Isolation and Screening of Lactic Acid Bacteria Strains With Antibacterial Properties From The Vagina of Healthy Cows

Background Lactic acid bacteria with probiotic and antibacterial properties were isolated from the vagina of healthy cows. The purpose of the study is to isolation and screening of lactic acid bacteria strains with antibacterial properties from the vagina of healthy cows, which could be used to treat cow vaginal inammation. Results Isolation and identication of eight dominant lactic acid bacteria strains from 55 isolates was performed using classic microbiology methods and fermentation engineering. Eight strains were selected that had no spores and capsules, exhibited strong acid production capacity (pH <4.5) and had a rapid acid production (time ≤ 12 h) at the lowest pH. These strains were screened using fermentation engineering, pharmacology, cell biology and molecular biology methods. Lactobacillus johnsonii (SQ0048) had the lowest pH (4.32) and shortest acid-producing time (8 h). L. johnsonii (SQ0048) could produce hydrogen peroxide, inhibit the growth of Staphylococcus aureus and Escherichia coli and adhere to the vaginal epithelial cells of cows. The average number adhering to each cell was 304±2.67. Bacteriocin genes were detected in L. johnsonii (SQ0048), and the bacteriocin gene of a positive clone of this strain was 100% similar to that of Lactobacillus johnsonii NCC 533 (NC_005362.1). Expression of the bacteriocin genes had inhibitory activity against S. aureus and E. coli. Conclusions These advantages indicate that SQ0048 is a promising candidate for use in antimicrobial preparations.


Background
Pathogenic microorganisms can enter the uterus of postpartum cows via the vagina because of cervix expansion. This can cause endometritis and reduce reproduction and milk production [1]. Veterinary medicine usually focuses on antibiotics for treatment of reproductive tract infections. However, the continued use of antibiotics can induce bacterial resistance to antibiotics. Also, the increasing amount of drug residues has become a problem that endangers human health. Probiotics could potentially be used as biological controls to reduce antibiotics in livestock production. Probiotics are environmentally friendly and more sustainable because of the slower pace of resistance development in biological control agents.
The normal microecological environment of the human vaginal system is a complex and dynamic ecosystem inhabited by lactic acid bacteria (LAB) [2][3][4]. LAB are generally composed of bene cial strains that protect the vaginal environment from invasion by potentially harmful microorganisms. Bene cial LAB could improve the disorder caused by BV (bacterial vaginosis) and prevent or treat VVC (vulvovaginal candidiasis). LAB, used as probiotics, can also increase the number of immune cells and enhance immune function [1]. However, the bacterial communities in the vagina of healthy cattle are not well known. LAB dominate the vaginal ora of dairy cows under normal conditions [3]. Bacteria infecting the vagina of dairy cows mainly include Escherichia coli and Staphylococcus aureus [4]. Ametaja. et al. (2014) perfused LAB (L. sakei FUA 3089, P. acidilactici FUA 3140, and P. acidilactici FUA 3138) isolated from the vagina of cows, and they reduced purulent vaginal secretions and improved animal health [5]. Deng et al. (2015) injected LAB into the vagina of cows to reduce uterine infection, regulate local and systemic immune responses and improve cow health [6]. LAB can inhibit uterine in ammation and reduce infection caused by E. coli and S. aureus. LAB can change the vaginal environment, limit the amount of pathogenic bacteria reaching the uterus and reduce the prevalence of uterine disease in cows. However, in one study, LAB did not reach the endometrium when they were injected into the vagina [7].
The method by which LAB inhibits pathogenic bacteria appears to be related to the production of lactic acid, hydrogen peroxide, bacteriocin and other active substances. It is also related to LAB adhesion and competitive adhesion to pathogenic bacteria. Lactic acid protects the body tissues by maintaining a pH value between 4 and 4.5 that creates an unsuitable environment for pathogenic microorganisms. Also, LAB can inhibit pathogenic bacteria by releasing bacteriocins and maintaining the dynamic balance of the microorganisms. Hydrogen peroxide (H 2 O 2 ) is also a defense factor used by LAB against pathogenic microorganisms [8]. LAB shows strong adhesion to strati ed non-keratinized epithelium, which can repel or inhibit pathogenic microorganisms such as Candida albicans, Gardnerella vaginalis, E. coli and S. aureus [9]. The screening process is a critical step in discovering probiotics with strong antibacterial activity. The present study focused on the screening and isolation of a LAB strain with superior antimicrobial qualities. Desirable LAB qualities include production of hydrogen peroxide, inhibition of pathogenic microorganisms, ability to adhere to vaginal epithelial cells of cows, possession of bacteriocin genes and production of high levels of acid. Our objective was to discover new LAB strains that could be used in microecological preparations for preventing and treating vaginal in ammation in cows.

