Detection of Staphylococcus aureus adhesion and biofilm-producing genes and their expression during internalization in bovine mammary epithelial cells

Pereyra, Elizabet Amanda Lorena; Picech, Florencia; Renna, María Sol; Baravalle, Celina; Andreotti, Carolina Soledad; Russi, Romina Cecilia; Calvinho, Luis Fernando; Diez, Cristina; Dallard, Bibiana Elisabet

doi:10.1016/j.vetmic.2015.12.002

Cited by 81 publications

(87 citation statements)

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“…Despite being the most prevalent gene, icaD was detected less frequently in NAS (62%) than from S. aureus (92%) isolates. Because overexpression of icaD improved internalization of S. aureus to mammary epithelial cells (Pereyra et al, 2016), higher carriage of icaD could make S. aureus more pathogenic in the udder than NAS.…”

Section: Discussionmentioning

confidence: 99%

Molecular epidemiology and distribution of antimicrobial resistance genes of Staphylococcus species isolated from Chinese dairy cows with clinical mastitis

Zhao

Nobrega

et al. 2019

Journal of Dairy Science

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Staphylococcus species, categorized into Staphylococcus aureus and non-aureus staphylococci (NAS), are frequent causes of mastitis in dairy cattle around the world. Current treatments using antimicrobials are under increasing scrutiny due to rising prevalence of multi-drug resistance in S. aureus. Objectives of this study were to determine: (1) genetic diversity of Staphylococcus species isolated from clinical mastitis in cows from large Chinese dairy farms; and (2) prevalence and distribution of antimicrobial resistance genes (ARG) in these isolates. Staphylococcus aureus (n = 96) were isolated from 26 herds located in 12 provinces of China, whereas NAS (n = 112) were isolated from 59 herds located in 18 provinces of China. The NAS were identified at the species level using a partial 16S rRNA sequencing method, whereas random amplification of polymorphic DNA (RAPD) PCR was done to determine genetic relationships of isolates. Finally, PCR was used to detect resistance and biofilm formation genes. Staphylococcus chromogenes (33%) was the most common NAS species, followed by Staphylococcus sciuri (17%) and Staphylococcus epidermidis (8%). Staphylococcus aureus was grouped in 12 genotypes, of which 2 types represented 56% of isolates. Staphylococcus chromogenes (n = 37) clustered into 8 RAPD types, with 2 prevalent types containing 73% of isolates. The most prevalent ARG in S. aureus isolates was blaZ (95%), followed by tetM (33%), tetK (31%), ermT (26%), and aacA-aphD (23%). The mecA and vanA were detected in 16 and 4% of isolates, respectively. In NAS, blaZ (100%), mecA (73%), tetK (79%), tetM (96%), mphC (63%), and msrA (54%) were frequently detected. Antimicrobial resistance genes mecA, tetK, tetL, tetM, dfrG, ermB, msrA, mphC, aadD, and aphA3 were more commonly detected in NAS than in S. aureus. Biofilm formation genes (icaA and icaD) were frequently detected in staphylococci isolated from bovine clinical mastitis. The existence of predominant RAPD types in S. aureus and S. chromogenes isolates across Chinese dairy farms indicated that specific genotypes had disseminated within herds and become more udder-adapted. High prevalence of ARG, especially in NAS, highlighted the risk of selection of multi-drug resistant staphylococci with potential as a reservoir of ARG.

