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Characterization of Streptococcus pyogenes from Animal Clinical Specimens, Spain.

Streptococcus pyogenes (group A Streptococcus) is a gram-positive bacterium that causes several diseases in humans. S. pyogenes usually colonizes the throat or skin epithelial surfaces and causes a wide variety of clinical manifestations, such as noninvasive pharyngitis, dermatitis, and scarlet fever (1,2). However, this pathogen is also responsible for deadly invasive systemic infections such as necrotizing fasciitis and streptococcal toxic shock syndrome (3).The ecologic niche of S. pyogenes appears to be quite narrow, with humans being the almost exclusive biologic host (4) and no animal or environmental reservoir of known importance contributing to its life cycle (2). Reports of isolation of S. pyogenes from sources other than humans are rare. S. pyogenes has recently been associated with an infection in a free-living European hedgehog (Erinaceus europaeus) (5). S. pyogenes has also been recovered from the feces of a dog with possible antibiotic-associated colitis (6) and from the eye discharge of a dog with conjunctivitis (7). We know of no other reports of isolation of this microorganism from animals.

We conducted a study to provide a detailed characterization of animal S. pyogenes isolates using emm typing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). We also investigated erythromycin resistance mechanisms and phenotypes, as well as virulence genes.

Materials and Methods

Origin and Identification of Bacterial Isolates

We analyzed 15 isolates of S. pyogenes obtained from rabbits (n = 14) and sheep (n = 1) in Spain during 2006-2014 (Table 1). Most rabbit isolates were from unrelated animals, located in different commercial farms (n = 14) and locations throughout Spain. Links between rabbit farms were not identified. The sheep included in this study was from a farm that had no rabbits. Human contact with animals was restricted to the personnel working in the rabbit farms and sheep flocks.

We recovered isolates from different clinical backgrounds: 8 from skin infections, 4 from genital tract infections, and 1 each from respiratory infections, mastitis, and otitis. We collected samples from skin and ear infections with sterile cotton swabs and collected the milk sample from the mastitis case aseptically in a sterile tube. Rabbits with genital tract or lung infections were euthanized, at farms or laboratories, and necropsied under aseptic conditions; clinical specimens were collected with forceps and scissors scrubbed in 70% ethanol. Samples taken at farms were transported to the laboratory in refrigerated polyethylene bags and processed within 24 hours after sampling.

Clinical specimens were sampled onto blood agar plates that were incubated at 37[degrees]C for 24-48 hours. Identification of isolates as S. pyogenes was based on colony morphology, P-hemolysis, and biochemical characteristics using the commercial identification system rapid ID 32 STREP (BioMerieux, Marcy L'Etoile, France). Biochemical identification was also confirmed by sequencing the 16S rRNA gene (8).

Antimicrobial Drug Susceptibility Tests

We performed drug susceptibility testing using the Clinical and Laboratory Standards Institute broth microdilution method (9) in Mueller-Hinton broth supplemented with 5% lysed horse blood. We determined the susceptibilities of the isolates with a commercially available susceptibility test (CMV3AGPF Sensititer standard panel; Trek Diagnostics, West Essex, UK) performed according to the manufacturer's instructions. The agents we tested were penicillin (0.25-16 [micro]g/mL), erythromycin (0.25-8 [micro]g/mL), vancomycin (0.25-32 [micro]g/mL), daptomycin (0.25-16 [micro]g/ mL), chloramphenicol (2-32 [micro]g/mL), linezolid (0.5-8 [micro]g/ mL), tetracycline (1-32 [micro]g/mL), quinupristin (0.5-32 [micro]g/ mL), tigecycline (0.05-0,5 [micro]g/mL), streptomycin (512-2048 [micro]g/mL), kanamycin (128-1024 [micro]g/mL), lincomycin (1-8 [micro]g/mL), and gentamicin (128-1024 [micro]g/mL). In addition, we determined MICs of clindamycin, erythromycin, and tetracycline by Etest (AB Biodisk, Solna, Sweden). We interpreted the results using the Clinical and Laboratory Standards Institute breakpoints for streptococci (9) for penicillin, erythromycin, vancomycin, daptomycin, chloramphenicol, tetracycline, and quinupristin; the European Committee on Antimicrobial Susceptibility Testing breakpoints for tigecycline and linezolid ( clinical_breakpoints); and the Comite de l'Antibiogramme de la Societe Francaise de Microbiologie breakpoints (10) for streptomycin, kanamycin, lincomycin, and gentamicin.

