In Vivo Efficacy of Bacillus velezensis Isolated from Korean Gochang Bokbunja Vinegar against Carbapenem-Resistant Klebsiella pneumoniae Infections

CRKP. CRKP was collected from the Pathogenic Resource Bank at Soonchunghyng University Hospital (Tajdozian et al. 2021). It was cultured in MacConkey broth (BD Difco, USA) and incubated at 37C for 18 h. Next, bacterial growth was determined using a spectrophotometer (DR 1900, HACH, USA), and optical density at 600 nm (OD_{600 nm}) was adjusted to 1.0. After that, the bacterial number was enumerated through a colony-forming unit (CFU) assay. For further uses, stock culture in 60% glycerol was prepared and kept at –80°C.

Isolation and identification of a probiotic strain from naturally fermented vinegar. The candidate probiotic was obtained from naturally 14-days-fermented Gochang Bokbunja vinegar from Gochang County, North Jeolla Province, South Korea. Upon receiving the samples, they were streaked onto an MRS agar (BD Difco, USA) plate and incubated at 37°C for 24 h. After that, single colonies were picked and subcultured in MRS broth (BD Difco, USA). After incubating in a shaking incubator overnight at 37°C under aerobic conditions, bacterial growth was determined using the spectrophotometer. Stock in 60% glycerol was prepared and stored at –80°C for further use. This probiotic strain was sent to a company (Biofact Co, Korea) for 16S rRNA sequencing and species identification.

Biochemical tests. A biochemical test was conducted using an API 50CH kit (bioMerieux, France) to observe the carbohydrate fermentation pattern of the probiotic strain compared to other reference strains. The candidate probiotic strain was cultured on an MRS agar plate. A single colony was then picked and mixed with 1 ml suspension medium. After that, the suspension turbidity was determined using 2 McFarland, and 0.1 ml of this suspension was diluted in 10 ml of CHB medium (bioMerieux, France). Next, the strip was removed from the API kit and placed in a tray. The final prepared bacterial suspension was transferred into the strip’s wells and incubated at 37°C for 48 h. Subsequently, color change in each well was observed, and an identification table was prepared as (+/–).

Quantitative RT-PCR assay. qRT-PCR is a well-established and robust approach for detecting and quantifying microorganisms (Kralik and Ricchi 2017). Thus, qRT-PCR was performed to identify the isolated strain. Genomic DNA was extracted from the isolated candidate probiotic strain using the QIAamp DNA Mini Kit (QIAGEN, Germany). Genomic DNAs were also extracted from B. velezensis KCTC 13417, Bacillus amyloliquefaciens KCTC 3002, Bacillus subtilis KCTC 3135, and Bacillus lichenoformis KCTC 1659. They were obtained from the Korean Collection for Type Cultures (KCTC) as reference organisms to help us to distinguish closely related species in the Bacillus genus. Extracted DNA was then diluted to run qRT-PCR using species-specific primers (Kwon et al. 2009; Huang et al. 2017a; Dunlap 2019; Bahuguna et al. 2020). A primer for the macrolactin gene was used to distinguish between B. velezensis and B. amyloliquefaciens (Fan et al. 2018; Li et al. 2021b). The used primers are listed in Table SI. We performed qRT-PCR in a final volume of 20 μl consisting of 10 μl of SYBER Green Supermix (BIO-RAD, USA), 4 μl nuclease-free water, 5 μl of genomic DNA, and 0.5 μl each of forward and reverse primers. Amplification used the following thermal cycling steps: an initial DNA denaturation step at 95°C for 3 min, followed by 39 cycles of 10 s at 95°C for denaturation, 10 s at 55°C for annealing, and 30 s at 72°C for extension.

Cultivation of B. velezensis using a lab-scale fermenter based on food-grade media. We used food-grade medium (FGM) to cultivate our strain as an anti-CRE therapeutic agent that could be developed in the future. FGM was used for cultivation because this strain could grow well in this medium. In addition, FGM is safe for human consumption, and it is composed of glucose, yeast peptone, Tween 80, and magnesium sulfate based on an MRS medium (Table SII). The pH of the optimized FGM was 7.5. We adjusted its pH to 6.3 using 6 N HCl. Before growing in a fermenter system, the strain was cultivated in 30 ml FGM. After incubation at 37°C for 18 h, the OD and pH of the culture broth were checked with a spectrophotometer and a pH meter (Mettler-Toledo, Switzerland), respectively, every 2 h during the incubation period. After confirming the bacterial growth and the pH, the probiotic strain was grown in a lab-scale fermenter system (FMT-ST-S07, Fermentec, South Korea). The fermenter was first filled up with 2 l of FGM. The FGM-filled fermenter was then autoclaved at 121°C for 15 minutes. After that, when the medium reached the optimal temperature, 1.0% of probiotic culture was aseptically added into the fermenter system and fermented at 37°C for 10 h using a fermenter impeller. After incubation, the culture was recovered in a sterile container, centrifuged, washed, and resuspended with the culture supernatant at 1.5 × 10⁹ CFU/200 μl for each mouse of the therapeutic model. FGM-cultured probiotics were used in the treatment model, but MRS‑cultured probiotics were used in the prevention model and toxicity tests.

