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Retrospective study of bacterial isolates and their antimicrobial susceptibilities in equine uteri during fertility problems
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Research in Veterinary Science 84 (2008) 1–6 www.elsevier.com/locate/rvsc
Retrospective study of bacterial isolates and their antimicrobial susceptibilities in equine uteri during fertility problems R. Frontoso a, E. De Carlo a, M.P. Pasolini b, K. van der Meulen U. Pagnini a, G. Iovane a, L. De Martino a
c,* ,
a
b
Department of Pathology and Animal Health, Infectious Disease Section, Faculty of Veterinary Medicine, University of Naples ‘‘Federico II’’, Via Delpino 1, 80137 Naples, Italy Department of Clinical Sciences, Surgical Section, Faculty of Veterinary Medicine,University of Naples ‘‘Federico II’’, Via Delpino 1, 80137 Naples, Italy c Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium Accepted 24 February 2007
Abstract Bacterial pathogens are a potential cause when a mare fails to conceive to a fertile stallion on a well-managed breeding farm on one or more cycles in the same season. Furthermore, emerging bacterial resistance to commonly used (topical) antibiotics has been demonstrated. In this study, a total of 586 uterine swabs from mares with fertility problems were evaluated and the bacterial isolates were identified and measured for resistance to 10 antibiotics most commonly used during bacterial equine infection. Forty-nine percent of the examined mares were positive at bacteriological investigations. Amongst 347 successful isolations, 31.7% were Streptococcus group C and 18.4% Escherichia (E.) coli, both considered frequently associated with fertility problems. Determination of the antibiotic susceptibility pattern of Streptococcus group C (110 organisms) revealed that only the amoxicillin/clavulanic acid was highly active with 82.7% of the isolates being inhibited. For E. coli, a major number of drugs displayed a high potency. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Mares; Fertility problems; Bacteria; Antimicrobial susceptibilities
Bacterial infections of the uterus are known to be an important cause of reduced fertility in the mare (Asbury, 1986). Potentially pathogenic organisms are introduced during natural breeding, artificial insemination, during and after parturition, during examination and as a result of failure of physical barriers to infection (ie. pneumovagina). When uterine defence mechanisms are functioning properly, they clear bacterial infection without interfering with reproduction (Asbury et al., 1982; Evans et al., 1986; Nikolakopoulos and Watson, 1999). Most cases of infertility can be attributed to a failure of the mare’s natural ability to remove contaminants from her uterus after
*
Corresponding author. Tel.: +32 9 264 7371; fax: +32 9 264 7495. E-mail addresses: [email protected] (K. van der Meulen), luisa. [email protected] (L. De Martino). 0034-5288/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2007.02.008
breeding or ‘‘delay in uterine clearance’’ (LeBlanc, 2003). Subfertile mares often have fluid remaining in their uterus at 24 or 48 h after breeding, while fertile mares have essentially cleared any excess fluid within 6 or 8 h after breeding (LeBlanc, 2003). Previous studies demonstrated that the most common bacterial causes of uterine infections include Streptococcus equi subsp. zooepidemicus, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Bacteroides fragilis and Bacteroides ureolyticus (Dhingra and Sandhu, 1987; Ricketts and Mackintosh, 1987; Fodor et al., 1995; Langoni et al., 1997; LeBlanc, 1999; Szeredi et al., 2003). The purpose of the present study is to provide an update on bacterial isolates in uteri of mares with fertility problems in Campania region, Italy. Therefore, 586 mares (502 Standardbred Trotter and 84 Italian Warmblood) with fertility problems were examined from 1998 to 2004.
2
R. Frontoso et al. / Research in Veterinary Science 84 (2008) 1–6
Mares included were aged from 2 to 23 years (average 11.9 years). They presented one or more of the following fertility problems: – – – –
Barren in preceding season (189 mares). Resorption/abortion in preceding season (78 mares). Resorption in present season (115 mares). Repeated breeding, defined as starting a new oestrus cycle after artificial insemination, in present season (257 mares). – Endometritis (inflammatory cells on cytological smear, fluid in uterine lumen during luteal phase and/or vulval discharge) (201 mares).
