Modulation of Drug Resistance by Furanochromones in NorA Overexpressing Staphylococcus Aureus.

Khellin and visnagin are natural furanochromones that photoreact with DNA. Khellin has been used in the treatment of vitiligo and psoriasis, as well as in the treatment of angina pectoris and asthma due to its potent action as a coronary vasodilator and antispasmodic agent. The present study aimed to investigate whether the compounds khellin and visnagin act as inhibitors of NorA protein, an efflux pump overproduced by the strain of Staphylococcus aureus SA-1199B that confers resistance to the fluoroquinolones, such as norfloxacin and ciprofloxacin. These substances alone did not show antibacterial activity against the strain tested. On the other hand, when these compounds were added to the culture medium at subinhibitory concentration, they were able to reduce the minimum inhibitory concentration (MIC) of norfloxacin, ethidium bromide, as well as berberine, suggesting that these compounds are modulating agents of norfloxacin resistance, possibly due to NorA inhibition. Molecular docking analysis showed that both khellin and visnagin form hydrogen bonds with Arg310, an important residue in the interaction between NorA and its substrates, supporting the hypothesis that these compounds are NorA inhibitors. These results suggest a possible application of khellin and visnagin as adjuvants to norfloxacin in the treatment of infections caused by strains of S. aureus that overproduce NorA.

Modulation of the Drug Resistance by Platonia insignis Mart. Extract, Ethyl Acetate Fraction and Morelloflavone/Volkensiflavone (Biflavonoids) in Staphylococcus aureus Strains Overexpressing Efflux Pump Genes.

BACKGROUND: Microbial resistance to antibiotics is a global public health problem, which requires urgent attention. Platonia insignis is a native species from the eastern Brazilian Amazon, used in the treatment of burns and wounds. OBJECTIVES: To evaluate the antimicrobial activity of the hydroalcoholic extract of P. insignis (PIHA), the ethyl acetate fraction (PIAE), and its subfraction containing a mixture of biflavonoids (BF). Moreover, the effect of these natural products on the antibiotic activity against S. aureus strains overexpressing efflux pump genes was also evaluated. METHODS: Minimal inhibitory concentrations were determined against different species of microorganisms. To evaluate the modulatory effect on the Norfloxacin-resistance, the MIC of this antibiotic was determined in the absence and presence of the natural products at subinhibitory concentrations. Inhibition of the EtBr efflux assays were conducted in the absence or presence of natural products. RESULTS: PIHA showed a microbicidal effect against S. aureus and C. albicans, while PIAE was bacteriostatic for S. aureus. PIAE and BF at subinhibitory concentrations were able to reduce the MIC of Norfloxacin acting as modulating agents. BF was able to inhibit the efflux of EtBr efflux in S. aureus strains overexpressing specific efflux pump genes. CONCLUSION: P. inignisis, a source of efflux pump inhibitors, including volkensiflavone and morelloflavone, which were able to potentiate the Norfloxacin activity by NorA inhibition, being also able to inhibit QacA/B, TetK and MsrA. Volkensiflavone and morelloflavone could be used as an adjuvant in the antibiotic therapy of multidrug resistant S. aureus strains overexpressing efflux pumps.

Inhibition of the TetK efflux-pump by the essential oil of Chenopodium ambrosioides L. and α-terpinene against Staphylococcus aureus IS-58.

The use of natural products is crucial to suppress the development of these micro-organisms and to reduce the concentration necessary to inhibit these microrganisms, reducing the toxicity risks also. In this study, the essential oil from Chenopodium ambrosioides Leaves and its main constituent α-Terpinene were used in the antibacterial and potentiating activity of antibiotics and ethidium bromide assays, against the bacterial strains Staphylococcus aureus IS-58, carriers of efflux pumps. The Minimum Inhibitory Concentration (MIC) was determined using a microdilution method. The capacity of the aforementioned was also tested in combination with tetracycline and ethidium bromide, with the aim of improving the activity of the antibacterials. The MIC of the C. ambrosioides L. essential oil and of α-Terpinene were above 1024 µg/mL, comprising a clinically irrelevant value. However, when associated with the antibiotics, the C. ambrosioides L. essential oil, significantly decreased the MIC of tetracycline and ethidium bromide. The efflux pump is the only mechanism the bacteria possesses to reduce the toxicity of ethidium bromide, and thus this reduction in the MIC demonstrates that the C. ambrosioides L. essential oil is an effective option in the inhibition of the efflux pump present in these micro-organisms.

