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In vitro antimicrobial susceptibility of equine clinical isolates from France, 2006–2016
Accepted Manuscript Title: In vitro antimicrobial susceptibility of equine clinical isolates from France, 2006–2016 Authors: Rachel DUCHESNE, Sophie CASTAGNET, Karine MAILLARD, Sandrine PETRY, Vincent CATTOIR, Jean-Christophe GIARD, Albertine LEON PII: DOI: Reference:
S2213-7165(19)30071-2 https://doi.org/10.1016/j.jgar.2019.03.006 JGAR 890
To appear in: Received date: Revised date: Accepted date:
20 September 2018 5 March 2019 6 March 2019
Please cite this article as: DUCHESNE R, CASTAGNET S, MAILLARD K, PETRY S, CATTOIR V, GIARD J-Christophe, LEON A, In vitro antimicrobial susceptibility of equine clinical isolates from France, 2006–2016, Journal of Global Antimicrobial Resistance (2019), https://doi.org/10.1016/j.jgar.2019.03.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
In vitro antimicrobial susceptibility of equine clinical isolates from France, 2006-2016
Running title: Antimicrobial susceptibility of equine pathogens from France
Vincent CATTOIR4,5, Jean-Christophe GIARD2, Albertine LEON1,2
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Rachel DUCHESNE1,2, Sophie CASTAGNET1,2, Karine MAILLARD1, Sandrine PETRY3,
LABÉO Frank Duncombe, 14053 Caen, France.
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Normandie Univ, UNICAEN, U2RM, 14033 Caen, France.
3
ANSES, Dozulé Laboratory for Equine Diseases, Bacteriology Unit, 14430 Goustranville,
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France.
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University Hospital of Rennes, Department of Clinical Microbiology, 35033 Rennes, France.
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National Reference Center for Antimicrobial Resistance (lab Enterococci), 35033 Rennes,
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France.
*Corresponding author: Dr Albertine LEON, 1 route de Rosel-Saint Contest, 14 053 Caen 4,
France.
Phone:
+33-2-31-47-19-39;
Fax:
+33-2-31-47-19-19;
Email:
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Cedex
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[email protected]
Highlights
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First study of antimicrobial susceptibility of 25 813 equine strains in France. High level of MDR strains in equine clinical samples as reservoir for human infection. Need to promote more appropriate use of antibiotics by veterinarians.
Abstract Objectives: The aim of this study was to analyze the evolution of the antimicrobial susceptibility of equine pathogens isolated from clinical samples from 2006 to 2016. -1-
Methods: A collection of 25,813 bacterial isolates were studied, clustered according to their origins (respiratory tract, cutaneous, genital and other) and analyzed for their antimicrobial susceptibility using the disk diffusion method. Results: The most frequently isolated pathogens were group C Streptococci (27.6%), Escherichia coli (20.0%), Staphylococcus aureus (7.8%), Pseudomonas aeruginosa (4.0%),
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Enterobacter spp (3.4%), Klebsiella pneumoniae (2.4%) and Rhodococcus equi (1.8%). 9,512
isolates were from respiratory samples (36.8%), 7,689 from genital origin (29.8%) and 4,083
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from cutaneous samples (15.8%). Over the 11-year period, the frequency of multidrugresistant (MDR) strains fluctuated between 6.4% and 20.4% for group C Streptococci and between 17% and 37.7% for K. pneumoniae. From 2006 to 2009, 24.5% to 43.0% of S.
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aureus isolates were MDR, after 2009 the level did not exceeded 27.6%. For E. coli and
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decreased thereafter (22.5% to 26.3%).
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Enterobacter spp, these levels were mostly higher than 30.0% until 2012 but significantly
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Conclusions: This study is the first large scale analysis of equine pathogens both by the
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number of samples and the duration of the study. Our results pointed out the presence of high level of MDR strains and the need to support veterinary antimicrobial stewardship to
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encourage proper use of antibiotic.
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Keywords: antimicrobial susceptibility testing, horse pathogens, epidemiology, multidrug
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resistance
1. Introduction The overuse and misuse of antimicrobials in both human and veterinary medicines have contributed to the emergence and spread of multidrug-resistant (MDR, bacteria resistant to three or more antimicrobial classes) bacterial pathogens around the world. Since the end of
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the 20th century, this phenomenon has become a major public health problem and a priority for international institutions such as the World Health Organization (WHO, www.who.int), the Food and Agriculture Organization of the United Nations (FAO, www.fao.org) and the World Organization for Animal Health (OIE, www.oie.int), which published a list of recommendations and guidelines to counteract MDR emergence. This problem is all the more
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alarming since the transmission of MDR pathogenic strains from animal to human has also been observed [1,2].
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In this context, several studies aiming to describe the evolution and the prevalence of
resistance in equine pathogens have been conducted in different countries, such as South Africa [3], Canada [4,5], Switzerland [6] and the United Kingdom [7]. These studies reported
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a high level of MDR among horse pathogens, which complicates therapy with commonly
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used antimicrobial drugs (i.e., penicillins, cephalosporins, gentamicin or cotrimoxazole). This
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underlines the importance to perform antimicrobial susceptibility testing, in order to optimize
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antimicrobial therapy in horses and to reduce the risk of development of resistance. Moreover,
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because of the close relationships between horses and humans, these bacteria may act as a reservoir of MDR bacteria.
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In France, a national surveillance network for antimicrobial resistance in pathogenic bacteria of animal origin (RESAPATH, www.resapath.anses.fr), which has already been in place for
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cattle since 1982 and has been extended to horses in 2007, provides annual data about antimicrobial susceptibility. Moreover, a French national plan (named ECOANTIBIO) was
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conducted over the period 2012-2016 with the objectives to reduce the exposure of animals to veterinary antibiotics by 25.0% and then to preserve therapeutic arsenal of antibiotics in a durable way. The data recently collected showed a decrease in the exposure of animals to antibiotics of 37.0% over the period 2012-2016 (http://agriculture.gouv.fr/ecoantibio). The decline was even more marked for critical antibiotics (-75.0% for fluoroquinolones and -
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81.0% for last-generation cephalosporins), and resulted in regulatory restrictions for their prescription since 2016. Following this positive dynamic, the ECOANTIBIO 2 Plan has been set up for the period 2017-2021 (http://agriculture.gouv.fr/le-plan-ecoantibio-2-2017-2021). Nevertheless, data on MDR strains isolated from horses in France are rarely available. The objective of this work was to describe the antimicrobial susceptibility profiles of equine
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pathogens over an 11-year period (2006-2016). For this purpose, more than 25,000 isolates recovered from different sources of infection from horses in France were collected and
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phenotypically analyzed.
2. Materials and methods
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2.1. Bacterial isolates
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From January 2006 to December 2016, 25,813 bacterial isolates were retrospectively included
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in the study (2006: n=1,582; 2007: n=1,923; 2008: n=2,281; 2009: n=2,392; 2010: n=2,605;
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2011: n=2,915; 2012: n=3,058; 2013: n=2,477; 2014: n=2,373; 2015: n=2,312; 2016:
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n=1,895). These isolates were issued from samples of horses collected by numerous practitioners all over France and all analyses were carried out in the veterinary microbiology
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diagnostic’s unit of our lab. They were all recovered from horses with a suspected bacterial infection (with no prior antimicrobial treatment) and were classified into four groups
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according to the origin of infection: respiratory (including nasal, nasopharyngeal, tracheal, bronchial and guttural pouch washes, swabs, secretions or aspirations), cutaneous, genital and
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others.
2.2. Phenotypic identification To isolate and identify strains, non-selective (Columbia agar with 5 % sheep blood) and selective media (Columbia CNA agar with 5 % sheep blood and Eosin methylene blue agar) were used. When more than three different species were isolated in a sample, it was
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considered as “contaminated” and no further analysis was performed. Isolates were identified definitively by Gram staining, followed by either standard biochemical tests or commercially available identification systems, such as API and VITEK 2 Compact® systems (bioMérieux, Marcy l’Étoile, France), or MALDI-TOF mass spectrometry technology (Microflex; Bruker
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Daltonics, Bremen, Germany) according to the manufacturers' instructions.
2.3. Antimicrobial susceptibility testing
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Antimicrobial susceptibility testing (AST) was performed using the disc diffusion method on Mueller-Hinton agar (enriched with 5.0% sheep blood for Streptococcus spp.) according to the recommendations of the European Committee on Antimicrobial Susceptibility Testing
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(EUCAST; http://www.eucast.org). After 18-24 hours of incubation at 37°C, diameters of
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growth inhibition around the discs were measured using OSIRIS (Bio-Rad, Marnes-la-
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Coquette, France) or SIRSCAN (I2A, Montpellier, France) and interpreted as susceptible,
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intermediate or resistant according to EUCAST clinical breakpoints. Of note, bacteria that
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were categorized as “intermediate” were further considered as “resistant” in our study. Bacterial isolates were evaluated for their susceptibilities to β-lactams, polymyxins,
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aminoglycosides, tetracyclines, macrolides, rifampicin, sulphonamides and fluoroquinolones (Table S2). Until November 2011, AST was performed without Gram distinction whereas
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from December 2011, two different panels have been tested, one for Gram-positives and another for Gram-negatives (Table S2). The sulphonamides were tested alone just from
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December 2011 to December 2012 because the combination of trimethoprim and sulfamethoxazole was privileged in the field conditions. Amikacin was systematically tested until July 2016 then only if other antimicrobial compounds were inactive on Gram-negative bacteria.
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2.4. Statistical analysis Statistical analysis was performed using the XLStat software. The chi-square test was undertaken to test for significant changes in antimicrobial resistance among each bacterial species over the period. The temporal trends in the prevalence of antimicrobial resistance were investigated for each antimicrobial agent using the Cochran Armitage trend test. For
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these analyses, P values <0.05 were considered as significant.