The external genitalia were washed twice with iodo povidone 0.1% and dried. Double-guarded, occluded swabs (Equi-Vet Kruuse, Marsley, Denmark) were introduced in the uterus through the cervix by a gloved hand to minimize the risk of contamination by perineal and vaginal bacteria. For conventional bacteriological detection, samples were cultured on Columbia agar base supplemented with 5% sterile defibrinated sheep blood, both with and without colistin (0.015 g/L) and naladixic acid (0.01 g/L) (Oxoid, Milan – Italy). These agar plates were used for isolating, cultivating and determining haemolytic reactions of pathogenic micro-organisms. Plates were incubated under humid, atmospheric (aerobic) conditions at 37 °C for up to 48 h. Cultures were examined at 24 and 48 h. Columbia-based chocolate agar containing 7% equine blood and incubated at 37 °C in 10% CO2 atmosphere for 48 h, were used to isolate Taylorella equigenitalis. Growth of Pseudomonas, K. pneumoniae, haemolytic E. coli and b-haemolytic Streptococci was always considered significant (Shin et al., 1979; Ricketts et al., 1993). Other bacterial isolates were typed and considered as significant if growth was in pure culture or dominating on the agar plate (Langoni et al., 1997). Micro-organisms were identified by standard laboratory methodologies using the API system (BioMe´rieux, Milan – Italy) according to the manufacturer’s instructions. E. coli ATCC 25922, P. aeruginosa ATCC 27853, S. aureus ATCC 29213 and S. equi subsp. zooepidemicus ATCC 53698 were included as quality control micro-organisms. The samples were also cultured for fungi on Sabouraud dextrose agar (Oxoid, Milan – Italy) incubated under normal atmospheric conditions at 37 °C for up to 4 days. No species identification of fungus yeast was performed except for Candida albicans (McGinnis, 1980). Of the 586 examined mares, 287 (or 49%) showed positive results. From 49 of them (17.1%), more than one bacterial species was isolated. A mixed culture of Streptococcus dysgalactiae subsp. equisimilis and E. coli was most common. T. equigenitalis was not detected in any of the mares examined. Table 1 presents an overview of the bacteria that were isolated as well as their frequency of isolation. The bacte-
rial species most frequently isolated was b-haemolytic Streptococcus group C, (110 isolates, 31.7%) (including S. equi subsp. zooepidemicus, S. dysgalactiae subsp. equisimilis and Streptococcus equi subsp. equi), followed by E. coli, (68 isolates, 19.6%), of which four were haemolytic. Other Streptococcus spp., S. aureus, Enterobacteriaceae, P. aeruginosa, Bacillus spp., and Staphylococcus spp. were found at lower frequencies as presented in Table 1. From 7 mares, Candida spp. was isolated in mixed culture with bacteria. No correlation was found between the type of isolated bacterial species and mares with characteristic clinical signs or the age of the mares. Antimicrobial drugs to which susceptibilities were tested, were those for routine testing by veterinary microbiology laboratories regarding horses, as recommended by CLSI-NCCLS (M31-A2, 2002). Results were categorised by using the guidelines recommended by the CLSI/NCCLS for veterinary pathogens (M31-A2, 2002). However, antibiotics characterized as moderately susceptible or intermediately susceptible were included with those characterized as susceptible. For the entire collection of Streptococcus group C, a clear and marked resistance (i.e. only 4.5–8.4% of bacterial strains being inhibited) was observed for kanamycin, gentamicin and enrofloxacin (Table 2a). The highest susceptibility was observed for amoxicillin/clavulanic acid with 82.7% of the isolates being inhibited. The amoxicillin/clavulanic acid also inhibited 89.2% of other Streptococcus spp. Surprisingly, these Streptococcus isolates exhibited variable susceptibility to b-lactam antimicrobial agents, with 57.2% and 75.1% being susceptible to penicillin and ampicillin, respectively. Similar unusual phenotypes being penicillinresistant but ampicillin-susceptible were also reported amongst clinical Enterococcus faecalis isolates (Metzidie et al., 2006). For S. aureus, amoxicillin/clavulanic acid was the most effective antimicrobial agent inhibiting 96.2% of the isolates. Among the 64 E. coli isolates, 73.5%, 71.9% and 78.1% of bacterial strains were inhibited by enrofloxacin, trimethoprim/sulphamethoxazole and amoxicillin/clavulanic acid, respectively. Furthermore, E. coli was susceptible to antibiotics of the aminoglycoside family, e.g. kanamycin (67.2% isolates inhibited) and gentamicin (73.5% isolates inhibited). The four haemolytic E. coli isolates were all highly and uniformly susceptible (100%) to 4 (enrofloxacin, kanamycin, gentamicin, trimethoprin/sulphamethoxazole) of 10 tested antibiotics, while highly resistant to ampicillin and penicillin. The 14 P. aeruginosa isolates were resistant to the majority of the tested antimicrobial agents except to gentamicin (Table 2b). P. aeruginosa is notorious for developing antibiotic resistance and has proven to be difficult to treat in other species (Sauer et al., 2003; Carmen et al., 2005). In our study, almost half of the mares presented a significant growth of micro-organisms that could be responsible for the observed fertility problems. In previous studies, the percentage of mares with fertility problems related to bacterial infection were lower. Redaelli and
R. Frontoso et al. / Research in Veterinary Science 84 (2008) 1–6
3
Table 1 Micro-organisms isolated from uterine swabs between 1998–2004 and their frequency of isolation Micro-organism*
Number of times isolated
Actinobacillus spp. Aeromonas hidrophila Other Aeromonas spp. Bacillus cereus Bacillus licheniformis Other Bacillus spp. Bordetella spp. Chryseomonas luteola Citrobacter gillenii Corynebacterium comparalensis Other Corynebacterium spp. Escherichia coli non-haemolytic Escherichia coli haemolytic Enterobacteriacae Klebsiella mobilis Klebsiella oxytoca Klebsiella pneumonite Other Klebsiella spp. Pasteurella caballi Pasteurella multocida Other Pasteurella spp. Proteus mirabilis Pseudomonas aeruginosa Pseudomonas fluorescens Other Pseudomonas spp. Ralstonia eutropha Shighella spp. Staphylococcus auretis Staphylococcus coagulasi+ Other Staphylococcus spp. Streptococcus group B Streptococcus agalactiae Streptococcus group C** Streptococcus group D Streptococcus group G Streptococcus uberis Other Streptococcus spp. Vibrio spp. Candida spp.
1 3 3 1 6 11 1 1 1 1 1 64 4 19 1 2 2 3 1 1 1 2 14 2 1 1 1 26 4 10 2 5 110 5 1 1 28 1 7
Total
347
Frequency of isolation (%) 0.3 0.9 0.9 0.3 1.7 3.2 0.3 0.3 0.3 0.3 0.3 18.4 1.2 5.5 0.3 0.6 0.6 0.9 0.3 0.3 0.3 0.6 4.0 0.6 0.3 0.3 0.3 7.5 1.2 2.9 0.6 1.4 31.7 1.4 0.3 0.3 8.1 0.3 2.0 100
*
Comprehensive list of antimicrobial agents that could be considered for routine testing by veterinary laboratories regarding horses (CLSI-NCCLS (M31-A2, 2002)). ** Streptococcus group C includes the following species: Streptococcus equi subsp. zooepidemicus, Streptococcus dysgalactiae subsp. equisimilis and Streptococcus equi subsp. equi.
