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Article

Antimicrobial Susceptibility of Bacterial Isolates from Donkey Uterine Infections, 2018–2021

by
Yufei Zhao
1,†,
Yiping Zhu
1,†,
Bo Liu
1,†,
Junpeng Mi
2,
Nan Li
1,
Weisen Zhao
3,
Rongzheng Wu
1,
Gilbert Reed Holyoak
4,
Jing Li
1,*,
Dejun Liu
1,*,
Shenming Zeng
3 and
Yang Wang
1
1
College of Veterinary Medicine, China Agriculture University, Beijing 100193, China
2
School of Veterinary Science, University of Sydney, Sydney, NSW 2000, Australia
3
College of Animal Science and Technology, China Agriculture University, Beijing 100193, China
4
College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078, USA
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Vet. Sci. 2022, 9(2), 67; https://doi.org/10.3390/vetsci9020067
Submission received: 4 January 2022 / Revised: 27 January 2022 / Accepted: 3 February 2022 / Published: 5 February 2022
Versions Notes

Abstract

:
Background: Endometritis is a common reproductive disease in equine animals. No investigation about the bacterial characteristics and antimicrobial susceptibility pattern of donkeys with endometritis has thus far been reported. Objectives: To determine the common uterine bacterial isolates from donkeys with endometritis and to evaluate their susceptibility to antimicrobials used for the treatment thereof. Study design: Retrospective case-series. Methods: Medical records at an equine clinical diagnostic center were retrospectively reviewed to identify submissions from donkeys with bacterial endometritis between 2018 and 2021. Data were extracted and analyzed descriptively in terms of the frequency of bacterial species, susceptibility to antimicrobials and multidrug resistance. Results: A total of 73 isolates were identified from 30 donkeys, of which 92% of the isolates were Gram-negative bacteria. Mixed cultures were found in 90% of the donkeys. The most common isolates were Escherichia coli (31.5%) and Acinetobacter spp. (21.9%). Susceptibility testing revealed that amikacin (98%), cefoxitin (95%), trimethoprim-sulfamethoxazole (78%) and gentamicin (74%) were the most efficient agents for donkeys. Multidrug resistance (MDR) was found in 20% of all bacterial isolates, of which all Pseudomonas aeruginosa isolates showed a multidrug resistance profile. Main limitations: The sample size was relatively small, which means a bias of selection may exist. The antimicrobial resistance and MDR of agents without break points were not calculated, which means the relative results may be underestimated in our study. Conclusions: Severe infections were detected in donkeys with endometritis. Antimicrobial resistance and MDR bacteria are not rare in our study. This study demonstrated that bacteria identification and antimicrobial susceptibility testing are highly recommended before the treatment of uterine infections in donkeys. Further studies, including the epidemiological investigation of bacterial endometritis of donkeys, should be conducted to provide a better understanding of this critical problem.

1. Introduction

The Nubian donkey (E. asinus africanus) and the Somali donkey (E. asinus somaliensis) were two original ancestors of today’s donkey species (Equus Asinus). These species accompanied the development of human civilization from Africa to Europe and Asia, over 10,000 years ago [1]. Donkey breeds and breeding populations have declined worldwide over the past century, due to a loss of their roles in human society. However, there has been a renewed interest and demand for donkeys (E. Asinus) in recent years, for their novel and evolving role in meat, milk, and skin production [2]. Increasing the reproductive efficiency of donkeys is essential for achieving sustainable populations and economic value.
Endometritis is the third most common equine disease [3], and has long been recognized as one of the major concerns of the equine breeding industry [3,4]. Although endometritis can be associated with a number of causes, bacterial infection, especially aerobic bacteria, is regarded as the main cause of endometritis [5], occurring in 25% to 60% of mares with infertility [6,7]. In mares, retrospective studies reported in several regions over the years have shown that Streptococcus equi subsp zooepidemicus (SEZ), Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), and Pseudomonas aeruginosa (P. aeruginosa) are the most common pathogenic microorganisms of bacterial endometritis [8,9]. This disease has caused significant financial losses, mainly due to failed conception and early embryonic death [7,10].
The presence of an endometrial microbiome in the equine uterus has been reported. The endometrial microbiome refers to the genome of the microorganisms that were discovered from endometrial samples. Over 200 bacterial species have been discovered from equine endometrial samples in recent years [11,12,13]. The potential connection between intrauterine and extrauterine microbiomes make it possible for pathogenic microorganisms to enter the uterus by various means, including mating, artificial insemination or urogenital veterinary examinations, and cervical defects or other failures of physical barriers to infection [7,14]. Transient endometritis can be seen as a normal physiological reaction of mares to eliminate pathogenic bacteria from the uterine lumen [15,16]. Physiologically, mares can autonomously clear the intrauterine bacteria and inflammatory responses within 48 h, while the “susceptible” mares may fail to do so, leading to dysbiotic bacterial endometritis [17].
The consensus statement of antimicrobial drugs used in veterinary medicine has recommended that any use of antimicrobials should be based on the results of laboratory bacteriology and antimicrobial susceptibility testing [18]. Meanwhile, empirical antibiotic treatments based on early studies are often administrated without laboratory diagnoses in the clinic [19], as laboratory diagnoses often take several days, and equine practitioners need to treat infections adequately while the animal is still in estrus [20]. In such cases, treatments often have a lower than desired efficacy. Several studies have been reported on horses confirming that pathogenic species and their antimicrobial susceptibility patterns vary greatly over time and across geographic locations [19,21,22,23,24].
Donkeys share many similarities with horses in reproductive features. For example, the length of uterine body to uterine horn are similar in donkeys and mares, and the shape and structure of the ovaries also resemble each other. Yet, donkeys have longer and narrower cervixes, and uterine edema are less commonly seen in donkeys than in mares [2]. From a reproductive physiology aspect, mares are a seasonal polyestrous animal, while donkeys are a non-seasonal species. In mares, two follicular waves can be seen before ovulation, whereas in donkeys, there appears to be only one follicular wave [2]. Differences here make it even more unreliable to treat endometritis in donkeys with data referenced exclusively from horses. As far as we can assess, information on bacteria prevalence and the antimicrobial susceptibility of the causative organisms of endometritis in donkeys is sparse, and there is insufficient information to provide a valid clinical reference for empirical treatment.
The aim of this study was to identify the bacterial characteristics in samples submitted from donkeys with endometritis, and the antimicrobial susceptibility patterns of the isolated bacteria. A potential goal of reporting these data is to establish a preliminary basis for the empirical treatment of endometritis in donkeys.

