Abstract
Vaginitis, cervicitis and bovine uterine infections are the causes of early death of the embryo, abortions, premature delivery, stillbirths and death of newborn calves. A better understanding of the bacterial cause of vaginal and uterine infections in cows may provide clues for early treatment and prevention of these reproductive problems. Vaginitis, which may follow trauma or may be secondary to other infections, is inflammation of the mucous membrane of the vagina (Y o u n g q u i s t, 1993). Vaginitis is most often caused by microorganisms such as Arcanobacterium pyogenes, Escherichia coli, Streptococcus sp. a Corynebacterium sp., and Haemophilus somnus (C o h e n, C o l o d n e r, Z i v, K e n e s s, 1996; D o h m e n, J o o p, S t u r k, B o l s, 2000). In this study was searched the inhibition activity of the selected strains obtained from vaginal swab against pathogenic strains obtained from the mare with chronic endometritis. The ability of bacterial species to adhere and survive in the ecological niche is strictly connected with its origin. Autochthons cultures have higher ability to colonize vaginal epithelial cells, than allochthon microorganisms, which are under the regular conditions easily drifted away, not incorporated into the ecological niche and inhibited by the interactions within microbial community or are eliminated by self - defense immune mechanisms of the host (N e m c o v á, 1997). This host specificity was reported in the studies of the several authors on gnothobiological animals and as it was established in the further studies, it is closely related to the presence of specific molecule receptors on the host cells, which are recognized by specific molecules of bacterial cells. We obtained ten different types of strains from three dairy herds. These strains were tested for the capability to inhibit the growth of the pathogenic microorganisms (CHE1, CHE2 E. coli, Staphylococcus aureus, Staphylococcus agalactiae) obtained from the mare with chronic endometritis. We reported, that one selected strain was able to inhibit the growth of the E.coli. Inhibition was probably induced by production of the organic acids, which led to decrease in pH.
Introduction
The majority of the uterine infections is caused by low pathogenic bacteria, which are often part of the normal vaginal microflora ( L a r s e n, 1977). The composition of the vaginal microflora is in general within females of one specie the same (L á z n i č k a, 1995). It is composed to lesser extent with Lactobacilli strains and opportune pathogenic species, which are usually a part of the mixed cultures. The most commonly cultivated bacterial species from the vagina of the mares are Arcanobacterium pyogenes, Escherichia coli, Streptococcus sp., Corynebacterium sp. and Haemophilus somnus (C o h e n, C o l o d n e r, Z i v, K e n e s s, 1996; D o h m e n, J o o p, S t u r k, B o l s 2000). Lactobacilli have an important function in the regulation of the vaginal microflora. Due to its beneficial properties for the host (F u l l e r, 1992; N e m c o v á, 1997) are the most commonly used probiotic microorganisms to restore the vaginal microflora. Growth of the suitable lactobacillus colonies is inhibiting the pathogenic microflora. E. coli growth is inhibited by lactobacilli produced organic acids. A l a k o m i et al. (2000) reported, that the outer membrane of E. coli was destroyed by lactic acid and this permeabilization of gram - negative wall increased her permeability against antimicrobial substances.
Material and Methods
Sampling
Before taking samples external genitals were washed with hypermangan dilution.
Samples were obtained by gentle swabbing of the vaginal wall and contamination was prevented by avoiding contact with the vulva. Vaginal swabs were taken with the carbon tampon Dispolab. After the arrival at the laboratory was each tampon put into the tube with 5 ml of PBS and vortexed for 30 s to suspend attached bacteria. Cultivation was done on the MRS agar. Samples were incubated in the thermostat during 48 hours at 39 °C. Cultivations were evaluated according to selection criteria.
In this study were used selected strains isolated from vaginal swab: LACS7, LAC1S7, LAC4S7, LAC5S7, LABS7, LAB3S7, LCCS7, LECS7, LCHA1S7, LCHA2S7. Pathogenic strains were obtained from the vaginal swab of the mare with chronic endometritis.
Growth substrates
For preparation of the 1 litre of PYG agar with 1% glucose was used 2,5 g of Pepton for bacteriology (Imuna, Šarišské Michaľany, SR), 5 g of yeast autolysate ((Imuna Pharm, Šarišské Michaľany, SR), 2,5 g Trypticase Peptone (Becton Dickinson and co., USA), 20g agar bacteriological (Unipath LTD., BABINGSTOKE, Hampshire, UK) and 10 g of glucose.
Bacterial strains
Selected strains were revitalized in LS broth. Pathogenic strains obtained from the mare with chronic endometritis were cultivated on the blood agar. Colonies were multiplied in PYG broth.
Determination of the inhibition activity
Sterile discs (Becton Dickinson, Inc., Cockeysvile, MD 21030, 0,6 cm in width) were put on the Petri plates which contained 15 ml of PYG agar with 1% glucose. Discs were inoculated with 10 µl of the selected strains, which were cultivated for 48 hours. As a blank control was used PY agar without substrate. Plates were incubated anaerobically for 48 hours at 37 °C. After that, discs with selected strains were removed. PY agar with 1% glucose was prepared. It was cooled to 45 °C and the pathogenic strain was poured. Plates were poured with 3 ml of agar with pathogenic strain.
