Revista de Salud Animal Vol. 45, January-December, 2023, ISSN: 2224-4700
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CU-ID: https://cu-id.com/2248/v45e02
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Detection of multidrug-resistant Streptococcus equi subsp. zooepidemicus in Cuba

Detección de Streptococcus equi subsp. zooepidemicus multirresistente a antimicrobianos en Cuba

iDAnia Ramón Martínez1Bacteriology-Parasitology Group, National Center for Animal and Plant Health (CENSA), San José de las Lajas, Cuba.*✉:aniaramon95@gmail.com

iDJosé B. Poveda2Epidemiology and Preventive Medicine Unit, University Institute of Animal Health and Food Safety (IUSA), University of Las Palmas de Gran Canaria (ULPGC), Arucas, Spain.

iDAna S. Ramírez2Epidemiology and Preventive Medicine Unit, University Institute of Animal Health and Food Safety (IUSA), University of Las Palmas de Gran Canaria (ULPGC), Arucas, Spain.

iDJosé Luis Martín-Barrasa3Facility for animals, Research Unit of the University Hospital of Gran Canaria, Doctor Negrín, Canary Islands Health Research Institute Foundation (FIISC), Las Palmas de Gran Canaria, Spain.4Fish Health and Infectious Diseases Group, University Institute of Animal Health and Food Safety (IUSA), University of Las Palmas de Gran Canaria (ULPGC), Arucas, Spain.5Canarian Foundation of the Canary Islands Health Research Institute (FIISC), Las Palmas de Gran Canaria, Spain.6CIBER of Infectious Diseases (CIBERINFEC), Carlos III Health Institute, Madrid, Spain.

iDLaura Alonso-Acero7Microbiology Service. Doctor Negrín University Hospital of Gran Canaria, Las Palmas de Gran Canaria, Spain.

iDAleinys Corteguera-Díaz1Bacteriology-Parasitology Group, National Center for Animal and Plant Health (CENSA), San José de las Lajas, Cuba.

iDYuset Fonseca-Rodríguez8Trial Center for Quality Control of Food (CENLAC), National Center for Animal and Plant Health (CENSA), San José de las Lajas, Cuba.

iDEvelyn Lobo-Rivero1Bacteriology-Parasitology Group, National Center for Animal and Plant Health (CENSA), San José de las Lajas, Cuba.

iDIvette Espinosa-Castaño1Bacteriology-Parasitology Group, National Center for Animal and Plant Health (CENSA), San José de las Lajas, Cuba.


1Bacteriology-Parasitology Group, National Center for Animal and Plant Health (CENSA), San José de las Lajas, Cuba.

2Epidemiology and Preventive Medicine Unit, University Institute of Animal Health and Food Safety (IUSA), University of Las Palmas de Gran Canaria (ULPGC), Arucas, Spain.

3Facility for animals, Research Unit of the University Hospital of Gran Canaria, Doctor Negrín, Canary Islands Health Research Institute Foundation (FIISC), Las Palmas de Gran Canaria, Spain.

4Fish Health and Infectious Diseases Group, University Institute of Animal Health and Food Safety (IUSA), University of Las Palmas de Gran Canaria (ULPGC), Arucas, Spain.

5Canarian Foundation of the Canary Islands Health Research Institute (FIISC), Las Palmas de Gran Canaria, Spain.

6CIBER of Infectious Diseases (CIBERINFEC), Carlos III Health Institute, Madrid, Spain.

7Microbiology Service. Doctor Negrín University Hospital of Gran Canaria, Las Palmas de Gran Canaria, Spain.

8Trial Center for Quality Control of Food (CENLAC), National Center for Animal and Plant Health (CENSA), San José de las Lajas, Cuba.