Light-mediated antibacterial activity of Lippia origanoides H.B.K. in vitro.

An ethanol extract and different partition fractions obtained from Lippia origanoides H.B.K. were assayed for light-mediated activity against strains of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Identical assays were conducted with and without exposure to UV-A (400-315 nm) light to test for light enhanced activity. The ethanol extract and dichloromethane fraction showed light-mediated activity against the S. aureus strain, but not against the E. coli strain. The dichloromethane fraction was more active than the ethanol extract. Naringenin did not display light-mediated activity against the tested bacteria, indicating that the light-mediated antimicrobial activity of the dichloromethane fraction is not due to its major component. The results represent the first report of light-mediated antimicrobial activity of Lippia origanoides and show that its phytochemicals could be used as light-mediated antimicrobial agents.

Modulation of the norfloxacin resistance in Staphylococcus aureus by Cordia verbenaceae DC.

BACKGROUND & OBJECTIVES: Several chemical compounds isolated from natural sources have antibacterial activity and some enhance the antibacterial activity of antibiotics reversing the natural resistance of bacteria to certain antibiotics. In this study, the hexane and methanol extract of Cordia verbenaceae were assessed for antibacterial activity alone and combinated with norfloxacin against the Staphylococcus aureus strain SA1199B. METHODS: The minimum inhibitory concentration (MIC) of extracts was assayed using microdilution assay and the modulatory activity was evaluated using plate diffusion assay. RESULTS: The MIC observed varied between 256 to >1024 µg/ml. However, the antibiotic activity of norfloxacin was enhanced in the presence of subinhibitory concentrations of hexane extract of C. verbenaceae (HECV). INTERPRETATIONS & CONCLUSIONS: Our results indicate that Cordia verbenaceae DC. can be a source of plant derived products with antibiotic modifying activity.

Modulation of the norfloxacin resistance in Staphylococcus aureus by Croton campestris A. and Ocimum gratissimum L.

INTRODUCTION: Some species of Staphylococcus are often recognized as etiological agents of many animal and human opportunistic infections. This study is the first test of change in resistance of antibiotic activity by Croton campestris A. and Ocimum gratissimum L. against multiresistant strains of Staphylococcus aureus. OBJECTIVE: In this study, the hexane and methanol extract of Croton campestris A. and Ocimum gratissimum L. was tested for antibacterial activity alone and in combination with norfloxacin against the strain SA1199B. MATERIALS AND METHODS: The minimum inhibitory concentration (MIC) and the modulatory effect of extracts was assayed using microtitre assay. RESULTS: By the fact of the MIC observed was not clinically relevant (MIC= 512 to ≥1.024 µg/ml), the antibiotic activity of norfloxacin was enhanced when this antibiotic was combined with sub-inhibitory concentrations of extracts, mainly the hexane extracts. CONCLUSIONS: These results indicate that the assayed extracts present compounds that can be used as a putative efflux pump inhibitor, indicating that Croton campestris A. and Ocimum gratissimum L. can be a source of plant derived products with antibiotic modifier activity.

In vitro additive effect of Hyptis martiusii in the resistance to aminoglycosides of methicillin-resistant Staphylococcus aureus.

