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Inhibicja patogennych szczepów pozyskanych od klaczy z przewlekłym zapaleniem macicy przez szczepy Lactobacillus


H. Morvayová, E. Styková,  F. Novotný, J. Pošivák, Ľ. Šťavová, I. Valocký
Klinika dla Koni, Uniwersytet Medycyny Weterynaryjnej, Koszyce, Słowacja

Streszczenie


   Zapalenie pochwy, szyjki macicy oraz zakażenia macicy u krów są przyczyną wczesnej śmierci zarodków, poronień, przedwczesnych porodów, rodzenia martwych cieląt oraz śmierci cieląt nowonarodzonych. Lepsze rozumienie bakteryjnych przyczyn zakażeń pochwy oraz macicy u krów może dostarczyć wskazówek pomocnych we wczesnym leczeniu i zapobieganiu tym problemom reprodukcyjnym. Vaginitis, które może nastąpić w wyniku urazu lub wtórnie do innych zakażeń, to zapalenie błony śluzowej pochwy (Y o u n g q u i s t, 1993). Zapalenie pochwy jest najczęściej wywoływane przez mikroorganizmy takie jak Arcanobacterium pyogenes, Escherichia coli, Streptococcus sp. Corynebacterium sp. oraz 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). W eksperymencie tym badano aktywność inhibicyjną pałeczek kwasu mlekowego otrzymanych z wymazów pochwowych oraz szyjkowych na patogenne szczepy otrzymane od klaczy z przewlekłym zapaleniem macicy. Zdolność poszczególnych gatunków bakterii do adhezji oraz przeżywania w niszy ekologicznej wiąże się ściśle z ich pochodzeniem. Autochtoniczne hodowle mają większą zdolność do kolonizacji pochwowych komórek nabłonkowych, niż organizmy allochtoniczne, które w normalnych warunkach zostają łatwo wypłukiwane i nie dochodzi do ich włączenia w niszę ekologiczną, a ich wzrost jest hamowany przez interakcje w obrębie lokalnego środowiska mikrobiologicznego, bądź dochodzi do ich eliminacji przez mechanizmy obronne gospodarza (N e m c o v á, 1997). Taka specyfika gospodarza została określona  przez kilku autorów w badaniach na zwierzętach gnotobiotycznych i jak ustalono we wcześniejszych badaniach jest ona blisko związana z obecnością specyficznych receptorów molekularnych na komórkach gospodarza, które są rozpoznawane przez specyficzne molekuły komórek bakteryjnych. Otrzymaliśmy dziesięć różnych rodzajów pałeczek kwasu mlekowego z trzech stad krów mlecznych. Szczepy te były następnie testowane pod kątem ich zdolności do hamowania wzrostu patogennych mikroorganizmów  (CHE1, CHE2, E. coli,  Staphylococcus aureus, Staphylococcus agalactiae) otrzymanych od klaczy z przewlekłym zapaleniem macicy. Stwierdziliśmy, iż pałeczki kwasu mlekowego hamowały wzrost patogennych mikroorganizmów. Hamowanie było prawdopodobnie indukowane produkowanymi przez pałeczki kwasami organicznymi, których działanie prowadziło do spadku pH.

Inhibition of the pathogenic strains obtained from the mare with chronic endometritis by Lactobacillus strains

H. Morvayová, E. Styková,  F. Novotný, J. Pošivák, Ľ. Šťavová, I. Valocký
Horse Clinic, University of  the Veterinary medicine, Košice, Slovakia


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

Pathogenic strain CHE2 E. coli makes the inhibition zone around LABS7 under aerobic conditions on the blood agar.


 

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.

Discussion
    

   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

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Acknowledgements

 This work was supported by Ministry of Education of Slovak Republic,
VEGA 1/3484/06 and AV 4/0009/07




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