The influence of antimicrobial agents on the development of antibiotic resistance in the vaginal bacterial flora of artificially inseminated mares : a study on mares in a stud environment

Sammanfattning: In Sweden and in many other countries, artificial insemination (AI) is frequently used in horse breeding. The technology makes it possible to use genetic material from horses in different geographical locations in an effective way, minimizing the risk of infection or even injury to both horses and stud personnel. Semen collection is performed with a high hygienic standard but despite this, it is seldom possible to obtain an ejaculate that does not contain bacteria. Bacteria in the ejaculate can cause disease in the mare, or result in a deterioration in sperm quality, thereby reducing the chance of the mare becoming pregnant after insemination. To ensure disease control and maintain sperm quality, addition of antibiotics to the semen is therefore required to reduce the number of bacteria in the ejaculate. To protect the spermatozoa during storage, a semen extender containing both buffering and nutrient-rich components, but also antimicrobial substances (AMS) is added. During insemination, the semen dose is deposited in the mare's uterus, which initiates a response from the local immune system. This results in an immunological and mechanical cleansing of the deposited material through the cervix uteri, which is an important function for maintaining a healthy uterine environment and enabling a pregnancy. The mare's vaginal bacterial flora is therefore exposed to the antibiotics contained in the extender, which could theoretically increase the risk of developing antibiotic resistance in the bacterial flora. In this study, vaginal swabs were obtained from 39 mares of varying breeds and ages in northern Sweden, with the aim of investigating the normal vaginal bacterial flora and how its resistance pattern is affected by the exposure to the antibiotics (penicillin and gentamicin) contained in the semen extender INRA-96, and if resistant bacteria are present in the normal vaginal flora of mares. The mares were categorized into either an exposed group consisting of breeding mares at a stud farm, or a control group consisting of mares that had never been inseminated. Swabs were taken from a predetermined area in the cranial vagina just before the first insemination of the season (D0), with a follow-up sample after three days (D3). The control group was sampled only once, as far as possible in connection with estrus. The samples were sent by post to the Department of Biomedical Science and Veterinary Public Health, SLU, Uppsala for analyses. Isolated bacteria were identified by Matrix-Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS). Bacterial species that could be isolated from both D0 and D3 in the same mare were analysed regarding antimicrobial resistance. A comparison was also made between the bacteria and resistance pattern of bacteria from mares that had never been inseminated. A total of 971 bacterial isolates were isolated from the 39 mares, with E. coli being by far the most common isolated bacterium (48.6%). The bacteria included in the antimicrobial susceptibility testing were Staphylococcus simulans, Streptococcus equi ssp. zooepidemicus, Streptococcus dysgalactiae and Enterococcus faecalis. Resistant isolates were identified in all species except Enterococcus faecalis. These isolates showed resistance to penicillin, oxacillin, fusidic acid, trimethoprim, erythromycin, clindamycin, nitrofurantoin and tetracycline, depending on species. Exposure to antibiotics did not affect the resistance pattern in the vaginal bacterial flora in this study, but there was a difference in the bacterial species obtained before and after insemination and between the exposed group and the control group. Resistant bacteria were found in the vaginal flora of both groups.