A Comprehensive Analysis of Cytotoxicity of Genetically Modified Lactobacillus plantarum 8РАЗ (B-11007) Strain Including Identification and Evaluation of Virulence Factors

Introduction. Асtive development of biotechnologies and genetic engineering techniques broadens the horizons for engineering the probiotic strains with tailored functional properties. In this context, identification and description of the potential factors of virulence are performed using the specialized databases and taxonomies [1]. Modern understanding of the role of probiotics in maintaining human health and regulating metabolic processes fosters the relevance of targeted modification of bacteria designated to enhance their beneficial properties [2]. At the same time, the results of the phenotypic and genomic analysis of Lactobacillus reuteri strains demonstrate the need for a comprehensive safety assessment even for commonly used probiotics [3]. Similar data on the importance of comparing the genomic markers and phenotypic characteristics in the context of assessing safety of a given strain are presented in publication [4]. General reviews emphasize that expansion of the range of probiotic products requires strict control of their biological safety and pathogenic potential absence [5].

A comprehensive approach to assessment of the safety of probiotic cultures, including genotypic and phenotypic analysis, was implemented in a study of Lacticaseibacillus paracasei NTU 101, demonstrating the relevance of multi-level testing of new strains [6]. Similar methodological principles were applied in the analysis of Lactobacillus bulgaricus IDCC 3601, where potential risks to humans were assessed [7]. A detailed description of the phenotypic and genotypic characteristics of Levilactobacillus brevis KU15006 has also proved the need to exclude the factors of virulence and antibiotic resistance [8]. It is known that molecular signaling pathways regulating cell proliferation and survival in condition of inflammatory stress can be modified by microbial factors, which emphasises the importance of studying the cellular mechanisms of the host response [9].

The problem of safety of genetically modified microorganisms is particularly relevant for veterinary practice, where probiotics are used to normalize the microbiocenosis and increase animal resistance to infections, as confirmed by the studies of Lactococcus lactis IDCC 2301 [10]. The assessment of four lactobacilli strains with probiotic properties showed that even closely related microorganisms require individual analysis of genetic stability and the absence of undesirable determinants [11]. The activity of enzymes, such as bile salt hydrolases, is based on the interaction of microbiota and the host organism, and has significant importance [12]. Studies of the Lactobacillus delbrueckii subsp. lactis strain CKDB001 confirm the need to evaluate not only safety but also functional metabolic effects [13].

In vitro experimental models are widely used to evaluate the cytotoxicity and safety of encapsulated probiotic cultures, as demonstrated for Lactiplantibacillus plantarum CRD7 and Lacticaseibacillus rhamnosus CRD11 [14]. Changes in intestinal microbiota and metabolic parameters after administration of Lacticaseibacillus paracasei AO356 confirm the importance of studying the systemic effect of probiotics [15]. Analysis of the phenotypic and genotypic characteristics of Weissellacibaria JW15 also indicates the need for a comprehensive assessment of safety of the new cultures [16]. An additional stage of preclinical testing is in silico analysis of potential genes of virulence and resistance, as shown for representatives of the genus Bacillus isolated from the bee products [17].

Particular attention is paid to the ability of strains to produce biogenic amines, which can have a toxic effect when accumulated in food products, as demonstrated for lactic acid bacteria isolated from fish products [18]. Genomic sequencing and evaluation of the probiotic potential of Lacticaseibacillus paracasei strain LC86 and Lacticaseibacillus casei strain confirm the importance of a comprehensive analysis of their safety [19]. The reviews investigating the biogenic amines in the fermented products emphasise the potential risks of uncontrolled microbial activity for the health [20]. Current data on the oral cavity microbiome and its impact on systemic health also indicate the need for rigorous evaluation of probiotic interventions [21].

Experimental studies using a Clostridioides difficile infection model demonstrate that probiotic strains can reduce expression of inflammation and restore barrier function of the intestines, thus, confirm their therapeutic potential subject to proven safety [22]. However, even opportunistic lactobacilli strains can, under certain conditions, exhibit cytopathic effect on intestinal epithelium. Therefore, comprehensive analysis of cytotoxicity is a necessary step in preclinical studies of new probiotic strains.

The aim of the study is to conduct a comprehensive analysis of the cytotoxic properties of the genetically modified strain Lactobacillus plantarum 8РАЗ (B-11007), aiming to identify and evaluate the virulence factors responsible for inducing the cell death and dysfunction of cellular mechanisms.

Materials and Methods. The experimental part of the study was performed at the Genome Center of the North-Caucasus Federal University (Stavropol) and at the bacteriological laboratory of the Department of Epizootology and Microbiology of Stavropol State Agrarian University in the period from January 23 to February 17, 2026.

