This study aimed to describe bacteria isolated from the reproductive tract of mares and to identify changes in antimicrobial susceptibility patterns to those antibiotics commonly used for the treatment of equine endometritis. A total of 4122 equine uterine swabs were collected from mares suffering from reproductive tract disorders in the period 2010–2017. Aerobic culture and antimicrobial susceptibility testing using agar disc diffusion were performed on each sample. Aerobic bacteria were isolated from 3171 of 4122 (76.9 per cent) samples. The most frequently isolated microorganisms were Escherichia coli (885/3171, 27.9 per cent) and Streptococcus equi subspecies zooepidemicus (791/3171, 24.9 per cent), confirming previous findings from the literature. Antimicrobial susceptibility patterns of E coli, S equi subspecies zooepidemicus and Klebsiella pneumoniae changed over time. A statistically significant decrease in antimicrobial efficacy of cefquinome against E coli was observed over the years, as well as of ampicillin, cefquinome and penicillin against S equi subspecies zooepidemicus. The high frequency of resistant bacteria isolated in the present work proceeds in the same way as indicated by surveillance data on the huge antibiotic use in Italy. As a result, testing and monitoring programmes of antimicrobial efficacy are crucial to consciously using antibiotics and preserving their effectiveness both for veterinary and human medicine.
- equine endometritis
- bacterial culture
- uterine swab
- antimicrobial susceptibility
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Endometritis is the third most common pathology observed by equine practitioners,1 occurring in 25–60 per cent of mares with fertility disorders.2 Endometrial infections cause reduced fertility and contribute to severe economic losses, mainly due to early embryonic death.2 3 Indeed, in the critical period between insemination and pregnancy diagnosis, the gestation rates fall from 80 per cent to 20 per cent among subfertile mares with endometrial infection.4 5
The anatomical and physiological uterine clearance normally ensures bacterial expulsion without negative effects on reproduction.6–8 However, opportunistic bacterial microorganisms that are common residents of the genital caudal tract are able to penetrate into the uterus during natural or artificial insemination, parturition, and as a result of the breakdown of the physical barriers to infection.9 This penetration by microorganisms results in inflammation and impaired reproductive performance. Escherichia coli, Streptococcus equi subspecies zooepidemicus, Klebsiella pneumoniae, Staphylococcus aureus and Pseudomonas subspecies are among the most common bacteria isolated from equine uteri.10–19
Guidelines concerning the gold standard treatment of bacterial infections suggest that every antibiotic prescription has to be based on the results of bacteriological culture and antimicrobial susceptibility testing.20 Conversely, there is a real need for rapid microbiological diagnosis in order to complete an adequate therapeutic regimen while the mare is still in oestrus.10 21 For that reason, the antimicrobial administration often is empirical and based on data from earlier studies.18 19 However, the pathogenic bacterial species along with their antimicrobial susceptibility patterns appear to vary over time and between equine populations.22
This study aimed to describe the pathogenic bacterial species isolated from the reproductive tract of mares and to identify changes in antimicrobial susceptibility patterns over an eight-year period with the goal of determining the efficacy of the antimicrobials commonly used in the treatment of equine endometritis of bacterial origin.
Materials and methods
A retrospective study of laboratory records was performed on aerobic cultures from equine uteri. Samples were collected between January 2010 and December 2017 by veterinary clinicians who specialise in equine reproduction. Samples of the uterus consisted of double-guarded swabs to minimise the risk of contamination. Most of the samples were collected for aetiological diagnosis purposes from subfertile mares, those with clinical endometritis or those animals predisposed to fluid accumulation. The mares undergoing the examination more than once a year varied among the period (2010–2017) and ranged from 40 per cent to 55 per cent of the total; however, only data from the first sampling of the year were included in the study.
Microbiological examination and antimicrobial susceptibility test
All uterine samples were delivered to the microbiology laboratory of the Department of Veterinary Medicine at the University of Perugia, in Central Italy. Uterine swabs were cultured onto 5 per cent defibrinated sheep blood agar, MacConkey’s agar and Mannitol salt agar (Oxoid, Italy). The plates were incubated at 37°C in aerobic conditions and examined at 24 and 48 hours. Bacteria were identified by evaluating colony characteristics, Gram stain reaction, sorbitol fermentation, catalase and oxidase tests, and biochemical characteristics through commercially available API identification systems (bioMérieux, France). Samples were also cultured on Sabouraud dextrose agar with 0.05 mg/ml chloramphenicol (Oxoid) for fungal culture, and plates were incubated at 30°C and 37°C for up to five days.