Codazza (1977), Shin et al. (1979), and Ricketts et al. (1993), reported values of 30%, 32%, and 39%, respectively. More recent studies by Baranski et al. (2003) reported values of 66.2%. The variation in the above-mentioned percentages may be due to the increasing use of antibiotics that, in turn, can increase the number of resistant bacterial strains. Antibiotic resistance more and more becomes a health problem of major importance (Oliver et al., 2000; De Graef et al., 2004). In our study, most bacterial isolates were identified as bhaemolytic Streptococcus (group C), followed by E. coli. This is in agreement with previous observations (Koskinen and Katila, 1987; Purswell et al., 1989; Waelchli et al., 1993; Langoni et al., 1997) but in contrast to a more recent
study of Albihn et al. (2003) that demonstrated the overall dominance of E. coli relative to b-haemolytic Streptococci in uterine swab samples. These contrasting data may be due to the different study area and different horse population. Interestingly, among 68 E. coli isolates we only found 4 haemolytic isolates, even though Barrelet (1995) noticed that especially the haemolytic E. coli is considered to be an important cause of fertility problems in the equine uterus. However, as already suggested by Albihn et al. (2003), the diffuse presence of non-haemolytic E. coli strains as a cause of reproductive problems should not be underrated. Similarly, also the well-known uterine pathogens such as P. aeruginosa and K. pneumoniae (Atherton and
4
Table 2 Microbial susceptibility* of the six major bacterial species isolated from uteri of mares in (a) gram-positive bacteria and in (b) gram-negative bacteria Antibiotic tested (1998–2004)
Streptococcus spp. (28 isolated)
S
R
NT
S
R
NT
S
R
NT
82.7 67.7 8.4 39.7 4.5 6.7 65.2 51.3 17.0 15.1
13.9 25.9 71.0 30.1 75.5 67.5 28.6 22.1 74.3 61.1
3.4 6.4 20.6 30.2 20.0 25.8 6.2 26.6 8.7 23.8
89.2 75.1 14.3 53.5 21.4 – 57.2 35.7 14.3 35.6
3.7 7.1 50.0 7.1 21.4 71.4 21.4 28.6 35.7 21.4
7.1 17.8 35.7 39.4 57.2 28.6 21.4 35.7 50.0 42.8
96.2 61.5 92.4 88.5 92.3 92.3 65.4 88.6 88.5 69.2
3.8 30.8 3.8 3.8 – – 30.8 – 3.8 23.2
– 7.7 3.8 7.7 7.7 7.7 3.8 11.4 7.7 7.6
Escherichia coli (64 isolated)
Panel b. Gram-negative bacteria Amoxixillin/clavulanic acid Ampicillin Enrofloxacin Erythromycin Kanamycin Gentamycin Penicillin Rifampicin Trimethoprirn/sulphamethoxazole Tetracyclin * **
Staphylococcus aureus (26 isolated)
Haemolytic Escherichia coli (4 isolated)
Pseudomonas aeruginosa (14 isolated)
S
R
NT
S
R
NT
S
R
NT
71.9 37.5 78.1 3.2 67.2 73.5 12.5 6.3 73.5 39.0
15.6 48.4 9.4 81.3 18.8 15.6 73.4 79.7 15.5 43.8
12.5 14.1 12.5 15.5 14.0 10.9 14.1 14.0 11.0 17.2
50.0 – 100.0 50.0 100.0 100.0 – 50.0 100.0 75.0
50.0 100.0 – 50.0 – – 100.0 50.0 – 25.0
– – – – – – – – – –
– – 35.7 – 28.5 78.6 – – – –
92.9 92.9 57.2 92.9 64.4 21.4 100.0 92.9 92.9 100.0
7.1 7.1 7.1 7.1 7.1 – – 7.1 7.1 –
Zone diameter interpretive standards and minimal inhibitory concentration (MIC) breakpoints for veterinary pathogens as recommended by CLSI-NCCLS (M31-A2, 2002). Streptococcus group C includes: S. equi subsp. equi, S. equi subsp. zooepidemicus and S. dysgalactiae subsp. equisimilis.
