Pimenta pseudocaryophyllus inhibits virulence factors and promotes metabolic changes in Candidayeast

IntroductionThis is the first study to examine the in vitrosusceptibility and the expression of virulence factors in Candida species in the presence of Pimenta pseudocaryophyllus (Gomes) L.R. Landrum (Myrtaceae), a Brazilian plant known as paucravo. Additionally, the mechanisms of action of the crude ethanol extract and the ethyl acetate and aqueous fractions of this plant were investigated.MethodsThe in vitro susceptibility of Candida was tested using the broth microdilution method, whereas an XTT reduction assay was used for biofilms. Adherence was determined by counting the number of yeast cells that adhered to 100 oral epithelial cells, and hyphal formation was verified in the hyphal induction medium M199. Flow cytometry with propidium iodide and FUN-1 was performed to assess the mechanism of action.ResultsThe results revealed that the crude ethanol extract and the ethyl acetate and aqueous fractions of P. pseudocaryophyllusinhibited the growth of Candida isolates at a minimal inhibitory concentration (MIC) ranging from 64 to 256µg/mL, whereas the 50% sessile minimal inhibitory concentration (SMIC50) ranged from 512 to >1,024µg/mL. Adherence and hyphal formation were significantly reduced in the presence of the crude ethanol extract and both fractions. Although cell membrane injury was detected, the predominant mechanism of action appeared to be the alteration of yeast metabolism, as demonstrated by flow cytometry.ConclusionsOur results indicated that antifungal activity reduced the expression of virulence factors in yeast via the alteration of yeast metabolism, suggesting that the crude extract of P. pseudocaryophyllus and its fractions may contain novel antifungal agents.

Chemical composition and antifungal activity of essential oil from Eucalyptus smithii against dermatophytes

ABSTRACT INTRODUCTION: In this study, we evaluated the chemical composition of a commercial sample of essential oil from Eucalyptus smithii R.T. Baker and its antifungal activity against Microsporum canis ATCC 32903, Microsporum gypseum ATCC 14683, Trichophyton mentagrophytes ATCC 9533, T. mentagrophytes ATCC 11480, T. mentagrophytes ATCC 11481, and Trichophyton rubrum CCT 5507. METHODS: Morphological changes in these fungi after treatment with the oil were determined by scanning electron microscopy (SEM). The antifungal activity of the oil was determined on the basis of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values. RESULTS: The compound 1,8-cineole was found to be the predominant component (72.2%) of the essential oil. The MIC values of the oil ranged from 62.5μg·mL−1 to >1,000μg·mL−1, and the MFC values of the oil ranged from 125μg·mL−1 to >1,000μg·mL−1. SEM analysis showed physical damage and morphological alterations in the fungi exposed to this oil. CONCLUSIONS: We demonstrated the potential of Eucalyptus smithii essential oil as a natural therapeutic agent for the treatment of dermatophytosis.

Penicillin tolerance among Beta-hemolytic streptococci and production of the group carbohydrates, hemolysins, hyaluronidases and deoxyribonucleases

Penicillin tolerance among 67 strains of beta-hemolytic streptococci was examined by determining the ratio of the minimal bactericidal concentration to the minimal inhibitory concentration as 32 or greater. Tolerance was demonstrated in 15 group A strains and in 11,7, and 4 of groups B, C and G, respectively. Thereafter the effects of a subminimal inhibitory concentration (1/2MIC) of penicillin on the bacterial products of four tolerant and four nontolerant strains (two of each Lancefield group) were analyzed and compared. The antibiotic caused a marked increase in the expression of the group carbo-hydrates for strains of group B. Penicillin was found to reduce the cell-bound hemolysin activities of the four tolerant strains and to increase the activity of the other (free) form of nontolerant groups A, C and G hemolysins. Penicillin caused an increase in the extracellular hyaluronidase activities of one group A and groups B, C and G streptococci. With added antibiotic the production of deoxyribonuclease by tolerant groups A, C and G was greatly enhanced and that of the group B streptococcus was arrested.

The effects of sevoflurane and halothane anesthesia on cerebral blood flow velocity in children.