2. Materials and Methods

2.1. Data Collection

Medical records of submitted uterine samples obtained from donkeys that had positive bacteriologic culture, cytological evaluation, and antimicrobial susceptibility results to the Equine Clinical Diagnostic Center (ECDC), China Agricultural University, between 1 June 2018 and 15 June 2021, were reviewed. Thirty donkeys with a clinical diagnosis of bacterial endometritis were included and sampled. Breeds represented included “Yangyuan donkeys” (n = 27) and “Dezhou donkeys” (n = 3). The average age was 4.1 years (range, 2 to 7 years old). Among the donkeys included, 14 donkeys (14/30, 46.7%) showed purulent vulvar/vulvovaginal discharge; intrauterine fluid was detected by ultrasonography from 16 donkeys’ uterine (16/30, 53.3%). All included donkeys were nulliparous, and they were all reported to have failed artificial insemination with fresh semen within a year.
According to the protocols of ECDC, all samples were collected by double-guarded uterine swabs. To avoid contamination as much as possible, the donkey’s perineum and vulva were washed with soap and water until clean. When sampling, the operator was asked to wear sterile rectal examination gloves (Jiangs, Nanning, China) and use disposable double-guarded swabs (IMV, Legler, France) to collect endometrial samples [25,26]. Each sample was immediately placed into Amies transport medium (Hopebio, Qingdao, China) and sent back to ECDC laboratory at room temperature within 24 h [19,22]. Other signalments such as age, breed, estrus period and pregnancy history of the included donkeys were also extracted. A written consent for the use of the data was obtained from each owner.

2.2. Bacterial Culturing and Identification

In the ECDC laboratory, bacterial culture and isolation procedures were carried out in accordance with Clinical and Laboratory Standards Institute (CLSI) documents M100-ED30 and VET04 [27,28]. Swabs sampled were streaked directly on Columbia blood agar (CBA) and MacConkey agar (Land Brigde, Beijing, China), intended for the culture of aerobic bacteria. The agar plates were incubated at 37 °C and examined at 24 h and 48 h to obtain individual colonies [22]. The microorganisms obtained were initially identified by the morphology of colonies, time of growth, hemolysis on the plates, and Gram stain microbiology. Bacteria growth was reported as slight (10–20 colony forming unit (CFU)/plate), moderate (20–100 CFU/plate), or abundant (>100 CFU/plate). Further identifications were performed by both matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS), and polymerase chain reaction (PCR) [29,30].

2.3. Antimicrobial Susceptibility Test

Based on CLSI guidelines [27,28], an antimicrobial susceptibility test (AST) was performed using broth microwell dilution. The antimicrobials tested were amoxicillin/clavulanic acid, cefazolin, cefoxitin, ceftiofur, cefepime, gentamicin, amikacin, kanamycin, tetracycline, tigecycline, enrofloxacin, trimethoprim-sulfamethoxazole (TMPS), meropenem and rifampicin. Erythromycin and vancomycin were used only for Gram-positive bacteria. Based on the European Committee on Antimicrobial Sensitivity Testing’s (EUCAST) expert rules in antimicrobial susceptibility testing [31], if a certain bacterial isolate was intrinsically resistant to an antimicrobial, then this agent would not be evaluated for this bacteria species. After the bacterial isolates were suspended to approximately 5 × 105 CFU/mL, they were added in cation-adjusted Mueller–Hinton broth (Land Brigde, Beijing, China), together with antimicrobials in 96-well panels, and incubated at 37 °C for 16–18 h. American Type Culture Collection (ATCC) bacterial strains were used for quality control.
The minimal inhibitory concentration (MIC) was recorded for each row on the panel. According to CLSI’s break points of the selected antimicrobials for each group of bacteria, the results of MIC were classified as sensitivity (S), intermediate (I) and resistant (R). When calculating susceptible rate, intermediate and resistant categories were grouped together. If breakpoints for equines were not available, human breakpoints were referred. Data were presented in MIC50 and MIC90 when there was no break point to refer to, according to CLSI [27]. Multi-drug resistance (MDR) was also recorded if one bacterial isolate was considered resistant to three or more antimicrobials [32].

2.4. Statistical Analysis

Basic data including clinical signalment (age, breed, clinical sign, history of pregnant, treatment), bacterial identification results, susceptibility results and endometrial cytological evaluation were documented in EXCEL (Microsoft, Redmond, WA, USA) to analyze the bacterial isolation rate, antimicrobial resistance rate and MDR rate. Descriptive analysis was performed by using the percentages and counts. The difference of relative frequencies between groups was compared using the Chi-square test and T test in R statistical computing software (Rstudio, Boston, MA, USA). For all comparisons, a value of p < 0.05 was considered significant.