Macrolide Resistance Phenotype

To identify macrolide resistance phenotypes, we used a double-disk diffusion test (D-zone test) using erythromycin (15 pg) and clindamycin (2 pg) disks, as described by Hasenbein et al. (11). Isolates with blunting of the clindamycin inhibition zone around the disk adjacent to the erythromycin disk were considered to have an [iMLS.sub.B] phenotype (erythromycin resistant and clindamycin inducible). Clindamycin-susceptible isolates without blunting indicated an M phenotype (erythromycin resistant and clindamycin susceptible). Isolates that were resistant to both antimicrobial drugs were considered to have a [cMLS.sub.B] phenotype (constitutive erythromycin and clindamycin resistant).

Detection of Macrolides and Tetracycline

Resistance Genes

We extracted DNA according to the protocol in the US Centers for Disease Control and Prevention (CDC) S. pyogenes sequence database ( We screened all erythromycinresistant isolates by PCR for the erythromycin resistance genes ermB (12), ermA (13), mefA (14), and msrD (15). We tested tetracycline-resistant isolates for the tetracycline resistance genes tetM and tetO (14).

Detection of Virulence Genes

We tested the S. pyogenes isolates for the presence of the virulence genes speA, speB, speC, speF, speG, speH, speJ, speM, ssa, and smeZ by PCR. We used primers and conditions described previously (16,17).

PFGE Analysis, MLST, and emm Typing

For PFGE analysis, genomic DNAs of the S. pyogenes isolates were prepared and digested with SmaI restriction enzyme (MBI Fermentas, Vilnius, Lithuania) following a previously published protocol (18). We performed MLST following the method established by Enright et al. (19) and assigned the allele and sequence type (ST) according to the PubMLST website ( We amplified and sequenced the emm gene according to the protocol of the CDC International Streptococcal Reference Laboratory ( We compared the sequences of the emm genes with those in the CDC database using BLAST analysis ( for type assignment.


We observed 2 emm types (Table 2): emm 12 was the most frequent (11 isolates), followed by emm77 (4 isolates). Two pulsotypes (A and B) were generated after typing the isolates by PFGE with the restriction enzyme SmaI; 11 isolates were pulsotype A and 4 isolates pulsotype B (Figure). Similarly, we observed 2 genetic linages (ST26 and ST63) after MLST analysis.

All 15 S. pyogenes isolates were susceptible to penicillin (MIC [less than or equal to]0.25 mg/L), vancomycin (MICs [less than or equal to]0.25 to 0.5 mg/L), daptomycin (MIC [less than or equal to]0.25 mg/L), chloramphenicol (MICs [less than or equal to]2 to 4 mg/L), tigecycline (MICs [less than or equal to]0.015 to 0.12 mg/L), and gentamicin (MIC [less than or equal to]128 mg/L). Additionally, all isolates but 1 were susceptible to kanamycin (MIC [less than or equal to]128 mg/L), and 12 isolates showed susceptibility to linezolid (MICs <2 mg/L), streptomycin (MICs [greater than or equal to]2,048 mg/L), and lincomycin ([greater than or equal to]8 mg/L). On the other hand, all isolates were resistant to tetracycline, with MICs ranging from 24 to 96 mg/L using Etest (Table 2). Eleven isolates showed tetracycline-resistant genotype tetM/tetO, 2 isolates tetO, and 1 isolate tetM (Table 2).

Most isolates (7/15) exhibited the M phenotype, 2 isolates the phenotype [cMLS.sub.B], and 1 the phenotype [iMLS.sub.B] (Table 2). The macrolide-resistant genotype mefA/ermB was the most frequently observed, seen in all isolates but 1 with the M phenotype and in the isolate with phenotype [cMLS.sub.B]. The genotype ermB was observed alone in 1 isolate of each phenotype. No isolate carried the msrD or ermA macrolide-resistant determinants.