Kirby-Bauer agar disc diffusion was performed as recommended by the Clinical and Laboratory Standards Institute (CLSI). Briefly, the inoculum was a 0.5 McFarland saline suspension of 24-hour-old colonies. Immediately after standardisation, a sterile swab was moistened with the suspension and inoculated evenly on the surface of a Müller-Hinton agar plate (Oxoid). Antimicrobial discs (Oxoid) were aseptically placed onto the inoculated surface, and the plates were read after 24 hours of incubation at 37°C. Susceptibility testing was performed for the following antimicrobial drugs: amikacin (AMK), gentamicin (GEN), ampicillin (AMP), cefazolin (CFZ), cefquinome (CEF), ceftiofur (CTF), penicillin (PEN), rifampin (RIF), thiamphenicol (TAP), enrofloxacin (ENR) and marbofloxacin (MRB). Zone diameters were manually measured and interpreted as susceptible, intermediate or resistant to the antibiotics according to the Clinical and Laboratory Standards Institute guidelines (2002, 2008, 2012, 2015). Organisms categorised as intermediate were considered to be resistant in order to statistically describe the results.18
The most frequently isolated bacterial species, such as E coli and S equi subspecies zooepidemicus, as well as sexually transmitted bacteria, P aeruginosa and K pneumoniae,23 were selected to assess the statistically significant variation of efficacy of the antimicrobials over time. Statistical analysis was performed using dedicated statistical software package R Statistical Software V.3.3.3 (The R Foundation for Statistical Computing, Vienna, Austria). Isolates were divided into two groups: the first including those detected during the first four years of the study period (2010–2013), and the second those isolated during the last four years (2014–2017), as done in a previous study.24 The proportion of bacterial species susceptible to each antimicrobial was compared between the two periods using the chi-squared test, as appropriate. Cochran-Armitage trend test was used to make sure the antimicrobial susceptibility trends among the years of the study (2010–2017). Results were considered significant if P<0.05.
One or more bacterial species were isolated from 3171 out of 4122 uterine swabs (76.9 per cent). The number of positive samples each year is detailed in figure 1 and ranged from 67 per cent to 81 per cent. More than one bacterial species were detected in 467 of 3171 positive samples (14.7 per cent).
Table 1 describes the bacterial species isolated from uterine swabs between 2010 and 2017. A total of 14 organisms were isolated, with E coli and S equi subspecies zooepidemicus being the most common (27.9 per cent and 24.9 per cent, respectively). Among the non-bacterial organisms, yeasts were detected in 48 out of 3171 uterine swabs (1.5 per cent), while fungi were isolated from 26 of 3171 (0.8 per cent). Fungal organisms were isolated from samples that were also positive for bacteria.
The changing antimicrobial susceptibility pattern of E coli over the years is reported in table 2. A significant increase of antimicrobial susceptibility was found for AMP (P<0.05), CTF (P<0.05) and RIF (P<0.05), while there was a reduction of E coli susceptibility to CEF (P<0.01) (figure 2). Resistance to AMK, GEN, CFZ, PEN, TAP, ENR and MRB was not statistically different between the eight years. The susceptibility pattern of S equi subspecies zooepidemicus is summarised in table 3. No statistically significant change of susceptibility was detected for AMK, GEN, CFZ, CTF, ENR and MRB, while AMP, CEF and PEN were significantly less susceptible over time (P<0.01). On the other hand, S equi subspecies zooepidemicus proved to be more susceptible to RIF (P<0.01) and TAP (P<0.01) compared with the previous years (figure 3). A significant decrease of antimicrobial susceptibility was found for AMK (P<0.05) against K pneumoniae, unlike what happened for CFZ (P<0.05). No statistically significant differences in susceptibility to any of the antimicrobials were found over time for Pseudomonas aeruginosa.
The antimicrobial susceptibility trend analysed by Cochran-Armitage trend test was significant for CEF in E coli and for RIF and TAP in S equi subspecies zooepidemicus However, there were no susceptibility changes over the study period for the remaining antimicrobial molecules.