R. Frontoso et al. / Research in Veterinary Science 84 (2008) 1–6
Panel a. Gram-positive bacteria Amoxixillin/clavulanic acid Ampicillin Enrofloxacin Erythromycin Kanamycin Gentamycin Penicillin Rifampicin Trimethoprim/sulphamethoxazole Tetracyclin
Streptococcus group C** (110 isolated)
R. Frontoso et al. / Research in Veterinary Science 84 (2008) 1–6
Pitt, 1982; Brown et al., 1979) were only observed in a low percentage of mares in the present study. S. aureus, which is reported to be rather frequently present in the healthy equine uterus (Ricketts et al., 1993), was isolated at a high frequency from mares with fertility problems (20.5%). Therefore, we suggest that S. aureus may cause fertility problems in the equine uterus. Further studies in healthy and diseased mares are necessary to strengthen our suggestion. The examination for fungi resulted in seven isolates, nearly always in mixed culture with bacteria. It is difficult to determine which infection occurred first, bacterial or fungal. We believe that primal fungal infections are more common because of the wide use of antibiotics and the increasingly intensive management and manipulation of reproduction in mares (Blue, 1987; LeBlanc, 1997). Even though differences were observed in the antimicrobial sensitivity of the isolated bacteria, our study could clearly indicate a marked resistance of many isolates to more than one of the antibiotics used. This is in agreement with other studies that reported a constantly increasing number of micro-organisms resistant to more than one antibiotic (Cohen, 1992; Gibbons, 1992; Siu, 2002). Further studies are needed to elucidate the relative importance of this antibiotic resistance in view of infertility problems. In this context, we would also like to mention a variation existing between susceptibility profiles of pathogens isolated from the mares in different countries (Ensink et al., 1993; Albihn et al., 2003; Sauer et al., 2003). One may speculate that country or even horse population-specific chemotherapeutic susceptibility testing is necessary to determine efficacy of antimicrobial agents in the treatment of uterine infections in mares. We conclude that, in order to allow a rapid and effective antimicrobial treatment, it is crucial that the prevalence of resistance is continuously monitored. As such, veterinarians can choose the appropriate antimicrobial drug, thereby increasing the possibility of resolution of an infection and, consequently, increasing the pregnancy rate in mares. References Albihn, A., Baverud, V., Magnusson, U., 2003. Uterine microbiology and antimicrobial susceptibility in isolated bacteria from mares with fertility problems. Acta Vet. Scand. 44, 121–129. Asbury, A.C. 1986. Endometritis in the mare. In: Morrow, D.A., Sounders, W.B. Current Therapy in Theriogenology, Philadelphia, USA, pp. 718–722. Asbury, A.C., Schultz, K.T., Klesius, P.H., Foster, G.W., Washburn, S.M., 1982. Factors affecting phagocytosis of bacteria by neutrophils in the mare’s uterus. J. Reprod. Fertil. Suppl. 32, 151–159. Atherton, J.G., Pitt, T.L., 1982. Types of Pseudomonas aeruginosa isolated from horses. Equine Vet. J. 14, 329–332. Baranski, W., Janowski, T., Ras, A., Podhalicz-Dziegie, M.R., Stre Zek, R., 2003. Relationship between bacteriological and cytological examination of the mares’ uterus during foal heat and fertility rate. Bull. Vet. Inst. Pulawy. 47, 427–433. Barrelet, A., 1995. Laboratory aids to routine gynaecological management. Proc. Equin e Stud. Medicine and AI Course British Equine Vet. Assoc. New-market, UK, 52–56.
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Blue, M.G., 1987. Mycotic endometritis in mares. Review and clinical observations. N.Z. Vet. J. 35, 181–183. Brown, J.E., Corstvet, R.E., Stratton, L.G., 1979. A study of Klebsiella pneumoniae infection in the uterus of the mare. Am. J. Vet. Res. 40 (11), 1523–1530. Carmen, J.C., Roeder, B.L., Nelson, J.L., Robison Ogilvie, R.L., Robison, R.A., Bruce Schaalje, G., Pitt, W.G., 2005. Treatment of biofilm infections on implants with low-frequency ultrasound and antibiotics. Am. J. Infect. Control 33 (2), 78–82. CLSI-NCCLS (National Committee for Clinical Laboratory Standards), 2002. Zone diameter interpretive standards and minimal inhibitory concentration (MIC) breakpoints for veterinary pathogens. M31-A2. Cohen, M.L., 1992. Epidemiology of drug resistance: implications for a post-antimicrobial era. Science 257, 1050–1055. De Graef, E.M., Decostere, A., Devriese, L.A., Haesebrouck, F., 2004. Antibiotic resistance among fecal indicator bacteria from healthy individually owned and kennel dogs. Microb. Drug Resist. 10, 65– 69. Dhingra, P.N., Sandhu, K.S., 1987. Culture and sensitivity of aerobic bacteria isolated from mares suffering from reproductive disorders. Indian J. Anim. Sci. 57, 525–527. Ensink, J.M., van Klingeren, B., Houwers, D.J., Klein, W.R., Vulto, A.G., 1993. In-vitro susceptibility to antimicrobial drugs of bacterial isolates from horses in The Netherlands. Equine Vet. J. 25, 309–313. Evans, M.J., Hamer, J.M., Gason, I.M., Graham, C.S., Asbury, A.C., Irvine, C.H.G., 1986. Clearance of bacteria and non-antigenic markers following intrauterine inoculation into maiden mares: effect of steroid hormone environment. Theriogenology 26, 37–50. Fodor, L., Szenci, O., Peters, M., Varga, J., Szemeredi, G., Wyszoczky, F., 1995. Isolation of Bacteroides ureolyticus from vaginal discharge of mares. Zentralbl. Veterinarmed. B 42, 415–420. Gibbons, A., 1992. Exploring new strategies to fight drug-resistant microbes. Science 257, 1036–1038. Koskinen, E., Katila, T., 1987. Uterine involution, ovarian activity and fertility in the post-partum mare. J. Reprod. Fert. 35, 733–734. Langoni, H., Alvarenga, M.A., Papa, F.O., Sakamoto, C., Baldini, S., Listoni, F.J.P., 1997. Aerobic, microaerobic ana anaerobic bacteria in equine endometritis. Pferdeheilkunde 13, 548. LeBlanc, M.M., 1997. The equine endometrium and the pathophysiology of endometritis. Proc. Reprod. Pathol., 78–84. LeBlanc, M.M., 1999. Diseases of the uterus. In: Colahan, P.T., Merrit, M., Moore, J.N., Mayhew, I.G.J. (Eds.), Equine Medicine and Surgery. W.B. Saunders Company, Philadelphia, pp. 1165–1173. LeBlanc, M.M., 2003. Persistent mating induced endometritis in the mares: pathogenesis, diagnosis and treatment. In: Ball, B.A. (Ed.), Recent Advances in Equine Reproduction. International Veterinary Information Service, Ithaca, New York, USA (www.ivis.org). McGinnis, M.R., 1980. Yeast Identification. Laboratory Handbook of Medical Mycology. Academic Press Inc Ltd., London.GB, pp. 37–411. Metzidie, E., Manolis, E.N., Pournaras, S., Sofianou, D., Tsakris, A., 2006. Spread of an unusual penicillin- and imipenem-resistant but ampicillin-susceptible phenotype among Enterococcus faecalis clinical isolates. J. Antimicrob. Chemother. 