Introduction

Bovine mastitis is the inflammation of the lactating cows’ mammary glands. This disease can cause both clinical and subclinical symptoms, resulting in decrease of yield and quality of milk as well as alteration of milk biochemical property¹. Because of this devastating disease, the dairy farmers also have to experience economic loss due to high treatment cost, inevitable animal culling and incidence of resistance against common antimicrobial agents^2,3. The common pathogens that caused cow mastitis are coagulase-negative staphylococci, Bacillus spp., Streptococcus spp., Staphylococcus aureus, Escherichia coli⁴, Enterococcus faecalis⁵ and Klebsiella pneumoniae^6,7,8. Hence, the economic burden of bovine mastitis on dairy industry has prompted research into the development of safe, effective treatments. Some studies have focused on using alternative approaches for treatment of mastitis due to the rising prevalence of drug resistance, for example, the use of medicinal plants⁹, endophytic fungi¹⁰ and lactic acid bacteria¹¹ . In particular, the secondary metabolites derived from bacteria with broad antibiotic property were found to have anti-mastitis activity. For instance, bacteriocin-like substances produced by lactic acid bacteria were shown to suppress mastitis-causing isolates of Listeria innocua, L. monocytogenes Scott A and Streptococcus dysgalactiae¹¹. Moreover, there are some approaches that using Xenorhabdus bacteria isolated from entomopathogenic nematodes as sources of antibiotic substances. These Xenorhabdus bacteria are symbiotic to the nematodes of the family Steinernematidae which have been used as biological control agents for a number of agricultural insect pests. When the nematodes infect the insect hosts, Xenorhabdus bacteria are released to insect’s haemocoel and resulting in the death of insect hosts. After that, Xenorhabdus bacteria can produce the secondary metabolites that control the growth of a variety of surrounding bacterial or fungal competitors¹². Therefore, it is interesting to apply the various substances of Xenorhabdus sp. for controlling the clinical isolates of mastitis-causing bacteria. Xenorhabdus bacteria, including Xenorhabdus nematophila, X. szentirmaii and X. budapestensis, were found to inhibit mastitis-causing isolates of S. aureus, E. coli and K. pneumoniae, whereby the anti-matitis activity was highest against S. aureus and lowest against K. pneumoniae¹³. A number of substances were isolated from Xenorhabdus bacteria and investigated for their potentials to be used as antimicrobial agents, including benzylideneacetone (trans-4-phenyl-3-buten-2-one) derived from X. nematophila¹⁴, xenematide from X. nematophila¹⁵, xenocoumacins 1 and 2 which were derivatives of benzopyran-1-one from X. nematophila¹⁶, xenoxides from X. bovienii¹⁷, and peptides (GP-19 and EP-20) from X. budapestensis NMC-10¹⁸ . In this study, the wild-type X. stockiae PB09 and X. stockiae SS7 isolated from the entomopathogenic nematodes (Steinernema siamkayai) and the mutant X. stockiae UV58 (obtained by UV treatment of X. stockiae PB09) were investigated for their activity against mastitis-causing bacterial isolates, including Bacillus subtilis, Staphylococcus aureus, Staphylococcus intermedius, Streptococcus agalactiae, Enterococcus faecalis, Klebsiella pneumoniae, and Escherichia coli. The knowledge from this study could be beneficial for future development of safe and effective approach for treatment of bacterial mastitis infection.

Materials and Methods

Culture of Xenorhabdus stockiae bacteria

stockiae PB09 and X. stockiae SS7 were wild type isolates derived from the entomopathogenic nematodes (Steinernema siamkayai) according to Kaya and Stock (1997)¹⁹. The nematodes were kindly provided by the Department of Agriculture, Ministry of Agriculture. X. stockiae PB09 was further mutated by UV treatment that resulting in X. stockiae UV58. All bacterial strains were maintained on Luria Bertani (LB) agar slants at 28°C and subcultured every month. X. stockiae bacteria that were used in the further experiments must be in the phase I which could be confirmed by growing their colonies on Nutrient Bromothymal blue Triphenyltetrazolium chloride agar (NBTA) at 28°C in the dark for 48 h. The bacterial colonies in the phase I could be detected by their absorption of bromothymol blue to produce the blue colonies that were surrounded by clear zones on NBTA. A single blue colony was inoculated to 100 ml of LB in 250 ml-flask, then incubated in the dark at 28°C and 200 rpm for 16-24 h, and used as seed culture in the further experiments.