Samples resource
The normal ora changes in the birth canal with cow growth. There are relatively few LABs during the juvenile period, but LAB populations signi cantly increase in adults. The rst 10-45 months of dairy cow life are the most fertile period. Therefore, 50 healthy dairy cows aged 10-15 months without any disease were selected as experimental samples from different dairy farms in Hohhot, Inner Mongolia.

Isolation and diversity identi cation of the LAB strains
Isolation of the LAB strains The LAB strains were isolated from the experimental treatments. First, mucus of the samples was prepared in PBS (phosphate buffer solution) and plated in LAPTg [10] (containing 0.2% bromocresol purple) agar pH 6.6 medium for 12-24 h at 37°C under anaerobic conditions. Then, isolated strains were picked from each plate and transferred to the same medium. The growth observed in pure cultures was recorded. Strains were freeze-dried and stored in milk yeast extract (10% (w/v) skim milk powder, 0.5% (w/v) yeast extract) at -80°C. (Strain activity degrades 40% after 1 year at room temperature.)

Identi cation of the LAB strains
The dominant LAB strains in this study were rst screened according to Chinese Pharmacopoeia [11]. Strain morphology was selected using Gram staining. Acid-producing properties were measured using acidity testing [12]. The probiotic strains that were previously freeze-dried were tested for stability after passage by subjecting to culture characteristics, bacterial morphology, water activity and purity testing, in accordance with the requirements of the third part of the Chinese Pharmacopoeia (2010) [11].
The dominant LAB was taxonomically identi ed by phenotype tests. Biochemical and sugar fermentation tests were performed by an API50 CHL test strip (BioMérieux, France) according to the Bergey's Manual of Determinative Bacteriology.
The dominant LAB strains were transferred into LAPTg agar, and then incubated at 37°C under the anaerobic condition for 24 h before DNA extraction. The DNA extraction of the isolated LAB strains was done using a Bacterial Genomic DNA extraction kit (Sigma, Germany). Ampli cation of 16S rRNA was carried out using the forward primer 27F (5′AGAGTTTGATCCTGGCTCAG3′) and the reverse primer 1492R (5′GGCTACCTTGTTACGACTT3′). The obtained single positive clones were sequenced, and then identi ed through the Genbank database using the BLAST algorithm. The phylogenetic status of the isolated LAB strains was established through analysis with software MEGA 5.0 version.
Acid production analysis of candidate strain Acid production was measured using the acidity method [12]. The dominant strains identi ed were inoculated from stage 1 to 4 in LAPTg broth medium under anaerobic conditions. The pH of samples was taken at 0, 3, 5, 8, 10, 12, 16, and 18 h at stage 4.
Hydrogen peroxide production analysis of candidate strains Hydrogen peroxide production was determined using the catalase test [13]. TMB (3,3', 5,5'-Tetramethyl-Benzidine) (Gibco, France) was added to 100% ethanol, heated and dissolved in a water bath, and then added to LAPTg agar medium, sterilized at 121°C for 15 min. Later, when the temperature was ~50°C, peroxidase (Gibco, France) was dissolved and added to the plate by ltering with a bacterial lter. After solidi cation, the plate was cultured at 37°C for 12 h. Single colonies of the dominant strains were picked to draw a line in the prepared plates and placed at 37°C for 36 h in an anaerobic environment. The plates were then taken out and left for 30 min to observe the color change of the colonies under aerobic conditions. Based on the color intensity, the strains were classi ed as strong (blue), medium (brown), weak (light brown) or negative (white colonies) producers of hydrogen peroxide [13].

