Strain-Dependent In Vitro Susceptibility of Leptospira spp. to Isoquinoline Alkaloids - Chelerythrine and Sanguinarine

1. Introduction

As pathogens become increasingly resistant to antibiotics, active plant-derived compounds are emerging as a promising area of research to solve this problem [1,2,3,4,5,6,7]. Compounds with such potential are isoquinoline alkaloids, particularly sanguinarine and chelerythrine, which are prominent bioactive compounds biosynthesized in various plants of the Papaveraceae family, including Chelidonium majus, Sanguinaria canadensis, and Coptis chinensis [8,9,10]. These alkaloids are recognized for their broad spectrum of pharmacological activities, such as antimicrobial, an-ti-inflammatory, antifungal, and antitumor effects [8,11,12]. Sanguinarine and chelerythrine are most abundantly found in the roots and aerial parts of these plants, with sanguinarine generally present at higher concentrations than chelerythrine [8].

Both sanguinarine and chelerythrine have been reported to exhibit antimicrobial activity against a variety of mi-croorganisms [8,9,11,12,13]. However, most studies have focused on a limited number of model organisms, and to date, there are no published reports investigating their effects on Leptospira bacteria. Sanguinarine, in particular, has been shown to be more active than chelerythrine in several antimicrobial and anti-inflammatory assays, alt-hough exceptions exist [8,13].The antimicrobial properties of these alkaloids are attributed to their ability to inhibit the multiplication of microorganisms and disrupt cellular processes such as membrane integrity and cell prolifera-tion [13,14]. For example, sanguinarine has been reported to cause cell membrane damage and inhibit spore germi-nation in plant pathogenic fungi [14]. The biosynthesis of sanguinarine and chelerythrine in Papaveraceae plants involves conserved enzymatic pathways, starting from tyrosine derivatives and proceeding through a series of plant-specific enzymes [10]. These alkaloids are often co-extracted as natural fractions from medicinal plants such as Coptis chinensis, which is known to contain both sanguinarine and chelerythrine along with other isoquinoline alkaloids [10].

Although isoquinoline alkaloids, such as sanguinarine and chelerythrine, have demonstrated antimicrobial activity against selected microorganisms, their activity against Leptospira spp. remains poorly characterized. Importantly, the activity of such compounds cannot be directly extrapolated to Leptospira spp., as these bacteria exhibit distinct structural and physiological features that may influence their interaction with exogenous agents.

Leptospira spp. are spirochete bacteria responsible for leptospirosis, a globally distributed zoonotic disease main-tained in animal reservoirs and transmitted through environmental exposure [15,16]. Pathogenic leptospires estab-lish infection through rapid dissemination in the host and subsequent colonisation of target tissues, particularly the kidneys, where they may persist and be shed into the environment [16,17]. The genus Leptospira exhibits substantial genetic and serological diversity. In addition to genomic species classification, strains are grouped into numerous serovars (hereafter abbreviated as sv.) based on antigenic differences associated with lipopolysaccharide (LPS) structure. Importantly, serovar classification does not correspond directly to phylogeny, and closely related strains may differ in phenotypic traits, host adaptation, and environmental survival [16,18,19]. A characteristic feature of pathogenic leptospires is the structure of the outer membrane, which contains atypical LPS and a large number of surface-exposed lipoproteins. This outer membrane represents a dynamic interface involved in host interaction, adaptation, and survival under changing environmental conditions [15,16,20,21]. Its composition may vary be-tween strains and is subject to regulation in response to external stimuli. Given these features, the interaction of Leptospira with exogenous compounds cannot be assumed to follow patterns observed in other bacteria. The per-meability of the outer membrane, together with strain-specific differences in its composition, may influence the observed effects of bioactive compounds [15,16,20]. Therefore, responses to such compounds may be variable and dependent on the strain studied. The aim of this study was to evaluate the in vitro effects of selected isoquinoline alkaloids on different Leptospira strains, with particular attention to potential strain-dependent variability in the response under defined conditions. To our knowledge, this is one of the first systematic evaluations of isoquinoline alkaloids in Leptospira spp.