Culture of mastitis-causing bacteria      

The clinical strains of mastitis-causing bacteria, including Bacillus subtilis, Staphylococcus aureus, Staphylococcus intermedius, Streptococcus agalactiae, Enterococcus faecalis, Klebsiella pneumoniae, and Escherichia coli were isolated and maintained on Mueller Hinton (M-H) agar slants at 4°C. To prepare each bacteria as seed culture, a loopful of bacterial colonies was inoculated into 15 ml of  M-H broth in 25 ml-flask, then incubated at 37°C and 150 rpm for 16-24 h, and finally adjusted to have the concentration of approximately 10⁷-10⁸ cfu/ml.

Evaluation of antibacterial activity by paper disc diffusion assay

The antibacterial activities of X. stockiae PB09,  X. stockiae SS7 and X. stockiae UV58 against 7 mastitis bacteria were determined by paper disc diffusion assay by using the method modified from Wang et al. (2008)²⁰. Briefly, 1 ml of X. stockiae seed culture at the concentration of 10⁸ cfu/ml was added to 99 ml of tryptic soy broth (TSB) and incubated at 28°C and 200 rpm for 48, 72 and 96 h.  Each culture was then centrifuged at 10,000 rpm at 4°C for 5 min and filtered through 0.22 µm-filter kit to obtain cell-free supernatant²¹. Twenty µl of each cell-free supernatant of X. stockiae were applied to 6-mm filter paper disc (Whatman no.1) and let to dry. In the meantime, 100 µl of each mastitis bacterial culture at the concentration of approximately 10⁷-10⁸ cfu/ml in M-H broth were spread onto M-H agar and incubated at room temperature for 2 h. Each dried filter paper containing cell-free supernatant of X. stockiae was carefully placed onto M-H agar plate which had been spread with mastitis bacteria. The M-H plates were incubated at 37°C for 24 h. The antibacterial activity was determined by the size of clear zone surrounded the 6 mm-filter paper. The control groups were M-H broth and streptomycin at the concentration of 1 mg/ml.

Measurement of antibacterial activity by overlay bioassay

The overlay assay was modified from the method previously described by Furgani et al. (2007)¹³. This method was used to measure the antibacterial interaction between the live cells of X. stockiae and the tested mastitis bacteria. Briefly, X. stockiae was cultured in M-H broth at 28°C and 150 rpm overnight in the dark. After that, 5 µl of X. stockiae culture were dropped at the center of M-H agar plate and let to dry, followed by incubation at 28°C for 5 days. In the meantime, a single colony of each of the mastitis bacteria, including B. subtilis, S. aureus, S. intermedius, S. agalactiae, E. faecalis, K. pneumoniae, and E. coli, was separately grown in 15 ml of M-H broth for 24 h, and 100 µl of each culture were individually mixed with 3.5 ml of 50°C-melting, M-H agar (containing agar at 0.6% v/w). Each mastitis bacteria-melting agar mixture was poured on M-H plate that had grown X. stockiae for 5 days. After the plates became solidified, they were incubated at 37°C for 48 h, then measured for the sizes of clear zone surrounded X. stockiae colony, which representing the activity of live X. stockiae cells against mastitis bacteria.

Data analysis    

The obtained data from four replicates were analyzed and compared by One-way Analysis of Variance (One-Way ANOVA) and LSD (Fisher’s Least Significant Difference) test at p≤ 0.05 using SAS program (1990).

Results

The cell-free supernatants of X. stockiae isolates (PB09, SS7 and UV58) which were cultured for different durations (48, 72 and 96 hours) were evaluated against the mastitis-causing bacteria by paper disc diffusion assay. The results showed that cell-free supernatants of X. stockiae PB09 could inhibit all mastitis bacteria, but they were more likely to inhibit gram-positive ones (Table 1). In addition, cell-free supernatants of X. stockiae that were cultivated for 48, 72 and 96 hours could inhibit each mastitis bacteria at similar levels. However, for inhibiting S. agalactiae, the72- and 96-h supernatants were found to be significantly more effective than 48-h supernatant. The highest antibacterial activity of X. stockiae PB09 was found in 72-h supernatant when being used against S. agalactiae (12.00±2.16 mm), followed by 96-h supernatant when also being used against S. agalactiae  (11.75±1.50 mm); however, both of them were not significantly different (Table 1).