57 (1), 158–160. Nikolakopoulos, E., Watson, E.D., 1999. Uterine contractility is necessary for the clearance of intrauterine fluid but not bacterial infusion in the mare. Theriogenology 52, 413–423. Oliver, A., Canto`n, R., Campos, P., Baquero, F., Blazquez, J., 2000. High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection. Science 288, 1251–1254. Purswell, B.J., Ley, W.B., Sriranganathan, N., Bowen, J.M., 1989. Aerobic and anaerobic bacterial flora in the postpartum mare. Equine Vet. Sci. 9, 141–144. Redaelli, G., Codazza, D., 1977. The incidence, pathogenicity and pathology of bacterial and fungal species in the mare’s uterus. Folia Vet. Lat. 8, 198–204. Ricketts, S.W., Mackintosh, ME., 1987. Role of anaerobic bacteria in equine endometritis. J. Reprod. Fertil. 35, 343–351.
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Ricketts, S.W., Young, A., Medici, E.B., 1993. Uterine clitorial cultures. In: Mckinnon, A.O., Voss, J.L. (Eds.), Equine Reproduction. Lea and Febringer, Philadelphia, pp. 234–245. Sauer, P., Andrew, S.E., Lassaline, M., Gelatt, K.N., Denis, H.M., 2003. Changes in antibiotic resistance in equine bacterial ulcerative keratitis (1991–2000): 65 horses. Vet. Ophthalmol. 6, 309–313. Shin, S.J., Lein, D.H., Anderson, A.L., Nusbaum, S.R., 1979. The bacteriological culture of equine uterine contents in vitro sensitivity of organism isolated and interpretation. J. Reprod. Fert. Suppl. 27, 307–315.
Siu, L.K., 2002. Antibiotics: action and resistance in gram-negative bacteria. J. Microbiol. Immunol. Infect. 35, 1–11. Szeredi, L., Tenk, M., Schiller, I., Revesz, T., 2003. Study of the role of Chlamydia, Mycoplasma, Ureaplasma and other microaerophilic and aerobic bacteria in uterine infections of mares with reproductive disorders. Acta Vet. Hung. 51, 45–52. Waelchli, R.O., Ka¨nzig, M., Gygax, A., Corboz, L., Ru¨sch, P., 1993. The relationship between cycle stage and results of uterine culture in the mare. Zentralb. Veterinarmed. 40, 569–575.
Research in Veterinary Science 84 (2008) 1–6 www.elsevier.com/locate/rvsc
Retrospective study of bacterial isolates and their antimicrobial susceptibilities in equine uteri during fertility problems R. Frontoso a, E. De Carlo a, M.P. Pasolini b, K. van der Meulen U. Pagnini a, G. Iovane a, L. De Martino a
c,* ,
a
b
Department of Pathology and Animal Health, Infectious Disease Section, Faculty of Veterinary Medicine, University of Naples ‘‘Federico II’’, Via Delpino 1, 80137 Naples, Italy Department of Clinical Sciences, Surgical Section, Faculty of Veterinary Medicine,University of Naples ‘‘Federico II’’, Via Delpino 1, 80137 Naples, Italy c Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium Accepted 24 February 2007
Abstract Bacterial pathogens are a potential cause when a mare fails to conceive to a fertile stallion on a well-managed breeding farm on one or more cycles in the same season. Furthermore, emerging bacterial resistance to commonly used (topical) antibiotics has been demonstrated. In this study, a total of 586 uterine swabs from mares with fertility problems were evaluated and the bacterial isolates were identified and measured for resistance to 10 antibiotics most commonly used during bacterial equine infection. Forty-nine percent of the examined mares were positive at bacteriological investigations. Amongst 347 successful isolations, 31.7% were Streptococcus group C and 18.4% Escherichia (E.) coli, both considered frequently associated with fertility problems. Determination of the antibiotic susceptibility pattern of Streptococcus group C (110 organisms) revealed that only the amoxicillin/clavulanic acid was highly active with 82.7% of the isolates being inhibited. For E. coli, a major number of drugs displayed a high potency. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Mares; Fertility problems; Bacteria; Antimicrobial susceptibilities
Bacterial infections of the uterus are known to be an important cause of reduced fertility in the mare (Asbury, 1986). Potentially pathogenic organisms are introduced during natural breeding, artificial insemination, during and after parturition, during examination and as a result of failure of physical barriers to infection (ie. pneumovagina). When uterine defence mechanisms are functioning properly, they clear bacterial infection without interfering with reproduction (Asbury et al., 1982; Evans et al., 1986; Nikolakopoulos and Watson, 1999). Most cases of infertility can be attributed to a failure of the mare’s natural ability to remove contaminants from her uterus after
*
Corresponding author. Tel.: +32 9 264 7371; fax: +32 9 264 7495. E-mail addresses: [email protected] (K. van der Meulen), luisa. [email protected] (L. De Martino). 0034-5288/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2007.02.008
breeding or ‘‘delay in uterine clearance’’ (LeBlanc, 2003). Subfertile mares often have fluid remaining in their uterus at 24 or 48 h after breeding, while fertile mares have essentially cleared any excess fluid within 6 or 8 h after breeding (LeBlanc, 2003). Previous studies demonstrated that the most common bacterial causes of uterine infections include Streptococcus equi subsp. zooepidemicus, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Bacteroides fragilis and Bacteroides ureolyticus (Dhingra and Sandhu, 1987; Ricketts and Mackintosh, 1987; Fodor et al., 1995; Langoni et al., 1997; LeBlanc, 1999; Szeredi et al., 2003). The purpose of the present study is to provide an update on bacterial isolates in uteri of mares with fertility problems in Campania region, Italy. Therefore, 586 mares (502 Standardbred Trotter and 84 Italian Warmblood) with fertility problems were examined from 1998 to 2004.