2. Results

2.1. Effects of Isoquinoline Alkaloids on Leptospira Growth and Viability

The effects of sanguinarine chloride, chelerythrine chloride, and their natural fraction on selected *Leptospira* strains were evaluated using complementary approaches, including flow cytometry (assessment of membrane integrity), total cell enumeration, and optical density measurements (Table 1, Table 2 and Table 3). For most tested strains—namely *L. interrogans* sv. Copenhageni (strain M20), *L. kirschneri* sv. Grippotyphosa (strain Moskwa V), *L. interrogans* sv. Pomona (strain Pomona), and *L. borgpetersenii* sv. Hardjo (strain KR39)—exposure to the tested compounds resulted in limited and variable changes in the analysed parameters. Total bacterial counts remained similar to control samples, and only minor variations in the proportion of live and dead cells were observed. Optical density values also did not show a consistent dose- or time-dependent trend, indicating that under the applied experimental conditions, the tested compounds did not induce a clear or reproducible inhibitory effect in these strains. In contrast, a distinct response was observed for *L. interrogans* sv. Australis (strain Balico), where increasing concentrations of the tested compounds led to reductions in total bacterial counts, the proportion of live cells, and optical density values, with effects more pronounced at higher concentrations and later time points. For sanguinarine chloride, moderate effects were observed in strain Balico at 5 and 10 µM, with decreases in cell counts and viability, particularly after 48–72 h, although some variability between time points was noted (Table 1). Chelerythrine chloride produced a more consistent effect in this strain, with reductions in bacterial counts and viability across all tested concentrations, and stronger effects at higher concentrations and longer incubation times (Table 2). The sanguinarine-chelerythrine fraction exhibited a weaker or intermediate profile, with reductions in bacterial counts and viability at higher concentrations, but the magnitude of the effect was generally lower than that observed for chelerythrine chloride and comparable to or slightly weaker than that observed for sanguinarine alone (Table 3).

2.1. Metabolic Activity (Alamar Blue Assay)

The Alamar Blue assay showed a pattern broadly consistent with the observations obtained using cytometric and growth-based methods. For most tested strains, metabolic activity remained relatively stable across the tested concentration range, and no consistent dose-dependent effect was observed. In contrast, in L. interrogans sv. Australis, strain Balico, a concentration-dependent decrease in metabolic activity was detected for all tested compounds. Statistical analysis was performed for all strains; however, statistically significant differences were observed only for this strain. Sanguinarine chloride induced a moderate reduction in metabolic activity, with statistically significant effects observed at 5 and 10 µM (p < 0.05), particularly at later time points. No consistent or significant effect was observed at 1 µM (Figure 1). Chelerythrine chloride produced a more pronounced response, with significant reductions in metabolic activity observed across the tested concentrations and time points (p < 0.01). The strongest effect was observed at the highest concentration and after prolonged incubation (72 h) (Figure 2). The sanguinarine–chelerythrine fraction induced a weaker effect compared to the individual compounds. Significant reductions in metabolic activity were observed primarily at higher concentrations (5 and 10 µM), while no significant effect was detected at 1 µM (Figure 3).

3. Discussion

The present study provides an in vitro evaluation of the effects of selected isoquinoline alkaloids on selected Lepto-spira strains. Using a combination of microscopic, cytometric, and metabolic assays, we observed that the effects of the tested compounds were limited and strongly strain-dependent. The most pronounced responses were detected in strain Balico, particularly at higher concentrations.

Effects of Sanguinarine Chloride

Among the tested compounds, sanguinarine chloride induced measurable changes in selected parameters, but these effects were not consistent across all strains and were primarily observed in strain Balico at higher concentrations and after prolonged incubation. Lower concentrations produced minimal or no detectable effects across most strains under the applied experimental conditions.

Previous studies have reported antimicrobial activity of sanguinarine against a range of microorganisms, however, direct comparison with Leptospira is limited due to major biological differences between these organisms [8,14,22,23,24]. Mechanistically, sanguinarine has been shown to disrupt membrane integrity, interfere with ion transport, inhibit key enzymes, and induce oxidative stress, leading to impaired cellular metabolism and eventual cell death, however, their relevance to Leptospira remains unclear [14,25,26,27]. The observed decrease in metabolic activity and membrane integrity in selected conditions suggests that the compounds may affect various aspects of leptospiral physiology; however, the underlying mechanism cannot be determined based on the present data.

Effect of Chelerythrine Chloride

Chelerythrine chloride showed a strain-dependent effect, with the most pronounced response observed in strain Balico. In most strains, the observed changes were limited, whereas in strain Balico more pronounced decrease in metabolic activity and viability was detected, even at lower concentrations.

Although chelerythrine is structurally related to sanguinarine and has demonstrated biological activity in other systems, differences observed between compounds in this study may reflect strain-specific properties of Leptospira as well as differences in compound interaction with the leptospiral cell envelop [12,28]. The outer membrane ar-chitecture of Leptospira may influence the interaction of exogenous compounds with the cell, potentially contrib-uting to strain-dependent differences in the observed response. Leptospira differs substantially from classical Gram-negative bacteria in terms of cell envelope organization and physiology, which may influence their interac-tion with exogenous compounds and limit direct comparison with data obtained for other bacterial species [15,16,20]. The atypical structure and variability of leptospiral lipopolysaccharide (LPS) and outer membrane components have been shown to play a key role in environmental adaptation and host interaction, which may also influence the response to external compounds [15,16,19,20].