The awareness on the emergence and spread of antimicrobial resistance has increased the demand for continuous microbiological monitoring and antimicrobial testing in both human and animal medicine. As such, this study was undertaken to see if the antimicrobial susceptibility testing results for bacterial isolates from equine uteri have changed over time.
Not surprisingly, E coli followed by S equi subspecies zooepidemicus were the most common bacteria isolated from the equine reproductive tract. These data confirm data from previous studies,13 18 25 26 although some investigations also reported the common isolation of β-haemolytic streptococci.3 15 19 27 28 E coli and S equi subspecies zooepidemicus are reported to be the cause of severe uterine inflammatory processes18 resulting in poor reproductive performance of mares. Sampling technique differences could explain the high prevalence of E coli detection in this study: uterine swabs come into contact only with the superficial component of the endometrium, while β-haemolytic streptococci are responsible for deeper infections.28 As a result, the prevalence of S equi subspecies zooepidemicus could be underestimated in this study without endometrial biopsy results.
The detection rate of P aeruginosa and K pneumoniae is similar to those obtained by Davis et al,18 but higher than those reported in other previous studies.13 15 19 The low prevalence of Staphylococcus aureus (table 1) is notable because it has been reported as being quite common in the uterus of healthy subjects29 and could be assumed to be even more common in a compromised uterus.
In spite of the fact that monitoring antimicrobial resistance among animal populations is recognised as a needed action for public health, few investigations have been carried out concerning the efficacy of commonly used antibiotics against bacteria infecting the equine reproductive tract.
It is reassuring that the susceptibility of E coli to the main antimicrobial drugs used for treatment changed minimally over the years, allowing veterinary practitioners to vary the therapeutic regimen as necessary. On the other hand, the authors observed a high resistance to AMP when compared with previous results.18 Additionally, the susceptibility of E coli isolates to PEN was significantly lower in this study (8.4 per cent) compared to an 86 per cent susceptibility reported by Albihn et al.13 The aminoglycoside and quinolone efficacy against E coli in this study and others ranges between 75 per cent and 96 per cent.13 15 18 The high susceptibility to these antimicrobials would imply that they are best used for these infections, despite such antibiotic classes are critically important for human treatment; in particular, quinolones are classified as prioritisation criterion 1 in human medicine,30 and thus should be administered exclusively in the case of antimicrobial resistance to other major antibiotics.
S equi subspecies zooepidemicus became significantly more resistant to CEF and PEN over time, particularly during the last year. This difference can be assumed to be because of frequent empirical administration. This assumption could not be tested in this study, but would be worthwhile to test prospectively. The antimicrobial susceptibility of RIF and TAP increased over all the years tested, potentially because such antimicrobials are less commonly used and are only administered to equines after laboratory results are reported.
This study’s antimicrobial susceptibility results are in line with previous findings in Italy15 31 but differ from those of Albihn et al 13 in Sweden. Indeed, the antimicrobial resistance of S equi subspecies zooepidemicus is 17.2–27.8 per cent higher to GEN, AMP and PEN when compared with those in Albihn et al.13 Additionally, susceptibility to fluoroquinolones (5.9–9.7 per cent) is rather different from that observed in previous studies,19 which had 83.1–84.4 per cent of isolates sensitive to quinolones.
This study confirmed that E coli and S equi subspecies zooepidemicus are the most commonly isolated bacterial species from equine uterus in Central Italy, as has been reported from populations in other countries. Other well-known pathogenic bacteria such as P aeruginosa and K pneumoniae are rather rare. Having categorised intermediate organisms as resistant, the authors sound a note of caution with regard to susceptibility profiles of the isolates. This notwithstanding, the high frequency of antimicrobial-resistant bacteria isolated in the present study proceeds in the same way as indicated by surveillance data on antibiotic use in Italy,32 a country with the highest volume of sales of veterinary antimicrobial agents. To tackle this alarming trend, treatment of equine endometritis should not be started before the results of antimicrobial susceptibility testing in order to prevent the overgrowth of resistant bacteria.
However, when empirical use of antibiotics is unavoidable, the prudent use of antimicrobials has to be based on antimicrobial susceptibility monitoring programmes, and balancing the professional obligations of veterinarians to the health of the animals and the prime importance of preserving the efficacy of critically important antibacterial agents for human medicine and public health.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement All data relevant to the study are included in the article.
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