INTRODUCTION

The microbiota is the population, resident or transitory, of microorganisms and viruses that live in the epithelia of animals, creating an ecosystem (^{Pascale et al., 2018}). In this ecosystem, prokaryotes (mainly bacteria, although there are also archaea) are the most abundant organisms. Generally, these microorganisms live in a state of symbiosis with the host. On the other hand, the imbalance of the environment and the immune system can trigger negative effects on the health of the hosts, and some microorganisms of the microbiota can become pathogenic (^{Maynard et al., 2012}; ^{Belkaid et al., 2013}). In ruminants, the role of the ruminal microbiota in the digestion of cellulose is well known, which allows obtaining energy for these animals (^{Henderson et al. 2015}).

It has been determined that the vaginal microbiota (VM) in ruminants varies according to the physiological/reproductive state, estrous cycle and to a lesser extent by breed (^{Giannattasio-Ferraz et al., 2019}; ^{Manes et al., 2018}). In sheep and goats, the VM analysis has gained relevance in recent years, due to the use of reproductive technologies (use of progestogens, prostaglandins and gonadotropins), mainly in intensive livestock (^{Suárez et al., 2006}; ^{Martins et al., 2009}; ^{Penna et al., 2013}; ^{Oliveira et al., 2013}; ^{Manes et al., 2013}; ^{Manes et al., 2018}). The composition of VM in goats is partially known. It has been reported that it is composed mainly of Gram positive bacteria (GP) and to a lesser extent Gram negative (GN) (^{Manes et al., 2013}). In goats, the change in VM composition due to the use of vaginal devices containing progestogens has been associated with vaginitis and infertility (^{Penna et al., 2013}).

In Mexico, goat production is linked to social classes with low economic income, following a primarily subsistence model (^{Pinos-Rodríguez et al., 2015}). In Tamaulipas, Mexico, goat production is aimed at the production of 21-day-old weaned kid. The type of production characteristic of northeastern Mexico is an extensive system dependent on natural resources in the region (^{Alva-Pérez et al., 2019}). Although goats are rustic animals, adaptable to different environmental conditions, fertility and conception problems in goats are common in production (^{Salinas-González et al., 2016}). Knowledge of VM in Creole goats can reveal the opportunistic bacterial population, which could trigger clinical and subclinical vaginitis. This knowledge can provide the basis for determining the degree of involvement of reproductive system infections in production problems in goats. In addition to this, knowledge of the chemotherapeutic susceptibility profile of the bacteria that make up the VM contributes to improving the treatment of these infections.

MATERIAL AND MÉTODOS

Animal management and sampling site. The present work was carried out in Jaumave municipality, Tamaulipas located between the parallels 23º 53´ and 23º 04´ north latitude, and the meridians 99º 41´ and 99º 10´ west longitude, with an average height of 735 meters above sea level. The orography is mainly mountainous, with a semi-dry semi-warm climate with rains in summer (^{INEGI, 2010}). The goat inventory in this municipality reported in 2018 was 3,931 heads (^{SIAP, 2019}). Five production units (PU) were sampled during June 2019. In each PU, 10 samples were taken, except for the last one, where 11 samples were taken, for a total of 51 vaginal swab samples. The goat herds present a population that mixes several races (Creole population), with predominant encastes of Boer, Alpino, Nubia and Toggenbourgh (^{Alva-Pérez et al., 2019}). The inclusion criteria were healthy females of reproductive age (2 to 4 years). The exclusion criterion was sick or pregnant females. The sampling was carried out under ethical standards of animal welfare, and was authorized by the Bioethics and Animal Welfare Committee of the Faculty of Veterinary Medicine and Zootechnics of the Autonomous University of Tamaulipas (official letter number CBBA_19_05).

Sample collection and processing. In the selected females, momentary physical restraint was carried out with the minimum possible stress, to insert a sterile swab in the vaginal vestibule. The swabs were gently rubbed on the walls of the vaginal mucosa to later be placed in a sterile transport medium (Dehydrated culture media: transport medium amies; BD Difco, Maryland EU). The swabs were kept refrigerated until processing in a period no longer than 12 hours. All the bacteriological processing of the samples was carried out in the Diagnostic Laboratory of the Faculty of Veterinary Medicine and Zootechnics of the Autonomous University of Tamaulipas. For the isolation and identification of the microorganisms, the swabs were sown on blood agar (Blood agar base; Becton Dickinson-Bioxon, Querétaro Mexico) and trypticasein soy agar (BD Difco, Maryland USA). The samples were incubated under aerobic conditions, 37º C for 24 to 48 hours.