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Section: Discussionmentioning

confidence: 99%

Molecular epidemiology and distribution of antimicrobial resistance genes of Staphylococcus species isolated from Chinese dairy cows with clinical mastitis

Zhao

Nobrega

et al. 2019

Journal of Dairy Science

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“…Adhesion assay of clinical N. cyriacigeorgica to bMECs was performed according to previously described protocols (Pöhlmann-Dietze et al, 2000; Pereyra et al, 2016), with slight modifications as bMECs were infected with N. cyriacigeorgica at a multiplicity of infection (MOI, ratio of N. cyriacigeorgica to cells) of 50:1 for 10 min, 1, 2, and 3 h at 37°C with 5% CO 2 . After incubation, the supernatants of infected cells were removed and cells were washed three times with phosphate buffer saline (PBS, pH 7.4) to remove non-adherent bacteria.…”

Section: Methodsmentioning

confidence: 99%

“…As a result, we found that 50 μg/mL of amikacin was enough to kill all Nocardia within 2 h; thus, this concentration of amikacin and incubation time was used for the following invasion assay. Invasion assay of N. cyriacigeorgica was performed according to the previous method with minor modifications (Beaman and Beaman, 1998; Pereyra et al, 2016). The bMECs were infected with N. cyriacigeorgica at a MOI of 50:1 for 10 min, 1, 2, and 3 h. Following incubation, cells were washed three times with PBS and treated with amikacin (50 μg/mL) for 2 h to kill extracellular Nocardia .…”

Section: Methodsmentioning

confidence: 99%

Nocardia cyriacigeogica from Bovine Mastitis Induced In vitro Apoptosis of Bovine Mammary Epithelial Cells via Activation of Mitochondrial-Caspase Pathway

Chen

Liu

Zhang

et al. 2017

Front. Cell. Infect. Microbiol.

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Nocardia is one of the causing agents of bovine mastitis and increasing prevalence of nocardial mastitis in shape of serious outbreaks has been reported from many countries. However, the mechanisms by which this pathogen damages the bovine mammary epithelial cells (bMECs) is not yet studied. Therefore, this study was designed with the aim to evaluate the apoptotic effects elicited by Nocardia and to investigate the pathway by which the Nocardia induce apoptosis in bMECs. Clinical Nocardia cyriacigeorgica strain from bovine mastitis was used to infect the bMECs for different time intervals, viz. 1, 3, 6, 12, and 18 h, and then the induced effects on bMECs were studied using adhesion and invasion assays, release of lactate dehydrogenase (LDH), apoptosis analysis by annexin V and propidium iodide (PI) double staining, morphological, and ultrastructural observations under scanning electron microscope (SEM) and transmission electron microscope (TEM), mitochondrial transmembrane potential (ΔΨm) assay using flow cytometry, and the protein quantification of mitochondrial cytochrome c and caspase-9 and caspase-3 by western blotting. The results of this study showed that N. cyriacigeorgica possessed the abilities of adhesion and invasion to bMECs. N. cyriacigeorgica was found to collapse mitochondrial transmembrane potential, significantly (p < 0.05) release mitochondrial cytochrome c and ultimately induce cell apoptosis. Additionally, it promoted casepase-9 (p < 0.01) and casepase-3 (p < 0.05) levels, significantly (p < 0.01) increased the release of LDH and promoted DNA fragmentation which further confirmed the apoptosis. Furthermore, N. cyriacigeorgica induced apoptosis/necrosis manifested specific ultrastructure features under TEM, such as swollen endoplasmic reticulum, cristae degeneration, and swelling of mitochondria, vesicle formation on the cell surface, rupturing of cell membrane and nuclear membrane, clumping, fragmentation, and margination of chromatin. The present study is the first comprehensive insight into patho-morphological ultrastructural features of apoptosis/necrosis induced by N. cyriacigeorgica, which concluded that the clinical N. cyriacigeorgica induced apoptotic changes in the bMECs through mitochondrial-caspase dependent apoptotic pathway.