 

*Correspondence author: Ania Ramón-Martínez. Email: aniaramon95@gmail.com

ABSTRACT

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is an opportunistic pathogen capable of causing various infections in the respiratory, genital, and urinary tracts of horses and other animals. This species is considered responsible for many emerging zoonotic diseases. There has been an increasing circulation of multidrug-resistant S. zooepidemicus strains in horses, however, there is no information on S. zooepidemicus and its antimicrobial susceptibility profile in Cuban horses. Therefore, the objectives of this study were to report the isolation of S. zooepidemicus in a horse and to determine its antimicrobial susceptibility profile. A mare with pale mucous membranes, owned by a private producer in Melena del Sur, Mayabeque, was sampled, and a swab was taken from the genital tract. The isolate obtained was identified using the analytical profile index and mass spectrometry. The minimal inhibitory concentration (MIC) of 11 antibiotics (penicillin G, ampicillin, amoxicillin-clavulanate, cefquinome, imipenem, gentamicin, enrofloxacin, doxycycline, erythromycin, chloramphenicol, and vancomycin) was determined for the isolate. The isolate was identified as S. zooepidemicus and it was susceptible to all β-lactam, erythromycin, chloramphenicol, and vancomycin. The isolate presented a multidrug resistance profile to gentamicin, enrofloxacin, and doxycycline with MIC values of 16 μg/mL, 2 μg/mL, and 2 μg/mL, respectively. For the first time in Cuba, multidrug-resistant S. zooepidemicus was detected in the genital mucosa of a mare. The close interaction between humans and horses increases the risk of acquiring these multiresistant microorganisms or favoring their dissemination, thus this result should be considered in staff training.

Key words: 
Streptococcus equi subsp. zooepidemicus, antimicrobial resistance, mare
RESUMEN

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) es un patógeno oportunista capaz de causar diversas infecciones en los tractos respiratorio, genital y urinario en caballos y otros animales. Esta especie se considera responsable de muchas enfermedades zoonóticas emergentes. Recientemente, existe una creciente circulación de cepas de S. zooepidemicus multirresistentes a antimicrobianos en caballos, sin embargo, no existe información sobre S. zooepidemicus y su perfil de susceptibilidad a antimicrobianos, en caballos cubanos. Por tanto, los objetivos de este estudio fueron reportar el aislamiento de S. zooepidemicus en un caballo y determinar su perfil de susceptibilidad a antimicrobianos. Se muestreó una yegua perteneciente a un productor privado de Melena del Sur, Mayabeque, con mucosas pálidas y se tomó un hisopado del tracto genital. El aislado obtenido se identificó mediante el índice de perfil analítico y espectrometría de masas. Se determinó la concentración mínima inhibitoria (CMI) de 11 antibióticos (penicilina G, ampicilina, amoxicilina-clavulanato, cefquinoma, imipenem, gentamicina, enrofloxacina, doxiciclina, eritromicina, cloranfenicol y vancomicina) para el aislado. El aislado se identificó como S. zooepidemicus y fue sensible a todos los betalactámicos, eritromicina, cloranfenicol y vancomicina. Presentó un perfil de multirresistencia a gentamicina, enrofloxacina y doxiciclina con valores de CMI de 16 μg/mL, 2 μg/mL y 2 μg/mL, respectivamente. Por primera vez en Cuba se detecta S. zooepidemicus multirresistente a antimicrobianos en la mucosa genital de una yegua. La estrecha interacción entre humanos y caballos, aumenta el riesgo de adquirir estos microorganismos multirresistentes o favorecer su diseminación, por lo que este resultado debe ser considerado en la capacitación del personal con vínculo ocupacional.

Palabras clave: 
Streptococcus equi subsp. zooepidemicus, resistencia a antimicrobianos, yegua

Received: 30/5/2023; Accepted: 05/6/2023

Conflicts of interest: The authors declare no conflicts of interest.

Authors contributions: Ania Ramón-Martínez: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Visualization, Writing-original draft. José B. Poveda: Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision. Ana S. Ramírez: Investigation, Methodology, Resources, Validation. José Luis Martín-Barrasa: Investigation, Resources. Laura Alonso-Acero: Investigation. Aleinys Corteguera-Díaz: Investigation. Yuset Fonseca-Rodríguez: Investigation. Evelyn Lobo-Rivero: Conceptualization, Funding acquisition, Project administration, Resources. Ivette Espinosa-Castaño: Conceptualization, Methodology, Project administration, Resources, Supervision, Writing-review & editing.