Effects of the Sanguinarine-Chelerythrine Fraction

The sanguinarine–chelerythrine fraction did not show a stronger effect than the individual compounds. At higher concentrations, the fraction was associated with a decrease in metabolic activity and bacterial counts, however, the overall effect remained weaker than that observed for the individual compounds. Under the tested conditions, the combined fraction did not show a stronger effect than the individual compounds. This observation highlights the importance of experimentally evaluating the effects of compound mixtures rather than assuming enhanced activity based on individual components. However, antagonistic or neutral interactions among alkaloids have been report-ed previously [10,29,30,31], highlighting the importance of experimentally validating the activity of compound mix-tures rather than inferring efficacy from individual components.

Strain-Dependent Responses of Leptospira

A key finding of this study is the variability in response among different Leptospira strains. Most tested strains showed limited or no consistent changes across the applied conditions, whereas a more pronounced response was observed only in strain Balico. Taken together, the results of this study indicate that the effects of the tested iso-quinoline alkaloids on Leptospira are limited and strongly strain-dependent, and therefore should be interpreted as preliminary observations rather than evidence of consistent antimicrobial activity.

Implications and Limitations

The observed effects were limited to in vitro conditions and were most evident at relatively high concentrations, which restricts broader interpretation of their biological relevance. [28]. The present results indicate that the tested compounds may affect selected Leptospira strains under defined experimental conditions, however, they do not support conclusions regarding potential therapeutic applications at this stage [32]. This is particularly important in the context of leptospirosis, where pathogenic Leptospira rapidly disseminate within the host and may colonize tis-sues such as the kidneys. Any potential therapeutic compound would therefore require adequate systemic activity and tissue penetration, which were not assessed in the present study. The biological relevance of the observed ef-fects in vivo remains unknown.

4. Materials and Methods

4.1. Bacterial Strains and Culture Conditions

Five reference strains of Leptospira were used in this study: Leptospira interrogans serovar Copenhageni (strain M20), Leptospira kirschneri serovar Grippotyphosa (strain Moskwa V), Leptospira interrogans serovar Pomona (strain Pomona), Leptospira interrogans serovar Australis (strain Balico), and Leptospira borgpetersenii serovar Hardjo (strain KR39). All strains were obtained from reference collections as indicated above. Leptospires were cultivated in Ellinghausen–McCullough–Johnson–Harris (EMJH) liquid medium under standard laboratory conditions at 28 °C without agitation. Cultures were maintained in exponential growth phase and adjusted to an initial density of approximately 1 × 10⁷ cells/mL prior to each experiment. Following treatment, cultures were incubated under identical conditions and sampled after 24, 48, and 72 h. All analyses were performed at each time point.

4.2. Compounds and Treatment Conditions

Sanguinarine chloride and chelerythrine chloride (Extrasynthese, Genay, France; HPLC purity ≥ 90–95%) were used as model isoquinoline alkaloids. Compounds were dissolved in dimethyl sulfoxide (DMSO) to obtain stock solutions at a concentration of 1 mM. A natural fraction containing sanguinarine and chelerythrine, previously described, was also included in the analysis [13]. Working solutions were prepared by diluting stock solutions in EMJH medium immediately prior to use. The final concentration of DMSO in all experimental conditions did not exceed 1% (v/v).

Sanguinarine chloride was tested at final concentrations of 1, 5, and 10 µM. The selection of concentrations was based on previously published studies [23] as well as preliminary experiments. Initial testing included lower concentrations (e.g., 0.5–2 µM), corresponding to ranges reported in the literature; however, under the applied experimental conditions for Leptospira spp., these concentrations did not result in detectable changes in the applied assays (data not shown). Therefore, the concentration range was extended to include higher doses to enable the assessment of potential biological effects.

Chelerythrine chloride was tested at final concentrations of 1, 5, and 10 µM. Concentration ranges were selected based on previously published data [33] and preliminary experiments. As in the case of sanguinarine, lower concentrations corresponding to literature reports did not result in detectable changes under the applied experimental conditions (data not shown), and therefore higher concentrations were included in further analyses.

The sanguinarine–chelerythrine fraction was tested at final concentrations of 1, 5, and 10 µM (expressed as total alkaloid content). The selection of concentrations followed the same rationale as for individual compounds, including preliminary testing of lower doses that did not produce measurable effects (data not shown).

