The Effect of an Oral Probiotic Containing Lactobacillus, Bifidobacterium, and Bacillus Species on the Vaginal Microbiota of Spayed Female Dogs
- This work was performed at the College of Veterinary Medicine, North Carolina State University, Raleigh, NC.
Recurrent urinary tract infections (UTIs) are often difficult to treat. Vaginal colonization with lactic acid-producing bacteria (LAB) is associated with reduced frequency of recurrent UTIs in women. Oral probiotics might help increase the prevalence of vaginal LAB and decrease the frequency of recurrent UTIs in dogs.
Administration of an oral probiotic supplement containing Lactobacillus, Bifidobacterium, and Bacillus species will increase the prevalence of LAB in the vagina of dogs.
Thirty-five healthy, spayed female dogs without history of recurrent UTIs.
Prospective, controlled study. Enrolled dogs received an oral probiotic supplement for 14 or 28 days. A vaginal tract culture was obtained from each dog before and after oral probiotic administration. Twenty-three dogs received the oral probiotic supplement daily for a period of 14 days and 12 dogs received the oral probiotic supplement daily for a period of 28 days.
Lactic acid-producing bacteria were isolated from 7 of 35 dogs prior to probiotic administration. After the treatment course, 6 of 35 dogs had LAB isolated. Only one of these dogs had LAB (Enterococcus canintestini) isolated for the first time. Enterococcus canintestini was the most common LAB isolated from all dogs in this study, although it was not included in the probiotic supplement.
Conclusions and Clinical Importance
Lactic acid-producing bacteria are not a common isolate from the vaginal vault of dogs. Administration of this oral probiotic supplement for a 2- or 4-week period did not increase the prevalence of vaginal LAB in dogs.
lactic acid-producing bacteria
de Man Rogosa Sharpe
urinary tract infection
Uncomplicated urinary tract infections (UTIs) in dogs are common and estimated to occur in 14% of all dogs during their lifetime.[1, 2] Although most UTIs occur as single episodes, recurrent UTIs are not uncommon. Recurrent UTIs are defined by the presence of 3 or more UTI during a 12-month period or 2 or more UTI during a 6-month period.[3, 4] Because recurrent UTIs frequently involve bacteria that are resistant to commonly used antibiotics, and extended treatment courses are frequently recommended, these infections are often difficult and expensive to eradicate.
The resident bacteria creating the vaginal microbiota might help prevent recurrent UTI. Studies in women have demonstrated that patients with recurrent UTIs often have depletion of the normally predominant Lactobacillus species. Conversely, women with increased vaginal colonization with Lactobacillus species have a reduction in the episodes of recurrent UTIs.[5-9] As lactic acid producing bacteria (LAB), including Lactobacillus and Enterococcus species, metabolize glycogen and subsequently produce lactic acid, it is thought that the presence of these bacteria could decrease vaginal pH, inhibiting colonization by uropathogenic strains of bacteria.
A study investigating the benefit of probiotics in women indicated that oral administration of lyophilized L. rhamnosus and L. reuteri capsules once daily for 14 days results in increased lactobacilli and fewer pathogenic bacteria. These findings suggest that administration of oral probiotics might be a viable alternative or complement to treatment with antimicrobials in human patients with recurrent UTI.
As in human patients, it is well recognized that the vaginal mucosa of dogs is not sterile and bacteria are often identified in vaginal cytology of healthy female dogs during proestrus and estrus. Vaginal bacteria isolated in these dogs typically consist of mixed populations of aerobic and anaerobic microorganisms that include opportunistic pathogens.[11, 12] LAB, including Lactobacillus and Enterococcus species, have been identified within the vaginal microbiota of healthy intact dogs. These vaginal LAB isolates inhibit growth of pathogenic bacteria in vitro, including Escherichia coli, Proteus mirabilis, and Staphylococcus aureus. These data suggest that vaginal LAB might also have a beneficial and protective role within the urogenital tract of female dogs.
