Accepted Manuscript Infection of human keratinocytes by Streptococcus dysgalactiae subspecies dysgalactiae isolated from milk of the bovine udderInfection of human keratinocytes by Streptococcus dysgalactiae subspecies dysgalactiae isolated from milk of the bovine udder Catarina Roma-Rodrigues, Cynthia Alves-Barroco, Luís R. Raposo, Mafalda N. Costa, Elvira Fortunato, Pedro Viana Baptista, Alexandra R. Fernandes, Ilda SantosSanches

PII:

S1286-4579(15)00253-1

DOI:

10.1016/j.micinf.2015.11.005

Reference:

MICINF 4351

To appear in:

Microbes and Infection

Received Date: 10 November 2015 Revised Date:

16 November 2015

Accepted Date: 21 November 2015

Please cite this article as: C. Roma-Rodrigues, C. Alves-Barroco, L.R Raposo, M.N Costa, E. Fortunato, P.V. Baptista, A.R Fernandes, I. Santos-Sanches, Infection of human keratinocytes by Streptococcus dysgalactiae subspecies dysgalactiae isolated from milk of the bovine udderInfection of human keratinocytes by Streptococcus dysgalactiae subspecies dysgalactiae isolated from milk of the bovine udder, Microbes and Infection (2015), doi: 10.1016/j.micinf.2015.11.005. 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.

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Infection of human keratinocytes by Streptococcus dysgalactiae subspecies dysgalactiae isolated from milk of the bovine udder

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Catarina Roma-Rodriguesa, Cynthia Alves-Barrocoa, Luís R Raposoa,b, Mafalda N Costa3, Elvira Fortunatoc, Pedro Viana Baptistaa, Alexandra R Fernandesa,b,*, Ilda Santos-Sanchesa,*

Affiliations:

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a – UCIBIO, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal b – CQE, Centro de Quimica Estrutural, Complexo 1, Instituto Superior Técnico, Universidade de Lisboa, Avenida Rovisco Pais, 1049-001 Lisboa, Portugal

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c – i3N/CENIMAT, Departamento de Ciência dos Materiais, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal

Corresponding authors:

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Alexandra Fernandes

Email: [email protected]

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Ilda Santos-Sanches

Email: [email protected]

Adress: Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal;

Phone: +351 212 948 530 Fax: +351 212 948 530

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Abstract (94/100) Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) are considered exclusive animal pathogens; however, a putative zoonotic upper limb cellulitis, a prosthetic joint infection and an infective endocarditis were described in humans. To unravel if bovine SDSD isolates are able to infect human cells, the adherence and internalization to human primary keratinocytes of two

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bovine SDSD strains isolated from milk collected from udder were analyzed. Bacterial adhesion assays and confocal microscopy indicate a high adherence and internalization of SDSD isolates

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to human cells, suggesting for the first time the ability of bovine isolates to infect human cells.

List of abbreviations

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Keywords: adhesion; bovine; host; human keratinocytes; internalization; Streptococcus dysgalactiae subspecies dysgalactiae

CFUs – Colony Forming Units

DMEM – Dulbecco’s Modified Eagle Medium supplemented with 10% (v/v) Foetal Bovine Serum

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PBS – Phosphate Buffer Saline

SDSD – Streptococcus dysgalactiae subps. dysgalactiae

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THB-5YE – Todd Hewitt Broth supplemented with 5% (w/v) yeast extract

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1. Introduction

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Streptococcus dysgalactiae subspecies dysgalactiae (SDSD) are alpha-hemolytic or non-

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hemolytic streptococci belonging to the Lancefield group C [1, 2]. They are considered as

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exclusive animal pathogens, despite three sporadic cases of infection in humans being reported,

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namely an ascending upper limb cellulitis in a woman that contacted raw fish [3], a prosthetic joint

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infection after total knee arthroplasty [4], and very recently, an infective endocarditis in a male

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patient [5]. Previous investigations in our group evidenced that SDSD strains recovered from

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subclinical bovine mastitis carried and expressed phage-encoded virulence genes including

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genes that code for the exotoxins SpeK, SpeC, SpeL and SpeM, genes coding for DNase I

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(Spd1), and streptodornase gene (sdn) of the strictly human pathogen Streptococcus pyogenes