3. Results

3.1. Bacteriologic Description

Of the 30 donkeys, three (3/30, 10%) yielded a single organism, while 27 (27/30, 90%) had two or more bacterial species cultured from submitted samples. In six donkeys (6/30, 20%), both Gram-positive and Gram-negative bacteria were identified. Twenty-four donkeys (24/30, 80%) had only Gram-negative isolates, while no donkeys (0%) yielded a Gram-positive isolate only. The complete bacterial isolation results are presented in Table 1.
A total of 73 bacterial isolates were obtained, of which six (6/73, 8%) were Gram-positive and 67 (67/73, 92%) were Gram-negative bacteria. The proportion of Gram-negative bacterial isolates was significantly higher than that of Gram-positive bacteria (p < 0.01). The most common isolated Gram-negative organism was E. coli (23/73, 31.5%), followed by Acinetobacter spp. (16/73, 21.9%) and P. aeruginosa (8/73, 11%). As for Gram-positive bacteria, Streptococcus spp. were the most common family, including three SEZ isolates (3/73, 4.1%).
E. coli was isolated from ten donkeys (10/14, 71.4%) with purulent vulvar discharge and from 13 donkeys (13/16, 81.3%) with intrauterine fluid detected by ultrasonography. Acinetobacter spp. was cultured from eight donkeys (8/14, 57.1%) with vulvar discharge and four donkeys (4/16, 25%) with intrauterine fluid. P. aeruginosa was only cultured from seven donkeys (7/14, 50%) with vulvar discharge. SEZ was isolated from two (2/14, 14.3%) donkeys with purulent vulvar discharge and one donkey (1/16, 6.3%) with fluid in intrauterine. K. pneumonia was cultured from two donkeys (2/16, 12.5%) with intrauterine fluid, while Klebsiella oxytoca was isolated from one donkey with discharge (1/14, 7.1%) and one donkey with fluid (1/16, 6.3%), respectively. The proportion of representative pathogenic bacterial isolates showed a significant difference between donkeys with vulvar discharge and those with intrauterine fluid (p < 0.05).

3.2. Antimicrobial Susceptibility Testing

Five species of isolated bacteria considered as potential pathogens of equine endometritis were included for antimicrobial susceptibility testing. Fifty strains of potentially pathogenic bacteria (50/73, 68.5%) isolated from 26 donkeys had antimicrobial susceptibility testing performed, yielding 19 antimicrobial susceptibility patterns. Complete results of AST are presented in Table 2 and Table 3. There was no break point of some antimicrobials, in those situations, MIC50 and MIC90 values were given. For example, cefoxitin MICs values showed <0.25 μg/mL to SEZ and >128 μg/mL to P. aeruginosa isolates. Kanamycin MICs values showed 32 μg/mL to SEZ, while they showed 128 μg/mL to P. aeruginosa. Complete results of these data are presented in Table 4.
Both cefepime and meropenem had the highest antimicrobial susceptibility frequencies, with all bacterial isolates (100%) being susceptible to them. Amikacin (98%), cefoxitin (95%), trimethoprim-sulfamethoxazole (78%) and gentamicin (74%) were also efficient agents. In addition to cefepime and meropenem, for E. coli, the proportion of resistant isolates was greatest for cefazolin (13%) and tetracycline (13%). All 23 E. coli isolates (100%) were classified as susceptible to cefoxitin, amikacin, and kanamycin. For the 13 Acinetobacter spp. isolates (including nine Acinetobacter lwoffii (A. lwoffii) isolates, three Acinetobacter schindleri (A. schindleri) isolates, and one Acinetobacter baumannii (A. baumannii) isolate), resistance was most common to cefazolin (84.6%). All the isolates (100%) were classified as susceptible to aminoglycosides. The seven P. aeruginosa isolates were classified as not susceptible to most of the selected antibacterial agents, except amikacin (100%) and TMPS (85.7%). For the three SEZ isolates, the proportion of resistant isolates was greatest to gentamicin (66.7%). They were classified as susceptible to all the other antimicrobials.
Antimicrobial susceptibility patterns revealed that 19 (19/26, 73.1%) of these donkeys had bacterial isolates resistant to the tested antimicrobials. Ten (10/12, 83.3%) had isolates resistant to cefazolin, and nine (9/12, 75%) had isolates resistant to gentamicin. Resistance to amoxicillin/clavulanic acid was found in seven donkeys (7/12, 58.3%). Five donkeys (5/12, 41.7%) had bacteria isolates resistant to kanamycin, while other isolates were resistant to tetracycline (3/12, 25%), ceftiofur (2/12, 16.7%), cefoxitin (1/12, 8.3%), tigecycline (1/12, 8.3%) and TMPS (2/12, 16.7%), respectively.
Of the donkeys with intra uterine fluid, seven (7/14, 50%) had bacterial isolates resistant to cefazolin. Some bacteria isolated from a few donkeys were also resistance to amoxicillin/clavulanic acid (2/14, 14.3%), gentamicin (1/14, 7.1%) and tetracycline (1/14, 7.1%). There was a significant difference (p < 0.05) found in the frequency of resistance for amoxicillin/clavulanic acid and gentamicin between donkeys with vulvar discharge and intrauterine fluid.

3.3. Multidrug Resistance

Ten (10/50, 20%) bacteria isolates were found to be multidrug-resistant based on the results of antimicrobial susceptibility testing. All the MDR bacteria found in this study were Gram-negative bacteria, including seven strains of P. aeruginosa (7/50, 14%), two strains of E. coli (2/50, 4%) and one strain of A. lwoffii (1/50, 2%).
Of the 26 donkeys that had antimicrobial susceptibility testing performed, nine (9/26, 34.6%) were infected with multidrug-resistant bacteria, all of them were those donkeys with purulent vulvar discharge. One donkey (1/26, 3.8%) with discharge was infected with more than one MDR bacteria. The frequency of MDR was more significant in donkeys with vulvar discharge than those with intrauterine fluid in the uterine (p = 0.011).