This article is under license Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

CONTENT

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a β-hemolytic Streptococcus belonging to the Lancefield group C (11. De Vos P. e. Bergey´s manual of systematic bacteriology. The Firmicutes. 2nd ed. New York, USA: Springer; 2009. 1450 p.). S. zooepidemicus is one of the most common pathogens infecting horses (22. Li J, Zhao Y, Gao Y, Zhu Y, Holyoak GR, Zeng S. Treatments for endometritis in mares caused by Streptococcus equi subspecies zooepidemicus: A structured literature review. J Equine Vet Sci. 2021;102:1-10.). It is also commonly isolated from bacterial infections in a wide range of animal species such as dogs (33. Day MJ, Carey S, Clercx C, Kohn B, MarsilIo F, Thiry E, et al. Aetiology of canine infectious respiratory disease complex and prevalence of its pathogens in Europe. J Comp Pathol. 2020;176:86-108.), felines (44. Sykes JE. Pediatric feline upper respiratory disease. Vet Clin North Am Small Anim Pract. 2014;44(2):331-342.), swine (55. Costa MO, Lage B. Streptococcus equi subspecies zooepidemicus and sudden deaths in swine, Canada. Emerg Infect Dis. 2020;26(10):2522-2524.), guinea pigs (66. Gruszynski K, Young A, Levine SJ, Garvin JP, Brown S, Turner L, et al. Streptococcus equi subsp. zooepidemicus infections associated with guinea pigs. Emerg Infect Dis. 2015;21(1):156-158.), ruminants (77. Pisoni G, Zadoks RN, Vimercati C, Locatelli C, Zanoni MG, Moroni P. Epidemiological investigation of Streptococcus equi subspecies zooepidemicus involved in clinical mastitis in dairy goats. J Dairy Sci. 2009;92(3):943-951.), camelids (88. Corpa JM, Carvallo F, Anderson ML, Nyaoke AC, Moore JD, Uzal FA. Streptococcus equi subspecies zooepidemicus septicemia in alpacas: three cases and review of the literature. J Vet Diagn Invest. 2018;30(4):598-602.), and non-human primates (99. Mätz-Rensing K, Winkelmann J, Becker T, Burckhardt I, van der Linden M, Köndgen S, et al. Outbreak of Streptococcus equi subsp. zooepidemicus infection in a group of rhesus monkeys (Macaca mulatta). J Med Primatol. 2009;38(5):328-334.).

S. zooepidemicus is a commensal bacteria residing in the skin and mucous membranes of horses. This species is an opportunistic pathogen frequently implicated as the cause of respiratory infections in both foals and adults, as well as genital and urinary tract infections (1010. EFSA AHAW Panel (EFSA Panel on Animal Health and Welfare), Nielsen SS, Bicout DJ, Calistri P, Canali E, Drewe JA, et al. Assessment of animal diseases caused by bacteria resistant to antimicrobials: Horses. EFSA J. 2021;19(12):1-43.). Many commensal bacteria including, S. zooepidemicus, colonize the exterior of the stallion penis, but they are not regarded as pathogenic and may be cultured from an ejaculate. However, alterations of the normal bacterial microbiota on the external genitalia may favor the growth of opportunistic bacteria such as S. zooepidemicus, which, if inseminated, may cause infertility in susceptible mares (1111. Samper JC, Tibary A. Disease transmission in horses. Theriogenology. 2006;66(3):551-559.).

S. zooepidemicus is considered responsible for many emerging zoonotic diseases. S. zooepidemicus-associated infections could be considered as zoonoses, since its transmission has been documented by the consumption of unpasteurized milk and dairy products (1212. Bordes-Benítez A, Sánchez-Oñoro M, Suárez-Bordón P, García-Rojas AJ, Saéz-Nieto JA, González-García A, et al. Outbreak of Streptococcus equi subsp. zooepidemicus infections on the island of Gran Canaria associated with the consumption of inadequately pasteurized cheese. Eur J Clin Microbiol Infect Dis. 2006;25(4):242-246.), but also due to direct contact with infected horses (1313. Toraño G, Arias I, Castillo A, Brossard G. Primer caso de meningitis por Streptococcus equi subsp. zooepidemicus en Cuba. Rev Cub Salud Pública. 2015;41(1):165-168., 1414. Høyer-Nielsen AK, Gaini S. Sepsis, endocarditis, and purulent arthritis due to a rare zoonotic infection with Streptococcus equi subspecies zooepidemicus. Case Rep Infect Dis. 2018;2018:1-8.) or infected dogs (1515. Zahlanie Y, Almatrafi M, Filkins L, Hsiang MS. Possible canine source of Streptococcus equi subspecies zooepidemicus causing meningitis in an infant. IDCases. 2019;17:1-3.). The possible effects of S. zooepidemicus infection in humans include nephritis, arthritis, meningitis, pneumonia, infected aortic aneurysm, infective endocarditis, and sepsis (1515. Zahlanie Y, Almatrafi M, Filkins L, Hsiang MS. Possible canine source of Streptococcus equi subspecies zooepidemicus causing meningitis in an infant. IDCases. 2019;17:1-3.).