In this study, the Lactobacillus plantarum strain 8РАЗ (B-11007) was genetically modified by introducing a recombinant plasmid containing functionally independent expression cassettes encoding heterologous proteins under the control of constitutive promoters.¹ The cytotoxicity of the genetically modified L. plantarum strain 8РАЗ (B-11007) was assessed in compliance with the author’s method of A.V. Frolov “Method for determining the cytotoxicity of bacteria”.²

The study was conducted using the genetically modified (GM) L. plantarum strain 8РАЗ (B-11007) and its native analogue as a control. Such an approach makes it possible not only to identify differences in the cytopathic effect between the GM form and the control one but also to assess the preservation or enhancement of strain’s beneficial properties, which is extremely important for the objective assessment of the modified strain’s safety and functional activity.

Bacterial strains were cultured in the standard MRS nutrient medium (Difco™) in under anaerobic conditions at 37°C using AnaeroGen gas bags (Oxoid, UK). Bacterial suspensions were prepared in sterile phosphate-buffered saline (PBS), adjusted to optical density of 0.5 McFarland standard, corresponding to a concentration of approximately 1.5×10⁸ CFU/ml, and then diluted to a working concentration of 1×10⁹ CFU/ml.

Cytotoxicity was assessed using two parallel approaches. In the first step, jejunal organ culture from clinically healthy 10-day-old calves (n=10) were placed in sterile containers over a cover slip with liquid nutrient medium (RPMI-1640) containing a suspension of the studied Lactobacillus strain at a final concentration of 1×10⁹ CFU/ml. Incubation with the bacterial suspension was carried out for 30 min at 37°C in an atmosphere of 5% CO₂ and 95% humidity using a CO₂ incubator (Binder GmbH, Germany). After exposure, the containers were centrifuged at the rate of 10 000 rpm for 10 min to sediment the cells detached as a result of the cytopathic effect. Each experiment was performed in 5 biological replicates.

In parallel, cytotoxicity was assessed in primary culture of enterocytes that were isolated from the jejunum of 10-day-old calves. Cells were seeded in 96-well plates coated with type I collagen at density of 3 × 10⁴ cells per well and cultured in RPMI-1640 medium supplemented with 10% fetal calf serum until a monolayer formed. Cytotoxicity was assessed using the MTT assay with 24-hour incubation and subsequent measurement of optical density (OD) at 570 nm. Intestinal barrier integrity was assessed by measuring transepithelial electrical resistance (TEER) using an EVOM2 Volt/Ohm meter, recording readings every 4 hours for 24 hours.

To determine the mechanisms of cytotoxicity, fluorescence microscopy was performed using annexin V-FITC and propidium iodide staining to differentiate apoptosis from necrosis. Caspase 3/7 activity was also assessed using the Caspase-Glo 3/7 Assay kit.

Quantitative cytotoxicity (C) was assessed using the formula:

С= N/t (1- g1/g0), (1)

where N — the number of cells detached during centrifugation, counted per 1.96 mm² along the diagonals of the cover glass;

g0 — centrifugal acceleration of cell detachment, not treated with a cytotoxic bacterial suspension;

g1 — centrifugal acceleration of cell detachment, treated with a cytotoxic bacterial suspension;

t — exposure time to cytotoxic bacterial suspension, h.

To more thoroughly assess the impact on the cellular functions, an MTT assay was used on a monolayer of primary enterocytes for 24 hours. The assay is based on the Tetrazolium dye (MTT) reduction by mitochondrial dehydrogenases of viable cells. Cell viability percentage (VC) was calculated using the formula:

VC (%) = (OD570 experiment / OD570control) × 100 %, (2)

where OD570 experiment — optical density of formazan solution in wells with cells treated with bacteria;

OD570 control — optical density in wells with cells cultured in a pure nutrient medium.

All numerical data obtained were subjected to statistical processing. Results are presented as the arithmetic mean (M) ± standard deviation (SD) or standard error of the mean (SEM). The Shapiro-Wilk test was used to assess the normality of data distribution. Comparison of means between the experimental and control groups was performed using the Student’s t-test for independent samples using the Primer of Biostatistics 4.03 software (McGraw-Hill, USA). Differences were considered statistically significant at p ≤ 0.05.

Research Results. The primary assessment of the potential cytotoxic effect of the genetically modified L. plantarum strain 8РАЗ (B-11007) was performed in compliance with A.V. Frolov’s method on jejunal organ culture and revealed no statistically significant increase in cytotoxicity between the GM strain and its native analogue (Table 1).

