly, comparable patterns of activity were noted in earlier assessments, confirming strains dependability and consistency in displaying this characteristic. These results highlight the chosen strains functional potential in applications needing strong lactic acid production.
The chromium resistance of the six chosen probiotic strains against potassium dichromate at concentrations of 500, 250, 125, and 62.5 µg/ml was assessed using the minimum inhibitory concentration (MIC) test. The strains differing degrees of resistance were shown by the results. Across all tested concentrations, strains L1 and L2 demonstrated moderate to good resistance. At 125 µg/ml and 62.5 µg/ml, strain L3 showed good resistance; at higher concentrations (250 µg/ml and 500 µg/ml), moderate resistance was seen. Greater sensitivity to higher chromium concentrations was indicated by strain L4-low inhibition at 125 µg/ml and high inhibition at 62.5 µg/ml. Whereas strain L12 showed a pattern of weak to high resistance as the chromium concentration dropped, strain L8 showed weak to moderate resistance, especially at higher concentrations32. Chromium resistance was further examined using a diphenyl carbazide (DPC) assay stated by Lace et al., 2019. It made use of the DPC reagent’s capacity to bind unreduced hexavalent chromium (Cr (VI)) and generate a purple hue, which enabled the measurement of Cr (VI) levels. To make the analysis easier, a standard curve was created that connected chromium concentrations with optical density at 540 nm. These results highlight the probiotic strains potential for use in reducing chromium toxicity and offer important insights into their capacity for chromium resistance33,34.
Significant new information about the genetic and structural characteristics of the flavin reductase protein in relation to the reference (REF) gene from Lacticaseibacillus rhamnosus was revealed by the bioinformatics analysis in order to understand the structural homology of protein under study as stated by Amin et al., 2022. The six samples alignment and sequencing showed significant nucleotide changes from the reference sequence, but no distinct mutations were found. Despite multiple mutations, structural analysis revealed that the protein’s secondary and tertiary structures only slightly differed, indicating that the protein’s global fold and overall integrity were largely maintained. The FMN-binding motif was discovered through functional domain analysis, highlighting its catalytic function in chromium reduction. As seen in Fig. 8, conserved areas inside the binding pockets further emphasized their functional significance. Strong interactions between flavin mononucleotide (FMN) and the reference and variant proteins were found by molecular docking simulations. The REF protein showed slightly higher stability, suggesting its strong catalytic potential, even though both proteins showed high binding affinity. Together, these results highlight the flavin reductase protein’s functional robustness and crucial function in chromium detoxification procedures35.
Even though our study yielded important results, there is room for more research to improve comprehension and application of these findings. The identification and analysis of six chromium-resistant strains of Lacticaseibacillus rhamnosus provided a solid basis for future research examining the molecular mechanisms underlying their capacity for chromium detoxification. Furthermore, although in vivo assessments employing mouse models provided insightful information, additional research utilizing human models may be able to more accurately depict the intricacies of chromium-induced dermatitis in clinical settings. A more thorough grasp of these probiotic strains therapeutic potential may also be obtained by investigating their stability and long-term effectiveness in a range of physiological and environmental settings. Lastly, the bioinformatics analyses paved the way for experimental validation to completely clarify the catalytic activity and interaction dynamics of the flavin reductase protein, providing crucial insights into its function in chromium reduction. These directions for further study have the potential to improve the application of probiotics in the treatment of heavy metal toxicity and related illnesses.