2
R. Frontoso et al. / Research in Veterinary Science 84 (2008) 1–6
Mares included were aged from 2 to 23 years (average 11.9 years). They presented one or more of the following fertility problems: – – – –
Barren in preceding season (189 mares). Resorption/abortion in preceding season (78 mares). Resorption in present season (115 mares). Repeated breeding, defined as starting a new oestrus cycle after artificial insemination, in present season (257 mares). – Endometritis (inflammatory cells on cytological smear, fluid in uterine lumen during luteal phase and/or vulval discharge) (201 mares).
The external genitalia were washed twice with iodo povidone 0.1% and dried. Double-guarded, occluded swabs (Equi-Vet Kruuse, Marsley, Denmark) were introduced in the uterus through the cervix by a gloved hand to minimize the risk of contamination by perineal and vaginal bacteria. For conventional bacteriological detection, samples were cultured on Columbia agar base supplemented with 5% sterile defibrinated sheep blood, both with and without colistin (0.015 g/L) and naladixic acid (0.01 g/L) (Oxoid, Milan – Italy). These agar plates were used for isolating, cultivating and determining haemolytic reactions of pathogenic micro-organisms. Plates were incubated under humid, atmospheric (aerobic) conditions at 37 °C for up to 48 h. Cultures were examined at 24 and 48 h. Columbia-based chocolate agar containing 7% equine blood and incubated at 37 °C in 10% CO2 atmosphere for 48 h, were used to isolate Taylorella equigenitalis. Growth of Pseudomonas, K. pneumoniae, haemolytic E. coli and b-haemolytic Streptococci was always considered significant (Shin et al., 1979; Ricketts et al., 1993). Other bacterial isolates were typed and considered as significant if growth was in pure culture or dominating on the agar plate (Langoni et al., 1997). Micro-organisms were identified by standard laboratory methodologies using the API system (BioMe´rieux, Milan – Italy) according to the manufacturer’s instructions. E. coli ATCC 25922, P. aeruginosa ATCC 27853, S. aureus ATCC 29213 and S. equi subsp. zooepidemicus ATCC 53698 were included as quality control micro-organisms. The samples were also cultured for fungi on Sabouraud dextrose agar (Oxoid, Milan – Italy) incubated under normal atmospheric conditions at 37 °C for up to 4 days. No species identification of fungus yeast was performed except for Candida albicans (McGinnis, 1980). Of the 586 examined mares, 287 (or 49%) showed positive results. From 49 of them (17.1%), more than one bacterial species was isolated. A mixed culture of Streptococcus dysgalactiae subsp. equisimilis and E. coli was most common. T. equigenitalis was not detected in any of the mares examined. Table 1 presents an overview of the bacteria that were isolated as well as their frequency of isolation. The bacte-
rial species most frequently isolated was b-haemolytic Streptococcus group C, (110 isolates, 31.7%) (including S. equi subsp. zooepidemicus, S. dysgalactiae subsp. equisimilis and Streptococcus equi subsp. equi), followed by E. coli, (68 isolates, 19.6%), of which four were haemolytic. Other Streptococcus spp., S. aureus, Enterobacteriaceae, P. aeruginosa, Bacillus spp., and Staphylococcus spp. were found at lower frequencies as presented in Table 1. From 7 mares, Candida spp. was isolated in mixed culture with bacteria. No correlation was found between the type of isolated bacterial species and mares with characteristic clinical signs or the age of the mares. Antimicrobial drugs to which susceptibilities were tested, were those for routine testing by veterinary microbiology laboratories regarding horses, as recommended by CLSI-NCCLS (M31-A2, 2002). Results were categorised by using the guidelines recommended by the CLSI/NCCLS for veterinary pathogens (M31-A2, 2002). However, antibiotics characterized as moderately susceptible or intermediately susceptible were included with those characterized as susceptible. For the entire collection of Streptococcus group C, a clear and marked resistance (i.e. only 4.5–8.4% of bacterial strains being inhibited) was observed for kanamycin, gentamicin and enrofloxacin (Table 2a). The highest susceptibility was observed for amoxicillin/clavulanic acid with 82.7% of the isolates being inhibited. The amoxicillin/clavulanic acid also inhibited 89.2% of other Streptococcus spp. Surprisingly, these Streptococcus isolates exhibited variable susceptibility to b-lactam antimicrobial agents, with 57.2% and 75.1% being susceptible to penicillin and ampicillin, respectively. Similar unusual phenotypes being penicillinresistant but ampicillin-susceptible were also reported amongst clinical Enterococcus faecalis isolates (Metzidie et al., 2006). For S. aureus, amoxicillin/clavulanic acid was the most effective antimicrobial agent inhibiting 