As shown in Table 1, the cytotoxicity index of the GM strain did not differ statistically significantly (p>0.05) from that of the native control strain. The number of detached cells in both groups (corresponding to a cytotoxicity index in the range of 0.06–0.12 a.u.) was within the reference values established for this organ culture in the settings free of pathogenic influence (0.05–0.15 a.u.). This indicates the absence of cytopathic effect and disruption of tissue adhesive properties under the influence of the genetically modified strain, even under extreme stress testing conditions. It is important to emphasize that the method is based on a short-term (30 min) exposure to an extremely high bacterial concentration (1×10⁹ CFU/ml) followed by the application of centrifugal force, which simulates the tissue resistance limit. These conditions are stressful and extreme, simulating not a standard interaction, but rather the tissue’s “resistance limit”. In the context of safety assessment, identifying an effect under such conditions is a valuable indicator for further, more in-depth studies, but cannot directly indicate pathogenicity in vivo, where bacterial concentrations and interaction conditions are significantly milder.

Visual assessment of the organ cultures after the experiment confirmed the quantitative data obtained. The jejunal mucosa in both studied groups (both those exposed to the native and GM strains) retained characteristic morphological features consistent with the normal values. The surface of the samples had a uniform pale pink color, indicating preserved microcirculation and the absence of signs of ischemia or hemorrhagic lesions. Macroscopic examination revealed a smooth, moist mucosal surface, characteristic of healthy tissue. The absence of areas of turbidity, focal hyperemia, edema, or massive epithelial desquamation further demonstrates the absence of destructive effects from the GM strain.

The results of the MTT test on a monolayer of primary enterocytes showed that after 24 hours of incubation, the metabolic activity of cells treated with the GM strain was statistically significantly higher than that in the control group (Table 2).

The data obtained in Table 2 indicate that the GM strain has a moderate stimulating effect on enterocyte metabolic activity, significantly (p < 0.05) increasing it by 8.7% compared to the control strain. This effect may be associated with targeted changes in bacterial metabolism as a result of genetic modification, such as increased production of specific metabolites (short-chain fatty acids, B vitamins) that have a trophic effect on intestinal epithelial cells.

To assess safety at the level of cell death regulation, the analysis of apoptosis and the activity of executioner caspases 3/7 was performed (Table 3).

As shown in Table 3, the GM strain significantly (p<0.05) reduced the proportion of cells undergoing early apoptosis by 31% compared to the control, without affecting caspase 3/7 activity (p > 0.05). This indicates an anti-apoptotic effect unrelated to the classical caspase-dependent pathway and confirms the strain’s safety.

All quantitative data obtained, demonstrating the balance between the safety and beneficial properties of the GM strain, are summarized in Table 4.

Discussion and Conclusion. Based on the conducted comprehansive studies, it was established that the genetically modified L. plantarum strain 8РАЗ (B-11007) does not exhibit a cytotoxic effect on the calf jejunal organ culture. The cytotoxicity index of the GM strain (0.09±0.03 a.u.) did not statistically significantly differ (p>0.05) from the index of the native precursor strain (0.08±0.02 a.u.) and was within the physiological norm, indicating the preservation of cellular adhesion and the integrity of the histological structure of the tissue. Macroscopic assessment of the mucous membrane showed the preservation of normal characteristics — pale pink color and typical surface topography with physiological roughness, which further confirms the absence of pathological changes in the tissue.

The GM strain proved to have a positive effect on the functional activity of enterocytes. A statistically significant (p<0.05) increase in cellular metabolic activity by 8.7% compared to the that in the control was detected, as well as a reliable enhancement of barrier function, expressed as an increase in transepithelial electrical resistance by 15.2% by 24^th hour of coincubation. It was confirmed that the mechanism of interaction between the GM strain and host cells is not associated with the induction of apoptosis or necrosis. In the experimental group, a statistically significant decrease in the proportion of cells in early apoptosis was recorded (3.1% versus 4.5% in the control; p<0.05) with maintained background level of caspase 3/7 activity, indicating the safety of the strain in the context of cell death regulation.

The conducted studies allow us to conclude that the genetic modification of the L. plantarum strain 8РАЗ (B-11007) did not lead to the acquisition of cytopathic properties or virulence factors. The preservation of normal macroscopic characteristics of the intestinal mucosa, combined with a positive effect on cellular functions, indicates that the GM strain has positive effects, expressed in stimulating enterocyte metabolism and strengthening the intestinal epithelial barrier. The identified properties make this strain a promising candidate for the development of new probiotic medications based on it. To fully understand the mechanisms of the observed positive effects and identify specific bacterial metabolites responsible for the identified effects, it is advisable to conduct further proteomic and metabolomic studies. The results obtained are the basis for moving to the next stage of preclinical testing in laboratory animals.