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3. Results 3.1. Origin and identification of equine clinical isolates
Out of 25,813 isolates, respiratory isolates represented the most important group (n=9,512;
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36.8%), followed by genital (n=7,689; 29.8%), cutaneous (n=4,083; 15.8%) and other isolates
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(n=4,529; 17.6%).
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The most frequently encountered bacterial species responsible for horse infections were group
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C Streptococci (27.6%), Escherichia coli (20.0%), Staphylococcus aureus (7.8%),
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Pseudomonas aeruginosa (4.0%), Enterobacter spp. (3.4%), Klebsiella pneumoniae (2.4%) and Rhodococcus equi (1.8%) (Figure 1A). Their distributions according to the source of
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infection group are presented in Figure 1B.
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3.2. Antimicrobial susceptibility of equine pathogens Group C Streptococci were the most frequent bacteria isolated (27.6%) mainly from genital
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(41.1%) and respiratory (30.6%) samples (Figure 1). Among the 7 124 clinical isolates, we observed that penicillins remained active against all streptococcal isolates since no resistant phenotype was observed over this period (Table 1A). Tetracycline was the only antimicrobial for which the number of resistant bacteria significantly increased between 2006 and 2016 with 22.0% and 82.0% of resistant strains, respectively (Table 1A). Moreover, as shown in Figure
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2A, most of these tetracycline-resistant strains came from the genital origin. Moreover, less than 23.0% of the group C Streptococci were resistant to aminoglycosides, macrolides, rifampicin and sulphonamides. Because Streptococci are intrinsically resistant to low levels to aminoglycosides, we used high concentration (HC) discs of aminoglycosides for the AST and less than 3.0% of group C Streptococci were categorized as highly resistant to gentamicin HC.
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As for tetracycline resistant isolates, we observed that strains resistant to aminoglycosides and
macrolides were mainly isolated from genital samples, 54.8% (397/724) and 57.3% (351/612)
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respectively (Figure 2A, Table S1).
R. equi represented 1.8% of total bacteria and was as expected mainly isolated from respiratory (61.3%) samples (Figure 1). Note that in R. equi strains from the group “Other”,
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more than 47.0% were from digestive origin (Table S1). As the treatment of R. equi infection
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is based on the use of the combination of macrolides and rifampicin, only the resistance of
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these two antibiotics has been studied (Figure 2B) and no increase of resistance of R. equi to
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erythromycin and rifampicin has been observed over the 11-years study period (Table 1B).
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S. aureus represented near 8.0% of total bacteria isolated in infected horses between 2006 and 2016 (Figure 1A). As shown in Figure 1B, most of S. aureus were isolated from cutaneous
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(41.5%), genital (18.8%) and respiratory (12.8%) samples. Thirty-two to 55.0% of our samples were resistant to penicillin and ampicillin according to the period whereas 99 to
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77.0% remained susceptible to ampicillin when clavulanic acid was present (Table 1C). The level of S. aureus resistant to oxacillin and cefoxitin (used as markers for methicillin
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resistance from 2012 and extended to other ß-lactams) were around 17.0% (except in 2012 with 30.0%). Thus, it appeared that this evolution stopped in 2013 followed by a stable level of MRSA around 17.0% (Table 1C). In addition, except the high level of streptomycin resistant strains isolated between 2006 and 2010 (more than 30.0%), less than 30.0% of the strains were resistant to the other aminoglycosides, macrolides, rifampicin, sulphonamides
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and quinolones (Table 1C). Based on the sample’s origin, those from the respiratory tract contained less resistant-strains of S. aureus (12.8%; 257/2012) than the others, with 18.8% (379/2012) and 41.5% (835/2012) for genital and cutaneous tract, respectively (Figure 2C, Table S1). P. aeruginosa represents 4.0% of total bacteria and was mainly isolated from respiratory (39.9%) and genital (28.9%) samples (Figure 1). As only cefquinome (C4G),
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gentamicin, amikacin and marbofloxacin are clinically relevant, only these four antimicrobial agents have been included in our study (Table 1D). As shown in Figure 2D and Table S1, the
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majority of resistant strains have been obtained from respiratory tract samples (39.9%; 413/1034) followed by genital swabs (28.9%; 299/1034) and cutaneous samples (18.8%; 194/1034).
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E. coli was the second most frequent bacteria isolated (20%) mainly from genital (51.4%),
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respiratory (23.7%) and cutaneous (12.7%) samples (Figure 1). The majority of resistant E.
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coli strains have been isolated from genital samples (51.4%; 2653/5159) (Figure 2E, Table
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S1). Streptomycin was the antimicrobial for which the highest number of resistant bacteria
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has been observed among the eleven years. However, this number decreased rising from 76.0% of E. coli resistant to streptomycin in 2012 to 33.0% in 2016 (Table 1E). Overall, less
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than 12.0% of E. coli strains were resistant to Ceftiofur (C3G), cefquinome (C4G) and fluoroquinolones; and around 40.0% of E. coli strains were resistant to amoxicillin.
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Enterobacter spp represents 3.4% of total bacteria and was mainly isolated from respiratory (48.0%), genital (24.0%) and cutaneous (15.0%) samples (Figure 1). Among the
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aminoglycosides, streptomycin was the antimicrobial for which a largest number of resistant strains have been observed. Indeed, between 2006 and 2012 the level of streptomycin resistant Enterobacter spp oscillated between 32.0% and 64.0% after which time it stayed under 30.0% (Table 1F). Tetracyclines were the second antimicrobials for which an important level of resistance was observed, especially in 2008-2010 and 2012-2014. During these periods, the
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percentages of resistant strains could reach approximately 37.0% to 56.0% (Table 1F). Moreover, less than 10.0% of the strains were resistant to amikacine and marbofloxacine. When we looked at the distribution of resistant strains according to the samples origins, we observed that the cutaneous group contained the least number of resistant bacteria (15.4%; 135/878) compared to the respiratory (47.9%; 421/878) and genital samples (23.8%; 208/878)
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(Figure 2F, Table S1).
K. pneumoniae represented 2.4% of all the bacteria and were mainly isolated from genital
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(43.0%) and respiratory (40.0%) samples (Figure 1). Among the K. pneumoniae isolated, the level of resistant bacteria was never more than 24.0% for amoxicillin-clavulanic acid and 10.0% for cephalosporins and aminoglycosides with the exception for streptomycin (Table
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1G). As for Enterobacter, streptomycin was the only aminoglycoside for which a high level
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of resistance has been observed. In 2009 this level reached 72.0% but remained under 30.0%
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since 2011. Sporadic high level of resistances (>30.0%) were observed for tetracyclines (2008
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and 2009), sulphonamides (2016) and nalidixic acid (2013). Genital and respiratory samples
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(Figure 2G, Table S1).
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contained most of the resistant strains, 43.1% (270/626) and 40.4% (253/626) respectively
3.3. Multidrug resistant bacteria
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Table 2 shows the overall level and trend of the presence of MDR bacteria (defined as nonsusceptible to at least three different classes of antibiotic usually efficient) according to
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bacterial species. Because few antimicrobial compounds have been tested against P. aeruginosa and R. equi, these species were excluded from the analysis. It is worth noting that for E. coli and Enterobacter spp, the level of MDR remained similar during the 2006-2012 period (average of resistance was 35.7% and 36.1% respectively), followed by a decrease from 2013 to 2016 (average of resistance was 23.3% and 25.3% respectively). For K.
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pneumoniae, the level of MDR varied from 17% to 39.7% within the 2006-2016 period with a relative stability around 21% with the exception of 2009 (39.7%), 2013 (35.5%) and 2016 (38.7%). Moreover, for group C Streptococci, the level of MDR decreased from 20.4% in 2006 to 9% in 2011 and then to 7% in 2013-2015 period, with an increase observed in 2012 (13.8%) and 2016 (10.7%) only. Finally, for S. aureus, the percentage of MDR was 43% in
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2006, after a decrease to 24.5% in 2007, this level increased to 41.1% in 2009. After 2009,
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from 18% to 27% of S. aureus strains were MDR.
4. Discussion
Our large scale study including more than 25,000 clinical isolates from equine origin
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harvested over eleven years brings an important panorama of the antimicrobial resistance of
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group C Streptococci, Rhodococcus equi, Pseudomonas aeruginosa, Enterobacteria and
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Staphylococcus aureus. Group C Streptococci identified include Streptococcus dysgalactiae
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subsp. equisimilis and the two most common streptococcal species from horses, Streptococcus
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equi subsp. zooepidemicus (which is part of the upper respiratory tract microbiota and can be virulent when it invades the lower respiratory tract), and Streptococcus equi subsp. equi (the
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causative agent of Strangles) [8].Our observations are in accordance with those made by Johns and Adams (2015) where no resistance to penicillins and increased resistance to
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tetracyclines have been observed [7]. In contrast with the South African and English studies performed between 1999-2012 and 2007-2013 respectively [3,7], no increase of gentamicin
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resistance was observed over our 11-years study period. Less than 3.0% of group C Streptococci were categorized as resistant in our study while more than 70.0% of streptococcal isolates were resistant in the South-African study (n=286) [3]. The reason explaining this important discordance need to be still elucidate.
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R. equi is a soil organism carried in the gut of herbivores. This facultative intracellular pathogen is the most common cause of severe pneumonia in foals [9]. In the past two decades, R. equi has emerged as an opportunistic human pathogen that affects immunocompromised patients [10]. Infections caused by this species are clinically well characterized in horses and the treatment is based on the use of the combination of macrolides and rifampicin.
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Interestingly, as opposed to the literature [11-13], no increase of resistance of R. equi to these two antimicrobial agents has been observed in our study.