4. Discussion

To the best of our knowledge, this is the first reported antimicrobial susceptibility information from uterine infections of donkeys. The species and proportion of bacteria isolated in our study were different from those reported in mares [16,17,19,21,23]. Most of the included donkey samples (90%) yielded a mixed culture. Larger scale epidemiological investigations should be conducted in the future to increase the number of donkeys. Different geographical locations, the population of animals included, and the antimicrobials used before have been suggested to be the general causes [21,24].
E. coli and Acinetobacter spp. were the most common pathogenic bacterial species in this study. The frequency of E. coli being isolated was consistent with previous results (27.9–30%) in mares [21,22,23], but was a little higher than some others (17.3–19.3%) [17,24]. E. coli, along with P. aeruginosa and K. pneumoniae, have been regarded as biofilm-producing microorganisms in equine endometritis [33,34,35]. All these species showed high prevalence in our study compared with previous studies reported in mares [16,17], implying that biofilm may be a potentially serious problem in endometritis in donkeys. For Acinetobacter spp., the A. lwoffii and A. schindleri were the predominant species. Though no retrospective study reported these two species obtained from the mare or donkey uterus, there are reports of these species isolated from the feces of both horses and donkeys [36,37]. Additionally, these have been isolated from catheters used to treat endometritis of equines [38,39]. A possible explanation may be that the fecal contaminants were introduced into the uterus during the breeding process, causing opportunistic infections in these donkeys.
Gram-positive bacteria were not common isolates in our study. We only identified three SEZ isolates, which have always been reported as the most common Gram-positive pathogen causing endometritis in mares [6,21,22]. In research conducted in the middle-east of China, the infection rate of SEZ in donkeys with endometritis was 57%, which was much higher than the current result [40]. In contrast, in a donkey vaginal bacterial microbiota study conducted in Portugal, the isolate rate of SEZ was only 2% (3/140) [41]. SEZ often colonize deeply in the endometrium, which may make it more difficult for the swab to collect them [42]. However, given that current studies have reported an inconsistent prevalence of SEZ using swabs for sampling, it is difficult to quantitatively determine the relative importance of SEZ in causing endometritis in donkeys [16,17,23]. Meanwhile, Staphylococcus aureus, as another identifiable pathogen in mares [16,23], was negative from the samples submitted from these donkeys.
While there is growing concern worldwide about the public health impact of antimicrobial resistance (AMR) in livestock [43,44], few studies have investigated the efficacy of antimicrobials used for infections of the equine reproductive tract and uterus [45]. To provide up-to-date data on donkeys, nineteen antimicrobials were used in our study. Cefepime and meropenem were the most effective agents, which were 100% effective against both Gram-positive and Gram-negative bacteria in our study. They are all used as first-line antimicrobials in human hospitals [46,47]. However, cefepime has proven to have adverse effects on the gastrointestinal tract of horses [48]; thus, the further evaluation of the pharmacokinetics of cefepime in donkeys prior to clinical administration is necessary. Although meropenem is safe for use in equine animals, it is regarded as the last resort for the treatment of infections [49]. Therefore, the public health significance of the two most effective antimicrobials outweighs their practical clinical application in this study.
For Gram-negative bacteria, amikacin (98%) and cefoxitin were highly effective. The results agree with those reported previously [24]. Considering the absolute predominance of Gram-negative bacteria among pathogens in this study, we cautiously recommend these drugs for the clinical management of endometritis in donkeys. Gentamicin and TMPS are also commonly used for the treatment of equine bacterial infections [22]. The susceptibility of gentamicin (74%) in our study was not as pronounced as has been reported before (83–96%) [16,19]. Although the use of gentamicin is still recommended, an increasing resistance to gentamicin, due to empirical treatment without the identification of antimicrobial susceptibility, should be noted. Our results for TMPS (78%) were similar to those reported with mares in Sweden (2003) and Italy (2008) [17,19], but higher than those in other European and American countries over the past ten years [16,21,24]. The often-empirical use of gentamicin and TMPS in the treatment of endometritis may lead to increasing resistance of equine endometritis pathogens to them [22]. However, in donkeys, it seems that gentamicin and TMPS still have good antibacterial activity.
Rifampicin seemed to be an undesirable agent in our study, though there was no break point to refer to. Gram-negative bacteria were more resistant to RIF, and in this respect our results were consistent with those of others [17,23]. Reports of RIF resistance began in the time period when it was used in combination with macrolides [50]. The mutation in the β subunit of bacterial RNA polymerase (RNAP) may attribute to the resistance of RIF in bacteria [51]. The first-generation of cephalosporins have been reported to be less effective [23,24], which was supported by our results in the case of cefazolin. It has been recommended to use a combination of enrofloxacin and polymyxins against extensive drug-resistant (XDR) P. aeruginosa [52].
Analysis of multi-drug resistant bacteria from the reproductive tract, especially the uterus, has not previously been reported, yet a general trend of increasing MDR bacteria has been reported over time [29,53]. The classical definition of MDR was followed in our study [32]. The most common MDR isolates in our study were identified as P. aeruginosa, which account for 70% of all MDR bacteria. The results here raise a concern and suggest an urgent need to re-evaluate current practices and empirical treatment on donkey farms. Furthermore, although the proportion of MDR bacteria showed no difference between donkeys with vulvar discharge and those with intrauterine fluid, the composition of bacterial isolates and the effective agents of the in vitro AST between the donkeys with two clinical signs were different. This re-emphasizes the importance of identifying antimicrobial susceptibility before treatment.
In general, the level of medical care of donkeys has yet to be improved. Considering the donkey population and the farm scale in our surrounding area, it is very likely that the number of donkeys with bacterial endometritis is higher than the number presented to the ECDC laboratory. Therefore, one limitation of the current study was the relatively small size of sampled donkeys. In addition, since the antimicrobial resistance and MDR of agents without break points were not calculated, the relative results may also be underestimated in our study.
In conclusion, severe infections were detected in donkeys with endometritis. Antimicrobial resistance and MDR bacteria are not rare in uterine infections in donkeys. This study demonstrated that bacteria identification and antimicrobial susceptibility testing are highly recommended before any treatment of uterine infections in donkeys. Further studies, including the epidemiological investigation of bacterial endometritis in donkeys, should be conducted to provide a better understanding of this critical problem.