Investigation of the effect of B. velezensis in a CRKP-infected mouse model. In this study, preventive and therapeutic models were applied to evaluate the effectiveness of probiotics in a CRKP-infected mouse model, and 9-week-old female BALB/c mice (Dooyeol Biotech, Korea) were used. The prevention model was used to see preventive effects by administering probiotics before infection. In the treatment model, probiotics were administered the day after inducing infection to see the therapeutic effect. At the time of treatment with probiotics in both models, the probiotic-treatment group received B. velezensis. However, the infection and control groups received distilled water through oral gavage for making the same treatment condition. In both models, gastric neutralization was induced by oral administration of sodium bicarbonate (NaHCO₃) to increase animal severity through the increased intestinal reach of live pathogens (Czuprynski and Faith 2002). In addition, cyclophosphamide was intraperitoneally injected 3 days before infection to induce neutropenia (Pan et al. 2015). 5-Fluorouracil (5-FU) was also administered intraperitoneally to induce additional immunosuppression (VanderVeen et al. 2020). Disease severity score, body weight, and survival of mice were checked in both models during the experimental period.

We examined the preventive effect of our candidate probiotic strain, B. velezensis, in CRKP‑infected BALB/c mice. The probiotic strain was administered at 1.5 × 10⁹ CFU/mouse in 200 μl daily as a single dose by oral gavage for three days before infection. Cyclophosphamide (Sigma Aldrich, USA) at 450 mg/kg (200 μl) was also injected intraperitoneally three days before infection. Three days after neutropenia, infection was induced twice per week through oral administration at a dose level of 6.7 × 10⁹ CFU/200 μl/mouse on day 0 and 9 × 10⁹ CFU/200 μl/mouse on days 2, 10, and 12. NaHCO₃ (0.2 M, 200 μl) (Sigma Aldrich, USA) was administered along with the infection. 5‑FU (50 mg/kg, 200 μl) (Sigma Aldrich, USA) was administered intra-peritoneally on days 13 and 15 after inducing infection.

We investigated the therapeutic potential of our candidate probiotic strain, B. velezensis, in CRKP-infected BALB/c mice. In the therapeutic mouse model, 200 μl of cyclophosphamide (450 mg/kg) was injected intra-peritoneally three days before infection. Infection was then induced by oral administration of 200 μl CRKP at 9 × 10⁹ CFU/mouse on days 0, 2, and 6 with pretreatment of NaHCO₃ (0.2 M, 200 μl). Mice were treated with B. velezensis at 1.5 × 10⁹ CFU/200 μl/mouse twice the next day after the infection treatment. They were intraperitoneally injected on days 10 and 14 with one dose of 5‑FU (50 mg/kg, 200 μl).

Evaluation of repeated oral acute toxicity of probiotics in guinea pigs. A 2-week repeated oral acute toxicity evaluation of probiotics in guinea pigs was performed according to a previous report (Lee et al. 2021). Adult male guinea pigs (weight range 1,000 to 1,280 g) were used and administered orally, and sterile water was used as the vehicle. In the case of the treatment group, probiotics (2 × 10⁸ CFU/ml/200 μl) were administered once a day, and clinical symptoms, mortality, and weight changes were observed over the entire period.

Review of animal experiment ethics. All animal experiments were conducted in a biosafety level 2 facility (LML 20–591) of Soonchunhyang University, according to the Ministry of Food and Drug Safety (Registration, MFDS, No. 657). This study’s animal experiments protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Soonchunhyang University.

CRKP strains are causing a significant public health concern across the globe because of their capacity to spread quickly in the hospital setting with a high mortality rate and their extensive antimicrobial resistance characteristics (Brink 2019). Currently, infections with CRE have a limited number of therapeutic options, which have been utilized rarely due to concerns about their effectiveness and toxicity (Morrill etal. 2015). Therefore, new anti-CRE drug agents that differ from existing drug regimens are desperately needed. Under such circumstances, probiotics can be a potential option as their effectiveness against different infectious diseases has already been well established.