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S. aureus is part of the normal cutaneous flora in human and animals. However, S. aureus is a major cause of nosocomial and community acquired infection in France and worldwide [14]. This opportunistic pathogen induces to several pathologies with various degree of severity
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[14]. Our results were lower than those reported in the literature where more than 60.0% of
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staphylococci from equine samples were resistant to penicillin [3,6]. In a recent study, 1,396
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S. aureus strains isolated from horses between 2007 and 2013 have been genetically analyzed.
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In this population, 6% of the strains were resistant to methicillin [15]. Over the period
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analyzed (2007 to 2013) an increased rate of Staphylococcus resistant to methicillin (MRSA) has been observed with 1.0% in 2007 to 11.0% in 2011 [15]. Since 2013, our results revealed
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that the level of such resistant was relatively stable around 17%. P. aeruginosa is a major opportunistic pathogen that causes many infections in
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immunosuppressed humans and in people suffering from cystic fibrosis [16]. In horses, it is often associated to endometritis in mares and represents a serious problem due to its ability to
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form biofilm as well as its multi resistance to antimicrobials [17-19]. As described in the literature, we observed over the eleven years that few antimicrobial agents remained active against this bacterium as cefquinome (C4G), gentamicin, amikacin and marbofloxacine [3,6]. E. coli is naturally present in the digestive tract of humans and animals and may be responsible for gastro-intestinal disorders, urinary tract infection and septicemia in rare cases
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[20]. In our study, an important decrease in resistant to streptomycin was observed (from 76% in 2012 to 33% in 2016). Moreover, level of resistance to amoxicillin (around 40%), which was similar to what observed for human clinical samples [21,22]. Note that less than 10% of E. coli strains were resistant to cephalosporins and/or fluoroquinolones. The use of these last drugs is regulated since their designation as “critically important” by the World Health
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Organization and French government in order to limit their consumption. Resistance to third
and fourth generation cephalosporins may be related to the production of cephalosporinase,
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extended spectrum β-lactamase (ESBL) or carbapenemase and became an increasing
therapeutic problem in equine infection by enterobacteria such as E. coli in the same way as for humans [21,22]. In this context, it could be interesting to determine the incidence of
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enzyme-producing E. coli in our cephalosporins resistant strains.
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In a large lower proportion than E. coli, two other Enterobacteria were also analyzed:
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Enterobacter spp. (3.4%) and K. pneumoniae (2.4%).
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Enterobacter species, especially E. aerogenes and E. cloacae have been associated with
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nosocomial infections and can cause numerous pathologies such as pneumonia, septicemia, and urinary tract and wound infections [23]. Against these isolates, amikacine and
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marbofloxacine remained the two anti-microbial compounds for which bacteria have the least resistance (less than 10%).
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K. pneumoniae is part of the normal urogenital and intestinal flora of humans and animals [24]. In horses, this bacterium can be responsible of metritis, infertility and pneumonia [25].
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In the literature, data on susceptibility of K. pneumoniae to common used antimicrobials in equine medicine are contradictory. Our data were in agreement with the German survey where isolates from mare uterine infections were very sensitive to commonly used antimicrobials [26], but were in contrast with two other studies that found an important prevalence of multidrug-resistant Klebsiella [3,27].
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By pooling all the data, an overview of MDR was shown. For Enterobacteria, according to the strain and the period, the rate of MDR were from 17% to 39%. On the other hand, the percentages of MDR strains of group C Streptococci decreased until around 7% (in 20132015). This level was relatively low but should be maintained with the rational use of antimicrobial drugs. Interestingly, 43% of S. aureus were MDR in 2006 versus 18% to 25%
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after 2012. Except erythromycin, rifampicin and amoxicillin-clavulanate, French equine
practitioners use all other antimicrobial compounds usually without any previous
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microbiological characterization increasing the risk of non-adapted treatment.
It may be hypothesized that the reduction of MDR strains of Staphylococcus over the period studied may be linked to the French national ECOANTIBIO Plan (2012-2016) aiming to the
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sensitization of practitioners to a reasoned use of antimicrobials and leading to an important
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decrease (37%) in the exposure of animals to antibiotics. In 2013, Hughes and collaborators
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showed that in UK only 0.8% (2/281) equine veterinarians had antimicrobial use guidelines
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and that 56% of licensed antimicrobial prescriptions were over the recommended dose
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rate [28]. It is legitimate to believe that empirical practices may, in large part, contribute to the high prevalence of MDR and our results argue for the crucial role of information as well as
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the warning against the problem of pathogens that will be difficult to fight. To our knowledge, this work is the first large scale retrospective study conducted in France over an eleven years
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period with more than 25,000 samples analyzed for their antimicrobial susceptibilities.
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Acknowledgements
We would like to thank the technical staff at the Veterinary Microbiology unit of LABÉO Frank Duncombe for the achievement of antimicrobial susceptibility tests.
Declarations
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Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Competing Interests: All other authors declare no competing interest. Ethical Approval: Not required.
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[17] Allen JL, Begg AP, Browning GF. Outbreak of equine endometritis caused by a genotypically identical strain of Pseudomonas aeruginosa. J Vet Diag Invest 2011; 23: 1236– 39. doi: 10.1177/1040638711425589. [18] Ferris RA, McCue PM, Borlee GI, Glapa KE, Martin KH, Mangalea MR, Hennet ML, Wolfe LM, Broeckling CD, Borlee BR. Model of chronic equine endometritis involving a aeruginosa
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[22] Maddox TW, Williams NJ, Clegg PD, O’Donnell AJ, Dawson S, Pinchbeck GL. Longitudinal study of antimicrobial-resistant commensal Escherichia coli in the faeces of in
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systems of horses, dogs and cats as determined in the BfT-GermVet monitoring program 2004-2006. Berl Munch Tierarztl Wochenschr 2007; 120: 402–11.
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[28] Hughes L., Pinchbeck G., Callaby R., Dawson S., Clegg P., Williams N. Antimicrobial
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prescribing practice in UK equine veterinary practice. Equine Vet. J. 2013; 45: 141-147. doi:
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10.1111/j.2042-3306.2012.00602.x.
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Legends Figure 1: Distribution of 25,813 antimicrobial susceptibility tests (2006-2016) in relation
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A
N
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with the principal microorganisms identified (A) and sample group (B).
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Figure 2: Total (blue) and resistant (red) relevant Gram-positive (A, B, C) and Gram-negative (D, E, F, G) bacteria isolated per year and sample group. PEN: penicillin; AMP/AMX: ampicillin or amoxicillin; AMC: amoxicillin-clavulanic acid; CEF: ceftiofur; CEQ: cefquinome; STR: streptomicin; KAN: kanamicin; GEN: gentamicin; TET/OT: tetracyclin or oxytetracyclin; ERY: erythromycin; RIF: rifampicin; SUL/SXT:
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sulphonamide or trimethoprim-sulfamethoxazole; ENO: enrofloxacin; MAR: marbofloxacine;
FLU: flumequine; OXA*: oxacillin, marker of methicillin resistance; FOX*: cefoxitin,
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marker of cephalosporin resistance; NAL*: nalidixic acid, marker of fluoroquinolone
A
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ED
M
A
N
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resistance.
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A ED
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N
A
M
A
% of resistant Streptococcus (group C)
Aminoglycosides
Tetracyclines Macrolides
ERY RIF**
ERY** RIF
C
Antibiotic category
Year
Number of strains (n) PEN AMP/AMX Penicillins OXA AMC** 2nd FOX** Cephalosporins 3rd CEF** 4th CEQ** STR** KAN Aminoglycosides GEN** AMK TET/OT** Tetracyclines
A
0.0 0.0 NT 0.0 0.0 0.0 7.0 NT 1.0
0.4 0.2 NT 0.2 0.2 0.0 7.3 NT 2.4 33.4 * 14.4 *
0.2 0.2 NT 0.2 0.2 0.2 6.1 NT 1.6
0.0 0.0 NT 0.0 0.0 0.0 5.9 NT 0.8
0.0 0.0 NT 0.0 0.0 0.0 7.0 NT 2.7 25.8 *
22.2
33.8
1.0
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671
34.0
201 5
2016
703
811
680
768
771
692
0.1 0.1 NT 0.1 0.0 0.0 6.4 NT 2.7 44.1 *
0.1 0.0 0.1 0.2 0.0 0.2 4.9 4.7 2.3
0.0 0.0 0.0 0.0 0.0 0.1 5.9 5.3 1.8 75.7 *
0.1 0.0 0.1 0.0 0.0 0.0 3.4 3.6 1.2 61.5 *
0.0 0.0 0.0 0.0 0.0 0.0 6.2 6.9 0.8
0.1 0.7 0.1 0.7 0.4 0.1 5.5 5.3 0.6 82.1 *
45.3
63.9
5.6*
13.2 *
6.7*
9.1*
6.0
6.5
6.6
8.8
11.1
1.1
0.5
0.6
0.4
1.0
0.1*
0.0
0.3
0.4
15.5 *
6.0*
14.8 *
1.4*
0.6
1.6
23.1 *
0.1*
1.0*
0.0*
4.8*
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Number of strains (n)
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Macrolides Rifampicin
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Year
Antibiotic category
627
0.5
SUL/SXT**
B
554
8.6
Rifampicin Sulphonamides
464
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3rd 4th
AMP/AMOX OXA AMC CEF CEQ STRHC KANHC GENHC TET/OT**
383
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Penicillins
Number of strains (n) PEN
2007 2008 2009 2010 2011 2012 2013 2014
A
Antibiotic category
200 6
M
Year
Cephalosporin s
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Table 1 (continued). Percentage of resistant bacteria per year. (A,B,C) percentage of resistant bacteria for relevant Gram-positive species. (D,E,F,G) percentage of resistant bacteria for relevant Gram-negative species. PEN: penicillin; AMP/AMX: ampicillin or amoxicillin; AMC: amoxicillin-clavulanic acid; CEF: ceftiofur; CEQ: cefquinome; STR: streptomicin; KAN: kanamicin; GEN: gentamicin; TET/OT: tetracyclin or oxytetracyclin; ERY: erythromycin; RIF: rifampicin; SUL/SXT: Sulphonamides or trimethoprim-sulfamethoxazole; ENO: enrofloxacin; MAR: marbofloxacine; FLU: flumequine; OXA: oxacillin, marker of methicillin resistance; FOX: cefoxitin, marker of cephalosporin resistance; NAL: nalidixic acid, marker of fluoroquinolone resistance. * Chi-square test, p.value <0.05; ** CochranArmitage trend test, p.value <0.05; NT: Not tested; NR: Not registered; HC: High concentration.