Author Contributions

Conceptualization, J.L.; Methodology, Y.Z. (Yiping Zhu), B.L., J.M., N.L., G.R.H., D.L. and Y.W.; Software, R.W.; Validation, Y.Z. (Yiping Zhu), B.L. and J.L.; formal analysis, Y.Z. (Yufei Zhao) and B.L.; investigation, Y.Z. (Yiping Zhu), J.M., N.L., W.Z. and J.L.; Resources, S.Z.; Data curation, Y.Z. (Yufei Zhao), B.L., J.M. and W.Z.; Writing—original draft preparation, Y.Z. (Yufei Zhao); Writing—review and editing, G.R.H., J.L., D.L. and Y.W.; Visualization, R.W.; supervision, J.L., D.L., S.Z. and Y.W.; Project administration, J.L., D.L., S.Z. and Y.W.; Funding acquisition, J.L., D.L. All authors have read and agreed to the published version of the manuscript.

Funding

National Natural Science Foundation of China: 81991531.

Institutional Review Board Statement

The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of China Agricultural University Laboratory Animal Welfare and Animal Experimental Ethical Inspection Committee (protocol code: AW-311022-2-1, date of approval: 1 August 2021).

Informed Consent Statement

Informed consent was obtained from all owners involved in the study.

Data Availability Statement

Data available in a publicly accessible repository. The data presented in this study are openly available in FigShare at https://doi.org/10.6084/m9.figshare.18979376, accessed on 1 January 2022.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Beja-Pereira, A.; England, P.R.; Ferrand, N.; Jordan, S.; Bakhiet, A.O.; Abdalla, M.A.; Mashkour, M.; Jordana, J.; Taberlet, P.; Luikart, G. African origins of the domestic donkey. Science 2004, 304, 1781. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Canisso, I.F.; Panzani, D.; Miró, J.; Ellerbrock, R.E. Key aspects of donkey and mule reproduction. Vet. Clin. N. Am. Equine Pract. 2019, 35, 607–642. [Google Scholar] [CrossRef]
  3. Troedsson, M.H. Uterine clearance and resistance to persistent endometritis in the mare. Theriogenology 1999, 52, 461–471. [Google Scholar] [CrossRef]
  4. Troedsson, M.H.; Woodward, E.M. Our current understanding of the pathophysiology of equine endometritis with an emphasis on breeding-induced endometritis. Reprod. Biol. 2016, 16, 8–12. [Google Scholar] [CrossRef]
  5. Rasmusse, C.D.; Petersen, M.R.; Bojesen, A.M.; Pedersen, H.G.; Lehn-Jensen, H.; Christoffersen, M. Equine infectious endometritis—Cinical and subclinical cases. J. Equine Vet. Sci. 2015, 35, 95–104. [Google Scholar] [CrossRef]
  6. Casagrande Proietti, P.; Bietta, A.; Coppola, G.; Felicetti, M.; Cook, R.F.; Coletti, M.; Marenzoni, M.L.; Passamonti, F. Isolation and characterization of β-haemolytic-Streptococci from endometritis in mares. Vet. Microbiol. 2011, 152, 126–130. [Google Scholar] [CrossRef] [Green Version]
  7. Causey, R.C. Making sense of equine uterine infections: The many faces of physical clearance. Vet. J. 2006, 172, 405–421. [Google Scholar] [CrossRef] [PubMed]
  8. Benko, T.; Boldizar, M.; Novotny, F.; Hura, V.; Valocky, I.; Dudrikova, K.; Karamanova, M.; Petrovic, V. Incidence of bacterial pathogens in equine uterine swabs, their antibiotic resistance patterns, and selected reproductive indices in English thoroughbred mares during the foal heat cycle. Vet. Med. 2016, 60, 613–620. [Google Scholar] [CrossRef]
  9. Frontoso, R.; De Carlo, E.; Pasolini, M.P.; van der Meulen, K.; Pagnini, U.; Iovane, G.; De Martino, L. Retrospective study of bacterial isolates and their antimicrobial susceptibilities in equine uteri during fertility problems. Res. Vet. Sci. 2008, 84, 1–6. [Google Scholar] [CrossRef] [PubMed]
  10. Riddle, W.T.; Leblanc, M.M.; Stromberg, A.J. Relationships between uterine culture, cytology and pregnancy rates in a Thoroughbred practice. Theriogenology 2007, 68, 395–402. [Google Scholar] [CrossRef] [PubMed]
  11. Holyoak, G.R.; Lyman, C.C. The equine endometrial microbiome: A brief review. Am. J. Biomed. Sci. Res. 2021, 11, 532–534. [Google Scholar] [CrossRef]
  12. Rock, K.S.; Love, B.C.; DeSilva, U.; Rezabek, G.B.; Meijer, W.G.; Carrington, S.D.; Holyoak, G.R. Detectable differences in the endometrial microbiome between normal and susceptible mares using metagenomic profiling and conventional bacterial culture. Clin. Theriogenol. 2011, 3, 376. [Google Scholar]
  13. Holyoak, G.R.; Lyman, C.C.; Wieneke, X.; DeSilva, U. The equine endometrial microbiome. Clin. Theriogenol. 2018, 10, 273–278. [Google Scholar]
  14. Causey, R.C. Uterine therapy for mares with bacterial infections. In Current Therapy in Equine Reproduction; Elsevier: Amsterdam, The Netherlands, 2007; pp. 105–115. [Google Scholar]
  15. Troedsson, M.H. Breeding-induced endometritis in mares. Vet. Clin. N. Am. Equine Pract. 2006, 22, 705–712. [Google Scholar] [CrossRef] [PubMed]
  16. Li, J.; Zhao, Y.; Gao, Y.; Zhu, Y.; Holyoak, G.R.; Zeng, S. Treatments for endometritis in mares caused by streptococcus equi subspecies zooepidemicus: A structured literature review. J. Equine Vet. Sci. 2021, 102, 103430. [Google Scholar] [CrossRef]
  17. Rogan, D.; Fumuso, E.; Rodríguez, E.; Wade, J.; Sánchez Bruni, S.F. Use of a mycobacterial cell wall extract (MCWE) in susceptible mares to clear experimentally induced endometritis with streptococcus zooepidemicus. J. Equine Vet. Sci. 2007, 27, 112–117. [Google Scholar] [CrossRef]
  18. Morley, P.S.; Apley, M.D.; Besser, T.E.; Burney, D.P.; Fedorka Cray, P.J.; Papich, M.G.; Traub Dargatz, J.L.; Weese, J. Antimicrobial drug use in veterinary medicine. J. Vet. Intern. Med. 2005, 19, 617–629. [Google Scholar] [CrossRef]
  19. Albihn, A.; Båverud, V.; Magnusson, U. Uterine microbiology and antimicrobial susceptibility in isolated bacteria from mares with fertility problems. Acta. Vet. Scand. 2003, 44, 121–129. [Google Scholar] [CrossRef]
  20. Ricketts, S. The barren mare: Diagnosis, prognosis, prophylaxis and treatment for genital abnormality. Practice 1989, 11, 156–164. [Google Scholar] [CrossRef]
