Additional Property of Xenorhabdus stockiae for Inhibiting Cow Mastitis-causing Bacteria

Xenorhabdusbacteria isolated from entomopathogenic nematodes have been found to produceseveral antimicrobial agents for inhibiting pathogenic bacteria. In this study,X. stockiae strains PB09, SS7 andUV58 (UV-mutant of PB09)were evaluated for antibacterial activities against cow mastitis-causing isolates of Bacillus subtilis, Staphylococcus aureus, Staphylococcus intermedius, Streptococcus agalactiae, Enterococcus faecalis, Klebsiella pneumoniae, and Escherichia coli. The paper disc diffusion showed thatcell-free supernatants of all X. stockiae strains cultivated for 48, 72 and 96 h had anti-mastitis activities, but 72and 96-h supernatants were more effective andtended to inhibitgram-positive bacteria. The maximal activities were found when X. stockiae PB09 were evaluated against S. agalactiae(12.00±2.16mm), X. stockiae SS7andX. stockiae UV58 against S. aureus(11.75±1.71 mmand 12.50±0.58 mm, respectively).The overlay assay also showed thatX. stockiae colonies were more likely to inhibit grampositive bacteria.Also, liveX. stockiae UV58 colonies were more effective than X. stockiae PB09 and X. stockiae SS7 colonies for suppressing S. intermedius,S. agalactiaeand E. coli(31.33±1.53, 22.67±1.53, 18.33±0.58 mm, respectively). This study may suggest that X. stockiae can be used for future effective suppression of mastitis infection in dairy cows.

Bovine mastitisis 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 property 1 .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 coli 4 , Enterococcus faecalis 5 andKlebsiella 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 plants 9 , endophytic fungi 10 and lactic acid bacteria 11 .In particular, the secondary metabolites derived from bacteria with broad antibiotic property were found to have antimastitis 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 dysgalactiae 11 .Moreover, there are some approaches that using Xenorhabdus bacteria isolated from entomopathogenic nematodes as sources of antibiotic substances.These Xenorhabdusbacteria 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 competitors 12 .Therefore, it is interesting to apply the various substances of Xenorhabdus sp. for controlling the clinical isolates of mastitis-causing bacteria.Xenorhabdus bacteria, including Xenorhabdusnematophila, X. szentirmaiiandX.budapestensis, were found to inhibit mastitiscausing isolates ofS.aureus, E. coli and K. pneumoniae, whereby the anti-matitis activity was highest against S. aureusand lowest against K. pneumoniae 13 .A number of substances were isolated from Xenorhabdus bacteria and investigated for their potentials to be used asantimicrobial agents, including benzylideneacetone (trans-4-phenyl-3-buten-2one) derived from X. nematophila 14 ,xenematide from X. nematophila 15 , xenocoumacins 1 and 2which were derivatives of benzopyran-1-onefromX.nematophila 16 , xenoxides fromX.bovienii 17 , and peptides(GP-19 and EP-20) from X. budapestensis NMC-10 18 .In this study, the wild-typeX.stockiae PB09andX.stockiae SS7 isolated from the entomopathogenic nematodes (Steinernemasiamkayai) and the mutantX.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, andEscherichia coli.The knowledge from this study could be beneficial for future development of safe and effective approach for treatment of bacterial mastitis infection.

