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

Abstract

Streptococcus 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.

Keywords: Streptococcus dysgalactiae subsp. dysgalactiae; bovine; host adhesion/internalization; systemic infection; zebrafish.

Figure 1

Infection potential of bovine Streptococcus dysgalactiae subspecies dysgalactiae isolates in human airway stem cell lines. Percentage of adherent and internalized SDSD VSD5, VSD9, VSD13 VSD21, VSD23, VSD24 and Streptococcus pyogenes GAP58, GAP90, and GAP447 isolates after the incubation for 2 hours in Detroit 562, A549, and BTEC human cell lines. Represented values are the average value with SEM. *p‐value < .005 for results obtained for A549 cells infection of VSD21, VSD23, VSD24 relative to GAP58. **p‐value < .05 for results obtained for BTEC infection of VSD5 and VSD24 relative to GAP58

Figure 2

Percentage of survival of zebrafish infected with S. dysgalactiae subsp. dysgalactiae VSD9 (blue line), VSD13 (purple line), VSD21 (green line), VSD23 (orange line), VSD24 (red line), S. pyogenes GAP58 (yellow line), and with growth medium TSB‐0.5YE (negative control) (black line). Results are the mean of at least four independent experiments with more than 10 zebrafish in each group and are significantly different with a p‐value < .001

Figure 3

Images of injected zebrafish with (a) no sign of infection/disease (control zebrafish that died 24 hours after inoculation), (b–d) Focal infection (zebrafish injected with SDSD VSD13, VSD24, and VSD21 isolates, respectively), and (e), (f) and (G1; G2) Gross pathology (zebrafish injected intraperitoneally with SDSD VSD21, VSD9, and VSD13, respectively)

Figure 4

(a) Relative number of CFUs in intramuscular and peritoneal region of zebrafish inoculated with SDSD isolates (VSD13, VSD9, VSD21, VSD23, and VSD24) or S. pyogenes GAP58. For control purposes, zebrafish were inoculated with 10 μl of TSB‐0.5YE. The relative CFUs in both anatomic regions were scored as follows: 0, no colonies; 1: 1 to 50 colonies; 2: 51 to 200 colonies; 3: 201 to 500 colonies; 4: > 500 colonies. (b) Percentage of zebrafish that died before 15 days of experiment with mixed and pure colonies in the intramuscular region. (c) Percentage of zebrafish that died before 15 days of experiment with mixed and pure colonies in the peritoneal region. Pure culture: containing colonies of the respective SDSD isolate. Mixed culture: contains colonies from more than one type of bacteria

Figure 5

Zebrafish histology (Davidson, H&E). (a) Exemplificative section across the abdominal cavity of a control animal, highlighting hepatic tissue (hp) and interorgan adipose tissue (arrowheads), both devoid of any signs of infection or other pathological features. (b) Section of an animal inoculated with the GAP58 strain, exhibiting severe infection in the interorgan adipose tissue, leading to tissue liquefaction. Cocci can be seen in small clumps of chains, most of which inside the remnants of macrophages (arrowheads). Note that most of the adipose material has been washed‐off during sample processing, leading to potential under evaluation of infection. (c) Massive exudate holding defense cells and bacteria (between arrowheads) in the abdomen of a fish inoculated with the VSD23 strain. The alteration is formed between skeletal muscle and major visceral blood vessel. (d) Severely infected body (trunk) kidney of a zebrafish injected with VSD24 strain. The hematopoietic tissue (ht) shows liquefying necrosis, cocci and macrophage aggregates. Kidney tubules (tb) were obviously less affected but glomeruli (gl) were severely infected as well. Inset: Cocci between fascicles of skeletal muscle, with leukocytes infiltrating adjacently. (e) Section across the abdominal cavity of a fish inoculated with the VSD21 strain, showing severe infection in the interorgan adipose tissue and adjacent kidney. (f) Section of a zebrafish treated with the same strain as previous, revealing acute kidney infection affecting haematopoietic tissue and glomeruli, similarly to panel D. Scale bars: 50 μm