Antimicrobial resistance is one of the most important threats to global health, food safety, and development. Nowadays, this phenomenon has received much attention due to the potential exposure of humans to resistant bacteria through food chains and direct contact with production and companion animals. Antibiotic resistant strains of S. zooepidemicus have been reported in recent studies in Italy (1616. Nocera FP, D'Eletto E, Ambrosio M, Fiorito F. Occurrence and antimicrobial susceptibility profiles of Streptococcus equi subsp. zooepidemicus strains isolated from mares with fertility problems. Antibiotics. 2022;11(25):1-11.), United States of America (1717. Lord J, Carter C, Smith J, Locke S, Phillips E, Odoi A. Antimicrobial resistance among Streptococcus equi subspecies zooepidemicus and Rhodococcus equi isolated from equine specimens submitted to a diagnostic laboratory in Kentucky, USA. PeerJ. 2022;10:1-23.), Sweden (1818. Malaluang P, Wilén E, Frosth S. Vaginal bacteria in mares and the occurrence of antimicrobial resistance. Microorganisms. 2022;10(11):1-18.), and Australia (1919. Deniaud M, Tee E. Susceptibility pattern of bacterial isolates in equine ulcerative keratitis: Implications for empirical treatment at a university teaching hospital in Sydney. Aust Vet J. 2023;101(3):115-120.); however, there is no information about S. zooepidemicus and its susceptibility profile to antimicrobials, in Cuban horses. Therefore, the objectives of this study were to determine the occurrence of S. zooepidemicus in a horse with pale mucous membranes and its antimicrobial susceptibility profile.

A mare less than two years old, owned by a private producer in Melena del Sur, Mayabeque province, was sampled in 2021. The mare had her vaccination schedule up to date and had not recently been given a treatment with antibiotics, according to the owner. At the time of sample collection, the animal did not present any clinical signs with the exception of pale mucous membranes.

For sampling, the mare's external genitalia were rinsed with physiological saline and then dried. A sample was taken by rotating a sterile swab from the mare’s clitoral fossa (2020. Malaluang P, Wilén E, Lindahl J. Antimicrobial Resistance in Equine Reproduction. Animals. 2021;11:1-13.). The swab tip was placed in a tryptone soy agar medium (Merck, Darmstadt, Germany), with 0.5 % bovine albumin. The sample was labeled and transported within 24 hours to the Animal Bacteriology Laboratory of the Department of Animal Health of the National Center for Animal and Plant Health, where it was processed.

The sample was streaked on 5 % sheep blood Columbia agar (VWR, Leuven, Belgium) and incubated aerobically at 37°C for 24 hours. The isolate was identified using Gram staining. Biochemical characterization was assessed through oxidase and catalase tests. The isolate was stored in brain heart infusion (AppliChem, Darmstadt, Germany), with 20 % glycerol at -20 ˚C.

The pinpoint, circular, small, opaque to grayish, and β-hemolytic colonies 1 to 3 mm in diameter were observed. Furthermore, the recovered isolate resulted to be catalase and oxidase negative, and it appeared as Gram-positive cocci organized in chains on the smears. Pathogenic bacteria species are frequently isolated from uterine swabs, uterine lavage, vaginal swabs, and clitoral swabs (2020. Malaluang P, Wilén E, Lindahl J. Antimicrobial Resistance in Equine Reproduction. Animals. 2021;11:1-13.) in mares. S. zooepidemicus is usually responsible for a dormant subclinical infection, persisting in endometrium tissue and resisting the immune system and antimicrobial treatment. This is related to S. zooepidemicus ability to survive in epithelial cells in the form of “persister” cells (2121. Petersen MR, Skive B, Christoffersen M, Lu K, Nielsen JM, Troedsson MH, et al. Activation of persistent Streptococcus equi subspecies zooepidemicus in mares with subclinical endometritis. Vet Microbiol. 2015;179(1-2):119-125.).