2006
% of resistant Rhodococcus equi 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016
22
36
50
42
61
70
53
43
45
33
7
13.6 0.0
0.0* 0.0
0.0 2.0
4.8 0.0
1.6 0.0
1.4 4.3
1.9 1.9
0.0 0.0
0.0 0.0
0.0 9.1
0.0 0.0
% of resistant Staphylococcus aureus 2006 2007
2008 2009 2010
2011
2012
114 163 198 190 226 253 185 55.3 35.6* 46.5* 41.6 32.3 32.8 44.3* 55.3 35.6* 46.5* 41.6 32.3 32.8 44.3* NR NR NR NR NR NR 23.8 5.3 1.8 12.6* 14.2 12.4 15.8 23.8* NT NT NT NT NT NT 30.8 0.9 2.5 11.1* 13.2 11.5 16.2 24.3* 1.8 4.3 9.1 12.1 11.5 15.8 24.3* 38.6 31.3 57.6* 65.3 43.4* 27.3* 25.9 NT NT NT NT NT NT 24.3 10.5 7.4 15.2* 18.4 15.0 21.3 23.2 10.5 7.4 15.7 18.4 15.0 21.3 0.0 18.4 8.6* 18.7* 18.4 17.3 16.6 28.1*
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2013 2014 2015 2016 178 42.1 42.1 13.5* 13.5 17.4 13.5* 13.5* 15.2* 18.5 18.5 0.0 20.2
180 39.4 38.9 13.9 13.9 13.9 13.9 13.9 15.0 19.4 18.9 0.0 22.8
186 44.1 44.1 16.7 16.7 18.3 16.7 16.7 19.4 25.8 25.3 0.0 29.6
139 43.9 43.2 15.8 15.8 17.3 15.8 15.1 20.9 23.0 21.6 0.0 27.3
ERY** RIF** SUL/SXT ENO** MAR**
Macrolides Rifampicin Sulphonamides Fluoroquinolones
6.1 4.4 0.9 0.0 0.0
D
5.5 4.3 4.3 1.2 0.6
7.0 7.6 9.2 8.6* 2.2
3.4 2.8* 5.1 4.5 1.1
6.1 2.8 3.3 5.6 3.9
4.8 2.7 2.2 3.8 2.7
5.8 2.9 6.5* 1.4 1.4
2011 528 52.5* 43.6* 10.8 10.6 63.6* NT 15.9* 0.6* 23.9 29.2 NT 7.8 6.3 4.9
2012 577 62.7* 45.4 10.2 9.9 76.1* 25.3 11.8 0.5 24.1 62.7* 7.1 6.1 5.9 4.7
2013 2014 490 491 55.7* 39.7* 46.1 32.8* 9.8 11.2 9.2 11.0 65.1* 59.3 24.9 21.4 11.6 10.4 1.6 1.0 20.4 21.4 25.1* 30.1 10.0 8.1 6.3 6.5 5.5 6.1 3.5 5.1
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2010 467 31.3* 25.1* 8.8 7.9 90.4* NT 10.5 3.4 24.0 26.1 NT 8.1 6.0 4.3
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N
A
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F
2006 2007 2008 2009 350 419 479 521 40.6 33.9 54.1* 56.6 28.9 23.6 48.6* 53.0 3.4 6.7* 8.6 8.6 1.4 6.0* 8.4 8.3 77.1 81.9 88.3 94.8* NT NT NT NT 5.7 8.1* 9.4 11.5 3.4 3.8 2.7 3.8 21.4 20.8 22.8 26.3 24.6 26.7 27.1 28.4 NT NT NT NT 7.7 17.2* 6.1 7.7 4.0 5.0 5.0 4.6 3.4 3.8 3.5 3.8
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% of resistant Escherichia coli
Year Antibiotic category Number of strains (n) AMP/AMX Penicillins AMC CEF 3rd Cephalosporins CEQ** 4th STR** KAN Aminoglycosides GEN AMK** TET/OT Tetracyclines Sulphonamides SUL/SXT** NAL FLU** Quinolones/Fluoroquinolones ENO MAR
2015 493 40.0 34.9 6.7* 6.9* 36.1 8.9 6.9 0.4 24.5 28.2 6.7 6.1 5.3 3.7
2016 344 39.5 31.4 6.1 5.8 33.1 9.0 6.1 0.0 20.6 31.4 4.9 4.9 3.2 2.9
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% of resistant Enterobacter spp
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Year Antibiotic category Number of strains (n) CEF 3rd Cephalosporins CEQ 4th STR** KAN** Aminoglycosides GEN AMK TET/OT** Tetracyclines Sulphonamides SUL/SXT NAL FLU** Quinolones/Fluoroquinolones ENO MAR
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5.9 10.3 4.7 4.0 2.8
2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 50 93 75 102 90 127 153 94 95 96 59 44.0 52.7 34.7* 41.2 34.4 33.1 43.8 46.8 50.5 21.9* 11.9 14.0 12.9 36.0* 44.1 20.0* 51.2* 58.8 35.1* 20.0* 3.1* 10.2 10.0 7.5 6.7 14.7 6.7 8.7 8.5 10.6 1.1* 1.0 0.0 2.0 2.2 2.7 14.7* 8.9 6.3 24.2* 9.6* 3.2 3.1 1.7
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G
2006 59 6.8 3.4 32.2 NT 3.4 1.7 10.2 5.1 NT 23.7 1.7 0.0
2007 2008 113 103 18.6 18.4 8.0 9.7 39.8 62.1* NT NT 17.7* 23.3 4.4 3.9 19.5 43.7* 23.0* 21.4 NT NT 37.2 33.0 19.5* 14.6 0.9 2.9
2009 95 20.0 7.4 64.2 NT 18.9 9.5 40.0 18.9 NT 23.2 13.7 0.0
2010 80 10.0 8.8* 47.5 NT 20.0 5.0 37.5 18.8 NT 20.0 10.0 0.0
2011 75 18.7 8.0 33.3 NT 24.0 9.3 26.7 20.0 NT 21.3 14.7 2.7
2012 110 28.2 11.8* 50.0 37.3 35.5 8.2 56.4* 43.6* 23.6 24.5 20.0 1.8
2013 2014 75 69 12.0* 8.7 8.0 1.4 29.3 18.8 16.0* 10.1 16.0* 8.7 0.0* 1.4 38.7* 43.5* 16.0 15.9 20.0 20.3 18.7 18.8 10.7 8.7 2.7 0.0
2015 61 16.4 8.2 26.2 23.0 18.0 6.6 16.4 19.7 18.0 18.0 14.8 4.9
2016 38 15.8 13.2 23.7 18.4 18.4 5.3 21.1 21.1 21.1 21.1 7.9 2.6
% of resistant Klebsiella pneumoniae
Antibiotic category
Penicillins
Aminoglycosides
6.2 5.8 4.9 2.7 1.3
% of resistant Pseudomonas aeruginosa
Year Antibiotic category Number of strains (n) Cephalosporin 4th CEQ** GEN Aminoglycosides AMK** MAR Fluoroquinolones
Cephalosporins
12.6* 10.5 11.1* 7.4 11.1 7.9 2.5 2.6 0.5 1.1
3rd 4th
Year 2006 2007 2008 2009 Number of strains (n) 55 47 63 58 AMC** 18.2 23.4 14.3 24.1 CEF 9.1 6.4 3.2 10.3 CEQ 5.5 4.3 1.6 1.7 STR** 40.0 29.8 61.9* 72.4 KAN NT NT NT NT GEN 9.1 10.6 3.2 3.4 AMK 0.0 0.0 0.0 0.0
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2010 2011 2012 2013 64 61 66 62 14.1 8.2 10.6 9.7 7.8 1.6 6.1 4.8 6.3 1.6 4.5 3.2 56.3 23.0* 21.2 25.8 NT NT 6.1 1.6 4.7 3.3 9.1 3.2 0.0 0.0 1.5 0.0
2014 2015 2016 62 57 31 9.7 7.0 12.9 1.6 7.0 9.7 1.6 7.0 9.7 19.4 26.3 29.0 0.0 3.5 3.2 0.0 3.5 6.5 0.0 0.0 0.0
TET/OTC SUL/SXT** NAL FLU Quinolones/Fluoroquinolones ENO** MAR Tetracyclines Sulphonamides
12.7 18.2 NT 3.6 1.8 0.0
10.6 30.2* 41.4 15.6* 12.8 3.2 10.3 9.4 NT NT NT NT 12.8 1.6* 13.8* 7.8 2.1 0.0 3.4 1.6 0.0 0.0 0.0 1.6
16.4 8.2 NT 3.3 0.0 0.0
27.3 27.4 24.2 12.3 25.8 21.2 11.3 12.9 19.3 32.3 13.6 32.3* 12.9* 5.3 19.4* 9.1 8.1 6.5 3.5 12.9 9.1* 3.2 4.8 3.5 9.7 6.1 0.0 1.6 0.0 3.2
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R≤10% 1050%
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Table 2. Percentage of bacteria resistant to three or more antimicrobial classes. Staphylococcus aureus 43.0 24.5 38.4 41.1 27.0 27.3 27.6 18.0 20.0 25.3 24.5
Enterobacter spp 20.3 31.9 45.6 41.1 36.3 26.7 50.9 24.0 24.6 26.2 26.3
E. coli
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N A M ED PT CC E A
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Klebsiella pneumoniae 18.2 17.0 23.8 39.7 21.9 21.3 22.7 35.5 22.6 21.1 38.7
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34.0 39.6 34.2 38.8 33.4 34.1 31.4 24.3 23.6 22.5 22.7
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2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016
Streptococcus (group C) 20.4 14.0 12.3 11.6 6.3 9.0 13.8 6.8 6.4 7.5 10.7
S2213-7165(19)30071-2 https://doi.org/10.1016/j.jgar.2019.03.006 JGAR 890
To appear in: Received date: Revised date: Accepted date:
20 September 2018 5 March 2019 6 March 2019
Please cite this article as: DUCHESNE R, CASTAGNET S, MAILLARD K, PETRY S, CATTOIR V, GIARD J-Christophe, LEON A, In vitro antimicrobial susceptibility of equine clinical isolates from France, 2006–2016, Journal of Global Antimicrobial Resistance (2019), https://doi.org/10.1016/j.jgar.2019.03.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
In vitro antimicrobial susceptibility of equine clinical isolates from France, 2006-2016
Running title: Antimicrobial susceptibility of equine pathogens from France
Vincent CATTOIR4,5, Jean-Christophe GIARD2, Albertine LEON1,2
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Rachel DUCHESNE1,2, Sophie CASTAGNET1,2, Karine MAILLARD1, Sandrine PETRY3,
LABÉO Frank Duncombe, 14053 Caen, France.