Bacteriocins detection in candidate strains
The DNA of the strain was extracted (Sigma Bacterial Genomic DNA extraction kit (Sigma, Germany)). The primers were designed on the basis of the Lactobacillus johnsonii NCC 533 (NC_005362.1) sequence published on GenBank with 99% similarity to the strain. Forward Primer: 5′-GGGAATGTGACGATAATGAC-3′; Reverse Primer: 5′-AAATCCTACTTTCTTATCTTGC-3′. The DNA was ampli ed and transformed. The positive clones were sequenced and ligated with the pMD19-T vector (Takara, Japan) to form a recombinant plasmid. The primers were designed again according to the BamHI and XhoI (ThermoFisher, USA) restriction sites. Forward Primer: 5′-CGCGGATCCATGAAACAATTTAATTATTTATCACA-3′, Reverse Primer: 5′-CCGCTCGAGCTACTTTCTTATCTTGCCAAAA-3′. The recombinant plasmid and pET-28a (ThermoFisher, USA) were PCR ampli ed with primers to splice and transform. The constructed expression product was cultured in LB medium containing kanamycin at 37°C for 12 h, and the growth curve was monitored. At the pre-middle stage, the logarithmic growth of the bacterial solution was induced and cultured at 37°C for 3 h at 1 mM IPTG (Isopropyl β-D-Thiogalactoside) (Thermo, USA) [14]. Then, they were centrifuged at 6000 r/min for 10 min at 4°C. The bacteria were collected and added to a lysis buffer equivalent to 1/10 volume of the bacterial solution to resuspend. Cells of the re-suspended bacteria in the solution were broken by ultrasound. At this time, the strain of the bacterial solution was clari ed. Bacteriocins gene expression was detected using SDS-PAGE. The SDS-PAGE experiment was performed in accordance with Genetic Engineering Pharmaceuticals [15]. The main reagent manufacturer: SDS-PAGE Gel Preparation Kit (Thermo, USA).
Cell adhesion testing of the candidate strain BVECs (Primary bovine vagina epithelial cells) were cultured up to 3 passages and washed twice with DPBS before use. The LAB cultures were centrifuged at 5000 r/min for 5 mins and washed with DMEM/F12 (Dulbecco's Modi ed Eagle Medium: Nutrient Mixture F-12 (Gibco, France)) without any additions. Cell concentration was adjusted to n × 10 8 CFU / mL, and then we added bovine vaginal epithelial cells. They were cultivated together at 37°C for 4 h in a CO 2 incubator [16]. The cell culture solution was then discarded, and Wright-Giemsa staining was performed. The cells were observed under a microscope.
The cells with the bacteria were stained with Swiss-Giemsa. Twenty elds of view were randomly selected under the microscope. The number of bacteria on 50 cells was counted, and the average number of bacteria adhering to each cell was calculated. Lactobacillus delbrueckii (ATCC 11842) was used as a positive control. This experiment was repeated three times.

Antibacterial susceptibility and bacteriocins detected in the candidate strain
This experiment was performed using the Oxford Cup method [17]. The methods used for evaluating the antibacterial activity of LAB against pathogenic microbial strains were described by Shokryazdan et al. [18,19]. The bacterial liquids of LAB, E. coli and S. aureus were centrifuged, washed and adjusted to a concentration to n × 10 8 CFU/mL with PBS. One milliliter of E. coli or S. aureus was, respectively, added onto sterile plates. Then Mueller-Hinton Agar (containing 1% glucose) was poured and shaken well in sterile plates. After solidifying and drying, sterile Oxford cups were placed evenly at equal distances. A 0.2 mL amount of the LAB suspension was added into the cup and allowed to stand for 2 h, then incubated at 37°C for 36 h. The PBS phosphate buffer was used as a negative control, and streptomycin (10 µg/mL) (Batch number: 130307-201009) (Biological Product Testing Institute, China) or Penicillin G (0.12 μg/mL) (Batch number: 130437-201005) (Biological Product Testing Institute, China) [20] was used as a positive control. Vernier calipers were used to measure the diameter of the bacteriostatic zone. The judgment criteria were inhibition range <6 mm negative (-), 6-10 mm mild inhibition (+), 10-14 mm strong inhibition (++) and >14 mm very strong inhibition (+++) [18]. This experiment was repeated three times. The bacteriostatic performance of bacteriocins gene expression was tested. The LAB suspension was used ss a proxy for bacteriocin expression. Other experimental steps were the same as above. All experiments were repeated three times.

Results
Isolation and diversity identi cation of the LAB strains Among the initial 55 strains, 43 strains had no spores and no capsules. These 43 LABs were in continuous culture for 30 generations according to stability requirements of the Chinese Pharmacopoeia.
A total of 28 strains retained stable culture characteristics, morphology and water activity after passage. Eight strains were selected with strong acid-producing ability (pH<4.5) and a rapid acid-producing rate (time≤12 h) through acidity method requirements of the Chinese Pharmacopoeia from 28 strains. The eight strains were labelled SQ0012, SQ0015, SQ0030, SQ0041, SQ0045, SQ0048, SQ0049 and SQ0054 (Table 1).