For an initial identification, a biochemically-based commercial system, API 20 Strep (bioMériuex, Marcy L’Etoile, France) was used. For the interpretation of results, the manufacturer’s guidelines were followed. API 20 Strep identification result was Streptococcus equi subsp. zooepidemicus (probability, 99.9%) for the recovered isolate. Then, identification was confirmed by Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) (MALDI Biotyper®, Bruker Daltonics GmbH & Co.KG, Bremen, Germany). For MALDI-TOF MS identification, fresh colonies were used. Bacterial colonies were directly applied from the culture to the MS plate and left to dry, adding then 1 µL of Bruker Matrix HCCA (Bruker Daltonics GmbH & Co.KG, Bremen, Germany). The identification was based on the score values released by the equipment’s instructions. According to Bruker Biotyper´s guidelines, a score value ≥ 2 is interpreted as high-confidence identifications, which means reliable identification at the species level.

S. zooepidemicus isolate was identified at subspecies level with a log (score) of 2.25 by MALDI-TOF MS. S. zooepidemicus and Streptococcus equi subps. equi, the causative agent of strangles in horses, are closely related so they can be misidentified (2222. Boyle AG. Streptococcus equi subspecies equi. Vet Clin North Am Equine Pract. 2023;39(1):115-131.). MALDI-TOF MS can be considered a useful, reliable, and rapid technique for S. zooepidemicus identification, able to discriminate between S. equi subsp. equi (2323. Mani RJ, Thachil AJ, Ramachandran A. Discrimination of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. J Vet Diagn Invest. 2017;29(5):622-627.). Also, Uchida-fujii et at. (2424. Uchida-Fujii E, Niwa H, Kinoshita Y, Nukada T. Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) for identification of bacterial isolates from horses. J Equine Vet Sci. 2020;95:1-7.) reported that this method is applicable for the identification of equine bacterial isolates. For this reason, in this study, the biochemical API 20 Strep identification of the recovered isolate was confirmed by MALDI-TOF MS analysis.

The antibiotic minimal inhibitory concentration (MIC) was determined using broth microdilution method. The isolate was tested for its susceptibility to 11 antibiotics, belonging to nine different classes: penicillins alone or combined (penicillin G, ampicillin, and amoxicillin-clavulanate) (Sigma-Aldrich, St. Louis, USA), cephalosporins (cefquinome) (Sigma-Aldrich, St. Louis, USA), carbapenems (imipenem) (EDQM CS, Strasbourg Cedex, France), aminoglycosides (gentamicin) (SERVA, Heidelberg, Germany), fluoroquinolones (enrofloxacin) (BioChemika, Espoo, Finland), tetracyclines (doxycycline) (Sigma-Aldrich, St. Louis, USA), macrolides (erythromycin) (SERVA, Heidelberg, Germany), phenicols (chloramphenicol) (SERVA, Heidelberg, Germany), and glycopeptides (vancomycin) (Sigma-Aldrich, St. Louis, USA). Antibiotic choice was performed considering the antibiotics most commonly used in equine clinical practice described in the Clinical and Laboratory Standards Institute (CLSI) manual (2525. CLSI. VET08 Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. 4th ed. Wayne, USA: Clinical and Laboratory Standards Institute; 2018. 200 p.). The broth microdilution method and the stock solutions of antimicrobial agents were performed as described in the CLSI (2525. CLSI. VET08 Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. 4th ed. Wayne, USA: Clinical and Laboratory Standards Institute; 2018. 200 p.) manual.

The isolate was classified as susceptible (S), intermediate (I) or resistant (R), according to both the CLSI (2525. CLSI. VET08 Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. 4th ed. Wayne, USA: Clinical and Laboratory Standards Institute; 2018. 200 p.) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (2626. The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint tables for interpretation of MICs and zone diameters. Version 13.0. Available online: http://www.eucast.org: EUCAST; 2023. 110 p.) guidelines for Streptococcus spp. Breakpoint values to determine susceptibility were used according to EUCAST (2626. The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint tables for interpretation of MICs and zone diameters. Version 13.0. Available online: http://www.eucast.org: EUCAST; 2023. 110 p.). When reference breakpoints did not exist, in EUCAST (2626. The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint tables for interpretation of MICs and zone diameters. Version 13.0. Available online: http://www.eucast.org: EUCAST; 2023. 110 p.), CLSI (2525. CLSI. VET08 Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. 4th ed. Wayne, USA: Clinical and Laboratory Standards Institute; 2018. 200 p.), criteria were used (Table 1).