For each strain and each tested compound, the following experimental groups were included: a negative control containing only EMJH medium and Alamar Blue reagent, an untreated control containing bacterial culture without compound addition, and three treatment groups corresponding to the selected concentrations of the tested compound. All experimental conditions were maintained under identical incubation parameters. Samples were collected after 24, 48, and 72 h for further analyses. Each experiment was performed independently in triplicate (n = 3 biological replicates), and all assays were conducted under the same experimental conditions to ensure comparability between strains and treatments.

4.3. Assessment of Metabolic Activity Using Alamar Blue Assay

Metabolic activity of leptospiral cells was evaluated using the Alamar Blue^® assay (Thermo Fisher Scientific), as described previously [32] with modifications adapted for Leptospira. Briefly, 180 µL of bacterial culture was transferred into wells of a 96-well plate, followed by the addition of 20 µL of Alamar Blue reagent. Plates were incubated at 28–30 °C under standard conditions until a measurable color change from blue (oxidized resazurin) to pink (reduced resorufin) was observed. Quantitative measurements were performed by recording absorbance at 570 nm and 600 nm using a microplate reader. Wells containing medium without bacteria were used as negative controls. The percentage reduction of resazurin was calculated according to established equations based on molar extinction coefficients, as described by [32], and used as an indicator of cellular metabolic activity.

4.4. Flow Cytometry Analysis and Viability Assessment

Leptospira cell counts and viability were assessed using flow cytometry with the LIVE/DEAD BacLight™ Bacterial Viability Kit (Thermo Fisher Scientific), as previously described [21,33,34]. Samples were stained with SYTO9 and propidium iodide (PI) at a ratio of 1:1000, gently mixed, and incubated for 3 minutes in the dark at room temperature. Immediately prior to analysis, samples were vortexed briefly to ensure homogeneity. Flow cytometric analysis was performed using a BD Accuri™ C6 flow cytometer (BD Biosciences). SYTO9 fluorescence (live cells) was detected in the FL1 channel, while fluorescence (cells with compromised membrane integrity) was detected in the FL3 channel. Instrument settings included a threshold of 2000 on the FL1 channel. Control samples consisting of untreated live bacteria and heat-inactivated bacteria (90 °C) were used to establish gating parameters and to distinguish between intact and membrane-compromised cells. For each sample, 10,000–50,000 events were recorded. Data were analyzed using BD Accuri C6 software and FlowJo v10 (BD Biosciences).

4.5. Cell Enumeration

Total leptospiral cell counts were determined using flow cytometry and additionally verified by direct counting in a Petroff–Hausser counting chamber under dark-field microscopy, as previously described. This approach allowed cross-validation of quantitative data obtained from cytometric analysis.

4.6. Optical Density Measurements

Bacterial growth was additionally monitored by measuring optical density (OD) using a spectrophotometer at (420nm). Measurements were performed for all samples at each time point.

4.7. Dark-Field Microscopy

Bacterial morphology and motility were evaluated qualitatively using dark-field microscopy, as described in previous studies [18]. Observations were performed directly from culture samples without fixation to assess potential changes in cell morphology and motility under different treatment conditions.

4.8. Statistical Analysis

Statistical analysis was performed for the Alamar Blue assay data for all tested strains. Data are presented as mean values ± standard deviation (SD) of three independent experiments (n = 3 biological replicates). Statistical significance between treated samples and the corresponding untreated control was assessed using one-way analysis of variance (ANOVA), followed by an appropriate post hoc test. A p-value < 0.05 was considered statistically significant.

Although statistical comparisons were performed for all strains, statistically significant differences were observed only in one strain, while in the remaining strains no significant differences were detected.

5. Conclusions

The present study demonstrates that the effects of selected isoquinoline alkaloids on Leptospira are limited and strongly strain-dependent under the applied in vitro conditions. Across most tested strains, no consistent or pronounced changes in bacterial viability, abundance, or metabolic activity were observed. A measurable response was detected primarily in) L. interrogans sv. Australis (strain Balico), particularly at higher concentrations. Among the tested compounds, chelerythrine chloride showed the most consistent effect in this strain, whereas sanguinarine chloride and the sanguinarine–chelerythrine fraction exhibited weaker or less consistent responses. The combined fraction did not demonstrate a stronger effect than the individual compounds under the applied conditions. These findings indicate that the response of Leptospira to the tested compounds cannot be generalized and may vary substantially between strains. Overall, the data suggest that selected isoquinoline alkaloids may influence Leptospira under defined in vitro conditions, however, their effects are restricted, strain-dependent, and observed mainly at higher concentrations. Further studies, taking into account standard antimicrobial parameters such as MIC, and dedicated mechanistic analyses, are necessary to explain the mechanism of action of these compounds on individual Leptospira strains, and to determine their biological relevance.