2
Normandie Univ, UNICAEN, U2RM, 14033 Caen, France.
3
ANSES, Dozulé Laboratory for Equine Diseases, Bacteriology Unit, 14430 Goustranville,
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France.
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1
University Hospital of Rennes, Department of Clinical Microbiology, 35033 Rennes, France.
5
National Reference Center for Antimicrobial Resistance (lab Enterococci), 35033 Rennes,
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4
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France.
*Corresponding author: Dr Albertine LEON, 1 route de Rosel-Saint Contest, 14 053 Caen 4,
France.
Phone:
+33-2-31-47-19-39;
Fax:
+33-2-31-47-19-19;
Email:
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Cedex
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[email protected]
Highlights
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First study of antimicrobial susceptibility of 25 813 equine strains in France. High level of MDR strains in equine clinical samples as reservoir for human infection. Need to promote more appropriate use of antibiotics by veterinarians.
Abstract Objectives: The aim of this study was to analyze the evolution of the antimicrobial susceptibility of equine pathogens isolated from clinical samples from 2006 to 2016. -1-
Methods: A collection of 25,813 bacterial isolates were studied, clustered according to their origins (respiratory tract, cutaneous, genital and other) and analyzed for their antimicrobial susceptibility using the disk diffusion method. Results: The most frequently isolated pathogens were group C Streptococci (27.6%), Escherichia coli (20.0%), Staphylococcus aureus (7.8%), Pseudomonas aeruginosa (4.0%),
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Enterobacter spp (3.4%), Klebsiella pneumoniae (2.4%) and Rhodococcus equi (1.8%). 9,512
isolates were from respiratory samples (36.8%), 7,689 from genital origin (29.8%) and 4,083
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from cutaneous samples (15.8%). Over the 11-year period, the frequency of multidrugresistant (MDR) strains fluctuated between 6.4% and 20.4% for group C Streptococci and between 17% and 37.7% for K. pneumoniae. From 2006 to 2009, 24.5% to 43.0% of S.
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aureus isolates were MDR, after 2009 the level did not exceeded 27.6%. For E. coli and
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decreased thereafter (22.5% to 26.3%).
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Enterobacter spp, these levels were mostly higher than 30.0% until 2012 but significantly
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Conclusions: This study is the first large scale analysis of equine pathogens both by the
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number of samples and the duration of the study. Our results pointed out the presence of high level of MDR strains and the need to support veterinary antimicrobial stewardship to
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encourage proper use of antibiotic.
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Keywords: antimicrobial susceptibility testing, horse pathogens, epidemiology, multidrug
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resistance
1. Introduction The overuse and misuse of antimicrobials in both human and veterinary medicines have contributed to the emergence and spread of multidrug-resistant (MDR, bacteria resistant to three or more antimicrobial classes) bacterial pathogens around the world. Since the end of
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the 20th century, this phenomenon has become a major public health problem and a priority for international institutions such as the World Health Organization (WHO, www.who.int), the Food and Agriculture Organization of the United Nations (FAO, www.fao.org) and the World Organization for Animal Health (OIE, www.oie.int), which published a list of recommendations and guidelines to counteract MDR emergence. This problem is all the more
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alarming since the transmission of MDR pathogenic strains from animal to human has also been observed [1,2].
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In this context, several studies aiming to describe the evolution and the prevalence of
resistance in equine pathogens have been conducted in different countries, such as South Africa [3], Canada [4,5], Switzerland [6] and the United Kingdom [7]. These studies reported
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a high level of MDR among horse pathogens, which complicates therapy with commonly
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used antimicrobial drugs (i.e., penicillins, cephalosporins, gentamicin or cotrimoxazole). This
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underlines the importance to perform antimicrobial susceptibility testing, in order to optimize
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antimicrobial therapy in horses and to reduce the risk of development of resistance. Moreover,
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because of the close relationships between horses and humans, these bacteria may act as a reservoir of MDR bacteria.
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In France, a national surveillance network for antimicrobial resistance in pathogenic bacteria of animal origin (RESAPATH, www.resapath.anses.fr), which has already been in place for
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cattle since 1982 and has been extended to horses in 2007, provides annual data about antimicrobial susceptibility. Moreover, a French national plan (named ECOANTIBIO) was
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conducted over the period 2012-2016 with the objectives to reduce the exposure of animals to veterinary antibiotics by 25.0% and then to preserve therapeutic arsenal of antibiotics in a durable way. The data recently collected showed a decrease in the exposure of animals to antibiotics of 37.0% over the period 2012-2016 (http://agriculture.gouv.fr/ecoantibio). The decline was even more marked for critical antibiotics (-75.0% for fluoroquinolones and -
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81.0% for last-generation cephalosporins), and resulted in regulatory restrictions for their prescription since 2016. Following this positive dynamic, the ECOANTIBIO 2 Plan has been set up for the period 2017-2021 (http://agriculture.gouv.fr/le-plan-ecoantibio-2-2017-2021). Nevertheless, data on MDR strains isolated from horses in France are rarely available. The objective of this work was to describe the antimicrobial susceptibility profiles of equine
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pathogens over an 11-year period (2006-2016). For this purpose, more than 25,000 isolates recovered from different sources of infection from horses in France were collected and
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phenotypically analyzed.
2. Materials and methods
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2.1. Bacterial isolates
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From January 2006 to December 2016, 25,813 bacterial isolates were retrospectively included
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in the study (2006: n=1,582; 2007: n=1,923; 2008: n=2,281; 2009: n=2,392; 2010: n=2,605;
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2011: n=2,915; 2012: n=3,058; 2013: n=2,477; 2014: n=2,373; 2015: n=2,312; 2016:
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n=1,895). These isolates were issued from samples of horses collected by numerous practitioners all over France and all analyses were carried out in the veterinary microbiology
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diagnostic’s unit of our lab. They were all recovered from horses with a suspected bacterial infection (with no prior antimicrobial treatment) and were classified into four groups
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according to the origin of infection: respiratory (including nasal, nasopharyngeal, tracheal, bronchial and guttural pouch washes, swabs, secretions or aspirations), cutaneous, genital and
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others.
2.2. Phenotypic identification To isolate and identify strains, non-selective (Columbia agar with 5 % sheep blood) and selective media (Columbia CNA agar with 5 % sheep blood and Eosin methylene blue agar) were used. When more than three different species were isolated in a sample, it was
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considered as “contaminated” and no further analysis was performed. Isolates were identified definitively by Gram staining, followed by either standard biochemical tests or commercially available identification systems, such as API and VITEK 2 Compact® systems (bioMérieux, Marcy l’Étoile, France), or MALDI-TOF mass spectrometry technology (Microflex; Bruker
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Daltonics, Bremen, Germany) according to the manufacturers' instructions.
2.3. Antimicrobial susceptibility testing
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Antimicrobial susceptibility testing (AST) was performed using the disc diffusion method on Mueller-Hinton agar (enriched with 5.0% sheep blood for Streptococcus spp.) according to the recommendations of the European Committee on Antimicrobial Susceptibility Testing
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(EUCAST; http://www.eucast.org). After 18-24 hours of incubation at 37°C, diameters of
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growth inhibition around the discs were measured using OSIRIS (Bio-Rad, Marnes-la-
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Coquette, France) or SIRSCAN (I2A, Montpellier, France) and interpreted as susceptible,
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intermediate or resistant according to EUCAST clinical breakpoints. Of note, bacteria that
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were categorized as “intermediate” were further considered as “resistant” in our study. Bacterial isolates were evaluated for their susceptibilities to β-lactams, polymyxins,
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aminoglycosides, tetracyclines, macrolides, rifampicin, sulphonamides and fluoroquinolones (Table S2). Until November 2011, AST was performed without Gram distinction whereas
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from December 2011, two different panels have been tested, one for Gram-positives and another for Gram-negatives (Table S2). The sulphonamides were tested alone just from
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December 2011 to December 2012 because the combination of trimethoprim and sulfamethoxazole was privileged in the field conditions. Amikacin was systematically tested until July 2016 then only if other antimicrobial compounds were inactive on Gram-negative bacteria.
