Effects of Piper betle Extracts against Biofilm Formation by Methicillin-Resistant Staphylococcus pseudintermedius Isolated from Dogs

Abstract

Emergence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolated from dogs with cutaneous and wound infections has significantly impacted veterinary medicine. This study aimed to isolate S. pseudintermedius from canine pyoderma and investigate the effects of ethanolic extracts of Piper betle (PB), P. sarmentosum (PS), and P. nigrum (PN) on the bacterial growth and biofilm formation of S. pseudintermedius and MRSP. Of the isolated 152 isolates, 53 were identified as S. pseudintermedius using polymerase chain reaction, and 10 isolates (6.58%) were identified as MRSP based on the presence of mecA. Based on phenotype, 90% of MRSPs were multidrug-resistant. All MRSP had moderate (10%, 1/10) and strong (90%, 9/10) biofilm production ability. PB extracts were the most effective in inhibiting planktonic cells, and the minimum inhibitory concentration at which ≥50% of the isolates were inhibited (MIC₅₀) was 256 µg/mL (256-1024 µg/mL) for S. pseudintermedius isolates and 512 µg/mL (256-1024 µg/mL) for MRSP isolates. The MIC₉₀ for S. pseudintermedius and MRSP was 512 µg/mL. In XTT assay, PB at 4× MIC showed an inhibition rate of 39.66-68.90% and 45.58-59.13% for S. pseudintermedius and MRSP, respectively, in inhibiting biofilm formation. For PB at 8× MIC, the inhibition rates for S. pseudintermedius and MRSP were 50.74-81.66% and 59.57-78.33%, respectively. Further, 18 compounds were identified in PB using gas chromatography-mass spectrometry, and hydroxychavicol (36.02%) was the major constituent. These results indicated that PB could inhibit bacteria growth of and biofilm formation by S. pseudintermedius and MRSP isolated from canine pyoderma in a concentration-dependent manner. Therefore, PB is a potential candidate for the treatment of MRSP infection and biofilm formation in veterinary medicine.

Keywords: Piper betle; Piper nigrum; Piper sarmentosum; biofilm; ethanolic extracts; hydroxychavicol; pyoderma.

Figure 1

Antimicrobial susceptibility profiles of Staphylococcus aureus, methicillin-resistant S. aureus, methicillin-resistant Staphylococcus pseudintermedius, and S. pseudintermedius were determined using the disk-diffusion method. Red squares indicate resistance; green squares indicate susceptibility, and yellow squares indicate intermediate characteristics. Abbreviations: SA, Staphylococcus aureus ATCC 25923; MRSA, methicillin-resistant S. aureus ATCC 33591; W, weak biofilm producer; M, moderate biofilm producer; S, strong biofilm producer; OX, oxacillin (1 µg); OB, cloxacillin; AMP, ampicillin (10 µg); AMC, amoxicillin–clavulanic acid (30 µg); CL, cephalexin (30 µg); FOX, cefoxitin (30 µg); CRO, ceftriaxone (30 µg); CTX, cefotaxime (30 µg); NOR, norfloxacin (10 µg); ENR, enrofloxacin (5 µg); SXT, sulfamethoxazole/trimethoprim (25 µg); DO, doxycycline (30 µg); AK, amikacin (30 µg); CN, gentamicin (10 µg); E, erythromycin (15 µg); DA, clindamycin (2 µg).

Figure 2

Effects of Piper betle ethanolic extracts on the biofilm formation of Staphylococcus aureus, methicillin-resistant S. aureus, and Staphylococcus pseudintermedius after 24 h of treatment. Each bar shows the mean ± SD of three experiments per group. * indicates that the differences between the control and treatments were statistically significant (p < 0.05). Abbreviations: SA, Staphylococcus aureus ATCC 25923; MRSA, methicillin-resistant S. aureus ATCC 33591; W, weak; M, moderate; and S, strong biofilm producers.

Figure 3

Effects of Piper betle ethanolic extracts on the biofilm formation of Staphylococcus aureus, methicillin-resistant S. aureus, and methicillin-resistant Staphylococcus pseudintermedius after 24 h of treatment. Each bar shows the mean ± SD of three experiments per group. * indicates that the differences between the control and treatment groups were statistically significant (p < 0.05). Abbreviations: SA, Staphylococcus aureus ATCC 25923; MRSA, methicillin-resistant S. aureus ATCC 33591; W, weak; M, moderate; and S, strong biofilm producers.

Figure 4

Chromatogram of the main components of Piper betle ethanolic extracts determined through gas chromatography–mass spectrometry.

Figure 5

Leaves of Piper betle L. (a) and P. sarmentosum Roxb. (b) and dried seeds of P. nigrum L. (c).