  21. Davis, H.A.; Stanton, M.B.; Thungrat, K.; Boothe, D.M. Uterine bacterial isolates from mares and their resistance to antimicrobials: 8,296 cases (2003–2008). J. Am. Vet. Med. Assoc. 2013, 242, 977–983. [Google Scholar] [CrossRef]
  22. Ferrer, M.S.; Palomares, R. Aerobic uterine isolates and antimicrobial susceptibility in mares with post-partum metritis. Equine Vet. J. 2017, 50, 202–207. [Google Scholar] [CrossRef]
  23. Pisello, L.; Rampacci, E.; Stefanetti, V.; Beccati, F.; Hyatt, D.R.; Coletti, M.; Passamonti, F. Temporal efficacy of antimicrobials against aerobic bacteria isolated from equine endometritis: An Italian retrospective analysis (2010–2017). Vet. Rec. 2019, 185, 598. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Díaz-Bertrana, M.L.; Deleuze, S.; Pitti Rios, L.; Yeste, M.; Morales Fariña, I.; Rivera Del Alamo, M.M. Microbial prevalence and antimicrobial sensitivity in equine endometritis in field conditions. Animals 2021, 11, 1476. [Google Scholar] [CrossRef] [PubMed]
  25. Canisso, I.F.; Segabinazzi, L.G.; Fedorka, C.E. Persistent breeding-induced endometritis in mares-a multifaceted challenge: From clinical aspects to immunopathogenesis and pathobiology. Int. J. Mol. Sci. 2020, 21, 1432. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Katila, T. Evaluation of diagnostic methods in equine endometritis. Reprod. Biol. 2016, 16, 189–196. [Google Scholar] [CrossRef] [PubMed]
  27. CLSI. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals, 4th ed.; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2013. [Google Scholar]
  28. CLSI. Performance Standards for Antimicrobial Susceptibility Testing, 30th ed.; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2020. [Google Scholar]
  29. Patel, R. MALDI-TOF MS for the diagnosis of infectious diseases. Clin. Chem. 2015, 61, 100–111. [Google Scholar] [CrossRef] [Green Version]
  30. Johnston, G.; Lumsden, J.M. Antimicrobial susceptibility of bacterial isolates from 33 thoroughbred horses with arytenoid chondropathy (2005–2019). Vet. Surg. 2020, 49, 1283–1291. [Google Scholar] [CrossRef]
  31. Leclercq, R.; Cantón, R.; Brown, D.F.J.; Giske, C.G.; Heisig, P.; Macgowan, A.P.; Mouton, J.W.; Nordmann, P.; Rodloff, A.C.; Rossolini, G.M.; et al. EUCAST expert rules in antimicrobial susceptibility testing. Clin. Microbiol. Infect. 2013, 19, 141–160. [Google Scholar] [CrossRef] [Green Version]
  32. Magiorakos, A.P.; Srinivasan, A.; Carey, R.B.; Carmeli, Y.; Falagas, M.E.; Giske, C.G.; Harbarth, S.; Hindler, J.F.; Kahlmeter, G.; Olsson-Liljequist, B.; et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clin. Microbiol. Infect. 2012, 18, 268–281. [Google Scholar] [CrossRef] [Green Version]
  33. Høiby, N.; Bjarnsholt, T.; Moser, C.; Bassi, G.L.; Coenye, T.; Donelli, G.; Hall-Stoodley, L.; Holá, V.; Imbert, C.; Kirketerp-Møller, K. ESCMID guideline for the diagnosis and treatment of biofilm infections 2014. Clin. Microbiol. Infect. 2015, 21, S1–S25. [Google Scholar] [CrossRef] [Green Version]
  34. Ferris, R.A.; Mccue, P.M.; Borlee, G.I.; Loncar, K.D.; Hennet, M.L.; Borlee, B.R. In vitro efficacy of nonantibiotic treatments on biofilm disruption of gram-negative pathogens and an in vivo model of infectious endometritis utilizing isolates from the equine uterus. J. Clin. Microbiol. 2016, 54, 631–639. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Ferris, R.A.; Mccue, P.M.; Borlee, G.I.; Glapa, K.E.; Martin, K.H.; Mangalea, M.R.; Hennet, M.L.; Wolfe, L.M.; Broeckling, C.D.; Borlee, B.R. Model of chronic equine endometritis involving a Pseudomonas aeruginosa biofilm. Infect. Immun. 2017, 85, e317–e332. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  36. Maboni, G.; Seguel, M.; Lorton, A.; Sanchez, S. Antimicrobial resistance patterns of Acinetobacter spp. of animal origin reveal high rate of multidrug resistance. Vet. Microbiol. 2020, 245, 108702. [Google Scholar] [CrossRef]
  37. Smet, A.; Cools, P.; Krizova, L.; Maixnerova, M.; Sedo, O.; Haesebrouck, F.; Kempf, M.; Nemec, A.; Vaneechoutte, M. Acinetobacter gandensis sp. nov. isolated from horse and cattle. Int. J. Syst. Evol. Microbiol. 2014, 64, 4007–4015. [Google Scholar] [CrossRef] [Green Version]
  38. Weese, J.S. Antimicrobial therapy for multidrug resistant pathogens. Equine Vet. Educ. 2009, 21, 328–334. [Google Scholar] [CrossRef]
  39. Ebert, M.A.; Riese, R.L. Infertility in the mare. Iowa State Univ. Vet. 1986, 48, 4. [Google Scholar]
  40. Gao, N.; Du, Y.; Zheng, X.; Shu, S.; Suo, J.; Han, M.; Ma, X.; Huang, R.; Peng, W.; Fu, C. Endometritis in donkeys associated with Streptococcus equi subspecies zooepidemicus infection. Pak. Vet. J. 2020, 40, 537–539. [Google Scholar] [CrossRef]
  41. Carvalho, S.; Marinho, C.; Gonçalves, A.; Sousa, M.; Nóvoa, M.; Quaresma, M.; Igrejas, G.; Poeta, P. Vaginal bacterial microbiota of an endangered donkey breed: A comparison between Miranda donkey breed (Equus asinus) jennies with and without reproductive problems. J. Integr. OMICS 2016, 6, 193. [Google Scholar]
  42. Christoffersen, M.; Söderlind, M.; Rudefalk, S.R.; Pedersen, H.G.; Allen, J.; Krekeler, N. Risk factors associated with uterine fluid after breeding caused by Streptococcus zooepidemicus. Theriogenology 2015, 84, 1283–1290. [Google Scholar] [CrossRef]
  43. Bourély, C.; Cazeau, G.; Jarrige, N.; Haenni, M.; Gay, E.; Leblond, A. Antimicrobial resistance in bacteria isolated from diseased horses in France. Equine Vet. J. 2019, 52, 112–119. [Google Scholar] [CrossRef]
  44. Schmiedel, J.; Falgenhauer, L.; Domann, E.; Bauerfeind, R.; Prenger-Berninghoff, E.; Imirzalioglu, C.; Chakraborty, T. Multiresistant extended-spectrum beta-lactamase-producing Enterobacteriaceae from humans, companion animals and horses in central Hesse, Germany. BMC Microbiol. 2014, 14, 187. [Google Scholar] [CrossRef] [Green Version]