We compared cerebral blood flow velocity during anesthesia with sevoflurane and halothane in 23 children admitted for elective surgery (age, 0.4-9.7 yr; median age, 1.9 yr; ASA physical status I-II). Inhaled induction was performed in a randomized sequence with sevoflurane or halothane. Under steady-state conditions, cerebral blood flow velocity (systolic [V(s)], mean [V(mn)], and diastolic [VD]) were measured by a blinded investigator using transcranial pulsed Doppler ultrasonography. The anesthetic was then changed. CBFV measurements were repeated after washout of the first anesthetic and after steady-state of the second (equivalent minimal alveolar concentration to first anesthetic). The resistance index was calculated. VD and V(mn) were significantly lower during sevoflurane (V(mn) 1.35 m/s) than during halothane (V(mn) 1.50 m/s; P = 0.001), whereas V(s) was unchanged. The resistance index was lower during halothane (P < 0.001). Our results indicate lower vessel resistance and higher mean velocity during halothane than during sevoflurane. IMPLICATIONS: The mean cerebral blood flow velocity is significantly decreased in children during inhaled anesthesia with sevoflurane than during halothane. This might be relevant for the choice of anesthetic in children with risk of increased intracranial pressure, neurosurgery, or craniofacial osteotomies.

Antibacterial xanthones from Kielmeyera variabilis mart. (Clusiaceae)

The bioassay-guided fractionation of stems from Kielmeyera variabilis, traditionally used in Brazilian folk medicine, yielded assiguxanthone-B (1), kielcorin (4), 2,5-dihydroxybenzoic acid (3), and a mixture of xanthones containing assiguxanthone-B (1) and 1,3,5,6-tetrahydroxy-2-prenylxanthone (2) (1:1 w/w). The xanthone mixture inhibited Staphylococcus aureus and Bacillus subtilis at a concentration of 6.25 µg/ml. When tested alone, the minimal inhibitory concentration of assiguxanthone-B was 25 µg/ml against B. subtilis. Kielcorin and 2,5-dihydroxybenzoic acid were inactive against both strains. None of the fractions was active against Escherichia coli or Pseudomonas aeruginosa. Viable cells of S. aureus were reduced by a 1-3 log CFU/ml within 12 h after exposure of one to eight times the MIC of the xanthone mixture. It is not known whether the tetrahydroxy-2-prenylxanthone or other components of the xanthone mixture are responsible for the main antibacterial activity or whether additive or synergistic action is involved

In vitro susceptibility characteristics of Cryptococcus neoformans varieties from AIDS patients in Goiânia, Brazil

Sixty clinical isolates of Cryptococcus neoformans from AIDS from Goiânia, state of Goiás, Brazil, were characterized according to varieties, serotypes and tested for antifungal susceptibility. To differentiate the two varieties was used L-canavanine-glycine-bromothymol blue medium and to separate the serotypes was used slide agglutination test with Crypto Check Iatron. The Minimal Inhibitory Concentration (MIC) of fluconazole, itraconazole, and amphotericin B were determined by the National Committee for Clinical Laboratory Standards macrodilution method. Our results identified 56 isolates as C. neoformans var. neoformans serotype A and 4 isolates as C. neoformans var. gattii serotype B. MIC values for C. neoformans var. gattii were higher than C. neoformans var. neoformans. We verified that none isolate was resistant to itraconazole and to amphotericin B, but one C. neoformans var. neoformans and three C. neoformans var. gattii isolates were resistant to fluconazole. The presence of C. neoformans var. gattii fluconazole resistant indicates the importance of determining not only the variety of C. neoformans infecting the patients but also measuring the MIC of the isolate in order to properly orient treatment.

