Virulence gene detection and expression in Streptococcus Dysgalactiae subsp. Dysgalactiae Strains and evaluation of infection potential

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) é considerado um agente patogénico animal exclusivo e Streptococcus pyogenes (GAS) um agente patogénico humano exclusivo. Recentemente foram encontrados fatores de virulência fágicos de GAS em estirpes de SDSD de origem bovina e casos de infeção humana associada a SDSD têm vindo a ser reportados. Em consequência, o potencial zoonótico de SDSD foi sugerido, contudo o papel destes fatores de virulência na patogénese de SDSD não foi comprovado. Um dos objetivos desta tese foi detetar a presença e expressão de fatores de virulência de GAS, entre isolados de SDSD contemporâneos de origem portuguesa, isolados de amostras de leite de bovinos disgnosticados com mastite em herdades leiteiras portuguesas entre 2011-13 e comparar estes dados com os reportados de uma coleção portuguesa de SDSD previamente estudada de 2002-03. O potencial de infeção in vitro e in vivo foi também avaliado e comparado entre coleções. Determinantes genéticos de GAS (os genes de virulência speB, speC, speF, speH, speK, speL, speM, smeZ, spd1, sdn e o elemento quimérico Tn1207.3/Φ10394.4) foram pesquisados por PCR e a sua expressão averiguada por PCR após síntese de cDNA. A produção de DNases extracelulares foi avaliada e correlacionada com o perfil genotipico dos genes spd1 e sdn. Para estudar o potencial de infeção, in vitro, foram utilizadas linhas celulares repiratórias normais e tumorais humanas (BTEC e Detroit 562, respetivamente) e in vivo, o modelo animal zebrafish. Os resultados sugerem que os fatores de virulência pesquisados são característicos de SDSD de origem bovina e a produção de DNases extracelulares é independente dos genes spd1 e sdn. Os estudos de infeção in vitro e in vivo revelam que os potenciais de infeção de SDSD são específicos de estirpe e independentes dos genes de virulência pesquisados. O potencial zoonótico de SDSD é novamente sugerido uma vez que estirpes de origem bovina foram capazes de infetar linhas celulares humanas e o zebrafish.Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) is considered an exclusively animal pathogen and Streptococcus pyogenes (GAS) a strictly human pathogen. GAS phage virulence determinants were recently found in SDSD strains of bovine origin, and cases of human infection associated with SDSD have been recently reported. The SDSD zoonotic potential has been therefore suggested, however the role of those virulence genes in the pathogenesis of the bovine SDSD has not been proved. One of the objectives of this thesis was to detect the presence and expression of GAS virulence determinants, among contemporary SDSD strains, isolated from milk samples of bovines diagnosed with mastitis in Portuguese dairy herds between 2011-13 and compare the data with the one previously reported of a study of a Portuguese SDSD collection of 2002-03. In vitro and in vivo infection potential was also evaluated and compared between both collections. GAS genetic determinants (virulence genes speB, speC, speF, speH, speK, speL, speM, smeZ, spd1, sdn and the chimeric element Tn1207.3/Φ10394.4) were screened by PCR and their expression was assessed by PCR after cDNA synthesis. Extracellular DNase production was assessed and correlated with spd1 and sdn genotypic profile. To study the infection potential, in vitro, human normal and tumoral respiratory cell lines (BTEC and Detroit 562, respectively) were used, and in vivo, the zebrafish animal model was chosen. Results suggested that the virulence determinants screened are characteristic of SDSD of bovine origin and that extracellular DNase production was independent on the spd1 and sdn genes. In vitro and in vivo infection studies revealed that the infection potentials of SDSD are strain-specific and independent on the virulence genes screened. Zoonotic potential of SDSD is further suggested, as strains from bovine origin were able to infect human cell lines, as well as the zebrafish