We detected the chromosomal-encoded speB and speF genes in all isolates. We observed 2 different virulence gene profiles based on the presence/absence of the speG and speC genes. We detected the genotype speG in 11 isolates and the genotype speC in 4 isolates (Table 2).

We grouped the 15 S. pyogenes isolates into 2 different clones on the basis of emm-MLST-PFGE-virulence genes profile combinations. Clone 1 grouped isolates characterized by the combined genotype emm 12-ST36-pulsotype A-speB/speF/speG, whereas isolates of clone 2 were characterized by the genotype emm77-ST63-pulsotype B-speB/ speF/speC (Table 2). In addition, isolates of clone 1 were erythromycin resistant, mainly exhibiting an M phenotype, and isolates of clone 2 were erythromycin susceptible.


S. pyogenes is a human pathogen that has rarely been isolated from animals. It has been isolated from abscesses in cervical and mesenteric lymph nodes and liver of a free-living European hedgehog (E. europaeus) and from 2 dogs with severe colonic disease and conjunctivitis (5-7). Here we describe the detailed characterization of animal S. pyogenes isolates from different clinical specimens obtained from rabbits (n = 14) and sheep (n = 1) in Spain during 2006-2014. This pathogen was recovered mainly from noninvasive cases, with skin infections being the most common clinical presentation (n = 6), followed by genital tract infections (n = 4) (Table 1). S. pyogenes was isolated from all skin clinical samples together with Staphylococcus aureus, a well-recognized pathogen associated with different skin diseases in animals (20). These results indicate that although S. pyogenes should be able to colonize the skin of animals, it is difficult to ascertain its etiologic significance in skin infections. However, S. pyogenes was isolated in pure culture from clinical specimens of the genital tract, ears, mammary glands, and lungs in rabbits, indicating the potential role of S. pyogenes in these infections.

Most of the S. pyogenes isolates we tested (n = 11) exhibited the genotype emm12-ST36, which has been isolated repeatedly from humans in different countries (21-27), including Spain (28-30). This genotype can exhibit an M phenotype (31) and has been associated with skin and soft tissue infections (32), data that fit with our results, as more than half of the isolates with this genotype were isolated from abscesses and dermatitis (Table 2). The genotype emm77-ST63 that we identified in 4 animal isolates has also been detected in human S. pyogenes isolates (21,25,33), but unlike human isolates, the isolates in our study were erythromycin and clindamycin susceptible (Table 2).

All 11 isolates in clone 1 (pulsotype A) exhibited PFGE profiles that were indistinguishable from each other, and all 4 isolates in clone 2 also exhibited PFGE profiles that were indistinguishable PGFE from each other (pulsotype B; Figure). Isolates of S. pyogenes usually exhibit high levels of genetic diversity (4). Thus, the fact that we identified only 2 clones in different isolates collected over a period of 8 years was unexpected. The possibility of a common source of infection is very unlikely because all isolates were recovered at different times from different animals in farms located at geographically distant locations spread throughout Spain, without any epidemiologic relationship (Table 1). In addition, clinical specimens were processed independently in the same laboratory by highly qualified and trained personnel, which makes the possibility of a cross-contamination in the laboratory unlikely.

Under these conditions, multiple human-to-animal transmission events should be the most likely origin of these genotypes in sheep and rabbits. Another possible explanation could be that genotypes ST36 and ST63, although originating from humans, represent genetic linages with a specific host tropism, mainly for rabbits, which contributed to their successful dissemination in these animals, as observed with other streptococci (34). Cases of S. pyogenes infection were not recorded among the personnel working in the rabbit farms and sheep flock from which S. pyogenes was isolated. Asymptomatic human carriers have a key role in S. pyogenes transmission (35). For these reasons and even though screenings to identify asymptomatic S. pyogenes carriers were not carried out, we can speculate that asymptomatic employees were the most probable source of S. pyogenes in the animals included in the study. Although we cannot infer from the results of this study that animals, mainly rabbits, may represent a new reservoir of S. pyogenes, the results clearly indicate the ability of human-derived S. pyogenes isolates to colonize and infect animals, which could be more frequent than has been recognized until now.