96.2% of the isolates. Among the 64 E. coli isolates, 73.5%, 71.9% and 78.1% of bacterial strains were inhibited by enrofloxacin, trimethoprim/sulphamethoxazole and amoxicillin/clavulanic acid, respectively. Furthermore, E. coli was susceptible to antibiotics of the aminoglycoside family, e.g. kanamycin (67.2% isolates inhibited) and gentamicin (73.5% isolates inhibited). The four haemolytic E. coli isolates were all highly and uniformly susceptible (100%) to 4 (enrofloxacin, kanamycin, gentamicin, trimethoprin/sulphamethoxazole) of 10 tested antibiotics, while highly resistant to ampicillin and penicillin. The 14 P. aeruginosa isolates were resistant to the majority of the tested antimicrobial agents except to gentamicin (Table 2b). P. aeruginosa is notorious for developing antibiotic resistance and has proven to be difficult to treat in other species (Sauer et al., 2003; Carmen et al., 2005). In our study, almost half of the mares presented a significant growth of micro-organisms that could be responsible for the observed fertility problems. In previous studies, the percentage of mares with fertility problems related to bacterial infection were lower. Redaelli and
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3
Table 1 Micro-organisms isolated from uterine swabs between 1998–2004 and their frequency of isolation Micro-organism*
Number of times isolated
Actinobacillus spp. Aeromonas hidrophila Other Aeromonas spp. Bacillus cereus Bacillus licheniformis Other Bacillus spp. Bordetella spp. Chryseomonas luteola Citrobacter gillenii Corynebacterium comparalensis Other Corynebacterium spp. Escherichia coli non-haemolytic Escherichia coli haemolytic Enterobacteriacae Klebsiella mobilis Klebsiella oxytoca Klebsiella pneumonite Other Klebsiella spp. Pasteurella caballi Pasteurella multocida Other Pasteurella spp. Proteus mirabilis Pseudomonas aeruginosa Pseudomonas fluorescens Other Pseudomonas spp. Ralstonia eutropha Shighella spp. Staphylococcus auretis Staphylococcus coagulasi+ Other Staphylococcus spp. Streptococcus group B Streptococcus agalactiae Streptococcus group C** Streptococcus group D Streptococcus group G Streptococcus uberis Other Streptococcus spp. Vibrio spp. Candida spp.
1 3 3 1 6 11 1 1 1 1 1 64 4 19 1 2 2 3 1 1 1 2 14 2 1 1 1 26 4 10 2 5 110 5 1 1 28 1 7
Total
347
Frequency of isolation (%) 0.3 0.9 0.9 0.3 1.7 3.2 0.3 0.3 0.3 0.3 0.3 18.4 1.2 5.5 0.3 0.6 0.6 0.9 0.3 0.3 0.3 0.6 4.0 0.6 0.3 0.3 0.3 7.5 1.2 2.9 0.6 1.4 31.7 1.4 0.3 0.3 8.1 0.3 2.0 100
*
Comprehensive list of antimicrobial agents that could be considered for routine testing by veterinary laboratories regarding horses (CLSI-NCCLS (M31-A2, 2002)). ** Streptococcus group C includes the following species: Streptococcus equi subsp. zooepidemicus, Streptococcus dysgalactiae subsp. equisimilis and Streptococcus equi subsp. equi.
Codazza (1977), Shin et al. (1979), and Ricketts et al. (1993), reported values of 30%, 32%, and 39%, respectively. More recent studies by Baranski et al. (2003) reported values of 66.2%. The variation in the above-mentioned percentages may be due to the increasing use of antibiotics that, in turn, can increase the number of resistant bacterial strains. Antibiotic resistance more and more becomes a health problem of major importance (Oliver et al., 2000; De Graef et al., 2004). In our study, most bacterial isolates were identified as bhaemolytic Streptococcus (group C), followed by E. coli. This is in agreement with previous observations (Koskinen and Katila, 1987; Purswell et al., 1989; Waelchli et al., 1993; Langoni et al., 1997) but in contrast to a more recent
study of Albihn et al. (2003) that demonstrated the overall dominance of E. coli relative to b-haemolytic Streptococci in uterine swab samples. These contrasting data may be due to the different study area and different horse population. Interestingly, among 68 E. coli isolates we only found 4 haemolytic isolates, even though Barrelet (1995) noticed that especially the haemolytic E. coli is considered to be an important cause of fertility problems in the equine uterus. However, as already suggested by Albihn et al. (2003), the diffuse presence of non-haemolytic E. coli strains as a cause of reproductive problems should not be underrated. Similarly, also the well-known uterine pathogens such as P. aeruginosa and K. pneumoniae (Atherton and
4
Table 2 Microbial susceptibility* of the six major bacterial species isolated from uteri of mares in (a) gram-positive bacteria and in (b) gram-negative bacteria Antibiotic tested (1998–2004)
Streptococcus spp. (28 isolated)
S
R
NT
S
R
NT
S
R
NT
82.7 67.7 8.4 39.7 4.5 6.7 65.2 51.3 17.0 15.1
13.9 25.9 71.0 30.1 75.5 67.5 28.6 22.1 74.3 61.1
3.4 6.4 20.6 30.2 20.0 25.8 6.2 26.6 8.7 23.8
89.2 75.1 14.3 53.5 21.4 – 57.2 35.7 14.3 35.6