The objective of this study was to evaluate the effect of a 14-day and a 28-day course of an oral, commercially available probiotic supplement on the prevalence of LAB, specifically Lactobacillus species, in the vagina of healthy, spayed, female dogs. We hypothesized that administration of an oral, commercially available probiotic supplement labeled to contain Lactobacillus, Bifidobacterium, and Bacillus species would result in an increase in the prevalence of vaginal LAB in dogs with no history of recurrent UTI.
Materials and Methods
Case Selection Criteria
Spayed female dogs older than 6 months of age and without a history of recurrent UTI were recruited from employees and students at the North Carolina State University Veterinary Health Complex. Recurrent UTI was defined as greater than 2 episodes of UTI in a 6-month period or greater than 3 episodes of UTI in a 1-year period. Dogs were excluded from the study if they were currently receiving antibiotics or cranberry supplements.
Owners of each dog included in the study completed a questionnaire at the time of enrollment. Their answers provided signalment, pertinent medical history, including information regarding recent vomiting, diarrhea, changes in appetite, or changes in urination, or water consumption, as well as current medications, and current diet, including supplements and treats, for each dog. After completion of the study, owners responded to additional questions regarding how their dog tolerated the probiotic and if all doses of the probiotic were administered. Questions regarding changes in appetite, development of vomiting, diarrhea, development of changes in water consumption or urination were also included.
Culture of Probiotic
Before initiation of the study, the commerically available probiotic supplement1 given to enrolled dogs was cultured to confirm the presence of live, representative bacterial species. One dosing size (eg, capsule) was inoculated into an enrichment of thioglycollate broth2 and de Man Rogosa Sharpe (MRS) broth2 and incubated for 48 hours in 5% CO2 at 35°C. After enrichment, a swab of the thioglycollate broth2 was plated onto Columbia blood agar2 and incubated for up to 72 hours in 5% CO2 at 35°C. Individually isolated, morphologically distinct colonies were evaluated by commercial panels3 and Gram stain to describe bacterial species. A swab from the MRS broth2 was inoculated onto Rogosa agar2 for selective isolation of Lactobacillus species. Plates were incubated anaerobically for up to 72 hours at 35°C. Representative isolates were Gram stained to confirm that they were Gram-positive cocci and rods.
Administration of Probiotic
Each dog was given a commercially available probiotic supplement1 labeled to contain Lactobacillus species (>64 bil CFU/g and >7200 mil spores/g), Bifidobacterium species (30 bil CFU/g) and Bacillus species (24 bil CFU/g), along with a combination of yeast, enzymes, vitamins, and prebiotics, once daily for either 14 days or 28 days. The first 23 dogs enrolled in the study received the probiotic supplement daily for 14 days. The following 12 dogs enrolled in the study received the probiotic supplement daily for 28 days after preliminary results indicated rare isolation of LAB after administration of the probiotic supplement for 14 days. Each dog was given 1 g of the probiotic supplement per 10 pounds of body weight daily with food.
Two sterile samples were obtained from the cranial aspect of the vagina of each dog. The 1st sample was collected before administration of the oral probiotic, and the 2nd sample was collected within 24 hours of completion of the assigned course of oral probiotic administration. Samples were collected with a sterile, double-guarded culturette4 to minimize contamination from other areas of the genitourinary tract. The outer guard of the vaginal swab was appropriately positioned cranial to the vestibulovaginal junction in the vaginal vault, and then the inner guard and culturette were advanced as far cranially as possible with gentle pressure. The sterile swab was advanced beyond the inner guard and rotated several times and both the inner guard and swab were retrieved whereas the outer guard was held in place. The entire culturette was removed, with the sterile swab contained within the inner and outer guard, eliminating exposure to other areas of the genitourinary tract.