Antibacterial activity of extracts and neolignans from Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck

The evaluation of the activity of the aqueous and ethyl acetate extracts of the leaves of Piper regnellii was tested against gram-positive and gram-negative bacteria. The aqueous extractdisplayed a weak activity against Staphylococcus aureus and Bacillus subtilis with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of 1000 µg/ml. The ethyl acetate extract presented a good activity against S. aureus and B. subtilis with MIC and MBC at 15.62 µg/ml. In contrast to the relative low MICs for gram-positive bacteria, gram-negative bacteria were not inhibited by the extracts at concentrations < 1000 mg/ml. The ethyl acetate extract was fractionated on silica gel into nine fractions. The hexane and chloroform fractions were active against S. aureus (MIC at 3.9 µg/ml) and B. subtilis (MIC at 3.9 and 7.8 µg/ml, respectively). Using bioactivity-directed fractionation, the hexane fraction was rechromatographed to yield the antimicrobial compounds 1, 2, 5, and 6identified as eupomatenoid-6, eupomatenoid-5, eupomatenoid-3, and conocarpan, respectively. The pure compounds 1 and 2 showed a good activity against S. aureus with MIC of 1.56 µg/ml and 3.12 µg/ml, respectively. Both compounds presented MIC of 3.12 µg/ml against B. subtilis. The pure compound 6 named as conocarpan was quite active against S. aureus and B. subtilis with MIC of 6.25 µg/ml. The antibacterial properties of P. regnellii justify its use in traditional medicine for the treatment of wounds, contaminated through bacteria infections.

Antifungal activity from Ocimum gratissimum L. towards Cryptococcus neoformans

Cryptococcal infection had an increased incidence in last years due to the explosion of acquired immune deficiency syndrome epidemic and by using new and effective immunosuppressive agents. The currently antifungal therapies used such as amphotericin B, fluconazole, and itraconazole have certain limitations due to side effects and emergence of resistant strains. So, a permanent search to find new drugs for cryptococcosis treatment is essential. Ocimum gratissimum, plant known as alfavaca (Labiatae family), has been reported earlier with in vitro activity against some bacteria and dermatophytes. In our work, we study the in vitro activity of the ethanolic crude extract, ethyl acetate, hexane, and chloroformic fractions, essential oil, and eugenol of O. gratissimum using an agar dilution susceptibility method towards 25 isolates of Cryptococcus neoformans. All the extracts of O. gratissimum studied showed activity in vitro towards C. neoformans. Based on the minimal inhibitory concentration values the most significant results were obtained with chloroformic fraction and eugenol. It was observed that chloroformic fraction inhibited 23 isolates (92%) of C. neoformans at a concentration of 62.5 µg/ml and eugenol inhibited 4 isolates (16%) at a concentration of 0.9 µg/ml. This screening may be the basis for the study of O. gratissimum as a possible antifungal agent.

Antibacterial and cytotoxic activity of Brazilian plant extracts - Clusiaceae

Twelve extracts obtained from nine plants belonging to six different genera of Clusiaceae were analyzed against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and Enterococcus faecalis) bacteria using the microdilution broth assay. Tovomita aff. longifolia, T. brasiliensis, Clusia columnaris, Garcinia madruno, Haploclathra paniculata, and Caraipa grandifolia extracts showed significant results against the bacteria. The organic extract obtained from the leaves of T. aff. longifolia showed minimal inhibitory concentration (MIC) = 70 µg/ml and minimal bactericidal concentration (MBC) = 90 µg/ml against E. faecalis and the organic extract made with the stem of C. columnaris showed MIC = 180 µg/ml and MBC = 270 µg/ml against P. aeruginosa. None of the antibacterial extracts showed lethal activity against brine shrimp nauplii. On the other hand, both aqueous and organic extracts obtained from the aerial organs of Vismia guianensis that were cytotoxic to brine shrimp nauplii did not show a significant antibacterial activity in the assay.