The different isolates were identified both macroscopically (colonial morphology, pigment production, and hemolysis) and microscopically (Gram stain). Final bacterial identification was achieved through the following biochemical tests: catalase (Hydrogen peroxide; Merck, Darmstadt Germany), oxidase (n, n, n´, n´-tetramethyl-1,4-phenylenediamine, Biomerieux México, Estado de México), methyl red-Voges Proskauer (Becton Dickinson Bioxon, Querétaro México), nitrate reduction (BD Difco, Maryland EU), urease (Caldo urea; Becton Dickinson-Bioxon, Querétaro México), indole, hydrogen sulfide and motility (SIM medium; Dibico, Estado de México México), growth on McConkey agar (BD Difco, Maryland EU), triple sugar iron (Iron and triple sugar agar, Becton Dickinson-Bioxon, Querétaro México), citrate (BBL Simmons Citrate Agar ; Becton Dickinson, Le Point de Claix France) and use of the following carbohydrates: maltose, mannitol, xylose, lactose and sorbitol (all from Becton Dickinson Bioxon, Querétaro México, prepared with phenol red, phenol red base broth BD Difco, Maryland EU). Identification was carried out following the standards of the Cowan and Steel bacterial identification manual (^{Barrow and Feltman, 2004}).

Susceptibility to chemotherapeutics. The isolates were evaluated in different antibiotics, through the disk diffusion method (^{Humphries et al. 2018}). It is briefly described below. 3 CFUs were selected from each isolation in pure culture, which were seeded in trypticasein soy broth (BD Difco, Maryland EU) and incubated at 37 ºC with shaking (200 rpm). The incubation time varied for each isolate, until obtaining an inoculum equivalent to the 0.5 McFarland standard (0.05 ml of 1% BaCl2 [Sigma-Aldrich, St. Louis, Missouri United States] and 9.95 ml of 1% H2SO4 [Sigma- Aldrich, St. Louis, Missouri United States]) of turbidity, corresponding to 1 to 2×10⁶ CFU/ml in GP bacteria and 5 × 10⁸ CFU/ml in GN bacteria. 1ml of each isolate was seeded on Müller-Hinton agar (MH, BD Difco, Marylando EU). The following sensidisks (Diagnostic Research; Mexico City, Mexico) were used: ampicillin (10 µg, Staphylococcus spp.: resistant phenotype (RF): <28 mm; susceptible phenotype (FS):> 29 mm; other bacterial genera: FR: <21 mm; FS:> 22 mm), Cephalothin (30 µg, FR: <14 mm, FS:> 15 mm), Cefotaxime (30 µg, FR: <14 mm, FS:> 15 mm) , ciprofloxacin (5 µg, RF: <15 mm, FS:> 16 mm), clindamycin (30 µg, RF:<14 mm, FS:> 15 mm), dicloxacillin (1 µg, RF: <10 mm) , FS:> 11 mm), erythromycin (15 µg, RF: <13 mm, FS:> 14 mm), gentamicin (10 µg, FR: <12 mm, FS:> 13 mm), penicillin (10 U , Staphylococcus spp .: FR: <28 mm, FS:> 29 mm, other genera GP: FR: <14 mm; FS:> 15 mm), sulfamethoxazole-trimethoprim (25 μg, FR: <10 mm, FS: > 11 mm), tetracycline (30 µg, FR: <14 mm, FS:> 15 mm), vancomycin (30 g, FR: <14 mm, FS:> 15 mm), chloramphenicol (30 µg, FR : <12 mm, FS:> 13 mm), Carbenicillin (100 µg, FR:<18 mm, FS:> 19 mm), Netilmicin (30 µg, FR: <12 mm, FS: > 13 mm), nitrofurantoin (300 µg, FR: <14 mm, FS:> 15 mm), norfloxacin (100 µg, FR: <18 mm, FS:> 19 mm) and amikacin (30 µg, FR : <14 mm, FS:> 15 mm). The chemotherapeutics clindamycin, dicloxacillin, erythromycin, penicillin, tetracycline, and vancomycin were tested only in GP isolates; whereas chloramphenicol, carbenicillin, netilmicin, nitrofurantoin, norfloxacin, and amikacin were tested only in GN isolates. The zones of inhibition in each sensidisc were measured with a ruler after 16 to 18 h of incubation at 37 ºC. Isolates with intermediate susceptibility were considered resistant, since these bacterial populations present subpopulations of resistant bacteria that will transmit this phenotype to susceptible bacteria (^{Hombach et al. 2013}; ^{Maurer et al. 2014}).