Table 1: Antibacterial activities of cell-free supernatant of X. stockiae PB09 cultivated for 48, 72 and 96 hours against mastitis-causing bacteria by disc diffusion assay

Note:Data followed by the different upper-case letters (^{A,B,C …} ) in the same row were significantly different (p0.05) as compared by LSD

Data followed by the different lower-case letters (^a,b,c…) in the same column were significantly different (p0.05) as compared by LSD

TSB = tryptic soy broth

Strep. = streptomycin at the concentration of 1 mg/ml

Diameter of paper disc = 6 mm

The cell-free supernatant of X. stockiae SS7 (Table 2) was also found to inhibit all mastitis bacteria with similar antibacterial activities to X. stockiae PB09 (Table 1). Also, 48-, 72-, and 96-h cell-free supernatants of X. stockiae SS7 were not significantly different at suppressing each mastitis bacteria, except that 72- and 96-h supernatants that were significantly more effective than 48-h supernatant at inhibiting S. intermedius. The maximum antibacterial activity of X. stockiae SS7 against mastitis bacteria was found in 96-h supernatant when being used against S. aureus (11.75±1.71 mm), followed by 72-h and 48-h supernatants when also being used against  S. aureus (11.25±2.06 and 11.25±1.71 mm, respectively), but all of them were not significantly different (Table 2).

Table 2: Antibacterial activities of cell-free supernatant of X. stockiae SS7 cultivated for 48, 72 and 96 hours against mastitis-causing bacteria by disc diffusion assay

Note:     Data followed by the different upper-case letters (^{A,B,C …} ) in the same row were significantly different (p≤0.05) as compared by LSD

Data followed by the different lower-case letters (^a,b,c…) in the same column were significantly different (p≤0.05) as compared by LSD

TSB = tryptic soy broth

Strep. = streptomycin at the concentration of 1 mg/ml

Diameter of paper disc = 6 mm

The antibacterial activity of X. stockiae UV58 (Table 3), a mutant of X. stockiae PB09 by UV treatment, was similar to its wild type (Table 1). For X. stockiae UV58, the supernatants of 48-,72- and 96-h supernatants had similar activities against mastitis bacteria, with the exception  of 96-h supernatant that was more effective than 48- and 72-h supernatant s when being used against S. aureus. The highest antibacterial activities of X. stockiae UV58 were found in 96-h supernatant when being used against S. aureus (12.50±0.58 mm), followed by 48-h supernatant when being used against  E. faecalis (11.50±1.29 mm) (Table 3).

Table 3: Antibacterial activities of cell-free supernatant of X. stockiae UV58 cultivated for 48, 72 and 96 hours against mastitis-causing bacteria by disc diffusion assay

Note:Data followed by the different upper-case letters (^{A,B,C …} ) in the same row were significantly different (p≤0.05) as compared by LSD

Data followed by the different lower-case letters (^a,b,c…) in the same column were significantly different (p≤0.05) as compared by LSD

TSB = tryptic soy broth

Strep. = streptomycin at the concentration of 1 mg/ml

Diameter of paper disc = 6 mm

The live colonies of X. stockiae PB09, X. stockiae SS7 and X. stockiae UV58 were overlaid by mastitis bacteria to determine the antibacterial activities that live colonies could produce (Table 4). X. stockiae UV58 colonies were found to be significantly more effective than X. stockiae PB09 and X. stockiae SS7 colonies for suppressing S. intermedius, S. agalactiae and E. coli. For X. stockiae UV58 colonies, the largest inhibition zone was found when they were overlaid by S. intermedius (31.33±1.53 mm), followed by B. subtilis (23.67±1.53 mm) and S. agalactiae (22.67±1.53 mm). Moreover, X. stockiae PB09 and SS7 colonies were found to be most effective for inhibiting B. subtilis (22.33±1.53 and 21.00±1.00 mm, respectively), but both were not different to X. stockiae UV58 (23.67±1.53 mm). However, both gram-negative mastitis bacteria (K. pneumoniae and E. coli) were found to be resistant to X. stockiae colonies, with the exception that X. stockiae UV58 colonies when being evaluated against E. coli (18.33±0.58 mm) (Table 4.)