CONTEXT: Bacterial infectious agents represent a risk to populations, where they are responsible for the high morbidity and mortality. In combating these pathogens, our main line of defense is the use of antibiotics. However, the indiscriminate use of these drugs select resistant strains to these same drugs. OBJECTIVE: In this study the ethanol extract of Hyptis martiusii Benth. (EEHM) (Lamiaceae) was tested for its antimicrobial activity against aminoglycoside multi-resistant Staphylococcus aureus (MRSA). MATERIALS AND METHODS: In this study, the ethanol extract of H. martiusii was prepared and tested with chlorpromazine for its antimicrobial activity using the microdilution method. Chlorpromazine and the ethanol extract were used alone and also in combination with aminoglycosides against a MRSA strain resistant to these antibiotics to determine the participation of efflux systems in resistance mechanisms. The FIC index was calculated and evaluated by the checkerboard method. RESULTS: A potentiating effect between this extract and aminoglycosides was demonstrated. Similarly, a potentiating effect of chlorpromazine with kanamycin was detected, indicating the involvement of an efflux system in the resistance to this aminoglycoside. The checkerboard method with combinations of aminoglycosides and EEHM demonstrated additive effect with kanamycin and gentamicin. It is therefore suggested that extracts from H. martiusii could be used as a source of plant-derived natural products with resistance- modifying activity. CONCLUSION: This is the first report about the modifying antibiotic activity of Hyptis martiusii, constituting a new approach against bacterial resistance to antibiotics as aminoglycosides.

Potentiation of antibiotic activity by Eugenia uniflora and Eugenia jambolanum.

This is the first report about the modifying antibiotic activity of Eugenia uniflora L. and Eugenia jambolanum L. In this study the ethanol extract of E. uniflora and E. jambolanum was tested for their antimicrobial activity against strains of Escherichia coli. The growth of the two strains of E. coli bacteria tested was not inhibited in a clinically relevant form by the extract. The minimal inhibitory concentration was >or=1,024 microg/mL for both strains of E. coli assayed. Synergism between this extract and gentamicin was demonstrated. In the same extract synergism was observed between chlorpromazine and kanamycin and between amikacin and tobramycin, indicating the involvement of an efflux system in the resistance to these aminoglycosides. It is therefore suggested that extracts from E. uniflora L. and E. jambolanum L. could be used as a source of plant-derived natural products with modifying antibiotic activity to gentamicin.

Antimicrobial activity of Mimosa tenuiflora (Willd.) Poir. from Northeast Brazil against clinical isolates of Staphylococcus aureus / Atividade antimicrobiana de Mimosa tenuiflora (Willd.) Poir. do Nordeste do Brasil, contra isolados clínicos de Staphylococcus aureus

Mimosa tenuiflora is a native plant of Northeast Brazil where it is popularly known as ''jurema-preta'' and it is widely used in folk medicine. In this work the anti-Staphylococcus aureus activity of ethanol extract of M tenuiflora was evaluated by determination of minimum inhibitory concentration (MIC) of clinical isolates by the agar dilution method, and by time-kill assay using a reference strain. MIC values against 30 isolates were 0,18 mg/mL (16/30) or 0,36 mg/mL (14/30, and also the reference strain). In the reference strains, at concentrations up to 4x MIC, only bacteriostatic effect was observed, but at 8x MIC a fast bactericidal effect was observed.

A Mimosa tenuiflora é uma planta nativa da região Nordeste do Brasil onde é conhecida como jurema-preta sendo amplamente utilizada na medicina popular. No presente trabalho a atividade anti-Staphylococcus aureus do extrato etanólico da M. tenuiflora foi avaliada pela determinação da concentração inibitória mínima (CIM), pelo método da diluição em agar, em 30 isolados clínicos e pela cinética de inativação com a linhagem referência. Os valores da CIM foram 0,18 mg/mL em 16 isolados e 0,36 mg/mL nos demais, bem como na linhagem referência. A cinética de inativação mostrou apenas efeito bacteriostático nas concentrações do extrato até aquela correspondente a 4x CIM e um rápido efeito bactericida na concentração correspondente a 8x CIM.

Effect of Momordica charantia L. in the resistance to aminoglycosides in methicilin-resistant Staphylococcus aureus.

