Klebsiella pneumoniae isolated from bovine mastitis is cytopathogenic for bovine mammary epithelial cells

Abstract

Klebsiella pneumoniae, a common cause of clinical mastitis (CM) in dairy cows, can cause severe clinical symptoms. However, its pathogenicity in the bovine mammary gland is not well understood. Our objectives were to establish an in vitro infection model of K. pneumoniae on bovine mammary epithelial cells (bMEC) to assess (1) cytopathogenicity (adhesive and invasive ability, damage and apoptosis, pro-inflammatory effects) of K. pneumoniae on bMEC and (2) the role of hypermucoviscous (HMV) phenotype on cytopathogenicity. Two K. pneumoniae isolates from CM cows, 1 HMV and 1 non-HMV, were used to infect bMEC. Adhesion and invasion ability, release of lactate dehydrogenase (LDH), ultrastructural morphology, apoptosis, transcriptional expression of pro-inflammatory genes and production of pro-inflammatory cytokines were characterized at various intervals. Both K. pneumoniae isolates rapidly adhered to and invaded bMEC within 1 h post infection (pi), causing ultrastructural damage (swelling of mitochondria and vesicle formation on cell surface) after 3 h pi and apoptotic death after 9 h pi. In addition, K. pneumoniae promoted transcriptional expression of pro-inflammatory genes IL-6, IL-8, IL-1β, and tumor necrosis factor (TNF)-α and production of IL-8, IL-1β, and TNF-α cytokines. Compared with non-HMV K. pneumoniae, the HMV isolate had lower adhesive and invasive abilities but caused more serious cellular damage. In conclusion, K. pneumoniae was cytopathogenic on bMEC and induced a pro-inflammatory response; however, the HMV phenotype did not have a key role in pathogenicity. Therefore, more attention should be paid to milk loss, and targeted prevention and treatment strategies should be implemented in Klebsiella mastitis episodes.

Bacterial Interference With Lactate Dehydrogenase Assay Leads to an Underestimation of Cytotoxicity

“…In the present study, we evaluated the use of the LDH assay to quantify cytotoxicity induced by bacteria in airway epithelial cells after infection with eight bacteria encountered in the lower airways. Microscopic analysis of A549 monolayer integrity (reflected in the number of cells that remained attached to the culture flask) was consistent with previous studies reporting high cytotoxicity toward airway epithelial cells by P. aeruginosa, S. aureus, K. pneumoniae, and S. maltophilia (Hoskins et al, 2001;Hawdon et al, 2010;Karaba et al, 2013;Cheng et al, 2020) and low cytotoxicity for A. xylosoxidans, S. pneumoniae, and M. catarrhalis (Slevogt et al, 2007;Mantovani et al, 2012;Weight et al, 2019). Importantly, the results obtained indicate a discrepancy between cell detachment and cytotoxicity determined by the LDH assay for several bacterial species.…”

“…Human LDH has an optimal stability and function at a pH of 7.2–7.4 (Gay et al, 1967 ). Although changes in pH are already known to influence LDH activity (Vallee and Williams, 1975 ), the LDH assay is still used to assess cytotoxicity in the presence of pH-altering bacteria as K. pneumoniae and S. pneumoniae (Cano et al, 2009 ; Zahlten et al, 2015 ; Lee et al, 2017 ; Brissac et al, 2018 ; Cheng et al, 2020 ) In contrast to K. pneumoniae and S. pneumoniae, P. aeruginosa , and S. maltophilia did not acidify the cell culture medium, indicating an alternative mechanism. Inhibiting proteases by a bacterial protease inhibitor cocktail resulted in higher LDH activity after exposure to P. aeruginosa and S. maltophilia , indicating that protease(s) are causing, at least in part, the observed interference with LDH activity.…”

Klebsiella pneumoniae infection causes mitochondrial damage and dysfunction in bovine mammary epithelial cells

“…In our previous study, K. pneumoniae isolated from bovine mastitis caused apoptosis and pro-inflammatory responses of bMECs cultured in vitro [15]. K. pneumoniae isolates adhered to and invaded bMECs, causing ultrastructural damage, including swollen mitochondria and vesicle formation on the cell surface.…”

“…Both of these strains belong to the K57 capsule serotype and have entB, ybtS, iutA, and mrkD genes. However, HLJ-D2 is a hypermucoviscous (HMV) strain, whereas HB-AF5 is a non-HMV strain [15,16]. In our previous study, these two K. pneumoniae strains were cytopathogenic on bMECs and induced pro-inflammatory responses [15].…”

“…K. pneumoniae isolates adhered to and invaded bMECs, causing ultrastructural damage, including swollen mitochondria and vesicle formation on the cell surface. In addition, in infected bMECs, there were increases in transcriptional expression of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor-α (TNFα) genes and production of IL-1β, IL-8 and TNF-α [15]. However, whether mitochondrial damage has a critical role in pathogenicity of K. pneumoniae on bMECs has not been well established.…”