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2.4. Statistical analysis Statistical analysis was performed using the XLStat software. The chi-square test was undertaken to test for significant changes in antimicrobial resistance among each bacterial species over the period. The temporal trends in the prevalence of antimicrobial resistance were investigated for each antimicrobial agent using the Cochran Armitage trend test. For
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these analyses, P values <0.05 were considered as significant.
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3. Results 3.1. Origin and identification of equine clinical isolates
Out of 25,813 isolates, respiratory isolates represented the most important group (n=9,512;
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36.8%), followed by genital (n=7,689; 29.8%), cutaneous (n=4,083; 15.8%) and other isolates
N
(n=4,529; 17.6%).
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The most frequently encountered bacterial species responsible for horse infections were group
M
C Streptococci (27.6%), Escherichia coli (20.0%), Staphylococcus aureus (7.8%),
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Pseudomonas aeruginosa (4.0%), Enterobacter spp. (3.4%), Klebsiella pneumoniae (2.4%) and Rhodococcus equi (1.8%) (Figure 1A). Their distributions according to the source of
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infection group are presented in Figure 1B.
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3.2. Antimicrobial susceptibility of equine pathogens Group C Streptococci were the most frequent bacteria isolated (27.6%) mainly from genital
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(41.1%) and respiratory (30.6%) samples (Figure 1). Among the 7 124 clinical isolates, we observed that penicillins remained active against all streptococcal isolates since no resistant phenotype was observed over this period (Table 1A). Tetracycline was the only antimicrobial for which the number of resistant bacteria significantly increased between 2006 and 2016 with 22.0% and 82.0% of resistant strains, respectively (Table 1A). Moreover, as shown in Figure
-6-
2A, most of these tetracycline-resistant strains came from the genital origin. Moreover, less than 23.0% of the group C Streptococci were resistant to aminoglycosides, macrolides, rifampicin and sulphonamides. Because Streptococci are intrinsically resistant to low levels to aminoglycosides, we used high concentration (HC) discs of aminoglycosides for the AST and less than 3.0% of group C Streptococci were categorized as highly resistant to gentamicin HC.
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As for tetracycline resistant isolates, we observed that strains resistant to aminoglycosides and
macrolides were mainly isolated from genital samples, 54.8% (397/724) and 57.3% (351/612)
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respectively (Figure 2A, Table S1).
R. equi represented 1.8% of total bacteria and was as expected mainly isolated from respiratory (61.3%) samples (Figure 1). Note that in R. equi strains from the group “Other”,
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more than 47.0% were from digestive origin (Table S1). As the treatment of R. equi infection
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is based on the use of the combination of macrolides and rifampicin, only the resistance of
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these two antibiotics has been studied (Figure 2B) and no increase of resistance of R. equi to
M
erythromycin and rifampicin has been observed over the 11-years study period (Table 1B).
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S. aureus represented near 8.0% of total bacteria isolated in infected horses between 2006 and 2016 (Figure 1A). As shown in Figure 1B, most of S. aureus were isolated from cutaneous
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(41.5%), genital (18.8%) and respiratory (12.8%) samples. Thirty-two to 55.0% of our samples were resistant to penicillin and ampicillin according to the period whereas 99 to
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77.0% remained susceptible to ampicillin when clavulanic acid was present (Table 1C). The level of S. aureus resistant to oxacillin and cefoxitin (used as markers for methicillin
A
resistance from 2012 and extended to other ß-lactams) were around 17.0% (except in 2012 with 30.0%). Thus, it appeared that this evolution stopped in 2013 followed by a stable level of MRSA around 17.0% (Table 1C). In addition, except the high level of streptomycin resistant strains isolated between 2006 and 2010 (more than 30.0%), less than 30.0% of the strains were resistant to the other aminoglycosides, macrolides, rifampicin, sulphonamides
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and quinolones (Table 1C). Based on the sample’s origin, those from the respiratory tract contained less resistant-strains of S. aureus (12.8%; 257/2012) than the others, with 18.8% (379/2012) and 41.5% (835/2012) for genital and cutaneous tract, respectively (Figure 2C, Table S1). P. aeruginosa represents 4.0% of total bacteria and was mainly isolated from respiratory (39.9%) and genital (28.9%) samples (Figure 1). As only cefquinome (C4G),
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gentamicin, amikacin and marbofloxacin are clinically relevant, only these four antimicrobial agents have been included in our study (Table 1D). As shown in Figure 2D and Table S1, the
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majority of resistant strains have been obtained from respiratory tract samples (39.9%; 413/1034) followed by genital swabs (28.9%; 299/1034) and cutaneous samples (18.8%; 194/1034).
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E. coli was the second most frequent bacteria isolated (20%) mainly from genital (51.4%),
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respiratory (23.7%) and cutaneous (12.7%) samples (Figure 1). The majority of resistant E.
A
coli strains have been isolated from genital samples (51.4%; 2653/5159) (Figure 2E, Table
M
S1). Streptomycin was the antimicrobial for which the highest number of resistant bacteria
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has been observed among the eleven years. However, this number decreased rising from 76.0% of E. coli resistant to streptomycin in 2012 to 33.0% in 2016 (Table 1E). Overall, less
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than 12.0% of E. coli strains were resistant to Ceftiofur (C3G), cefquinome (C4G) and fluoroquinolones; and around 40.0% of E. coli strains were resistant to amoxicillin.
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Enterobacter spp represents 3.4% of total bacteria and was mainly isolated from respiratory (48.0%), genital (24.0%) and cutaneous (15.0%) samples (Figure 1). Among the
A
aminoglycosides, streptomycin was the antimicrobial for which a largest number of resistant strains have been observed. Indeed, between 2006 and 2012 the level of streptomycin resistant Enterobacter spp oscillated between 32.0% and 64.0% after which time it stayed under 30.0% (Table 1F). Tetracyclines were the second antimicrobials for which an important level of resistance was observed, especially in 2008-2010 and 2012-2014. During these periods, the
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percentages of resistant strains could reach approximately 37.0% to 56.0% (Table 1F). Moreover, less than 10.0% of the strains were resistant to amikacine and marbofloxacine. When we looked at the distribution of resistant strains according to the samples origins, we observed that the cutaneous group contained the least number of resistant bacteria (15.4%; 135/878) compared to the respiratory (47.9%; 421/878) and genital samples (23.8%; 208/878)
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(Figure 2F, Table S1).
K. pneumoniae represented 2.4% of all the bacteria and were mainly isolated from genital
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(43.0%) and respiratory (40.0%) samples (Figure 1). Among the K. pneumoniae isolated, the level of resistant bacteria was never more than 24.0% for amoxicillin-clavulanic acid and 10.0% for cephalosporins and aminoglycosides with the exception for streptomycin (Table
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1G). As for Enterobacter, streptomycin was the only aminoglycoside for which a high level
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of resistance has been observed. In 2009 this level reached 72.0% but remained under 30.0%
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since 2011. Sporadic high level of resistances (>30.0%) were observed for tetracyclines (2008
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and 2009), sulphonamides (2016) and nalidixic acid (2013). Genital and respiratory samples
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(Figure 2G, Table S1).
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contained most of the resistant strains, 43.1% (270/626) and 40.4% (253/626) respectively
3.3. Multidrug resistant bacteria
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Table 2 shows the overall level and trend of the presence of MDR bacteria (defined as nonsusceptible to at least three different classes of antibiotic usually efficient) according to
A
bacterial species. Because few antimicrobial compounds have been tested against P. aeruginosa and R. equi, these species were excluded from the analysis. It is worth noting that for E. coli and Enterobacter spp, the level of MDR remained similar during the 2006-2012 period (average of resistance was 35.7% and 36.1% respectively), followed by a decrease from 2013 to 2016 (average of resistance was 23.3% and 25.3% respectively). For K.
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pneumoniae, the level of MDR varied from 17% to 39.7% within the 2006-2016 period with a relative stability around 21% with the exception of 2009 (39.7%), 2013 (35.5%) and 2016 (38.7%). Moreover, for group C Streptococci, the level of MDR decreased from 20.4% in 2006 to 9% in 2011 and then to 7% in 2013-2015 period, with an increase observed in 2012 (13.8%) and 2016 (10.7%) only. Finally, for S. aureus, the percentage of MDR was 43% in
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2006, after a decrease to 24.5% in 2007, this level increased to 41.1% in 2009. After 2009,
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from 18% to 27% of S. aureus strains were MDR.
4. Discussion
Our large scale study including more than 25,000 clinical isolates from equine origin
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harvested over eleven years brings an important panorama of the antimicrobial resistance of
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group C Streptococci, Rhodococcus equi, Pseudomonas aeruginosa, Enterobacteria and
A
Staphylococcus aureus. Group C Streptococci identified include Streptococcus dysgalactiae
M
subsp. equisimilis and the two most common streptococcal species from horses, Streptococcus
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equi subsp. zooepidemicus (which is part of the upper respiratory tract microbiota and can be virulent when it invades the lower respiratory tract), and Streptococcus equi subsp. equi (the
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causative agent of Strangles) [8].Our observations are in accordance with those made by Johns and Adams (2015) where no resistance to penicillins and increased resistance to
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tetracyclines have been observed [7]. In contrast with the South African and English studies performed between 1999-2012 and 2007-2013 respectively [3,7], no increase of gentamicin
A
resistance was observed over our 11-years study period. Less than 3.0% of group C Streptococci were categorized as resistant in our study while more than 70.0% of streptococcal isolates were resistant in the South-African study (n=286) [3]. The reason explaining this important discordance need to be still elucidate.
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R. equi is a soil organism carried in the gut of herbivores. This facultative intracellular pathogen is the most common cause of severe pneumonia in foals [9]. In the past two decades, R. equi has emerged as an opportunistic human pathogen that affects immunocompromised patients [10]. Infections caused by this species are clinically well characterized in horses and the treatment is based on the use of the combination of macrolides and rifampicin.