Table 4: Inhibitory activities of X. stockiae colonies against mastitis-causing bacteria by overlay bioassay

Note: Diameter of X. stockiae ≈ 15 mm

Data followed by the different upper-case letters (^{A,B,C …} ) in the same row were significantly different (p0.05) as compared by LSD

Data followed by the different lower-case letters (^a,b,c…) in the same column were significantly different (p0.05) as compared by LSD

Discussion

The cell-free supernatants of X. stockiae PB09, SS7 and UV58 were shown to be effective at inhibiting the clinical isolates of both gram-positive and gram-negative mastitis-causing bacteria, particularly S. agalactiae and S. aureus, as shown by paper disc diffusion assay, and S. intermedius, B. subtilis and S. agalactiae,  as shown by overlay bioassay. The activities of X. stockiae strains against mastitis-causing bacteria were found to slightly increase with X. stockiae cultivation time. Moreover, type of cultivation media (TSB, LB and M-H broths) was found to have no effect on the anti-mastitis activities of X. stockiae isolates (data not shown). This may suggest that, in the enriched media, the secondary metabolites with anti-mastitis properties of X. stockiae strains could be produced, regardless of cultivation time. There are a number of reports that showing the abilities of Xenorhabdus sp. to produce antibiotics against mastitis-causing bacteria. Several species of  Xenorhabdus bacteria, including X. budapestensis, X. szentirmaii, X. innexi, X. nematophila, X. cabanillassii, were shown to have antibacterial activities against S. aureus, E. coli and K. pneumoniae²². However, in this study, the cell-free supernatants and live colonies of X. stockiae PB09, SS7 and UV58 were found to have antibacterial activities that were more likely to be specific to gram-positive bacteria, especially S. intermedius and S. aureus, than gram-negative ones. This was similar to the studies of X. bovienii and X. nematophila which could produce Xenorhabdins that inhibited gram-positive mastitis bacteria (Micrococcus luteus, B. subtilis, S. pyogenes and S. aureus) more effectively than gram-negative ones²³. In addition, although both cell-free supernatants and live colonies of X. nematophila, X. budapestensis X. szentirmaii were found to have antibacterial activities against mastitis-causing isolates of S. aureus, E. coli and K. pneumoniae, but the antibacterial activities were highest against S. aureus and lowest against K. pneumoniae¹³.

The activities of Xenorhabdus sp. against mastitis-causing bacteria may be due to their production of a variety of antibiotic derivatives. Interestingly, X. nematophila was one of the symbiotic bacteria that capable of generating several antibacterial metabolites with broad spectrum activities. For example, X. nematophila could produce Indole compounds which capable of inhibiting both gram-positive and gram-negative bacteria^24,25. It was also found to generate Xenocoumacins 1 and 2 that effectively inhibiting gram-positive bacteria, particularly Streptococci and Staphylococci groups, and also gram-negative bacteria, including some strains of E. coli¹⁶. In addition, its Nematophin compounds were shown to inhibit Staphylococci bacteria²⁶. Even though several reports have suggested that metabolites of X. nematophila were more likely to be effective against gram-positive bacteria, there were a number of gram-negative plant pathogenic bacteria that were found to be suppressed by its Benzylideneacetone compounds, including Agrobacterium vitis, Pectobacterium carotovorum subsp. atrosepticum, P. carotovorum subsp. carotovorum, Pseudomonas syringae pv. tabaci, and Ralstonia solanacearum¹⁴. Furthermore, both X. nematophila and X. bovienii were found to produce Xenorhabdins, the compounds that could effectively inhibit several gram-positive bacteria, including Micrococcus luteus, B. subtilis, S. pyogenes and S. aureus, but they had rather low efficacy specific to gram-negative bacteria¹³. Also, X. bovienii was also found to generate Xenoxides, the compounds that could inhibit several pathogenic strains of gram-positive bacteria, including B. subtilis, M. luteus and S. aureus¹⁷. Similarly, in this study, all three strains of X. stockiae were shown to suppress mastitis bacteria, especially the gram-positive ones. This requires further analysis of their secondary metabolites that are capable of effectively inhibiting the mastitis bacteria and can be developed into practical commercial products for dairy farming.

Conclusion

The cell-free supernatants and live colonies of three X. stockiae strains (PB09, SS7, UV58) were evaluated against mastitis-causing isolates of B. subtilis, S. aureus, S. intermedius, S. agalactiae and E. faecalis, K. pneumoniae and E. coli. The anti-mastitis activities of X. stockiae strains were higher against gram-positive bacteria rather than gram-negative ones and found to increase with their cultivation time. This may suggest that the anti-mastitis efficacy of X. stockiae strains was more likely to be due to their secondary metabolites. Further studies are required to determine which metabolites of X. stockiae that have anti-mastitis property and whether mutation of X. stockiae could result in increase of its anti-mastitis activity.

Acknowledgements

The authors would like to thank the National Research Council of Thailand for providing financial support and Mahasarakham University for providing laboratory facilities

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