Citral and carvone chemotypes from the essential oils of Colombian Lippia alba (Mill.) N.E. Brown: composition, cytotoxicity and antifungal activity

Two essential oils of Lippia alba (Mill.) N.E. Brown (Verbenacea), the carvone and citral chemotypes and 15 of their compounds were evaluated to determine cytotoxicity and antifungal activity. Cytotoxicity assays for both the citral and carvone chemotypes were carried out with tetrazolium-dye, which showed a dose-dependent cytotoxic effect against HeLa cells. Interestingly, this effect on the evaluated cells (HeLa and the non-tumoural cell line, Vero) was lower than that of commercial citral alone. Commercial citral showed the highest cytotoxic activity on HeLa cells. The antifungal activity was evaluated against Candida parapsilosis, Candida krusei, Aspergillus flavus and Aspergillus fumigatus strains following the standard protocols, Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing and CLSI M38-A. Results demonstrated that the most active essential oil was the citral chemotype, with geometric means-minimal inhibitory concentration (GM-MIC) values of 78.7 and 270.8 μg/mL for A. fumigatus and C. krusei, respectively. Commercial citral showed an antifungal activity similar to that of the citral chemotype (GM-MIC values of 62.5 μg/mL for A. fumigatus and 39.7 μg/mL for C. krusei). Although the citronellal and geraniol were found in lower concentrations in the citral chemotype, they had significant antifungal activity, with GM-MIC values of 49.6 μg/mL for C. krusei and 176.8 μg/mL for A. fumigatus.

Linezolid alone or combined with rifampin against methicillin-resistant Staphylococcus aureus in experimental foreign-body infection.

We investigated the activity of linezolid, alone and in combination with rifampin (rifampicin), against a methicillin-resistant Staphylococcus aureus (MRSA) strain in vitro and in a guinea pig model of foreign-body infection. The MIC, minimal bactericidal concentration (MBC) in logarithmic phase, and MBC in stationary growth phase were 2.5, >20, and >20 microg/ml, respectively, for linezolid; 0.01, 0.08, and 2.5 microg/ml, respectively, for rifampin; and 0.16, 0.63, >20 microg/ml, respectively, for levofloxacin. In time-kill studies, bacterial regrowth and the development of rifampin resistance were observed after 24 h with rifampin alone at 1x or 4x the MIC and were prevented by the addition of linezolid. After the administration of single intraperitoneal doses of 25, 50, and 75 mg/kg of body weight, linezolid peak concentrations of 6.8, 12.7, and 18.1 microg/ml, respectively, were achieved in sterile cage fluid at approximately 3 h. The linezolid concentration remained above the MIC of the test organism for 12 h with all doses. Antimicrobial treatments of animals with cage implant infections were given twice daily for 4 days. Linezolid alone at 25, 50, and 75 mg/kg reduced the planktonic bacteria in cage fluid during treatment by 1.2 to 1.7 log(10) CFU/ml; only linezolid at 75 mg/kg prevented bacterial regrowth 5 days after the end of treatment. Linezolid used in combination with rifampin (12.5 mg/kg) was more effective than linezolid used as monotherapy, reducing the planktonic bacteria by >or=3 log(10) CFU (P < 0.05). Efficacy in the eradication of cage-associated infection was achieved only when linezolid was combined with rifampin, with cure rates being between 50% and 60%, whereas the levofloxacin-rifampin combination demonstrated the highest cure rate (91%) against the strain tested. The linezolid-rifampin combination is a treatment option for implant-associated infections caused by quinolone-resistant MRSA.

High vancomycin resistance among biofilms produced by Staphylococcus species isolated from central venous catheters

Biofilm production is an important mechanism that allows microbes to escape host defences and antimicrobial therapy. Vancomycin has been used largely for the treatment of methicillin-resistant staphylococcal infections. Here, we determined the minimal inhibitory concentration (MIC) and minimal biofilm eradication concentration (MBEC) for 82 Staphylococcus species isolated from central venous catheters (CVC). Our results showed that the 41 strong and moderate-biofilm-producing isolates presented a higher MBEC/MIC ratio for vancomycin than the 24 weak-biofilm-producing isolates, illustrating the importance of biofilm production ability and the difficulty in treating biofilm-related infections. The MBEC was significantly higher in moderate-biofilm-producing isolates than in weak-biofilm-producing isolates (p < 0.001) and in strong-biofilm-producing isolates than in weak-biofilm-producing isolates (p = 0.001). The correlation between the MIC and the MBEC was poor. Based on our results, we recommend that bacterial biofilms be suspected in all cases of CVC infection.