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Interestingly, as opposed to the literature [11-13], no increase of resistance of R. equi to these two antimicrobial agents has been observed in our study.
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S. aureus is part of the normal cutaneous flora in human and animals. However, S. aureus is a major cause of nosocomial and community acquired infection in France and worldwide [14]. This opportunistic pathogen induces to several pathologies with various degree of severity
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[14]. Our results were lower than those reported in the literature where more than 60.0% of
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staphylococci from equine samples were resistant to penicillin [3,6]. In a recent study, 1,396
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S. aureus strains isolated from horses between 2007 and 2013 have been genetically analyzed.
M
In this population, 6% of the strains were resistant to methicillin [15]. Over the period
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analyzed (2007 to 2013) an increased rate of Staphylococcus resistant to methicillin (MRSA) has been observed with 1.0% in 2007 to 11.0% in 2011 [15]. Since 2013, our results revealed
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that the level of such resistant was relatively stable around 17%. P. aeruginosa is a major opportunistic pathogen that causes many infections in
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immunosuppressed humans and in people suffering from cystic fibrosis [16]. In horses, it is often associated to endometritis in mares and represents a serious problem due to its ability to
A
form biofilm as well as its multi resistance to antimicrobials [17-19]. As described in the literature, we observed over the eleven years that few antimicrobial agents remained active against this bacterium as cefquinome (C4G), gentamicin, amikacin and marbofloxacine [3,6]. E. coli is naturally present in the digestive tract of humans and animals and may be responsible for gastro-intestinal disorders, urinary tract infection and septicemia in rare cases
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[20]. In our study, an important decrease in resistant to streptomycin was observed (from 76% in 2012 to 33% in 2016). Moreover, level of resistance to amoxicillin (around 40%), which was similar to what observed for human clinical samples [21,22]. Note that less than 10% of E. coli strains were resistant to cephalosporins and/or fluoroquinolones. The use of these last drugs is regulated since their designation as “critically important” by the World Health
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Organization and French government in order to limit their consumption. Resistance to third
and fourth generation cephalosporins may be related to the production of cephalosporinase,
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extended spectrum β-lactamase (ESBL) or carbapenemase and became an increasing
therapeutic problem in equine infection by enterobacteria such as E. coli in the same way as for humans [21,22]. In this context, it could be interesting to determine the incidence of
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enzyme-producing E. coli in our cephalosporins resistant strains.
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In a large lower proportion than E. coli, two other Enterobacteria were also analyzed:
A
Enterobacter spp. (3.4%) and K. pneumoniae (2.4%).
M
Enterobacter species, especially E. aerogenes and E. cloacae have been associated with
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nosocomial infections and can cause numerous pathologies such as pneumonia, septicemia, and urinary tract and wound infections [23]. Against these isolates, amikacine and
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marbofloxacine remained the two anti-microbial compounds for which bacteria have the least resistance (less than 10%).
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K. pneumoniae is part of the normal urogenital and intestinal flora of humans and animals [24]. In horses, this bacterium can be responsible of metritis, infertility and pneumonia [25].
A
In the literature, data on susceptibility of K. pneumoniae to common used antimicrobials in equine medicine are contradictory. Our data were in agreement with the German survey where isolates from mare uterine infections were very sensitive to commonly used antimicrobials [26], but were in contrast with two other studies that found an important prevalence of multidrug-resistant Klebsiella [3,27].
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By pooling all the data, an overview of MDR was shown. For Enterobacteria, according to the strain and the period, the rate of MDR were from 17% to 39%. On the other hand, the percentages of MDR strains of group C Streptococci decreased until around 7% (in 20132015). This level was relatively low but should be maintained with the rational use of antimicrobial drugs. Interestingly, 43% of S. aureus were MDR in 2006 versus 18% to 25%
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after 2012. Except erythromycin, rifampicin and amoxicillin-clavulanate, French equine
practitioners use all other antimicrobial compounds usually without any previous
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microbiological characterization increasing the risk of non-adapted treatment.
It may be hypothesized that the reduction of MDR strains of Staphylococcus over the period studied may be linked to the French national ECOANTIBIO Plan (2012-2016) aiming to the
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sensitization of practitioners to a reasoned use of antimicrobials and leading to an important
N
decrease (37%) in the exposure of animals to antibiotics. In 2013, Hughes and collaborators
A
showed that in UK only 0.8% (2/281) equine veterinarians had antimicrobial use guidelines
M
and that 56% of licensed antimicrobial prescriptions were over the recommended dose
ED
rate [28]. It is legitimate to believe that empirical practices may, in large part, contribute to the high prevalence of MDR and our results argue for the crucial role of information as well as
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the warning against the problem of pathogens that will be difficult to fight. To our knowledge, this work is the first large scale retrospective study conducted in France over an eleven years
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period with more than 25,000 samples analyzed for their antimicrobial susceptibilities.
A
Acknowledgements
We would like to thank the technical staff at the Veterinary Microbiology unit of LABÉO Frank Duncombe for the achievement of antimicrobial susceptibility tests.
Declarations
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Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Competing Interests: All other authors declare no competing interest. Ethical Approval: Not required.
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Legends Figure 1: Distribution of 25,813 antimicrobial susceptibility tests (2006-2016) in relation
A
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M
A
N
U
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with the principal microorganisms identified (A) and sample group (B).
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Figure 2: Total (blue) and resistant (red) relevant Gram-positive (A, B, C) and Gram-negative (D, E, F, G) bacteria isolated per year and sample group. PEN: penicillin; AMP/AMX: ampicillin or amoxicillin; AMC: amoxicillin-clavulanic acid; CEF: ceftiofur; CEQ: cefquinome; STR: streptomicin; KAN: kanamicin; GEN: gentamicin; TET/OT: tetracyclin or oxytetracyclin; ERY: erythromycin; RIF: rifampicin; SUL/SXT:
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sulphonamide or trimethoprim-sulfamethoxazole; ENO: enrofloxacin; MAR: marbofloxacine;
FLU: flumequine; OXA*: oxacillin, marker of methicillin resistance; FOX*: cefoxitin,
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marker of cephalosporin resistance; NAL*: nalidixic acid, marker of fluoroquinolone
A
CC E
PT
ED
M
A
N
U
resistance.
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A ED
PT
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SC R
U
N
A
M
A
% of resistant Streptococcus (group C)
Aminoglycosides
Tetracyclines Macrolides
ERY RIF**
ERY** RIF
C
Antibiotic category
Year
Number of strains (n) PEN AMP/AMX Penicillins OXA AMC** 2nd FOX** Cephalosporins 3rd CEF** 4th CEQ** STR** KAN Aminoglycosides GEN** AMK TET/OT** Tetracyclines
A
0.0 0.0 NT 0.0 0.0 0.0 7.0 NT 1.0
0.4 0.2 NT 0.2 0.2 0.0 7.3 NT 2.4 33.4 * 14.4 *
0.2 0.2 NT 0.2 0.2 0.2 6.1 NT 1.6
0.0 0.0 NT 0.0 0.0 0.0 5.9 NT 0.8
0.0 0.0 NT 0.0 0.0 0.0 7.0 NT 2.7 25.8 *
22.2
33.8
1.0
SC R
671
34.0
201 5
2016
703
811
680
768
771
692
0.1 0.1 NT 0.1 0.0 0.0 6.4 NT 2.7 44.1 *
0.1 0.0 0.1 0.2 0.0 0.2 4.9 4.7 2.3
0.0 0.0 0.0 0.0 0.0 0.1 5.9 5.3 1.8 75.7 *
0.1 0.0 0.1 0.0 0.0 0.0 3.4 3.6 1.2 61.5 *
0.0 0.0 0.0 0.0 0.0 0.0 6.2 6.9 0.8
0.1 0.7 0.1 0.7 0.4 0.1 5.5 5.3 0.6 82.1 *
45.3
63.9
5.6*
13.2 *
6.7*
9.1*
6.0
6.5
6.6
8.8
11.1
1.1
0.5
0.6
0.4
1.0
0.1*
0.0
0.3
0.4
15.5 *
6.0*
14.8 *
1.4*
0.6
1.6
23.1 *
0.1*
1.0*
0.0*
4.8*
ED
Number of strains (n)
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Macrolides Rifampicin
PT
Year
Antibiotic category
627
0.5
SUL/SXT**
B
554
8.6
Rifampicin Sulphonamides
464
U
3rd 4th
AMP/AMOX OXA AMC CEF CEQ STRHC KANHC GENHC TET/OT**
383
N
Penicillins
Number of strains (n) PEN
2007 2008 2009 2010 2011 2012 2013 2014
A
Antibiotic category
200 6
M
Year
Cephalosporin s
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Table 1 (continued). Percentage of resistant bacteria per year. (A,B,C) percentage of resistant bacteria for relevant Gram-positive species. (D,E,F,G) percentage of resistant bacteria for relevant Gram-negative species. PEN: penicillin; AMP/AMX: ampicillin or amoxicillin; AMC: amoxicillin-clavulanic acid; CEF: ceftiofur; CEQ: cefquinome; STR: streptomicin; KAN: kanamicin; GEN: gentamicin; TET/OT: tetracyclin or oxytetracyclin; ERY: erythromycin; RIF: rifampicin; SUL/SXT: Sulphonamides or trimethoprim-sulfamethoxazole; ENO: enrofloxacin; MAR: marbofloxacine; FLU: flumequine; OXA: oxacillin, marker of methicillin resistance; FOX: cefoxitin, marker of cephalosporin resistance; NAL: nalidixic acid, marker of fluoroquinolone resistance. * Chi-square test, p.value <0.05; ** CochranArmitage trend test, p.value <0.05; NT: Not tested; NR: Not registered; HC: High concentration.