New Insights on Streptococcus dysgalactiae subsp. dysgalactiae Isolates

Funding Information: This work was supported by the Unidade de Ciências Biomoleculares Aplicadas-UCIBIO, which is financed by Funding Information: Funding. This work was supported by the Unidade de Ci?ncias Biomoleculares Aplicadas-UCIBIO, which is financed by national funds from FCT/MEC (UIDP/04378/2020 and UIDB/04378/2020) and also by projects PTDC/CVT-EPI/4651/2012 and PTDC/CVT-EPI/6685/2014. FCT-MEC is also acknowledged for grant SFRH/BD/118350/2016 to CA-B. Publisher Copyright: © Copyright © 2021 Alves-Barroco, Caço, Roma-Rodrigues, Fernandes, Bexiga, Oliveira, Chambel, Tenreiro, Mato and Santos-Sanches.Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) has been considered a strict animal pathogen. Nevertheless, the recent reports of human infections suggest a niche expansion for this subspecies, which may be a consequence of the virulence gene acquisition that increases its pathogenicity. Previous studies reported the presence of virulence genes of Streptococcus pyogenes phages among bovine SDSD (collected in 2002–2003); however, the identity of these mobile genetic elements remains to be clarified. Thus, this study aimed to characterize the SDSD isolates collected in 2011–2013 and compare them with SDSD isolates collected in 2002–2003 and pyogenic streptococcus genomes available at the National Center for Biotechnology Information (NCBI) database, including human SDSD and S. dysgalactiae subsp. equisimilis (SDSE) strains to track temporal shifts on bovine SDSD genotypes. The very close genetic relationships between humans SDSD and SDSE were evident from the analysis of housekeeping genes, while bovine SDSD isolates seem more divergent. The results showed that all bovine SDSD harbor Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas IIA system. The widespread presence of this system among bovine SDSD isolates, high conservation of repeat sequences, and the polymorphism observed in spacer can be considered indicators of the system activity. Overall, comparative analysis shows that bovine SDSD isolates carry speK, speC, speL, speM, spd1, and sdn virulence genes of S. pyogenes prophages. Our data suggest that these genes are maintained over time and seem to be exclusively a property of bovine SDSD strains. Although the bovine SDSD genomes characterized in the present study were not sequenced, the data set, including the high homology of superantigens (SAgs) genes between bovine SDSD and S. pyogenes strains, may indicate that events of horizontal genetic transfer occurred before habitat separation. All bovine SDSD isolates were negative for genes of operon encoding streptolysin S, except for sagA gene, while the presence of this operon was detected in all SDSE and human SDSD strains. The data set of this study suggests that the separation between the subspecies “dysgalactiae” and “equisimilis” should be reconsidered. However, a study including the most comprehensive collection of strains from different environments would be required for definitive conclusions regarding the two taxa.publishersversionpublishe

Streptococcus dysgalactiae subsp. dysgalactiae isolated from milk of the bovine udder as emerging pathogens: In vitro and in vivo infection of human cells and zebrafish as biological models

Fundacao para a Ciencia e a Tecnologia, Grant/Award Number: ERDF under the PT2020 Partnership Agreement (POCI-, IF/00265/2015, PTDC/CVT-EPI/6685/2014, SFRH/BD/118350/2016, UID/MAR/04292/2013 and UID/Multi/04378/2013; Unidade de Ciencias Biomoleculares Aplicadas-UCIBIO; FCT/MEC, Grant/Award Number: UID/Multi/04378/2013Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) is a major cause of bovine mastitis and has been regarded as an animal-restricted pathogen, although rare infections have been described in humans. Previous studies revealed the presence of virulence genes encoded by phages of the human pathogen Group A Streptococcus pyogenes (GAS) in SDSD isolated from the milk of bovine udder with mastitis. The isolates SDSD VSD5 and VSD13 could adhere and internalize human primary keratinocyte cells, suggesting a possible human infection potential of bovine isolates. In this work, the in vitro and in vivo potential of SDSD to internalize/adhere human cells of the respiratory track and zebrafish as biological models was evaluated. Our results showed that, in vitro, bovine SDSD strains could interact and internalize human respiratory cell lines and that this internalization was dependent on an active transport mechanism and that, in vivo, SDSD are able to cause invasive infections producing zebrafish morbidity and mortality. The infectious potential of these isolates showed to be isolate-specific and appeared to be independent of the presence or absence of GAS phage-encoded virulence genes. Although the infection ability of the bovine SDSD strains was not as strong as the human pathogenic S. pyogenes in the zebrafish model, results suggested that these SDSD isolates are able to interact with human cells and infect zebrafish, a vertebrate infectious model, emerging as pathogens with zoonotic capability.publishersversionpublishe