Isolates with the genotype mefA/ermB usually correlate with the [cMLS.sub.B] phenotype, but 5 of the 6 S. pyogenes isolates with the mefA/ermB genotype in our study exhibited M phenotype (Table 2), which agrees with previous observations (29). The erm gene usually confers co-resistance to macrolides, lincosamides, and streptogramins. Curiously, all M phenotype isolates in our study showed susceptibility to clindamycin and were positive for the emrB gene. This result, although unusual, has also been observed previously in S. pyogenes isolates from different countries (26,36-38). A possible explanation could be that the ermB gene was nonfunctional in the isolates with clindamycin-susceptible phenotypes. The isolate M72193 exhibited the [iMLS.sub.B] phenotype but was ermA-negative (Table 2). This result, although infrequent, has also been observed in previous studies (39). Isolates with the [iMLS.sub.B] phenotype have been further subdivided into 3 distinct types: type A, associated with the presence of the ermB gene; and types B and C, associated with the presence of the ermA gene (40,41). This isolate carried the ermB gene (Table 2), suggesting therefore an [iMLS.sub.B]-A phenotype.

Unlike most human S. pyogenes isolates, which usually carry either tetM or tetO genes, most of the isolates in this study (n = 11) carried both genes (Table 2). Human isolates with the combination of tetM and tetO tetracycline-resistance genes have been identified previously in Spain (29). Another uncommon result was the identification of 1 isolate (83553) that was resistant to tetracycline (MIC 64 mg/L) but lacked resistance tetM and tetO genes (Table 2) commonly associated with tetracycline resistance in S. pyogenes (42). However, tetracycline-resistant strains and negativity to these genes have also been reported (43). Further studies will be necessary to elucidate the precise mechanism of resistance to tetracycline in this strain.

In summary, this study provides a detailed characterization of animal S. pyogenes isolates associated with different clinical backgrounds. This pathogen should be considered by veterinary microbiologists when processing clinical material from animals.

Dr. Vela is an associate professor at the Animal Health Department, Veterinary Faculty, Complutense University, Madrid, Spain. Her research focuses on the characterization of relevant animal bacterial pathogens.


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Address for correspondence: Jose F. Fernandez-Garayzabal, Universidad Complutense de Madrid, Ftad. de Veterinaria-Patologia Animal I (Sanidad Animal), Avda. Puerta de Hierro s/n n/a, Madrid, Madrid 28040 Spain; email:

Ana Isabel Vela, Pilar Villalon, Juan Antonio Saez-Nieto, Gema Chacon, Lucas Dominguez, Jose Francisco Fernandez-Garayzabal

Author affiliations: Complutense University, Madrid, Spain (A.I. Vela, L. Dominguez, J.F. Fernandez-Garayzabal); Instituto de Salud Carlos III, Majadahonda, Madrid (P. Villalon, J.A. Saez-Nieto); Laboratorio Exopol San Mateo, Zaragoza, Spain (G. Chacon)


Caption: Figure. Pulsed-field gel electrophoresis patterns of Smal-digested DNA of clinical isolates of Streptococcus pyogenes from animal specimens, Spain, 2006-2014. Lanes 1 and 17, DNA molecular size marker; lanes 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, and 16, isolates M50163, M79144, M78761, M75791, M75539, M75533, M75123, M73512, M72636, M72193, and M83639, respectively (pulsotype A); lanes 2, 6, 14, and 16, isolates 83553, 85374, M75768, and M82209, respectively (pulsotype B).
Table 1. Features and disease manifestations of 15 animals
from which Streptococcus pyogenes isolates were collected,
Spain, 2006-2014

Isolate *   Animal       Clinical background       Specimen

M50163      Rabbit            Metritis              Uterus
M79144      Rabbit    Abscesses and dermatitis       Skin
M78761      Rabbit           Dermatitis              Skin
M75791      Rabbit            Abscesses              Skin
M75539      Sheep             Abscesses              Skin
M75533      Rabbit             Otitis                 Ear
M75123      Rabbit            Metritis              Uterus
M73512      Rabbit            Abortion              Uterus
M72636      Rabbit            Metritis              Uterus
M72193      Rabbit            Abscesses              Skin
83639       Rabbit    Abscesses and dermatitis       Skin
83553       Rabbit            Pneumonia              Lung
M82209      Rabbit            Abscesses              Skin
M75768      Rabbit            Mastitis               Milk
85374       Rabbit         Skin infection            Skin

