Antibacterial efficacy of Citrus hystrix (makrut lime) essential oil against clinical multidrug-resistant methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates.

The increasing incidence of methicillin-resistant S. aureus is a major public health concern. Recently, the performance of Citrus hystrix essential oil (CHEO) has been shown to contain broad-spectrum antibacterial activity. Therefore, this study aims to determine the antibacterial activity of CHEO alone and in combination with gentamicin against panels of clinical isolates of methicillin-susceptible S. aureus (MSSA, n = 45) and methicillin-resistant S. aureus (MRSA, n = 40). Antibiotic susceptibility testing revealed multidrug-resistant (MDR) patterns among 3 MSSA isolates and 39 MRSA isolates, indicating that the clinical MRSA isolates were associated with MDR (p < 0.05). For the drug resistant isolates, resistance was observed toward most antibiotics, except for chloramphenicol, trimethoprim-sulfamethoxazole, linezolid, and vancomycin. Antibacterial screening by disk diffusion demonstrated that CHEO alone had certain antibacterial activity toward all MSSA isolates (IZD: 16.0 ± 4.7 mm) and MRSA isolates (IZD: 16.5 ± 4.2 mm) (p > 0.05). The MIC values of CHEO are 18.3 ± 6.1 mg/mL in MSSA isolates and 17.9 ± 6.9 mg/mL in MRSA isolates (p > 0.05). The antibacterial activity of CHEO demonstrated the bactericidal effect with MIC index 1.0-1.4. Time-killing kinetics revealed that CHEO at 1 × MIC completely killed MSSA and MRSA within 12 h. Moreover, the checkerboard titration demonstrated the synergistic and additive interactions of CHEO with gentamicin with FIC index 0.012-0.625. CHEO against human epidermal keratinocyte; HaCaT cell line demonstrated the IC50 value at 2.15 mg/mL. The use of CHEO as an alternative antibacterial agent would reduce the emergence of resistant bacteria, especially MDR MRSA.

Antibacterial Activities and Synergistic Interaction of Citrus Essential Oils and Limonene with Gentamicin against Clinically Isolated Methicillin-Resistant Staphylococcus aureus.

Citrus reticulata Blanco and Citrus aurantifolia are the edible plants which contain several biological properties including antibacterial activity. The aims of the present study were to determine the chemical compositions and evaluate antibacterial activities of citrus essential oils extracted from the fruit peels of C. reticulata (CREO) and C. aurantifolia (CAEO), alone and in combination with gentamicin, against a panel of clinically isolated methicillin-resistant S. aureus (MRSA) (n = 40) and methicillin-susceptible S. aureus (MSSA) (n = 45). Gas chromatography-mass spectrometry analysis revealed that 12 and 25 compounds were identified in CREO and CAEO with the most predominant compound of limonene (62.9-72.5%). The antibacterial activities were determined by agar disk diffusion and resazurin-based microdilution methods. The results found that almost all MRSA isolates were resistant to ciprofloxacin, erythromycin, and clindamycin, and some isolates were resistant to gentamicin. CREO and CAEO exhibited inhibitory effects toward clinical isolates (MIC: 1.0-32.0 and 8.0-32.0 mg/mL, respectively), with a similar trend to limonene (MIC: 1.0-32.0 mg/mL). However, the higher antibacterial effects were found in CREO and limonene when compared to CAEO (p < 0.01). In combination effect, the results showed the synergistic interaction of gentamicin with CREO and limonene on the MRSA and MSSA isolates (FIC indexes: 0.012-0.258 and 0.012-0.375), but that interaction of gentamicin with CAEO was observed only on MRSA (FIC index: 0.012-0.016). These findings demonstrated the potential of these citrus essential oils as natural antibacterial agents that may contribute to reduce the emerging of antimicrobial-resistant bacteria.

HIV-1 neutralization by monoclonal antibody against conserved region 2 and patterns of epitope exposure on the surface of native viruses.

