QseBC is involved in the biofilm formation and antibiotic resistance in Escherichia coli isolated from bovine mastitis

Abstract

Background: Mastitis is one of the most common infectious diseases in dairy cattle and causes significant financial losses in the dairy industry worldwide. Antibiotic therapy has been used as the most effective strategy for clinical mastitis treatment. However, due to the extensive use of antibacterial agents, antimicrobial resistance (AMR) is considered to be one of the reasons for low cure rates in bovine mastitis. In addition, biofilms could protect bacteria by restricting antibiotic access and shielding the bacterial pathogen from mammary gland immune defences. The functional mechanisms of quorum sensing E. coli regulators B an d C (QseBC) have been well studied in E. coli model strains; however, whether QseBC regulates antibiotic susceptibility and biofilm formation in clinical E. coli strain has not been reported.

Methods: In this study, we performed construction of the qseBC gene mutant, complementation of the qseBC mutant, antimicrobial susceptibility testing, antibacterial activity assays, biofilm formation assays, real-time reverse transcription PCR (RT-PCR) experiments and electrophoretic mobility shift assays (EMSAs) to investigate the role of qseBC in regulating biofilm formation and antibiotic susceptibility in the clinical E. coli strain ECDCM2.

Results: We reported that inactivation of QseBC led to a decrease in biofilm formation capacity and an increase in antibiotic susceptibility of an E. coli strain isolated from a dairy cow that suffered from mastitis. In addition, this study indicated that QseBC increased biofilm formation by upregulating the transcription of the biofilm-associated genes bcsA, csgA, fliC, motA, wcaF and fimA and decreased antibiotic susceptibility by upregulating the transcription of the efflux-pump-associated genes marA, acrA, acrB, acrD, emrD and mdtH. We also performed EMSA assays, and the results showed that QseB can directly bind to the marA promoter.

Conclusions: The QseBC two-component system affects antibiotic sensitivity by regulating the transcription of efflux-pump-associated genes. Further, biofilm-formation-associated genes were also regulated by QseBC TCS in E. coli ECDCM2. Hence, this study might provide new clues to the prevention and treatment of infections caused by the clinical E. coli strains.

Keywords: Biofilm; Bovine mastitis; Efflux pump; Escherichia coli; QseBC two component system.

Figure 1. Growth curves of the wild-type strain WT/pSTV28, the mutant strain XL4/pSTV28, and the complement strain XL4/pCqseBC.

Strains WT/pSTV28, XL4/pSTV28, and XL4/pCqseBC were grown in LB broth with 16 µg/ml chloramphenicol at 37 °C for 26 h with shaking, and their growth curves were determined by measuring the cell density (OD) at 600 nm.

Figure 2. Antimicrobial activity assay of E. coli strain isogenic derivatives WT/pSTV28, XL4/pSTV28 and XL4/pCqseBC cultured with different antibiotic conditions.

The survival rate of the parental strain was designated as 100%. The colony counts of XL4/pSTV28 with different antibiotics (A, ciprofloxacin; B, gentamicin) were compared with that of the parental strain WT/pSTV28. Error bars indicate standard deviations. ∗ ∗ P < 0.01, means difference in WT/pSTV28, XL4/pSTV28 and XL4/pCqseBC.

Figure 3. Fluctuation of transcript levels of efflux-pump-associated genes.

Real-time reverse transcription PCR experiments were utilized to detect the transcript levels of efflux-pump -associated genes marA, acrA, acrB, acrD, emrD and mdtH in strains WT/pSTV28, XL4/pSTV28 and XL4/pCqseBC incubate in LB broth with 16 µg/ml chloramphenicol. Double asterisks (∗∗ P < 0.01) and single asterisk (∗P < 0.05) means difference in WT/pSTV28, XL4/pSTV28 and XL4/pCqseBC.

Figure 4. Detection of biomass of biofilm formed by WT/pSTV28, XL4/pSTV28 and XL4/pCqseBC on glass tubes by CV staining.

Biofilms were quantified after 72 h incubation at 37 °C without shaking. (A) Image of biofilm stained with 1% CV and adhered on glass tubes. (B) Biofilm were stained by 1% CV and dissolved the purple area with 33% glacial acetic acid and measured by optical density at a wavelength of 492 nm. Error bars indicate standard deviations. Double asterisks (∗∗ P < 0.01) means difference in WT/pSTV28, XL4/pSTV28 and XL4/pCqseBC.

Figure 5. Result of transcript levels of biofilm-associated genes.

Real-time reverse transcription PCR experiments were utilized to detect the transcript levels of biofilm-associated genes bcsA, csgA, fliC, motA, wcaF, and fimA in strains WT/ pSTV28, XL4/pSTV28 and XL4/pCqseBC incubate in LB broth with 16 µg/ml chloramphenicol. Double asterisks (∗ ∗ P < 0.01) and single asterisk (∗P < 0.05) means difference in WT/pSTV28, XL4/pSTV28 and XL4/pCqseBC.

Figure 6. Electrophoretic mobility shift assay for QseB.

Increasing amounts of QseB were incubated with Biotin-labeled qseB, marA, acrA, acrD, mdtH and emrD promoters (Biotin-p-qseB, Biotin-p-marA, Biotin-p-acrA, Biotin-p-acrD, Biotin-p- mdtH, Biotin-p- emrD). The lanes 1 to 5 were the DNA probe with increasing amounts of QseB (12, 0,3,6 and 12 µM); 150 fmol of Biotin-labeled probes was added to all lanes. And additional 1.5 pmol of unlabeled probe was added in lane 1 as the competitive control (Ctrl). (A) The positive control, demonstrating the binding ability of QseB to the qseB promoter; (B) the marA promoter; (C) the acrA promoter; (D) the acrD promoter; (E) the mdtH promoter; (F) the emrD promoter.