  45. Robinson, T.P.; Bu, D.P.; Carrique-Mas, J.; Fèvre, E.M.; Gilbert, M.; Grace, D.; Hay, S.I.; Jiwakanon, J.; Kakkar, M.; Kariuki, S.; et al. Antibiotic resistance: Mitigation opportunities in livestock sector development. Animal 2017, 11, 1–3. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Patel, H.B.; Lusk, K.A.; Cota, J.M. The role of cefepime in the treatment of extended-spectrum beta-lactamase infections. J. Pharm. Pract. 2019, 32, 458–463. [Google Scholar] [CrossRef] [PubMed]
  47. Salmon-Rousseau, A.; Martins, C.; Blot, M.; Buisson, M.; Mahy, S.; Chavanet, P.; Piroth, L. Comparative review of imipenem/cilastatin versus meropenem. Med. Mal. Infect. 2020, 50, 316–322. [Google Scholar] [CrossRef] [PubMed]
  48. Guglick, M.A.; Macallister, C.G.; Clarke, C.R.; Pollet, R.; Hague, C.; Clarke, J.M. Pharmacokinetics of cefepime and comparison with those of ceftiofur in horses. Am. J. Vet. Res. 1998, 59, 458–463. [Google Scholar]
  49. Langston, V.C.; Fontenot, R.L.; Byers, J.A.; Andrews, C.M.; Mochal King, C.A. Plasma and synovial fluid pharmacokinetics of a single intravenous dose of meropenem in adult horses. J. Ve.t Pharmacol. Ther. 2019, 42, 525–529. [Google Scholar] [CrossRef]
  50. Petry, S.; Sévin, C.; Kozak, S.; Foucher, N.; Laugier, C.; Linster, M.; Breuil, M.; Dupuis, M.; Hans, A.; Duquesne, F.; et al. Relationship between rifampicin resistance and RpoB substitutions of Rhodococcus equi strains isolated in France. J. Glob. Antimicrob. Resist. 2020, 23, 137–144. [Google Scholar] [CrossRef]
  51. Goldstein, B.P. Resistance to rifampicin: A review. J. Antibiot. 2014, 67, 625–630. [Google Scholar] [CrossRef] [Green Version]
  52. Lin, Y.; Han, M.; Zhao, J.; Zhu, Y.; Rao, G.; Forrest, A.; Song, J.; Kaye, K.S.; Hertzog, P.; Purcell, A.; et al. Synergistic combination of polymyxin B and enrofloxacin induced metabolic perturbations in extensive drug-resistant Pseudomonas aeruginosa. Front Pharmacol. 2019, 10, 1146. [Google Scholar] [CrossRef] [Green Version]
  53. Johns, I.C.; Adams, E.L. Trends in antimicrobial resistance in equine bacterial isolates: 1999–2012. Vet. Rec. 2015, 176, 334. [Google Scholar] [CrossRef]
Table
Table 1. Species and frequency of bacteria isolated from uterine swabs of 30 donkeys at ECDC between 2018 and 2021.
Table 1. Species and frequency of bacteria isolated from uterine swabs of 30 donkeys at ECDC between 2018 and 2021.
Micro-OrganismsNumber of IsolatesFrequency of Isolates (%)
Acinetobacter baumannii34.1
Acinetobacter lwoffii1013.7
Acinetobacter schindleri34.1
Other Acinetobacter spp.11.7
Aeromonas spp.11.7
Arthrobacter gandavensis11.7
Arthrobacter koreensis11.7
Bacillus cereus22.7
Burkholderia cepacia22.7
Corynebacterium spp.11.7
Enterobacter spp.34.1
Escherichia coli2331.5
Klebsiella pneumoniae22.7
Klebsiella oxytoca22.7
Pantoea agglomerans11.7
Proteus mirabilis22.7
Pseudomonas aeruginosa810.9
Other Pseudomonas spp.45.4
Streptococcus equi subsp. zooepidemicus34.1
Total73100
Table
Table 2. Antimicrobial susceptibility of Gram-negative bacteria isolated from the uterus of 30 donkeys at ECDC between 2018 and 2021.
Table 2. Antimicrobial susceptibility of Gram-negative bacteria isolated from the uterus of 30 donkeys at ECDC between 2018 and 2021.
Gram-Negative BacteriaE. coli
(23 Isolates)		Acinetobacter spp.
(13 Isolates)		P. aeruginosa
(7 Isolates)		Klebsiella spp.
(4 Isolates)
Antimicrobials aS (%)I (%)R (%)S (%)I (%)R (%)S (%)I (%)R (%)S (%)I (%)R (%)
Amoxicillin/clavulanic-acid91.3-8.792.37.7---100502525
Cefazolin65.321.7137.77.784.6--10025-75
Cefoxitin100--92.3-7.7M b--100--
Ceftiofur95.7-4.392.3-7.7M b--100--
Cefepime100--100--100--100--
Gentamicin874.38.7100----100100--
Amikacin100--100--100--100--
Kanamycin100--100--M b--7525-
Tetracycline87-1392.3-7.7NA cNA cNA c7525-
Tigecycline95.74.3-92.3-7.7NA cNA cNA c100--
EnrofloxacinM b--M b--M b--M b--
Trimethoprim-Sulfamethoxazole95.74.3-92.3-7.785.7-14.3100--
Meropenem100--100--100--100--
RifampicinM b--M b--M b--M b--
a, S = sensitive, I = intermediate, R = resistant. b, M = only MIC50 and MIC90 value were provided. Data were presented in this way when there was no break point to refer to, according to CLSI [27]. c, NA = not applicable. P. aeruginosa was intrinsically resistant to tetracyclines according to EUCAST. Antimicrobial susceptibility to these agents were not evaluated for P. aeruginosa [31].
Table
Table 3. Antimicrobial susceptibility of SEZ isolates collected from the uteruses of 30 donkeys at ECDC between 2018 and 2021.
Table 3. Antimicrobial susceptibility of SEZ isolates collected from the uteruses of 30 donkeys at ECDC between 2018 and 2021.
Gram-Positive BacteriaSEZ
(3 Isolates)
Antimicrobials aS (%)I (%)R (%)
Amoxicillin/clavulanic-acid100--
CefoxitinM b--
Ceftiofur100--
Cefepime100--
Gentamicin-33.366.7
Amikacin66.733.3-
KanamycinM b--
Tigecycline100--
Erythromycin100--
EnrofloxacinMb--
Trimethoprim-Sulfamethoxazole100--
Meropenem100--
Vancomycin100--
Rifampicin100--
a, S = sensitive, I = intermediate, R = resistant. b, M = only MIC50 and MIC90 value were provided. Data were presented in that way when there was no break point to refer to, according to CLSI.
Table
Table 4. MIC values of bacteria isolated from the uteruses of 30 donkeys at ECDC between 2018 and 2021.
Table 4. MIC values of bacteria isolated from the uteruses of 30 donkeys at ECDC between 2018 and 2021.
BacteriaE. coliAcinetobacter spp.P. aeruginosaKlebsiella spp.SEZ
MIC Values (μg/mL)MIC50MIC90MIC50MIC90MIC50MIC90MIC50MIC90MIC50MIC90
Cefoxitin---->128>128--<0.25<0.25
Ceftiofur----3232----
Kanamycin---->128>128--3232
Enrofloxacin<0.251<0.25188<0.25<0.2511
Rifampicin48<0.250.5>128>1281664--
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Zhao, Y.; Zhu, Y.; Liu, B.; Mi, J.; Li, N.; Zhao, W.; Wu, R.; Holyoak, G.R.; Li, J.; Liu, D.; et al. Antimicrobial Susceptibility of Bacterial Isolates from Donkey Uterine Infections, 2018–2021. Vet. Sci. 2022, 9, 67. https://doi.org/10.3390/vetsci9020067