Table 1.  Antimicrobial susceptibility profile of S. zooepidemicus. / Perfil de susceptibilidad a antimicrobianos de S. zooepidemicus.
Antimicrobial substance MIC breakpoints values (μg/mL) Reference MIC Value (μg/mL) Interpretation
S I R
Penicillin G ≤ 0.25 - > 0.25 (2626. The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint tables for interpretation of MICs and zone diameters. Version 13.0. Available online: http://www.eucast.org: EUCAST; 2023. 110 p.) < 0.25 S
Ampicillin ≤ 0.25 - > 0.25 (2626. The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint tables for interpretation of MICs and zone diameters. Version 13.0. Available online: http://www.eucast.org: EUCAST; 2023. 110 p.) < 0.25 S
Amoxicillin - clavulanate ≤ 0.25 - > 0.25 (2626. The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint tables for interpretation of MICs and zone diameters. Version 13.0. Available online: http://www.eucast.org: EUCAST; 2023. 110 p.) < 0.25 S
Cefquinome ≤ 0.25 - > 0.25 (2626. The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint tables for interpretation of MICs and zone diameters. Version 13.0. Available online: http://www.eucast.org: EUCAST; 2023. 110 p.) < 0.25 S
Imipenem ≤ 0.25 - > 0.25 (2626. The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint tables for interpretation of MICs and zone diameters. Version 13.0. Available online: http://www.eucast.org: EUCAST; 2023. 110 p.) < 0.25 S
Gentamicin ≤ 2 4 ≥ 8 (2525. CLSI. VET08 Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. 4th ed. Wayne, USA: Clinical and Laboratory Standards Institute; 2018. 200 p.) 16 R
Enrofloxacin ≤ 0.5 - > 0.5 (2626. The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint tables for interpretation of MICs and zone diameters. Version 13.0. Available online: http://www.eucast.org: EUCAST; 2023. 110 p.) 2 R
Doxycycline ≤ 1 - > 1 (2626. The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint tables for interpretation of MICs and zone diameters. Version 13.0. Available online: http://www.eucast.org: EUCAST; 2023. 110 p.) 2 R
Erythromycin ≤ 0.25 - > 0.25 (2626. The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint tables for interpretation of MICs and zone diameters. Version 13.0. Available online: http://www.eucast.org: EUCAST; 2023. 110 p.) < 0.25 S
Chloramphenicol ≤ 4 8 ≥ 16 (2525. CLSI. VET08 Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. 4th ed. Wayne, USA: Clinical and Laboratory Standards Institute; 2018. 200 p.) 4 S
Vancomycin ≤ 1 - - (2626. The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint tables for interpretation of MICs and zone diameters. Version 13.0. Available online: http://www.eucast.org: EUCAST; 2023. 110 p.) 0.5 S

S: susceptible, I: intermediate, R: resistant, “-”: not applicable. / S: susceptible, I: intermedio, R: resistente, “-”: no aplicable.

The isolate was susceptible to all β-lactam with MIC under 0.25 μg/mL (Table 1). Penicillins are recommended as the first-line of treatment for suspected S. zooepidemicus infections because in general, penicillin resistance among S. zooepidemicus has remained low (1717. Lord J, Carter C, Smith J, Locke S, Phillips E, Odoi A. Antimicrobial resistance among Streptococcus equi subspecies zooepidemicus and Rhodococcus equi isolated from equine specimens submitted to a diagnostic laboratory in Kentucky, USA. PeerJ. 2022;10:1-23., 2727. Awosile BB, Heider LC, Saab ME, McClure JT. Antimicrobial resistance in bacteria isolated from horses from the atlantic provinces, Canada (1994 to 2013). Can Vet J. 2018;59(9):951-957.). However, Nocera et al. (1616. Nocera FP, D&apos;Eletto E, Ambrosio M, Fiorito F. Occurrence and antimicrobial susceptibility profiles of Streptococcus equi subsp. zooepidemicus strains isolated from mares with fertility problems. Antibiotics. 2022;11(25):1-11.) reported an increase in penicillin and ampicillin resistance, and amoxicillin-clavulanate had the highest intermediate susceptibility value. The trend observed suggests that the susceptibility profile of S. zooepidemicus may be less predictable than previously reported, highlighting the importance of culture and susceptibility studies for guiding antimicrobial therapy when possible.