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[6, 7]. Despite these reported cases of human infections caused by SDSD, it has never been

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reported that animal SDSD isolates, particularly bovine mastitis subclinical strains, are capable to

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infect human cells. Nevertheless, the presence of S. pyogenes virulence factors in the genome of

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bovine SDSD has lead to the hypothesis of SDSD dissemination to other hosts. Hence, here the

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possibility of SDSD strains to adhere to and invade human cells was investigated. Adherence and

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invasion of SDSD isolates in epithelial cells has been described to occur in mammary cell lines

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and fish epithelial cell lines cultured in vitro [8-10]. The virulence of SDSD seems to be related to

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cell surface properties, such as high hemagglutination and hydrophobic properties that determine

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the main adhesion and invasive pathogenic mechanism of the species [8, 11-13].

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2. Materials and Methods

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2.1. Bacterial species and culture conditions

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ACCEPTED MANUSCRIPT Streptococcus dysgalactiae subsp. dysgalactiae strains VSD5 and VSD13 were isolated from

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milk collected from the udder of cows with mastitis [6, 7]. Strain identified as GCS-Si was

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responsible for an ascending upper limb cellulitis in a woman that was pricked by the fins and

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scales of a raw fish [3]. S. pyogenes GAP58 is an invasive strain isolated from the blood a patient

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and GAP8 is a strain isolated from the tonsils of an asymptomatic patient [14]. The

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characterization of each strain based on 16S sequencing, emm subtype and MLST was

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previously performed [7, 14]. Strains were cultivated in 20 mL Todd Hewitt broth supplemented

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with 5 % (w/v) yeast extract (THB-5YE) in 100 mL Erlenmeyer-flasks and grown at 37ºC until

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reach a standardized optical density at 600 nm (OD600) of 0.3 – 04 (5x107 – 1x108 cells/mL) .

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For each strain, an aliquot of 1 mL of cell suspension was collected and cells were washed 3

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times in fresh THB-5YE.

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2.2. Human Cells and culture conditions

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Human primary epidermal keratinocytes (ATCC-PCS-200-010, ATCC, Manassas, USA) were

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grown in keratinocyte growth media according to ATCC (Manassas, USA) instructions. For

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infection assays, cells were seeded in a 96-well culture plate at a density of 3 x 104 cells/well and

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incubated for 24 h at 37 ºC, 5 % (v/v) CO2 and 99 % (v/v) relative humidity. For confocal

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microscopy analysis 1x105 cells were placed over a Polylysine treated microscope slide and

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incubated for 24 h at 37 ºC, 5 % (v/v) CO2 and 99 % (v/v) humidity.

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2.3. Bacterial internalization and adherence assays

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The adhesion and internalization assays were based on previously described protocols [15, 16]

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with few modifications. Bacteria were grown at 37 ºC in Todd Hewitt Broth supplemented with 5%

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(w/v) yeast extract (THB-5YE) until the middle of the exponential phase. The infection was started

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by adding 106 bacterial cells in Dulbecco’s Modified Eagle’s Medium supplemented with 10%

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(v/v) Foetal Bovine Serum (DMEM) to 104 human primary epidermal keratinocytes (ATCC-PCS-

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ACCEPTED MANUSCRIPT 200-010, ATCC, Manassas, USA). After 2 h of incubation at 37 ºC, 5% (v/v) CO2 and 99% (v/v)

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relative humidity, cell monolayers were washed with Phosphate Buffer Saline (PBS) to remove

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unbound bacteria. To obtain the number of bacteria that adhere and internalize to keratinocytes

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[(Adh+Int)value] and the number of bacteria that internalize keratinocytes (Intvalue), infected

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keratinocyte cell monolayers were incubated for 30 min with DMEM or DMEM supplemented with

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Penicillin 100 units/mL, respectively. Keratinocytes were detached from the well, disrupted with

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Triton X-100 0.01% (v/v) (Sigma), and the bacterial colony-forming units (CFUs) were calculated

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using standard plate counting techniques. In parallel, the exact same procedure was performed in

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keratinocyte free wells in order to obtain procedural negative control values. The values obtained

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in the procedural control is subtracted from Intvalue and (Adh+Int)value. Intvalue was the averaged

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CFUs counted for the keratinocytes infected cells treated with Penicillin minus the averaged

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CFUs obtained in Intctrl. The (Adh+Int)value was the averaged CFUs counted for the keratinocytes

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infected cells minus the averaged CFUs obtained in (Adh+Int)ctrl. The Adhvalue of each strain was

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obtained by subtracting the Intvalue to the (Adh+Int)value. Every experiment was repeated at least 4

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times.