Culture of Xenorhabdus stockiae bacteria
X. stockiae PB09andX.stockiae SS7were wild type isolates derived from the entomopathogenic nematodes (Steinernemasiamkayai) 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 blueTriphenyltetrazolium 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 coliwere 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 10 7 -10 8 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 paperdisc diffusion assay by using the method modified fromWang et al. (2008) 20 .Briefly, 1 ml of X. stockiaeseed culture at the concentration of 10 8 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 supernatant 21 .Twenty µl of each cell-free supernatant of X. stockiaewere 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 10 7 -10 8 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. stockiaewas carefully placed onto M-H agar plate which had been spread with mastitis bacteria.The M-H plateswere 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 Furganiet 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, andE.coli, was separately grown in 15 ml of M-H broth for 24 h, and 100 µl of each culturewere individually mixed with 3.5 ml of50°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 pd" 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 eachmastitis bacteria at similar levels.However, for inhibitingS.agalactiae, the72-and 96-h supernatants werefound 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 againstS.agalactiae(12.00±2.16mm),followed by 96-h supernatant when also being used against S. agalactiae(11.75±1.50mm); however, both of them were not significantly different(Table 1).
The antibacterialactivity 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 96h supernatant that was more effective than 48-and 72-h supernatant s when being used againstS.aureus.The highest antibacterial activities of X. stockiae UV58 were found in 96-h supernatant when being used against S. aureus(12.50±0.58mm),  3).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. agalactiaeand E. coli.For X. stockiae UV58 colonies, the largest inhibition zone was found when they were overlaid byS.intermedius (31.33±1.53mm), followed byB.subtilis (23.67±1.53mm) and S. agalactiae (22.67±1.53mm).Moreover, X. stockiae PB09 and SS7 colonies were found to be most effective for inhibiting B. subtilis (22.33±1.53 and 21.00±1.00mm, respectively), but both were not different to X. stockiae UV58 (23.67±1.53mm).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±0.58mm) (Table 4).

DISCUSSION
The cell-free supernatants ofX.stockiaePB09, SS7 and UV58 were shown to be effective at inhibiting the clinical isolates of both gram-positive and gram-negative mastitis-causing bacteria, particularlyS.agalactiaeand S. aureus,as shown by paper disc diffusion assay, andS.intermedius,B.subtilis and S. agalactiae,as shown by overlay bioassay.The activities of X. stockiaestrains against mastitis-causing bacteria were found to slightly increase withX.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 withanti-mastitis properties of X. stockiaestrains could be produced, regardless of cultivation time.There are a number of reports that showing the abilities of Xenorhabdussp. 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 andK.pneumoniae 22 .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, especiallyS.intermediusand S.aureus, than gramnegative ones.This was similar to the studies of X. bovieniiandX.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 ones 23 .In addition, althoughboth cell-free supernatants and live colonies ofX.nematophila, X. budapestensisX.szentirmaiiwere found to have antibacterial activities against mastitis-causing isolates of S.aureus, E. coli andK.pneumoniae,but the antibacterial activities were highest against S. aureus and lowest against K. pneumoniae 13 .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 Indolecompounds 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 andStaphylococci groups, and also gram-negative bacteria, including some strains of E. coli 16 .In addition, its Nematophin compounds were shown to inhibit Staphylococci bacteria 26 .Even though several reports have suggested that metabolites ofX.nematophila were more likely to be effective against gram-positive bacteria, there were a number of gram-negativeplant pathogenic bacteria that were found to be suppressed by its Benzylideneacetone compounds, including Agrobacterium vitis, Pectobacteriumcarotovorum subsp.atrosepticum, P. carotovorum subsp.carotovorum, Pseudomonas syringaepv.tabaci, andRalstonia solanacearum 14 .Furthermore, both X. nematophila and X. bovieniiwere found to produce Xenorhabdins, the compounds that could effectively inhibit several gram-positive bacteria, including Micrococcus luteus, B. subtilis, S. pyogenesandS.aureus, but they hadrather low efficacy specific to gram-negative bacteria 13 .Also,X.bovieniiwas also found to generate Xenoxides, the compounds that could inhibit several pathogenic strains of gram-positive bacteria, includingB.subtilis,M. luteusand S. aureus 17 .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.

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

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

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

Table 4 .
Inhibitory activities of X. stockiae colonies against mastitis-causing bacteria by overlay bioassay Diameter of X. stockiae ≈ 15 mm 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 (p0.05) as compared by LSD