The isolate also presented susceptibility to erythromycin, chloramphenicol, and vancomycin. This result is in concordance with other studies reporting low resistance to macrolides in S. zooepidemicus (1717. Lord J, Carter C, Smith J, Locke S, Phillips E, Odoi A. Antimicrobial resistance among Streptococcus equi subspecies zooepidemicus and Rhodococcus equi isolated from equine specimens submitted to a diagnostic laboratory in Kentucky, USA. PeerJ. 2022;10:1-23., 2727. Awosile BB, Heider LC, Saab ME, McClure JT. Antimicrobial resistance in bacteria isolated from horses from the atlantic provinces, Canada (1994 to 2013). Can Vet J. 2018;59(9):951-957.). Although resistance to macrolides is low, a temporal increase in macrolide resistance over a longer-term period has been reported. Something similar happened with chloramphenicol, but with higher initial levels of resistance (1717. Lord J, Carter C, Smith J, Locke S, Phillips E, Odoi A. Antimicrobial resistance among Streptococcus equi subspecies zooepidemicus and Rhodococcus equi isolated from equine specimens submitted to a diagnostic laboratory in Kentucky, USA. PeerJ. 2022;10:1-23.). Susceptibility to vancomycin was found in S. zooepidemicus strains isolated from humans as well (2828. Kerdsin A, Chopjitt P. Zoonotic infection and clonal dissemination of Streptococcus equi subspecies zooepidemicus sequence type 194 isolated from humans in Thailand. Transbound Emerg Dis. 2022;69(4):554-565.).

According to EUCAST Expert Rules (2929. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) Expert Rules. Streptococcus. Available online: http://www.eucast.org: EUCAST; 2019. 3 p.), Streptococcus spp. exhibit low-level intrinsic resistance to aminoglycosides, so these drugs should not be assessed for S. zooepidemicus susceptibility. However, the CLSI (2525. CLSI. VET08 Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. 4th ed. Wayne, USA: Clinical and Laboratory Standards Institute; 2018. 200 p.) manual does no report intrinsic resistance to aminoglycosides for streptococci. In addition, several recent studies continue to evaluate the aminoglycoside susceptibility for streptococcal strains (1616. Nocera FP, D&apos;Eletto E, Ambrosio M, Fiorito F. Occurrence and antimicrobial susceptibility profiles of Streptococcus equi subsp. zooepidemicus strains isolated from mares with fertility problems. Antibiotics. 2022;11(25):1-11., 1818. Malaluang P, Wilén E, Frosth S. Vaginal bacteria in mares and the occurrence of antimicrobial resistance. Microorganisms. 2022;10(11):1-18., 3030. Léon A, Castagnet S, Maillard K, Paillot R, Giard JC. Evolution of in vitro antimicrobial susceptibility of equine clinical isolates in France between 2016 and 2019. Animals. 2020;10(812):1-11.). The isolate in the present study was resistant to gentamicin with a MIC of 16 μg/mL. There has been an increase in aminoglycoside resistance through time (1919. Deniaud M, Tee E. Susceptibility pattern of bacterial isolates in equine ulcerative keratitis: Implications for empirical treatment at a university teaching hospital in Sydney. Aust Vet J. 2023;101(3):115-120.). Aminoglycosides have reduced efficacy against streptococci when administered in low concentrations, which is due to their restricted drug uptake (3131. Jospe-Kaufman M, Siomin L, Fridman M. The relationship between the structure and toxicity of aminoglycoside antibiotics. Bioorg Med Chem Lett. 2020;30(13):1-6.). However, it has been reported that the combination of aminoglycosides with other antibiotics, such as penicillins and glycopeptides turn out to have a significant bactericidal synergy, especially for internal infection (3232. Haenni M, Lupo A, Madec JY. Antimicrobial resistance in Streptococcus spp. Microbiol Spectr. 2018;6(2):1-25.).