Identification of Lactic Acid-Producing Bacteria
Culture of lactic acid-producing bacteria, including Lactobacillus species, from vaginal samples was completed at the North Carolina State University Clinical Microbiology Laboratory. Vaginal swabs were plated onto Rogosa agar2 and incubated for a minimum of 120 hours at 35°C under anaerobic conditions. Swabs were also enriched in MRS broth2 and incubated at 35°C. If no colonies were obtained on the original Rogosa plate after 120 hours, the enrichment was inoculated onto Rogosa agar and again incubated for 120 hours at 35°C under anaerobic conditions. Any morphologically distinct colony was isolated and Gram stained; organisms that appeared Gram positive after Gram stain were frozen at −80°C for further analysis. After completion of sample collection, all frozen isolates were submitted for 16s rDNA sequencing at the National Veterinary Services Laboratory.5 A lack of isolates following these steps were considered to be consistent with an absence of viable LAB within the sample.
Thirty-five dogs without a history of recurrent UTI were included in the study. The median age was 5 years (range: 1–12 years). Breeds represented included 20 mixed breeds, 3 Labrador Retrievers, 2 Golden Retrievers, and 1 each of Australian Shepherd, Beagle, Catahoula Cur, Cocker Spaniel, Giant Schnauzer, Miniature Schnauzer, Papillon, Siberian Husky, Standard Poodle, and West Highland White Terrier. Two of 35 dogs were fed diets that contained dried probiotics (dried fermentation products of Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, and Bacillus subtilis).
Culture of the probiotic supplement confirmed the presence of viable organisms representative of LAB. None of the owners reported any adverse effects during the treatment period. All dogs voluntarily consumed the probiotic supplement daily.
During the study, LAB were isolated from a total of 8 dogs (22.9%). Four of the 8 dogs received the probiotic for 14 days and the remaining 4 dogs received the probiotic for 28 days. Seven of these dogs had LAB isolated before starting the probiotic. Of these dogs, 5 of 7 remained LAB culture positive at the completion of the probiotic trial. Only 1 dog did not have LAB in the presample but had LAB isolated after probiotic administration. Enterococcus canintestini was isolated in this dog, which was not a species included in probiotic supplement. LAB characterized in the remaining 7 dogs included E. canintestini (5/7), Enterococcus avium (1/7), and L. pentosus or plantarum (unable to distinguish; 1/7). LAB were not isolated from either of the dogs that were fed diets containing dried probiotics within the ingredients.
The results of this study suggest that LAB, particularly Lactobacillus species, do not appear to be a predominant isolate from the vaginal vault of spayed female dogs. In addition, the administration of an oral, commercially available probiotic supplement for a 2- or 4-week period does not increase the prevalence of vaginal Lactobacillus, or other LAB, in dogs.
Women with recurrent UTI have increased vaginal colonization by uropathogens, as well as significantly increased bacterial adherence to vaginal epithelial cells.[8, 15-18] In health, LAB, specifically lactobacilli, are the predominant colonizers of the vaginal tract of women.[7, 18] However, women with recurrent UTI often have alterations in vaginal flora, namely increased colonization of uropathogens and depletion of lactobacilli, and bacteriuria is typically preceded by establishment of the infecting strain within the vagina.[8, 9, 19]
Depletion of vaginal LAB is likely an important predisposing factor for recurrent UTI in women, as these bacteria have antimicrobial properties that have been shown to regulate other urogenital flora. Lactic acid-producing bacteria metabolize glycogen to form lactic acid, producing a low pH and thus inhibiting the growth of other bacteria, including common uropathogens. These organisms have additional antimicrobial properties that regulate the growth of uropathogens, including production of bacteriocin and hydrogen peroxide. Moreover, LAB prevent adhesion of uropathogens via coaggregation and production of biosurfactants. Therefore, depletion of LAB could result in increased vaginal colonization and adherence of uropathogens, with subsequent infection of the urethra and bladder. This conclusion is further supported by studies demonstrating that high-level vaginal colonization with lactobacilli achieved via vaginal administration of capsules containing Lactobacillus species results in a reduction of UTI frequency in women.