Isolate *    Geographic region     Isolation date

M50163           Valencia             2006 Jan
M79144          Valladolid            2013 Mar
M78761          Valladolid            2013 Feb
M75791           Valencia             2012 Apr
M75539           Zaragoza             2012 Mar
M75533           Valencia             2012 Mar
M75123           Castellon            2012 Feb
M73512           Zaragoza             2011 Aug
M72636           Zaragoza             2011 May
M72193           Valencia             2011 Apr
83639           Valladolid            2014 Mar
83553            Zaragoza             2014 Mar
M82209          Valladolid            2013 Dec
M75768           Zaragoza             2012 Mar
85374           Valladolid            2014 Aug

* Isolates M50163 and M73512 were recovered in pure culture.
The remaining isolates were recovered together with Staphylococcus

([dagger]) Except for isolates M79144 and M78761, which were isolated
in the same farm but at different times, all other isolates were
recovered from animals at different farms.

Table 2. Testing results for the 15 isolates characterized
in study of Streptococcus pyogenes from animal specimens,
Spain *

Isolate                                   MIC, mg/L
           emm      PFGE      MLST
           type    profile    type    ERY    CLIN   TET

M50163      12        A       ST36    >256    32    96
M79144      12        A       ST36    >256   0.75   48
M78761      12        A       ST36    >256   0.75   32
M75791      12        A       ST36     6     0.09   32
M75539      12        A       ST36     8     0.19   24
M75533      12        A       ST36     16    0.19   32
M75123      12        A       ST36     12    0.19   48
M73512      12        A       ST36    0.25   0.12   32
M72636      12        A       ST36    >256   >256   48
M72193      12        A       ST36    >256   1.5    96
83639       12        A       ST36    >256   0.38   48
83553       77        B       ST63    0.19   0.12   64
85374       77        B       ST63    0.12   0.09   64
M75768      77        B       ST63    0.12   0.09   32
M82209      77        B       ST63    0.19   0.12   64

Isolate       Macrolide resistance
                                         TET resistance
            Phenotype       Genotype          genes

M50163     [cMLS.sub.B]    mefA/ermB        tetM/tetO
M79144           M         mefA/ermB        tetM/tetO
M78761           M         mefA/ermB        tetM/tetO
M75791           M         mefA/ermB        tetM/tetO
M75539           M         mefA/ermB        tetM/tetO
M75533           M         mefA/ermB        tetM/tetO
M75123           M            ermB          tetM/tetO
M73512                                      tetM/tetO
M72636     [cMLS.sub.B]       ermB          tetM/tetO
M72193     [iMLS.sub.B]       ermB          tetM/tetO
83639            M            ermB            tetM
85374                                         tetO
M75768                                      tetM/tetO
M82209                                        tetO

Isolate     Virulence genes

M50163      speB/speF/speG
M79144      speB/speF/speG
M78761      speB/speF/speG
M75791      speB/speF/speG
M75539      speB/speF/speG
M75533      speB/speF/speG
M75123      speB/speF/speG
M73512      speB/speF/speG
M72636      speB/speF/speG
M72193      speB/speF/speG
83639       speB/speF/speG
83553       speB/speF/spec
85374       speB/speF/spec
M75768      speB/speF/speC
M82209      speB/speF/speC

* ERY, erythromycin; CLIN, clindamycin; MLST, multilocus
sequence typing; PFGE, pulsed-field gel electrophoresis;
ST, sequence type; TET, tetracycline.
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Title Annotation:RESEARCH
Author:Vela, Ana Isabel; Villalon, Pilar; Saez-Nieto, Juan Antonio; Chacon, Gema; Dominguez, Lucas; Fernand
Publication:Emerging Infectious Diseases
Geographic Code:4EUSP
Date:Dec 1, 2017
Previous Article:December 2012: Zoonotic Infections.
Next Article:July 2016: Zoonoses.

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