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“…Murine macrophages and bMECs were infected with P. zopfii for up to 8 h, washed with PBS (pH 7.4) and incubated for 2 h with gentamycin (200 μg/mL) to eliminate extracellular P. zopfii . Cells were washed with PBS to eliminate non-adherent bacteria and then lysed by 0.5% Triton X-100 (v/v) to determine CFU by 10-fold serial dilution [24]. Further confirmation of phagocytic activity of macrophages was conducted by actin inhibition (cytochalasin D; C8273, Sigma, USA; 1 h) before inoculation.…”

Section: Methodsmentioning

confidence: 99%

Prototheca zopfiiGenotype II induces mitochondrial apoptosis in models of bovine mastitis

Shahid

Cobo

Chen

et al. 2019

Preprint

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20Prototheca zopfii is an alga increasingly isolated from bovine mastitis. Of the two genotypes of 21 P. zopfii (genotype I and II (GT-I and II)), P. zopfii GT-II is the genotype associated with acute 22 mastitis and decreased milk production by unknown mechanisms. The objective was to 23 determine inflammatory and apoptotic roles of P. zopfii GT-II in cultured mammary epithelial 24 cells (from cattle and mice) and murine macrophages and using a murine model of mastitis. 25 Prototheca zopfii GT-II (but not GT-I) invaded bovine and murine mammary epithelial cells 26 (MECs) and induced apoptosis, as determined by the terminal deoxynucleotidyl transferase 27 mediated deoxyuridine triphosphate nick end labeling assay. This P. zopfii GT-II driven 28 apoptosis corresponded to mitochondrial pathways; mitochondrial transmembrane resistance 29 (ΔΨm) was altered and modulation of mitochondrion-mediated apoptosis regulating genes 30 changed (increased transcriptional Bax, cytochrome-c and Apaf-1 and downregulated Bcl-2), 31 whereas caspase-9 and -3 expression increased. Apoptotic effects by P. zopfii GT-II were more 32 pronounced in macrophages compared to MECs. In a murine mammary infection model, P. 33zopfii GT-II replicated in the mammary gland and caused severe inflammation with infiltration 34 of macrophages and neutrophils and upregulation of pro-inflammatory genes (TNF-α, IL-1β and 35 Cxcl-1) and also apoptosis of epithelial cells. Thus, we concluded P. zopfii GT-II is a mastitis-36 causing pathogen that triggers severe inflammation and also mitochondrial apoptosis. 37 38 39 40 3 41 Author summary 42 Bovine mastitis (inflammation of the udder) reduces milk production and quality, causing huge 43 economic losses in the dairy industry worldwide. Although the alga Prototheca zopfii is a major 44 cause of mastitis in dairy cows, mechanisms by which it damages mammary tissues are not well 45 known. Here, we used cell cultures and a mouse model of mastitis to determine how Prototheca 46 caused inflammation and cell death in mammary tissues. Prototheca invaded mammary gland 47 cells, from cattle and mice, as well as macrophages (white cells that take up and kill pathogens) 48 and caused cell death by interfering with mitochondria. Furthermore, Prototheca causes severe 49 inflammation and tissue damage when injected into the mammary glands of mice. Although 50 there are two genotypes of P. zopfii, only genotype II causes tissue damage, whereas gentotype I, 51 common in farm environments, does not damage mammary tissues. Since P. zopfii is an alga and 52 not a bacterium, antibiotic treatments, frequently used to treat mastitis in cattle, are not effective 53 against this organism. Understanding how P. zopfii damages mammary tissue and causes mastitis 54 is important new knowledge to promote future development of evidence-based approaches to 55 prevent and treat mammary gland infections with this organism. 56 57 4 63 protothecal mastitis can be clinical or subclinical. In clinical cases, symptoms include fever (up 64 to 40 ...

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Detection of Staphylococcus aureus adhesion and biofilm-producing genes and their expression during internalization in bovine mammary epithelial cells

Cited by 81 publications

References 34 publications

Molecular epidemiology and distribution of antimicrobial resistance genes of Staphylococcus species isolated from Chinese dairy cows with clinical mastitis

Molecular epidemiology and distribution of antimicrobial resistance genes of Staphylococcus species isolated from Chinese dairy cows with clinical mastitis

Nocardia cyriacigeogica from Bovine Mastitis Induced In vitro Apoptosis of Bovine Mammary Epithelial Cells via Activation of Mitochondrial-Caspase Pathway

Prototheca zopfiiGenotype II induces mitochondrial apoptosis in models of bovine mastitis

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