Although limited information regarding the role of LAB within the genitourinary tract of dogs is available, the normal flora of the vaginal tract of dogs includes opportunistic and uropathogenic bacteria.[6, 18] LAB are also a constituent of normal vaginal flora in dogs, being identified in 41 of 42 intact dogs. Enterococcus and Lactobacillus species are most commonly isolated in dogs, with Lactobacillus species present in 25 of 41 dogs. Data from the current study indicate that LAB are not a predominant isolate from the vaginal vault of spayed female dogs. The cause for this difference in results is not clear. One possible explanation of this discrepancy in results is the fact that an unguarded swab was used to obtain vaginal samples for culture in the previous study, allowing possible contamination from the vestibule or vulva. The use of a guarded swab in this study prevented contamination of the samples collected for culture.
In women, rectal lactobacilli could be a reservoir for vaginal lactobacilli, thereby contributing to the maintenance of vaginal microflora, and studies in women have demonstrated that lactobacilli can be delivered to the vagina through oral ingestion of probiotic capsules containing Lactobacillus species.[21, 22] Moreover, both vaginal and fecal colonization with LAB has been noted within 14 days of daily oral probiotic administration in women. Data from this study suggest that similar oral probiotic administration might not be an effective means of promoting vaginal colonization of LAB in dogs. The reason for this discordant result is unclear. One possibility is that in order to colonize the vaginal tract after oral ingestion, the lactobacilli must be able to maintain structural integrity while passing through the gastrointestinal tract. It is possible that the Lactobacillus species contained within the probiotic used in this study were not viable after digestion.[20, 22] The probiotic supplement utilized in this study was administered as a powder to facilitate administration and it is possible that a capsule formulation would have resulted in more effective transit through the gastrointestinal tract. Furthermore, it is known that lactobacilli have varying degrees of adherence to vaginal epithelial cells, with L. gasseri, L. brevis, and L. acidophilus having the greatest ability to adhere to these cells in women.[5, 23] Perhaps the more commonly isolated LAB of the vaginal tract of dogs, such as E. canintestini, might be more effective at colonizing and adhering to canine vaginal epithelial cells than the bacteria included in the administered probiotic.
Two dogs that were fed diets containing probiotic supplements were included in this study. These dogs were not excluded from enrollment because the objective of this study was to identify an increased prevalence in LAB within the vaginal tract after oral administration of a specific probiotic supplement. Because samples were collected from all dogs before being given the probiotic supplement, a direct comparison in the number of colonies of LAB identified before and after administration of the supplement could be ascertained for each dog. Thus, the identification of LAB within the vaginal tract of dogs before being given the probiotic supplement as a consequence of diet or normal flora of individual dogs did not preclude inclusion in the study.
In conclusion, the effects of administration of an oral probiotic product labeled to contain Lactobacillus, Bifidobacterium, and Bacillus species on the vaginal flora of dogs were investigated in this study because of the beneficial role of LAB in women with recurrent UTI, as well as the role of LAB in growth inhibition of common uropathogens. Although the prevalence of vaginal Lactobacillus species was not increased in dogs after the administration of the oral, commercially available probiotic supplement used in this study, further study of the role of probiotics for urogenital health in dogs is indicated.
The authors acknowledge the assistance of the pet owners who participated in the study. The authors also wish to thank Megan Fauls and Denise Crowell for their contributions to this work.
Grant support: Study supported by a grant from Rayne Clinical Nutrition, Kansas City, MO.
Conflict of Interest Declaration: Authors disclose no conflict of interest.
Y+ powder; Rayne Clinical Nutrition, Kansas City, MO
Remel; Thermo Fisher Scientific, Lenexa, KS
Vitek 2; Biomeriux, Durham, NC
14″ Canine Guarded Culture Swab; Reproduction Resources, Walworth, WI
National Veterinary Services Laboratory, Ames, IA