Identi cation of LAB strains
The eight strains were distinguished by morphology, biochemical characteristics, sugar fermentation and 16S rRNA gene sequencing. These isolated strains were classi ed as follows: SQ0012 L. plantarum, SQ0015 L. brevis, SQ0030 Enterococcus faecalis, SQ0045 L. kitasatonis, SQ0054 Lactococcus garvieae, SQ0048 L. johnsonii and SQ0049 L. amylovorus. Based on analysis of identi cation results, SQ0030 and SQ0041 were the same strain. So, only the SQ0030 strain was retained (Table 2). A phylogenetic tree was constructed based on the 16S rRNA gene, which established the classi cation status of the seven isolated LAB strains (Fig. 1).
Hydrogen peroxide performance SQ0048 had a strong positive reaction for H 2 O 2 and produced a blue color, while the other strains did not change color (Fig. 3).

Bacteriocins
The bacteriocin of SQ0048 was extracted because it had better acid-producing performance and hydrogen peroxide production than the other strains. A 400 bp fragment of SQ0048 bacteriocin was detected (Fig. 4). Sequencing results showed that the positive clone strain of SQ0048 was 100% similar to Lactobacillus johnsonii NCC 533 (NC_005362.1). Expression of the bacteriocin gene was detected by SDS-PAGE. SQ0048 had a clear target band at about 10 kD (Fig. 5).
Adhesion of bacteria to cells SQ0048 had obvious adhesion to the vaginal epithelial cells of cows (Fig. 6). The average number of SQ0048 adhering to each cell was 304±2.67, but the average number of L. delbrueckii adhering to each cell was 32±3.15. The average number of SQ0048 adhering to each cell was signi cantly greater than L. delbrueckii (P<0.01).
Antibacterial effect SQ0048 had a weak inhibitory effect on S. aureus and E. coli (Table 3, Fig. 7). However, the expression of the bacteriocin gene of SQ0048 had very strong inhibition on S. aureus and E. coli (Table 4, Fig. 8). The antibacterial expression of the SQ0048 bacteriocin gene was signi cantly greater than SQ0048, the positive control group and the blank control group (PBS) (P < 0.01).

Discussion
Pathogenic microorganisms can enter the uterus from the vagina, via procreation or the environment, and cause infection. Infections have the potential to affect reproduction and milk production of cows and cause economic losses. Antibiotic treatments are typically effective at killing or inhibiting the pathogenic microorganisms that cause vaginal in ammation in dairy cows. However, the persistence of antibiotic residues in milk and beef is a human health issue. Other methods for treating cow vaginal in ammation, which minimize antibiotic use, are needed.
Lactic acid bacteria (LAB) are fundamental in the production of fermented foods and several strains are regarded as probiotics [21]. Living bacteria and metabolites can function as bacteriostatic agents but also can enhance immunity. LAB do not have drug resistance issues and are not toxic to humans or animals [22]. LAB are recognized as the dominant species in healthy vaginal microbial ora [23]. A lack of LAB is believed to disrupt the balance of vaginal microbial ora and reduce the immune barrier function. It can lead to reproductive tract in ammation, parasitic diseases, fungal and chlamydial infections [24].
Micro-ecological preparations made by LAB have previously been used in clinical practice. They have played an important role in maintaining or adjusting the micro-ecological balance of the body and preventing diseases. However, research on the bacterial communities in the vagina of cattle is relatively limited.
The SQ0048 isolate produced the lowest pH (4. 31-4.33). The acid-production times of SQ0012, SQ0045, SQ0048 and SQ0049 were 8 h at the lowest pH. SQ0048 appeared to have better acid-producing ability than the other microbial strains. SQ0048 also had the highest hydrogen peroxide-producing ability of all LAB identi ed. A pH value between 4 and 4.5 and hydrogen peroxide production are both strong defense factors against pathogenic microorganisms. SQ0048 was therefore used as a candidate strain and studied to determine if it possessed other antibacterial properties. The bacteriocin gene of SQ0048 was successfully cloned and expressed. SQ0048 and its bacteriocin gene expression had inhibitory activity against S. aureus and E. coli, which was consistent with the activity of other LAB bacteriocins against pathogenic bacteria [31][32][33]. Moreover, the strains of L. plantarum showed signi cant inhibitory activity against various fungi that commonly contaminate food indicating their potential as a bio-preservative of food [34]. The average number of SQ0048 adhered to each cell was 304±2.67, which was signi cantly greater than L. delbrueckii. SQ0048 also appeared to have strong adhesion. These factors could repel and inhibit pathogenic microorganisms [30]. Our data showed that SQ0048 has the potential to become a strain in microecological preparations used to prevent and treat in ammation of the cow vagina. Additional research will be necessary, such as safety assessment and other related research to provide su cient technical and e cacy support for formulation development.

Conclusions
SQ0048 had antibacterial e cacy and pharmacological potential. It could potentially be used as a vaginal probiotic, through its toxicity to pathogenic microorganisms, for the prevention and treatment of bovine vaginal infections.