AMA Style

Zhao Y, Zhu Y, Liu B, Mi J, Li N, Zhao W, Wu R, Holyoak GR, Li J, Liu D, et al. Antimicrobial Susceptibility of Bacterial Isolates from Donkey Uterine Infections, 2018–2021. Veterinary Sciences. 2022; 9(2):67. https://doi.org/10.3390/vetsci9020067

Chicago/Turabian Style

Zhao, Yufei, Yiping Zhu, Bo Liu, Junpeng Mi, Nan Li, Weisen Zhao, Rongzheng Wu, Gilbert Reed Holyoak, Jing Li, Dejun Liu, and et al. 2022. "Antimicrobial Susceptibility of Bacterial Isolates from Donkey Uterine Infections, 2018–2021" Veterinary Sciences 9, no. 2: 67. https://doi.org/10.3390/vetsci9020067

APA Style

Zhao, Y., Zhu, Y., Liu, B., Mi, J., Li, N., Zhao, W., Wu, R., Holyoak, G. R., Li, J., Liu, D., Zeng, S., & Wang, Y. (2022). Antimicrobial Susceptibility of Bacterial Isolates from Donkey Uterine Infections, 2018–2021. Veterinary Sciences, 9(2), 67. https://doi.org/10.3390/vetsci9020067

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