BACKGROUND: Conserved neutralizing epitopes are considered to be a key role for eliciting broadly neutralizing antibody (NAb). Previously, two conserved neutralizing epitopes of HIV-1 CRF01_AE envelope were identified at amino acid 93-112 of the C1 (C1E) and at 218-239 of the C2 (C2E) regions. To access the potency of antibody directed against conserved epitopes, a monoclonal antibody (MAb) specific to the C2E region was developed and characterized. METHODS: The immunogenicity of two epitopes was examined by immunizing BALB/c mice with the matching synthetic peptides. One MAb, C2EB5, directed against peptide C2E, was generated by conventional methods, while C1E1 and C1E2 peptides induced slight antibody response in mice. The neutralizing activity of MAb C2EB5 was examined using a peripheral blood mononuclear cell (PBMC) based method and various HIV-1 subtypes including A, B, C, D, and CRF01_AE; C2EB5 was compared with other known neutralizing MAbs (4E10, 447-52D) and with sCD4. The exposure of the C2 epitope on native virus was investigated using virus capture by these MAbs. RESULTS: The MAb C2EB5 demonstrated cross-neutralization against various HIV-1 subtypes. The overall potency of MAb C2EB5 against 5 subtypes was ranked in the following order: subtype C> CRF01_AE> subtype D> subtype A> subtype B. The epitope exposure for MAb C2EB5 was also correlated with the neutralization properties of each subtype. CONCLUSION: This study demonstrates the cross-clade neutralizing activity of a MAb directed against an epitope located in the C2 region of the HIV-1 env and highlights differences in the exposure of antigenic epitopes on the surface of various HIV-1 subtypes. The epitope for this newly identified neutralizing MAb made against a subtype CRF01_AE peptide is particularly exposed in subtype C viral isolates.

Conserved neutralizing epitopes of HIV type 1 CRF01_AE against primary isolates in long-term nonprogressors.

Linear conserved B cell epitopes in envelope glycoprotein of long-term nonprogressors (LTNPs) HIV-1 CRF01_AE were determined. The envelope sequences of HIV-1 subtype E from Thailand were aligned to define consensus sequences. Then the peptides corresponding to these predicted regions were synthesized as peptides represent C1, C2, C3, C5, V2, V3, and gp41 regions. After that, the neutralizing B cell epitopes were determined by neutralized competitive assay with pool sera of typical progressor and LTNP HIV-1 CRF01_AE patients against HIV-1 CRF01_AE 24 primary isolates (PI) and laboratory strains (TCLA). We found that the strength and breadth of neutralization were greater for sera from LTNPs compared with sera from typical progressors. Peptides C1E and C2E could inhibit primary isolates but not the TCLA strain in LTNP sera. The new B cell epitopes, which were located in the C1 and C2 regions of CRF01_AE against primary HIV-1 isolates, were identified in HIV-1 CRF01_AE LTNPs. This may be important in HIV-1 vaccine development and trial.

Effect of dihydroartemisinin on the antioxidant capacity of P. falciparum-infected erythrocytes.

Many lines of evidence reveal that artemisinin, an antimalarial containing endoperoxide, generates free radicals to kill malaria parasites. The present study re-evaluated the antioxidants of P. falciparum-infected erythrocytes in the absence and presence of 0.25, 0.5 and 1.0 ng/ml of dihydroartemisinin (DHA), the active metabolite of artemisinin. The ratio of reduced to oxidized glutathione (GSH/GSSG) and activities of superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) were determined. The data indicated that malaria infection induced oxidative stress in erythrocytes that resulted in a significant lower GSH in parasitized cells compared to the non-parasitized. DHA showed no effect on the antioxidant levels of non-parasitized erythrocytes treated under similar conditions as P. falciparum-infected erythrocytes. However, significantly lower GSH as well as catalase and GPx activities in parasitized cells were seen at drug concentrations of 0.5 and 1.0 ng/ml (p < 0.05). GSH is the most sensitive indicator of oxidative stress in malaria-infected erythrocytes both in the absence and in the presence of DHA. Parasite GPx might play a more important role than catalase in the elimination of peroxide. Parasite viabilities in the presence of DHA were analyzed simultaneously and were affected to a greater extent than the antioxidant levels. The present observation showed that although DHA killed malaria parasites by generating free radicals from the endoperoxide bridge causing the reduction of antioxidants, but the depletion of parasite antioxidants is not a prerequisite for the parasite death.