3.7 7.1 50.0 7.1 21.4 71.4 21.4 28.6 35.7 21.4
7.1 17.8 35.7 39.4 57.2 28.6 21.4 35.7 50.0 42.8
96.2 61.5 92.4 88.5 92.3 92.3 65.4 88.6 88.5 69.2
3.8 30.8 3.8 3.8 – – 30.8 – 3.8 23.2
– 7.7 3.8 7.7 7.7 7.7 3.8 11.4 7.7 7.6
Escherichia coli (64 isolated)
Panel b. Gram-negative bacteria Amoxixillin/clavulanic acid Ampicillin Enrofloxacin Erythromycin Kanamycin Gentamycin Penicillin Rifampicin Trimethoprirn/sulphamethoxazole Tetracyclin * **
Staphylococcus aureus (26 isolated)
Haemolytic Escherichia coli (4 isolated)
Pseudomonas aeruginosa (14 isolated)
S
R
NT
S
R
NT
S
R
NT
71.9 37.5 78.1 3.2 67.2 73.5 12.5 6.3 73.5 39.0
15.6 48.4 9.4 81.3 18.8 15.6 73.4 79.7 15.5 43.8
12.5 14.1 12.5 15.5 14.0 10.9 14.1 14.0 11.0 17.2
50.0 – 100.0 50.0 100.0 100.0 – 50.0 100.0 75.0
50.0 100.0 – 50.0 – – 100.0 50.0 – 25.0
– – – – – – – – – –
– – 35.7 – 28.5 78.6 – – – –
92.9 92.9 57.2 92.9 64.4 21.4 100.0 92.9 92.9 100.0
7.1 7.1 7.1 7.1 7.1 – – 7.1 7.1 –
Zone diameter interpretive standards and minimal inhibitory concentration (MIC) breakpoints for veterinary pathogens as recommended by CLSI-NCCLS (M31-A2, 2002). Streptococcus group C includes: S. equi subsp. equi, S. equi subsp. zooepidemicus and S. dysgalactiae subsp. equisimilis.
R. Frontoso et al. / Research in Veterinary Science 84 (2008) 1–6
Panel a. Gram-positive bacteria Amoxixillin/clavulanic acid Ampicillin Enrofloxacin Erythromycin Kanamycin Gentamycin Penicillin Rifampicin Trimethoprim/sulphamethoxazole Tetracyclin
Streptococcus group C** (110 isolated)
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Pitt, 1982; Brown et al., 1979) were only observed in a low percentage of mares in the present study. S. aureus, which is reported to be rather frequently present in the healthy equine uterus (Ricketts et al., 1993), was isolated at a high frequency from mares with fertility problems (20.5%). Therefore, we suggest that S. aureus may cause fertility problems in the equine uterus. Further studies in healthy and diseased mares are necessary to strengthen our suggestion. The examination for fungi resulted in seven isolates, nearly always in mixed culture with bacteria. It is difficult to determine which infection occurred first, bacterial or fungal. We believe that primal fungal infections are more common because of the wide use of antibiotics and the increasingly intensive management and manipulation of reproduction in mares (Blue, 1987; LeBlanc, 1997). Even though differences were observed in the antimicrobial sensitivity of the isolated bacteria, our study could clearly indicate a marked resistance of many isolates to more than one of the antibiotics used. This is in agreement with other studies that reported a constantly increasing number of micro-organisms resistant to more than one antibiotic (Cohen, 1992; Gibbons, 1992; Siu, 2002). Further studies are needed to elucidate the relative importance of this antibiotic resistance in view of infertility problems. In this context, we would also like to mention a variation existing between susceptibility profiles of pathogens isolated from the mares in different countries (Ensink et al., 1993; Albihn et al., 2003; Sauer et al., 2003). One may speculate that country or even horse population-specific chemotherapeutic susceptibility testing is necessary to determine efficacy of antimicrobial agents in the treatment of uterine infections in mares. We conclude that, in order to allow a rapid and effective antimicrobial treatment, it is crucial that the prevalence of resistance is continuously monitored. As such, veterinarians can choose the appropriate antimicrobial drug, thereby increasing the possibility of resolution of an infection and, consequently, increasing the pregnancy rate in mares. References Albihn, A., Baverud, V., Magnusson, U., 2003. Uterine microbiology and antimicrobial susceptibility in isolated bacteria from mares with fertility problems. Acta Vet. Scand. 44, 121–129. Asbury, A.C. 1986. Endometritis in the mare. In: Morrow, D.A., Sounders, W.B. Current Therapy in Theriogenology, Philadelphia, USA, pp. 718–722. Asbury, A.C., Schultz, K.T., Klesius, P.H., Foster, G.W., Washburn, S.M., 1982. Factors affecting phagocytosis of bacteria by neutrophils in the mare’s uterus. J. Reprod. Fertil. Suppl. 32, 151–159. Atherton, J.G., Pitt, T.L., 1982. Types of Pseudomonas aeruginosa isolated from horses. Equine Vet. J. 14, 329–332. Baranski, W., Janowski, T., Ras, A., Podhalicz-Dziegie, M.R., Stre Zek, R., 2003. Relationship between bacteriological and cytological examination of the mares’ uterus during foal heat and fertility rate. Bull. Vet. Inst. Pulawy. 47, 427–433. Barrelet, A., 1995. Laboratory aids to routine gynaecological management. Proc. Equin e Stud. Medicine and AI Course British Equine Vet. Assoc. New-market, UK, 52–56.
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