Several human studies have recently reported the effectiveness of probiotics for various gut-related diseases, such as necrotizing enterocolitis and antibioticassociated diarrhea (Cremonini et al. 2002; AlFaleh andAnabrees 2014). Besides, probiotics can help maintain intestinal barrier integrity (Hemert et al. 2013). Moreover, probiotic administration can protect and enhance the gut microbiota of mice by improving the number of beneficial bacteria while decreasing the number of pathogenic bacteria (Li et al. 2019). With this background, our research team is interested in using probiotics to counter the threat to human health caused by CRE infections.

Our research team isolated various probiotic strains, such as Bacillus, during a vinegar microbiome study, and the Bacillus genus was applied to the CRE infection treatment study. Vinegar has long been considered fermented food for its many health benefits, including its antioxidant activity, ability to improve hypercholesterolemia, prevent metabolic syndromes, and regulate the gut immune system (Urtasun et al. 2020; Sui et al. 2021;). However, in vivo effects of probiotics isolated from vinegar on CRKP infection have not been reported yet. Here we report the efficacy of a probiotic strain isolated from Gochang Bokbunja vinegar against CRKP infections.

The strain discovered in the vinegar was confirmed as B. velezensis through 16S rRNA gene sequencing analysis and additional species-specific PCR. It was judged to be a new strain as it showed a different carbon use pattern from the existing standard B. velezensis strain. As a result of additional efficacy evaluation in vivo, this candidate probiotic strain's prophylactic and therapeutic efficacies were confirmed, similar to a recent study (Tajdozian et al. 2021) showing the infection prevention and therapeutic effect of probiotics in a mouse model. Such effects are believed to be related to their diverse ability to adhere to epithelial surfaces and induce immunological responses (Wagner et al. 1997). Moreover, it has recently been reported that Bacillus treatment might have a solid immunostimulatory effect on the host to defend against infections (Plaza-Diaz et al. 2014; Mazaya et al. 2015; Suva et al. 2016). According to the results of related studies on B. velezensis as a candidate strain in this study, B. velezensis naturally produces various antibiotic compounds such as bacteriocins, short-chain fatty acids, and organic acids that can protect the gastrointestinal tract from diseases (Lee et al. 2019). There have also been claims that the administration of B. velezensis in animals can increase intestinal Lactobacillus and Ruminococcus and decrease Acinetobacter and that the effectiveness of B. velezensis on CRE might be related to this phenomenon (Li et al. 2019).

Probiotics are classified as Generally Regarded as Safe (GRAS) by the United States Food and Drug Administration (FDA) (Martin and Langella 2019). Probiotics have been used for a long time, and their safety has been established. These findings allow their usage as food or supplements from a scientific perspective (Martin and Langella 2019). Although probiotics are commonly considered safe (Williams 2010), some strains can cause bacteremia and septicemia (Kulkarni 2019). In consideration of these safety issues, a repeated two-week oral acute toxicity test was performed using guinea pigs. No abnormal clinical symptoms, weight loss, or animal death were observed during the experiment period.

Concerning safety, this study used probiotic strains cultured in FGM. FGM is one medium allowed to come into direct contact with foods, and it does not cause any hazards or change the taste and flavor, making it suitable for human consumption (Sawatari et al. 2006). Besides, FGM is less expensive and easier for bulk production; therefore, many in vivo studies rely on FGM (Huang et al. 2016; 2017b).

Many studies have found that the gastrointestinal tract's stability and beneficial bacterial population enhance the host's immune system and antioxidant capacity and prevent the spread of pathogenic bacteria in the gut (Khalid et al. 2021; Li et al. 2019). Moreover, according to a report, the probiotic strain B. velezensis can also generate secondary metabolites such as polyketides, lipopeptides, and peptides that have antibacterial properties (Ye et al. 2018). However, here, the indepth mechanism of action based on the metabolite mentioned above and pathology studies to support the effectiveness of this probiotic strain remains a limitation.

In summary, in this study, a new probiotic strain, the B. velezensis strain, was isolated from Korean Gochang Bokbunja fermented vinegar. Its efficacy against CRKP infections was confirmed in vivo. Results of this study suggest the potential for developing B. velezensis as a live biotherapeutic agent for CRKP-associated infections. To develop CRKP therapeutics, extensive and indepth research is required, including extensive animal efficacy investigations, GLP toxicity studies, clinical trials, and research regarding synergistic effects with existing drugs.

Financial support for this study was provided by a grant (No. 2021040D288) from the Gochang Bokbunja vinegar Research Project through the Gochang Agricultural Technology Center, funded by Gochang County, Republic of Korea. This work was also supported by the Technology Innovation Program (20018499), funded By the Ministry of Trade, Industry & Energy (MOTIE, Korea). This research was also supported by the Soonchunhyang University Research Fund. Moreover, we would like to thank the officials of Gochanggun, the vinegar farmers in the Gochang area,