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2.4. Confocal laser scanning microscopy

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For confocal laser scanning microscopy analysis, pelleted bacteria were resuspended in THB-

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5YE supplemented with 0.2 mg/mL Hoechst 33258 (LifeTechnologies), incubated at 37 ºC for 45

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min, washed with PBS and resuspended in DMEM. Keratinocytes in microscope slides were

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washed 3 times with PBS and bacterial cells placed over the human cells. After 2 h of incubation

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at 37 ºC, 5 % (v/v) CO2 and 99 % (v/v) relative humidity, cells were fixed with 2 % (w/v)

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paraformaldehyde, lysed with 0.1 % (v/v) Triton X-100 and animal cells were stained with

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AlexaFluor 488 Phalloidin (LifeTechnologies) according to the manufacturer’s instructions. After

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washing 3 times with PBS, a drop of ProLong Diamond antifade mountant (LifeTechnologies) was

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placed on top of the cells, covered with a cover slip and sealed. A confocal laser scanning

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AlexaFluor 488 Phalloidin, and the 405 nm laser for visualization of bacterial cells stained with

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Hoechst 33258, and respective software (ZEN Black, 2011), was used for image acquisition. This

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approach only used the bovine SDSD VSD13 and the human S. pyogenes GAP58. A control

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sample was also prepared, consisting in keratinocytes stained for 2 h with 20 µg/mL Hoechst

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33258.

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3. Results

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To unravel if bovine SDSD isolates were able to interact with human cells, human keratinocytes

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were infected for 2 h with two bovine SDSD strains, VSD5 and VSD13, both isolated from milk

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collected from udder [6, 7], and the adherence and internalization assessed. For comparative

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purposes, adherence and internalization of the SDSD isolate that caused ascending upper limb

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cellulitis (SDSD GCS-Si) [3], an invasive human isolate S. pyogenes GAP58 collected from the

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blood of a patient, and a S. pyogenes GAP8 human isolate from the oropharynx of an

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asymptomatic carrier [14] were also assessed. Primary keratinocytes were selected due to the

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common colonization of non-intact skin by S. pyogenes [17] and the ascending upper limb

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cellulitis caused by SDSD GCS-Si.

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Figure 1 shows that there is 1 internalized/adhered VSD5 cell per 10 keratinocytes. Also, data

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show that 3 and 11 VSD13 cells are capable to internalize and adhere to a single keratinocyte,

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respectively. These results suggest a higher adherence and internalization of VSD13 strains to

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human keratinocytes. Also, higher adherence of the bovine isolate VSD13 and higher

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internalization values of the bovine isolates are observed when compared to the two GAS strains.

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ACCEPTED MANUSCRIPT The adherence and internalization of SDSD VSD13 was further confirmed by confocal

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microscopy. Results suggest that only the nuclear DNA of the epithelial cells was stained, as can

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be observed in the movie from additional file 1, which shows the X-Y images obtained along the

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Z-plan for the control sample.

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Confocal images analysis (Fig.2) corroborates adherence and internalization results. A higher

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number of SDSD VSD13 (Fig.2, A) than S. pyogenes GAP58 cells (Fig.2, B) were observed to be

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interacting with the human keratinocytes. Two additional movies show with more detail the X-Y

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images obtained along the Z plan, the additional file 2 shows the interaction of SDSD VSD13 with

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keratinocytes and additional file 3 show the adherence and internalization of S. pyogenes GAP58.

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4. Discussion

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This work describes for the first time that SDSD bovine isolates collected from the milk of udder

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are capable to adhere and internalize human cells. Remarkably, the adherence and

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internalization values of bovine SDSD isolates are higher than those of human streptococci

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isolates analyzed in this study.