Occurrence of blaOXA-23 gene in imipenem-susceptible Acinetobacter baumannii

The aim of the current study was to describe the occurrence of the blaOXA-23 gene and the ISAba1 element in imipenem-susceptible Acinetobacter baumannii strains. By performing the polymerase chain reaction mapping using combinations of ISAba1 forward primers and the blaOXA-23-like gene reverse primers, we demonstrated that the ISAba1 element did not occur upstream of the blaOXA-23 gene in five of 31 isolates, which explained the lack of resistance to imipenem despite the presence of the blaOXA-23 gene. All of the blaOXA-23-positive isolates were susceptible to imipenem and meropenem with minimal inhibitory concentration < 4 µg/mL. Pulsed-field gel electrophoresis analysis revealed four genotypes among the five blaOXA-23-positive isolates. The current report of the blaOXA-23 gene in imipenem-susceptible isolates provided evidence that this gene may be silently spread in a hospital environment and highlighted the threat of undetected reservoirs of carbapenemase genes.

Anti-Mycobacterium tuberculosis activity and cytotoxicity of Calophyllum brasiliense Cambess (Clusiaceae)

We evaluated the in vitro anti-Mycobacterium tuberculosis activity and the cytotoxicity of dichloromethane extract and pure compounds from the leaves of Calophyllum brasiliense. Purification of the dichloromethane extract yielded the pure compounds (-) mammea A/BB (1), (-) mammea B/BB (2) and amentoflavone (3). The compound structures were elucidated on the basis of spectroscopic and spectrometric data. The contents of bioactive compounds in the extracts were quantified using high performance liquid chromatography coupled to an ultraviolet detector. The anti-M. tuberculosis activity of the extracts and the pure compounds was evaluated using a resazurin microtitre assay plate. The cytotoxicity assay was performed in J774G.8 macrophages using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide colourimetric method. The quantification of the dichloromethane extract showed (1) and (2) at concentrations of 31.86 ± 2.6 and 8.24 ± 1.1 µg/mg of extract, respectively. The dichloromethane and aqueous extracts showed anti-M. tuberculosis H37Rv activity of 62.5 and 125 µg/mL, respectively. Coumarins (1) and (2) showed minimal inhibitory concentration ranges of 31.2 and 62.5 µg/mL against M. tuberculosis H37Rv and clinical isolates. Compound (3) showed no activity against M. tuberculosis H37Rv. The selectivity index ranged from 0.59-1.06. We report the activity of the extracts and coumarins from the leaves of C. brasiliense against M. tuberculosis.

Bioactive endophytic fungi isolated from Caesalpinia echinata Lam. (Brazilwood) and identification of beauvericin as a trypanocidal metabolite from Fusarium sp.

Aiming to identify new sources of bioactive secondary metabolites, we isolated 82 endophytic fungi from stems and barks of the native Brazilian tree Caesalpinia echinata Lam. (Fabaceae). We tested their ethyl acetate extracts in several in vitro assays. The organic extracts from three isolates showed antibacterial activity against Staphylococcus aureus and Escherichia coli [minimal inhibitory concentration (MIC) 32-64 μg/mL]. One isolate inhibited the growth of Salmonella typhimurium (MIC 64 μg/mL) and two isolates inhibited the growth of Klebsiella oxytoca (MIC 64 μg/mL), Candida albicans and Candida tropicalis (MIC 64-128 μg/mL). Fourteen extracts at a concentration of 20 μg/mL showed antitumour activities against human breast cancer and human renal cancer cells, while two isolates showed anti-tumour activities against human melanoma cancer cells. Six extracts were able to reduce the proliferation of human peripheral blood mononuclear cells, indicating some degree of selective toxicity. Four isolates were able to inhibit Leishmania (Leishmania) amazonensis and one isolate inhibited Trypanosoma cruzi by at least 40% at 20 μg/mL. The trypanocidal extract obtained from Fusarium sp. [KF611679] culture was subjected to bioguided fractionation, which revealed beauvericin as the compound responsible for the observed toxicity of Fusarium sp. to T. cruzi. This depsipeptide showed a half maximal inhibitory concentration of 1.9 μg/mL (2.43 μM) in a T. cruzi cellular culture assay.