Statistical analysis. The bacterial identification results of the different samples are presented through descriptive statistics and frequency tables. For the results of the chemotherapeutic susceptibility test, contingency tables were constructed with the chisquare test with a significance level of P <0.05 using the PROC FREQ procedure of the SAS program (2002, v9.0. SAS Institute Inc., Cary, NC., USA) to know the resistance percentages. Furthermore, an analysis of variance was performed in a generalized linear model using the PROC GLM and the least significant difference test (LSD, Fisher's exact test) for the comparison of the resistance profile between antibiotics with a P <0.05. For this analysis, the percentage values were converted into values in a range from 0 to 1.

RESULTS

Bacterial isolation and identification. From the 51 samples, only 21 (41.2%) were positive to bacteriological isolation. From these 21 samples, 32 bacteria were isolated and identified (4).

Susceptibility to chemotherapeutics. In the analysis of the antimicrobial resistance profile for GP bacteria, it is noteworthy that all isolates were resistant to erythromycin and tetracycline (Table 2). Isolates of Staphylococcus spp. had a general percentage of resistance of 87.5% (χ²= 18.51, p = 0.0704), with penicillin being the antibiotic with the lowest resistance (70%). The two isolates of S. chromogenes had the lowest resistance among the tested antibiotics (70.8%), with no difference between them (χ²=16.73, p=0.1158). The isolates of C. renale, S. xylosus and Streptococcus spp., had resistance percentages of 83.3% (χ²= 12.0, p = 0.3636), 91.7% (χ²= 12.0, p = 0.3636) and 83.3% (χ²= 12.0, p = 0.3636), respectively. On the other hand, isolates of the genus Aerococcus spp. (resistance 80.9%) showed a lower resistance against dicloxacillin (28.6%, χ2= 22.85, p=0.0185), compared to the other antibiotics. Likewise, isolates of C. jeikeium showed a lower percentage of resistance to cefotaxime and ampicillin (20% and 40%, respectively χ2= 34.9, p <0.001) in contrast to the other antibiotics. For these bacteria the resistance was 85%. Under the conditions of this work, the GP isolates had a resistance profile of 83.2%.

For Enterobacteriaceae (GN isolates) the general percentage of resistance was 95%, most of the antibiotics tested had a resistance of 100%, with the exception of ciprofloxacin (60%) and nitrofurantoin (80%), whose resistance was less than rest of antibiotics (p <0.05, Table 3).

The comparison between the resistance profiles shows that dicloxacillin was more effective among GP bacteria (0.59, Table 3), similar to the resistance profile of ampicillin (0.63) and cefotaxime (0.77). The overall average resistance for antibiotics tested against GP bacteria was 0.84, while the overall average resistance for antibiotics tested against GN bacteria was 0.95.

DISCUSSION

CONCLUSION

The aerobic VM of the Creole goats of Jaumave municipality, Tamaulipas is composed mainly of GP bacteria (population represented mainly by the genus Staphylococcus spp) and to a lesser extent by GN bacteria (Escherichia coli). The bacteriological isolation of the vaginal swabs represented 41.2% of the samples, indicating that other types of bacteria (nutritionally demanding) could be part of the VM. The aerobic VM GP found proved to be highly resistant to erythromycin, tetracycline and vancomycin, while the GN isolates were resistant to most of the chemotherapeutics evaluated, except for cryprofloxacin and nitrofurantoin (with a resistance profile of 60% and 80%, respectively). The high percentage of resistance found in this work highlights the importance of a responsible use of antibiotics in extensive goat production.