In this study the ethanol extract (EEMC) of Momordica charantia L. (Cucurbitaceae) was tested for its modifying antibiotic activity against a MRSA strain. The growth of an MRSA (SA358) in the absence and presence of aminoglycosides was evaluated. A potentiating effect between this extract and all aminoglycosides was demonstrated. Similarly, the same effect was shown by chlorpromazine on kanamycin, gentamicin and neomycin, indicating the involvement of an efflux system in the resistance to these aminoglycosides. Extracts from M. charantia could be used as a source of plant-derived natural products with resistance-modifying activity. This is the first report about the modifying antibiotic activity of M. charantia, constituting a new weapon against multi-resistant bacteria such as MRSA.

Increasing of the aminoglicosyde antibiotic activity against a multidrug-resistant E. coli by Turnera ulmifolia L. and chlorpromazine.

In this study, an ethanol extract of Turnera ulmifolia L. (EETU) and chlorpromazine (CPZ) were tested for their antimicrobial activity alone or in combination with conventional antibiotics against two strains of Escherichia coli (E. coli). The growth of neither E. coli strain was inhibited by the extract. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration values were > or =1 mg/ml for both the strains of E. coli. However, the extract did increase the antimicrobial effects of amikacin, neomycin, and tobramycin. A similar effect of CPZ on amikacin, kanamycin, and tobramycin indicated the involvement of an efflux system in the resistance to these aminoglycosides. Results suggest that extracts from T. ulmifolia could be used as a plant-derived natural product with resistance-modifying activity, constituting a new weapon against bacterial resistance to antibiotics.

In vitro interference of Hyptis martiusii Benth. & chlorpromazine against an aminoglycoside-resistant Escherichia coli.

The antibacterial and synergistic activity of the ethanol extract from Hyptis martiusii Benth. was assayed by microdillution. The growth of two isolates of Escherichia coli tested was inhibited by the extract. The minimum inhibitory and minimum bactericidal concentrations (MIC and MBC) values ranged from 512 and >1024 microg/ml for the E. coli 27 and 1024 and > 1024 microg/ml for the E. coli ATCC8539, respectively. A synergism between this extract and all aminoglycosides assayed was demonstrated. In the same form synergism between chlorpromazine and kanamycin, amikacin and tobramycin was observed, indicating the involvement of an efflux system. Extracts from H. martiusii could be used as a source of plant derived natural products with modifying antibiotic activity and these products may interact and affect multidrug resistance systems (MDR) as efflux pumps.

In vitro phototoxic activity of Eugenia jambolana L. and Hyptis martiusii Benth.

Ethanol extracts from Hyptis martiusii and Eugenia jambolana were assayed for light-mediated activity against strains of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Duplicate assays were conducted with and without exposure to UV-A radiation to test for light-activated or light-enhanced activity. Both extracts showed activity against at least two bacterial strains, but H. martiusii was the most active extract, being active against all strains of S. aureus and E. coli. The results represent a first report of the light-mediated antimicrobial activities of H. martiusii and E. jambolana and suggest that phytochemical investigations may be warranted.

Potentiating effect of Mentha arvensis and chlorpromazine in the resistance to aminoglycosides of methicillin-resistant Staphylococcus aureus.

BACKGROUND: This is the first report testing the antibiotic resistance-modifying activity of Mentha arvensis against MRSA (methicillin-resistant Staphylococcus aureus). MATERIALS AND METHODS: In this study an ethanol extract of Mentha arvensis L. and chlorpromazine were tested for their antimicrobial activity alone or in combination with conventional antibiotics against MRSA strains. RESULTS: A potentiating effect of this extract on gentamicin, kanamycin and neomycin was demonstrated. Similarly, a potentiating effect of chlorpromazine on the same aminoglycosides was observed, indicating the involvement of an efflux system in the resistance to these antibiotics. CONCLUSION: It is therefore suggested that extracts from M. arvensis could be used as a source of plant-derived natural products with resistance-modifying activity, such as in the case of aminoglycosides, constituting a new weapon against bacterial resistance to antibiotics, as with chlorpromazine.