2006
% of resistant Rhodococcus equi 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016
22
36
50
42
61
70
53
43
45
33
7
13.6 0.0
0.0* 0.0
0.0 2.0
4.8 0.0
1.6 0.0
1.4 4.3
1.9 1.9
0.0 0.0
0.0 0.0
0.0 9.1
0.0 0.0
% of resistant Staphylococcus aureus 2006 2007
2008 2009 2010
2011
2012
114 163 198 190 226 253 185 55.3 35.6* 46.5* 41.6 32.3 32.8 44.3* 55.3 35.6* 46.5* 41.6 32.3 32.8 44.3* NR NR NR NR NR NR 23.8 5.3 1.8 12.6* 14.2 12.4 15.8 23.8* NT NT NT NT NT NT 30.8 0.9 2.5 11.1* 13.2 11.5 16.2 24.3* 1.8 4.3 9.1 12.1 11.5 15.8 24.3* 38.6 31.3 57.6* 65.3 43.4* 27.3* 25.9 NT NT NT NT NT NT 24.3 10.5 7.4 15.2* 18.4 15.0 21.3 23.2 10.5 7.4 15.7 18.4 15.0 21.3 0.0 18.4 8.6* 18.7* 18.4 17.3 16.6 28.1*
- 21 -
2013 2014 2015 2016 178 42.1 42.1 13.5* 13.5 17.4 13.5* 13.5* 15.2* 18.5 18.5 0.0 20.2
180 39.4 38.9 13.9 13.9 13.9 13.9 13.9 15.0 19.4 18.9 0.0 22.8
186 44.1 44.1 16.7 16.7 18.3 16.7 16.7 19.4 25.8 25.3 0.0 29.6
139 43.9 43.2 15.8 15.8 17.3 15.8 15.1 20.9 23.0 21.6 0.0 27.3
ERY** RIF** SUL/SXT ENO** MAR**
Macrolides Rifampicin Sulphonamides Fluoroquinolones
6.1 4.4 0.9 0.0 0.0
D
5.5 4.3 4.3 1.2 0.6
7.0 7.6 9.2 8.6* 2.2
3.4 2.8* 5.1 4.5 1.1
6.1 2.8 3.3 5.6 3.9
4.8 2.7 2.2 3.8 2.7
5.8 2.9 6.5* 1.4 1.4
2011 528 52.5* 43.6* 10.8 10.6 63.6* NT 15.9* 0.6* 23.9 29.2 NT 7.8 6.3 4.9
2012 577 62.7* 45.4 10.2 9.9 76.1* 25.3 11.8 0.5 24.1 62.7* 7.1 6.1 5.9 4.7
2013 2014 490 491 55.7* 39.7* 46.1 32.8* 9.8 11.2 9.2 11.0 65.1* 59.3 24.9 21.4 11.6 10.4 1.6 1.0 20.4 21.4 25.1* 30.1 10.0 8.1 6.3 6.5 5.5 6.1 3.5 5.1
SC R
2010 467 31.3* 25.1* 8.8 7.9 90.4* NT 10.5 3.4 24.0 26.1 NT 8.1 6.0 4.3
U
N
A
M
F
2006 2007 2008 2009 350 419 479 521 40.6 33.9 54.1* 56.6 28.9 23.6 48.6* 53.0 3.4 6.7* 8.6 8.6 1.4 6.0* 8.4 8.3 77.1 81.9 88.3 94.8* NT NT NT NT 5.7 8.1* 9.4 11.5 3.4 3.8 2.7 3.8 21.4 20.8 22.8 26.3 24.6 26.7 27.1 28.4 NT NT NT NT 7.7 17.2* 6.1 7.7 4.0 5.0 5.0 4.6 3.4 3.8 3.5 3.8
IP T
% of resistant Escherichia coli
Year Antibiotic category Number of strains (n) AMP/AMX Penicillins AMC CEF 3rd Cephalosporins CEQ** 4th STR** KAN Aminoglycosides GEN AMK** TET/OT Tetracyclines Sulphonamides SUL/SXT** NAL FLU** Quinolones/Fluoroquinolones ENO MAR
2015 493 40.0 34.9 6.7* 6.9* 36.1 8.9 6.9 0.4 24.5 28.2 6.7 6.1 5.3 3.7
2016 344 39.5 31.4 6.1 5.8 33.1 9.0 6.1 0.0 20.6 31.4 4.9 4.9 3.2 2.9
ED
% of resistant Enterobacter spp
CC E
PT
Year Antibiotic category Number of strains (n) CEF 3rd Cephalosporins CEQ 4th STR** KAN** Aminoglycosides GEN AMK TET/OT** Tetracyclines Sulphonamides SUL/SXT NAL FLU** Quinolones/Fluoroquinolones ENO MAR
A
5.9 10.3 4.7 4.0 2.8
2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 50 93 75 102 90 127 153 94 95 96 59 44.0 52.7 34.7* 41.2 34.4 33.1 43.8 46.8 50.5 21.9* 11.9 14.0 12.9 36.0* 44.1 20.0* 51.2* 58.8 35.1* 20.0* 3.1* 10.2 10.0 7.5 6.7 14.7 6.7 8.7 8.5 10.6 1.1* 1.0 0.0 2.0 2.2 2.7 14.7* 8.9 6.3 24.2* 9.6* 3.2 3.1 1.7
E
G
2006 59 6.8 3.4 32.2 NT 3.4 1.7 10.2 5.1 NT 23.7 1.7 0.0
2007 2008 113 103 18.6 18.4 8.0 9.7 39.8 62.1* NT NT 17.7* 23.3 4.4 3.9 19.5 43.7* 23.0* 21.4 NT NT 37.2 33.0 19.5* 14.6 0.9 2.9
2009 95 20.0 7.4 64.2 NT 18.9 9.5 40.0 18.9 NT 23.2 13.7 0.0
2010 80 10.0 8.8* 47.5 NT 20.0 5.0 37.5 18.8 NT 20.0 10.0 0.0
2011 75 18.7 8.0 33.3 NT 24.0 9.3 26.7 20.0 NT 21.3 14.7 2.7
2012 110 28.2 11.8* 50.0 37.3 35.5 8.2 56.4* 43.6* 23.6 24.5 20.0 1.8
2013 2014 75 69 12.0* 8.7 8.0 1.4 29.3 18.8 16.0* 10.1 16.0* 8.7 0.0* 1.4 38.7* 43.5* 16.0 15.9 20.0 20.3 18.7 18.8 10.7 8.7 2.7 0.0
2015 61 16.4 8.2 26.2 23.0 18.0 6.6 16.4 19.7 18.0 18.0 14.8 4.9
2016 38 15.8 13.2 23.7 18.4 18.4 5.3 21.1 21.1 21.1 21.1 7.9 2.6
% of resistant Klebsiella pneumoniae
Antibiotic category
Penicillins
Aminoglycosides
6.2 5.8 4.9 2.7 1.3
% of resistant Pseudomonas aeruginosa
Year Antibiotic category Number of strains (n) Cephalosporin 4th CEQ** GEN Aminoglycosides AMK** MAR Fluoroquinolones
Cephalosporins
12.6* 10.5 11.1* 7.4 11.1 7.9 2.5 2.6 0.5 1.1
3rd 4th
Year 2006 2007 2008 2009 Number of strains (n) 55 47 63 58 AMC** 18.2 23.4 14.3 24.1 CEF 9.1 6.4 3.2 10.3 CEQ 5.5 4.3 1.6 1.7 STR** 40.0 29.8 61.9* 72.4 KAN NT NT NT NT GEN 9.1 10.6 3.2 3.4 AMK 0.0 0.0 0.0 0.0
- 22 -
2010 2011 2012 2013 64 61 66 62 14.1 8.2 10.6 9.7 7.8 1.6 6.1 4.8 6.3 1.6 4.5 3.2 56.3 23.0* 21.2 25.8 NT NT 6.1 1.6 4.7 3.3 9.1 3.2 0.0 0.0 1.5 0.0
2014 2015 2016 62 57 31 9.7 7.0 12.9 1.6 7.0 9.7 1.6 7.0 9.7 19.4 26.3 29.0 0.0 3.5 3.2 0.0 3.5 6.5 0.0 0.0 0.0
TET/OTC SUL/SXT** NAL FLU Quinolones/Fluoroquinolones ENO** MAR Tetracyclines Sulphonamides
12.7 18.2 NT 3.6 1.8 0.0
10.6 30.2* 41.4 15.6* 12.8 3.2 10.3 9.4 NT NT NT NT 12.8 1.6* 13.8* 7.8 2.1 0.0 3.4 1.6 0.0 0.0 0.0 1.6
16.4 8.2 NT 3.3 0.0 0.0
27.3 27.4 24.2 12.3 25.8 21.2 11.3 12.9 19.3 32.3 13.6 32.3* 12.9* 5.3 19.4* 9.1 8.1 6.5 3.5 12.9 9.1* 3.2 4.8 3.5 9.7 6.1 0.0 1.6 0.0 3.2
A
CC E
PT
ED
M
A
N
U
SC R
IP T
R≤10% 10
- 23 -
Table 2. Percentage of bacteria resistant to three or more antimicrobial classes. Staphylococcus aureus 43.0 24.5 38.4 41.1 27.0 27.3 27.6 18.0 20.0 25.3 24.5
Enterobacter spp 20.3 31.9 45.6 41.1 36.3 26.7 50.9 24.0 24.6 26.2 26.3
E. coli
SC R
N A M ED PT CC E A
- 24 -
Klebsiella pneumoniae 18.2 17.0 23.8 39.7 21.9 21.3 22.7 35.5 22.6 21.1 38.7
IP T
34.0 39.6 34.2 38.8 33.4 34.1 31.4 24.3 23.6 22.5 22.7
U
2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016
Streptococcus (group C) 20.4 14.0 12.3 11.6 6.3 9.0 13.8 6.8 6.4 7.5 10.7