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Since the classification of S. dysgalactiae by Vandamme et al [2], that human S. dysgalactiae G

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and C groups are classified as S. dysgalactiae subsp. equisimillis, while all strains of animal

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origin are classified as S. dysgalactiae subsp. dysgalactiae [1, 2]. In this work, the ability of

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bovine SDSD strains to adhere and internalize human primary keratinocyte cells is demonstrated,

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raising the question if this division is adequate. In fact, the correct identification of S. dysgalactiae

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species is paramount in clinical practice, since the information of the causative infection agent is

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vital for the adequate diagnosis and treatment of streptococcal infections [18].

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Acknowledgments

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We thank the Fundação para a Ciência e a Tecnologia/Ministério da Educação e Ciência

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(FCT/MEC) for financial support via PTDC/CVT-EPI/4651/2012, PTDC/BBB-NAN/1812/2012 and

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UID/CTM/50025/2013.

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Conflict of interests

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Authors declare no conflict of interests.

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References

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Figure Legends:

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Figure 1: Infection potential of bovine Streptococcus dysgalactiae subspecies

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dysgalactiae strains of human keratinocytes. Adhesion (open bars) and internalization (filled

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bars) in human primary epithelial keratinocytes of bovine isolates Streptococcus dysgalactiae

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subsp. dysgalactiae (SDSD) VSD5 and VSD13, and human isolates SDSD GCS-Si,

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Streptococcus pyogenes (SPYO) GAP58 and GAP8. Represented values are the average value

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with SD. Represented values were corrected by subtracting values of procedural control

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(keratinocyte-free wells). Results were only considered when procedural control values were

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significantly lower (p-value < 0.05) than adherence and internalization values. *;**;***,**** P value

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< 0.05.

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Figure 2: Infection of human keratinocytes by bovine Streptococcus dysgalactiae subsp.

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dysgalactiae. Representative confocal laser scanning ortho-image of Z-stacks of 2 h infected

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human keratinocytes (green, stained with AlexaFluor 488 Phaloidin, LifeTechnologies) with

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Streptococcus (blue, stained with Hoechst 33258, LifeTechnologies). The centered panel is the

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X-Y view of the image, corresponding to the blue line in the upper and right panels. The upper

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panel, with a green outline, is the X-Z cross section of the green line in the centered image. The

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image at the right, with a red outline, is the Y-Z cross section of the red line in the X-Y view. A)

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Human keratinocytes infected with S. dysgalactiae subps. dysgalactiae VSD13 collected from the

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milk of bovine udder. C) Human keratinocytes infected with the invasive human isolate S.

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pyogenes GAP58. Red arrows point to internalized bacteria; yellow arrows point to adhered

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bacteria.

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Supporting information

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Additional file 1

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File format: .MOV

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Title of data: Control sample.

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Description of data: Representative movie obtained using confocal laser scanning microscopy,

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of the X-Y images obtained along the Z-plane of human keratinocytes stained for 2 h with 20

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µg/mL Hoechst 33258 (blue, LifeTechnologies), fixed with 2 % paraformaldehyde and stained

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with AlexaFluor 488 Phaloidin (green, LifeTechnologies).

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Additional file 2

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File format: MOV

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Title of data: Infection of human keratinocytes by Streptococcus dysgalactiae subsp.

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dysgalactiae VSD13 collected from the milk of the bovine udder.

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Description of data: Representative movie obtained using confocal laser scanning microscopy,

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of the X-Y images obtained along the Z-plane, of human keratinocytes (green, stained with

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AlexaFluor 488 Phaloidin, LifeTechnologies) infected for 2 h with S. dysgalactiae subps.

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dysgalactiae VSD13 (blue, stained with Hoechst 33258, LifeTechnologies).

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Additional file 3

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File format: MOV

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ACCEPTED MANUSCRIPT Title of data: Infection of human keratinocytes by human Streptococcus pyogenes GAP58.

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Description of data: Representative movie obtained using confocal laser scanning microscopy,

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of the X-Y images obtained along the Z-plane, of human keratinocytes (green, stained with

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AlexaFluor 488 Phaloidin, LifeTechnologies) infected for 2 h with S. pyogenes GAP58 (blue,

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stained with Hoechst 33258, LifeTechnologies).

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Infection of human keratinocytes by Streptococcus dysgalactiae subspecies dysgalactiae isolated from milk of the bovine udder.

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) are considered exclusive animal pathogens; however, a putative zoonotic upper limb cellulitis, a...
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