There was also resistance to enrofloxacin and doxycycline with 2 μg/mL MIC values in both cases. Fluoroquinolones may be an alternative to beta-lactam antibiotics to treat streptococcal infections (3232. Haenni M, Lupo A, Madec JY. Antimicrobial resistance in Streptococcus spp. Microbiol Spectr. 2018;6(2):1-25.). However, fluoroquinolones, as well as third-generation cephalosporins and higher, are considered by World Health Organization as critically important antimicrobials in human medicine, classified with the priority criterion 1 (3333. World Health Organization & WHO Advisory Group on Integrated Surveillance of Antimicrobial Resistance (AGISAR). Critically important antimicrobials for human medicine: Ranking of antimicrobial agents for risk management of antimicrobial resistance due to non-human use. 6th ed. Geneva, Switzerland: World Health Organization; 2019. 52 p.). Therefore, their use in animals should be carried out with caution. Fluoroquinolone resistant strains of S. zooepidemicus have been reported in equine, although with a decreasing trend of resistance (1616. Nocera FP, D&apos;Eletto E, Ambrosio M, Fiorito F. Occurrence and antimicrobial susceptibility profiles of Streptococcus equi subsp. zooepidemicus strains isolated from mares with fertility problems. Antibiotics. 2022;11(25):1-11., 1717. Lord J, Carter C, Smith J, Locke S, Phillips E, Odoi A. Antimicrobial resistance among Streptococcus equi subspecies zooepidemicus and Rhodococcus equi isolated from equine specimens submitted to a diagnostic laboratory in Kentucky, USA. PeerJ. 2022;10:1-23.). S. zooepidemicus and streptococci often show high levels of resistance to tetracyclines (1616. Nocera FP, D&apos;Eletto E, Ambrosio M, Fiorito F. Occurrence and antimicrobial susceptibility profiles of Streptococcus equi subsp. zooepidemicus strains isolated from mares with fertility problems. Antibiotics. 2022;11(25):1-11.). Furthermore, Lord et al. (1717. Lord J, Carter C, Smith J, Locke S, Phillips E, Odoi A. Antimicrobial resistance among Streptococcus equi subspecies zooepidemicus and Rhodococcus equi isolated from equine specimens submitted to a diagnostic laboratory in Kentucky, USA. PeerJ. 2022;10:1-23.) reported that the vast majority of isolates (85.3%) were resistant to tetracycline, with significant temporal increase in their study.

A bacterium is considered to be multidrug-resistant if it is non-susceptible to at least one agent from three or more classes of antimicrobials (3434. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012;18(3):268-281., 3535. Sweeney MT, Lubbers BV, Schwarz S, Watts JL. Applying definitions for multidrug resistance, extensive drug resistance and pandrug resistance to clinically significant livestock and companion animal bacterial pathogens. J Antimicrob Chemother. 2018;73(6):1460-1463.). In this study, the isolate was resistant to three antimicrobial classes: aminoglycosides, fluoroquinolones, and tetracyclines. This is in agreement with Lord et at. (1717. Lord J, Carter C, Smith J, Locke S, Phillips E, Odoi A. Antimicrobial resistance among Streptococcus equi subspecies zooepidemicus and Rhodococcus equi isolated from equine specimens submitted to a diagnostic laboratory in Kentucky, USA. PeerJ. 2022;10:1-23.), who reported six types of antimicrobial combinations in multiresistant S. zooepidemicus strains. In all cases, fluoroquinolones and tetracyclines were part of the combinations. To cope with the alarming increasing circulation of multidrug-resistant strains, empirical equine therapy should be avoided and treatments based on the results of antimicrobial susceptibility testing should be preferred.

Multidrug-resistant S. zooepidemicus has been detected for the first time in Cuba. It is a species with pathogenic and zoonotic potential in horses, isolated from a mare genital mucosa. The close interaction between humans and horses increases the risk of acquiring these multiresistant microorganisms or favoring their dissemination, thus this result should be considered in staff training.

Therefore, it is strongly recommended to wash hands with soap and water after contact with horses or other animals. In addition, further studies on the risk factors for its zoonotic transmission and on the spectrum of human diseases associated with S. zooepidemicus are needed. The growing rate of antimicrobial resistance represents a serious public health problem with important implications for clinical practice in both human and veterinary medicine.

Funding

 ⌅

This work was supported by the International Atomic Energy Agency (IAEA) Marie Sklodowska-Curie Fellowship Programme (MSCFP) (EVT2100330) and by the Epidemiology and Preventive Medicine Unit, Institute of Animal Health and Food Safety (IUSA), University of Las Palmas de Gran Canaria (ULPGC).

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