Antibiotic Susceptibility and Differential expression of virulence genes in Staphylococcus aureus

Abstract

Staphylococcus aureus is one of the most common pathogens in biofilm-associated chronic infections. S. aureus that live within biofilms avoid the host's immune response and are more resistant to antibiotics than planktonic bacteria. The current study was conducted to evaluate the antibacterial, antibiofilm and antivirulence of seven antibiotics (Ciprofloxacin (CP), Gentamicin (GEN), Tetracycline (TET), Amikacin (AMK), Clindamycin (CLI), Erythromycin (Ery) and Vancomycin (VAN) against S. aureus. The effects of seven antibiotics (CP, AMK, VAN, TET GEN, Ery and CLI) on S. aureus planktonic and biofilm were determined via Antibiotic susceptibility test, Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), disruption of microcolony, biofilm inhibition and degradation (crystal violet staining) and RT-qPCR. Antibiotic susceptibility test showed that CP, AMK, VAN, TET GEN, Ery and CLI has antibacterial activity against S. aureus with an inhibition zone of 28 mm, 21 mm, 27 mm, 20 mm, 25 mm, 27 mm and 19 mm respectively. The results showed that CP and AMK possessed the lowest MIC value against S. aureus with 0.125 µg/mL and 0.25 µg/mL for VAN, TET and GEN and 1.0 for Ery and CLI. The recorded values for MBCs were 0.25 μg for CP and AMK for S. aureus, 0.5 μg for vancomycin, tetracycline and gentamicin for S. aureus and 1.0 μg for Ery and CLI for S. aureus. Notably, CP and AMK demonstrated considerable efficacy, as shown by the low values for MIC; 0.125 μg and MBC; 0.25 μg for S. aureus. All antibiotics were found to disrupt microcolony formation in S. aureus at MIC of each antibiotics. At 0.25 μg concentration to 8 μg concentration of each antibiotic were significantly found to degrade and inhibit biofilm formation of S. aureus. The RT-qPCR showed that four genes including argF, purC, adh, and fabG were downregulated, whilst, three genes including scdA, pykA and menB were upregulated after exposure to CP, AMK, VAN, TET GEN, Ery and CLI. This study showed the efficacy of seven antibiotics against planktonic, biofilm, gene expression and that different concentrations of antibiotics have different degrees of potential effect on established biofilm. In addition, a decreased expression of virulence genes in S. aureus will impact their pathogenicity. These results provide the theoretical parameters for the selection of effective antimicrobial in clinical therapy and demonstrate how to correctly use antibiotics at MIC and sub-MIC as preventive drugs.

Comparative <i>in vitro</i> activity of various antibiotic against planktonic and biofilm and the gene expression profile in <i>Pseudomonas aeruginosa</i>

“…Bacterial inhibition increases with antibiotic concentration and incubation time. A biofilm is an aggregation of one or more species of microorganisms adhered to a surface, as compared to planktonic bacteria, which exist as individual organisms [20] , [51] . Biofilm-forming bacteria are reported to be 100–1000 times more resistant to antibiotics than planktonic bacteria [13] , [14] .…”

“…The diameter of the zone of inhibition for each antibiotic was measured by digital venire caliper. The experiment was carried out in triplicate [20] , [22] , [23] .…”

“…The optical density at 540 nm (OD 540 nm) of each sample was measured at 2-h intervals using a microplate reader. The concentrations that exerted no significant effects on P. aeruginosa growth were considered as the sub-inhibitory concentrations (SICs) of CP, GEN, TET, AMK, CLI, Ery and VAN [20] , [23] , [28] , [29] .…”

“…Following 24 hrs of incubation, colony counting was performed by using colony counter. Time-kill curve was plotted by assessing the log 10 CFU (colony forming units) versus time [20] , [21] , [23] , [28] , [30] .…”

“…The percentage of biofilm inhibition was calculated by following formulas as mentioned below. The experiment was repeated in triplicate [20] , [21] , [23] , [31] , [32] .…”