Modulation of drug resistance in Staphylococcus aureus by a kaempferol glycoside from Herissantia tiubae (Malvaceae).

In an ongoing project to evaluate natural compounds isolated from plants from the Brazilian biodiversity as modulators of antibiotic resistance, kaempferol-3-O-beta-d-(6''-E-p-coumaroyl) glucopyranoside (tiliroside), isolated from Herissantia tiubae (Malvaceae) was investigated using the strain SA-1199B of Staphylococcus aureus, which overexpresses the norA gene encoding the NorA efflux protein which extrudes hydrophilic fluorquinolones and some biocides, such as benzalkonium chloride, cetrimide, acriflavine and ethidium bromide. The minimum inhibitory concentrations (MICs) of the antibiotics and biocides were determined by the microdilution assay in the absence and in the presence of sub-inhibitory concentration of tiliroside. Although tiliroside did not display relevant antibacterial activity (MIC = 256 microg/mL), it modulated the activity of antibiotics, i.e. in combination with antibiotics a reduction in the MIC was observed for norfloxacin (16-fold), ciprofloxacin (16-fold), lomefloxacin (four-fold) and ofloxacin (two-fold), and an impressive reduction in the MICs for the biocides (up to 128-fold). The results presented here represent the first report of a kaempferol glycoside as a putative efflux pump inhibitor in bacteria. The present finding indicates that H. tiubae (and broadly Malvaceae) could serve as a source of plant-derived natural products that modulate bacterial resistance, i.e. a source of potential adjuvants of antibiotics.

Atividade antimicrobiana do extrato de Anacardium occidentale Linn. em amostras multiresistentes de Staphylococcus aureus / Antimicrobial activity of the hydroalcoholic extract of Anacardium occidentale Linn. against multi-drug resistant strains of Staphylococcus aureus

Plantas medicinais com propriedades terapêuticas são de grande relevância em todo o mundo, principalmente em países em desenvolvimento. A planta Anacardium occidentale Linn. é largamente usada na medicina tradicional na nossa região, como antidiarréico, para amigdalite, bronquites, artrites, e antiiflamatório. No presente estudo avaliou-se a ação antimicrobiana do extrato hidroalcóolico da casca do caule do cajueiro frente a amostras de Staphylococcus aureus resistentes e sensíveis à meticilina, obtidas a partir de pacientes internados do Hospital Universitário/Universidade Federal da Paraíba. A atividade antimicrobiana foi determinada pelo método de difusão em meio sólido para a determinação da Concentração Inibitória Mínima do extrato, e foi observada no total de 30 amostras, pela presença ou não do halo de inibição. Todas as amostras ensaiadas mostraram-se sensíveis à ação do extrato do cajueiro, com diâmetros dos halos de inibição variando de 10 a 20 mm, demonstrando grande eficácia do cajueiro. Assim, o uso desta planta na nossa região pode inferir uma alternativa terapêutica eficiente e de baixo custo, contra infecções bacterianas causadas por Staphylococcus aureus.

Medicinal plants with therapeutical properties are of great significance in the whole world, especially in developing countries. Anacardium occidentale Linn. is a plant widely used in the traditional medicine in our region against diarrhea, tonsillitis, bronchitis, arthritis, and inflammation. In this paper, the antimicrobial activity of the hydroalcoholic extract of the cashew tree stem was evaluated against samples of meticillin-resistant and meticillin-sensible Staphylococcus aureus, attained from patients interned at Hospital Universitário/Universidade Federal da Paraíba. The antimicrobial activity was determined by the diffusion method in solid milieu to determine the Minimum Inhibitory Concentration (MIC) of the extract, and it was observed in 30 samples the presence or not of inhibition zone. All the assayed samples proved to be sensible to the activity of the extract of the cashew tree, with inhibition zones diameter of 10 to 20 mm, displaying consequently great effectiveness of the extract of the cashew tree stem. Thus, the use of this plant in our region can infer a low cost and efficient therapeutical alternative against bacterial infections caused by Staphylococcus aureus.