Determination of the antibiotic susceptibility
Pathogenic strains were tested for the antibiotic susceptibility (bacitracin, streptomycin, penicillin, erythromycin, tetracyclin, ampicillin). The ability of selected strains to inhibit the pathogenic strains was compared with the ability of the antibiotics.
10 µl of the pathogenic strains cultivated from the mare with the chronic endometritis was put from PYG broth to PYG agar and spread by loop. 6 antibiotic discs (BAC, STR, PEN, ERY, TET, AMP) were put on the plates and incubated for 48 hours at 39 °C. After that, the inhibition zones were measured.
Results
|
|
|
Table 1. Inhibition of the pathogenic strains by selected strains
AIZ= average inhibition zones |
|
|
|
Table 2. Inhibition of the pathogenic strains cultivated from the mare with chronic endometritis by antibiotics
AIZ= average inhibition zones
Only erythromycin was able to inhibit the pathogenic strains. |
Lactobacilli have been increasingly recognized as an important component of the normal vaginal flora that may help to repel invading pathogens. Thus, loss of vaginal lactobacilli may predispose a patient acquisition of genitourinary infections (R e i d et al., 1991; R e i d et al., 1990). For this reason, the use of probiotics, incorporating selected lactobacilli strains, may be an effective means of restoring the normal microbial flora in the vagina (M c G r o a r t y, M o o d y, 1993; R e i d et al., 1990)
Lactobacilli are widespread in nature. A number of species are present in alimentary products (milk, yogurt, fruits, vegetables), whereas only some strains are usual eubiotic saprophytes of the intestinal and/or urogenital flora. In the most cases known lactobacilli, when administered through the antimicrobial substances, are affected by a number of the technical factors such as the composition of the culture medium and the incubation conditions (temperature, pH, oxygen).
Lactobacilli administered orally or vaginally restore the physiological bacterial flora, but are usually not able to inhibit the growth of the pathogens or of exerting any microbicidal activity against them. Only some bacterial strains have recognizably developed this natural capability of inhibiting the growth of the pathogenic microorganisms when placed in the same culture. These properties are related to the ability of some lactobacilli to produce bacteriocins, organic acids and H2O2. However, such bacterial strains having these characteristics can effectively be used in therapy only if they keep and/or increase said properties in time. It is moreover desirable to induce the inhibitory and/or microbicidal activity against pathogens in lactobacilli strains which are not capable of exerting these activities.
The ability of selected strains to inhibit the pathogenic strains was proved only by LABS7 against the E.coli. Among the antibiotics only erythromycin was able to inhibit the growth of the pathogenic strains.
References
1.ALAKOMI, H. L.; SKYTTA, E.; SAARELA, M.; MATTILA- SANDHOLM, T.; LATVA- KALA, K.; HELANDER, L. M. (2000) Lactic acid permeabilizes gram- negative bacteria by disrupting the outer membrane. Appl. Environ. Microbiol 66, 2001- 2005
2.COHEN, R. O.; COLODNER, R.; ZIV, G.; KENESS, J. (1996) Isolation and antimicrobial susceptibility of obligate anaerobic bacteria recovered from the uteri of dairy cows with retained fetal membranes and postparturient endometritis. Zentralblatt Fuer Veterinaermedizin 43, 193- 199
3.DOHMEN, M. J.; W.; JOOP, K.; STURK, A., BOLS, P.E.J., LOHUIS, J.A.C.M. (2000): Relationship between intra-uterine bacterial contamination, endotoxin levels and the development of endometritis in postpartum cows with dystocia or retained fetal membranes. Theriogenology 54, 1019-1032
4.FULLER, R. (1992) The lactic acid bacteria. AS 3108, 171- 192
5.LARSEN, B.; MARKOVETZ, A. J.; GALASK, R.P. (1977) Relationship of vaginal cytology to alterations of vaginal microflora of rats during the estrous cycle. Appl. Environ. Microbiol. 33, 556- 562
6.LÁZNIČKA, A. (1995) Mikroflóra pohlavního aparátu fen a její vztah k reprodukčním poruchám. Vaginitis. Veterinářství 5, 210- 212
7.Mc GROARTY, J. A.; MOODY, K. J. (1993) In vitro inhibition of growth and adhesion of Candida albicans by vaginal isolates of Lactobacilli. Program and abstracts of the 33 rd Interscience Conference on Antimicrobial Agents and Chemotherapy (New Orleans). Washington, DC, American Society for Microbiology
8.NEMCOVÁ, R. (1997) Vet. Med.- Czech, 42, 19- 27
9.REID G, BRUCE AW, COOK R. L., LLANO M. (1990) Effect on urogenital flora of antibiotic therapy for urinary tract infection. Scand J Infect Dis 22, 43- 7
10.REID G, BRUCE AW, Mc GROARTY, J. A., CHENG K.J., COSTERSONJ.W. (1990) Is there a role for lactobacilli in prevention of urogenital and intestinal infections? Clin Microbiol Rev 3, 335- 44
11.REID G, BRUCE AW, UEHLING, D. T. (1991) Vaginal flora and urinary tract infections. Curr Opin Infect Dis 4, 37_ 41
12.YOUNGQUIST, R. S.; BRAUN, W. F. (1993) Abnormalities of the tubular genital organs. Veterinary Clinics of North America 9, 309-321
Acknowledgements
This work was supported by Ministry of Education of Slovak Republic,
VEGA 1/3484/06 and AV 4/0009/07
