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. 2025 Jul 16;13(7):1674.
doi: 10.3390/microorganisms13071674.

Virulence, Antibiotic Resistance and Cytotoxic Effects of Lactococcus lactis Isolated from Chinese Cows with Clinical Mastitis on MAC-T Cells

Tiancheng Wang  1 , Fan Wu  1 , Tao Du  1 , Xiaodan Jiang  1 , Shuhong Liu  1 , Yiru Cheng  1 , Jianmin Hu  1
Affiliations

Virulence, Antibiotic Resistance and Cytotoxic Effects of Lactococcus lactis Isolated from Chinese Cows with Clinical Mastitis on MAC-T Cells

Tiancheng Wang et al. Microorganisms. .

Abstract

Lactococcus lactis (L. lactis) is a pathogenic Gram-positive, catalase-negative coccobacillus (GPCN) associated with bovine mastitis. In this study, nine strains of L. lactis were successfully isolated and characterized from 457 milk samples from cows with clinical mastitis in China. All isolates exhibited a high degree of susceptibility to marbofloxacin and vancomycin. A series of molecular and cell biological techniques were used to explore the biological characteristics and pathogenicity of these isolates. The virulence gene profiles of the isolates were analyzed using whole genome resequencing combined with polymerase chain reaction (PCR) to elucidate the differences in virulence gene expression between isolates. To provide a more visual demonstration of the pathogenic effect of L. lactis on bovine mammary epithelial cells, an in vitro infection model was established using MAC-T cells. The results showed that L. lactis rapidly adhered to the surface of bovine mammary epithelial cells and significantly induced the release of lactate dehydrogenase, suggesting that the cell membranes might be damaged. Ultrastructural observations showed that L. lactis not only adhered to MAC-T cells, but also invaded the cells through a perforation mechanism, leading to a cascade of organelle damage, including mitochondrial swelling and ribosome detachment from the endoplasmic reticulum. The objective of this study was to provide strong evidence for the cytotoxic effects of L. lactis on bovine mammary epithelial cells. Based on this research, a prevention and treatment strategy for L. lactis as well as major pathogenic mastitis bacteria should be established, and there is a need for continuous monitoring.

Keywords: Lactococcus lactis; antimicrobial resistance; bovine clinical mastitis; pathogenicity; virulence gene.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Morphological characteristics of L. lactis. (A) Pinpoint colonies with α-hemolysis were observed on trypticase soy agar with 5% sheep blood; (B) Gram-positive cocci observed under the microscope; and (C,D) scanning electron microscopy of L. lactis.
Figure 2
Figure 2
16SrRNA identification and subsequent analysis of homology results. The first column is labeled ‘Mark2000’; the second column is labeled ‘Negative control’; and the third to seventh columns are labeled with the names of the isolated L. lactis strains (A). These nine L. lactis strains were more closely related to each other as a result of comparison with the exogenous control strains (B).
Figure 3
Figure 3
Growth curves of L. lactis, S. aureus, and E. faecalis isolates. Data are mean ± SD of OD600 nm values of three isolates. The curves were plotted using GraphPad Prism 8.
Figure 4
Figure 4
Identification of different virulence genes among L. lactis isolates. M: 100 bp DNA ladder. Lanes 1–16: 30S rRNA gene, AC1, PsaA, CGC C, CHP, eno, EpsA, EpsB, EpsC, EpsD, EpsL, EpsR, EpsX, hly 2, RIF, and SOD.
Figure 5
Figure 5
GO analysis of differentially expressed genes in L. lactis. LL1 GO results of differentially expressed genes, the size of the dots indicates the number of enriched genes and the color indicates significance.
Figure 6
Figure 6
KEGG analysis results of differentially expressed genes in L. lactis. The green part on the left side of the figure shows the p−value results, reflecting the significance level of each pathway. The black part on the right shows the number of genes involved in each pathway, which intuitively reflects the gene enrichment level of each pathway.
Figure 7
Figure 7
LL1 virulence factor annotated classification statistical results.
Figure 8
Figure 8
Cytotoxic effects of L. lactis infection on MAC-T cells. Effect of L. lactis on MAC-T cell viability (A). Effect of L. lactis on LDH of MAC-T cells (B). * indicates a significant different treatment groups (p < 0.05 by ANOVA test), and ** indicates a very significant difference between different treatment groups (p < 0.01 by ANOVA test).
Figure 9
Figure 9
In vitro pathogenic effect of L. lactis on bovine mammary epithelial cells (MAC-T). (A) Effect of MAC-T on adhesion by L. lactis isolates (within 3 h of infection). (B) Invasion of MAC-T by L. lactis. (within 3 h of infection). Data are mean ± SD of three independent experiments, bar graphs were calculated and plotted using GraphPad Prism 8.
Figure 10
Figure 10
Ultrastructural images of MAC-T infected with L. lactis created via scanning and transmission electron microscopy, where black arrows are bacteria, red arrows are mitochondria, yellow arrows are endoplasmic reticulum, and green arrows are cell membranes. LL1 infected with MAC-T (A,B) control, (C,D) 3 h after LL1 infection, (E,F) 6 h after LL1 infection, (G,H) 12 h after LL1 infection, and (I,J) 24 h after LL1 infection. M: mitochondria, RER: rough endoplasmic reticulum.
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