Broth Macrodilution Assays Research Articles

Value addition in the efficacy of conventional antibiotics by Nisin against Salmonella.

Frequent and indiscriminate use of existing battery of antibiotics has led to the development of multi drug resistant (MDR) strains of pathogens. As decreasing the concentration of the antibiotic required to treat Salmonellosis might help in combating the development of resistant strains, the present study was designed to assess the synergistic effects, if any, of nisin, in combination with conventional anti-Salmonella antibiotics against Salmonella enterica serovar Typhimurium. Minimum inhibitory concentrations (MICs) of the selected antimicrobial agents were determined by micro and macro broth dilution assays. In-vitro synergy between the agents was evaluated by radial diffusion assay, fractional inhibitory concentration (FIC) index (checkerboard test) and time-kill assay. Scanning electron microscopy (SEM) was also performed to substantiate the effect of the combinations. In-vivo synergistic efficacy of the combinations selected on the basis of in-vitro results was also evaluated in the murine model, in terms of reduction in the number of Salmonellae in liver, spleen and intestine. Nisin-ampicillin and nisin-EDTA combinations were observed to have additive effects, whereas the combinations of nisin-ceftriaxone and nisin-cefotaxime were found to be highly synergistic against serovar Typhimurium as evident by checkerboard test and time-kill assay. SEM results revealed marked changes on the outer membrane of the bacterial cells treated with various combinations. In-vivo synergy was evident from the larger log unit decreases in all the target organs of mice treated with the combinations than in those treated with drugs alone. This study thus highlights that nisin has the potential to act in conjunction with conventional antibiotics at much lower MICs. These observations seem to be significant, as reducing the therapeutic concentrations of antibiotics may be a valuable strategy for avoiding/reducing the development of emerging antibiotic resistance. Value added potential of nisin in the efficacy of conventional antibiotics may thus be exploited not only against Salmonella but against other Gram-negative infections as well.

Antimicrobial activity of Euclea undulata, Euclea divinorum and Diospyros lycioides extracts on multi- drug resistant Streptococcus mutans

The antimicrobial activities of three Zimbabwean chewing sticks (Euclea undulata, Euclea divinorum and Diospyros lycioides) on multi-drug resistant (MDR) Streptococcus mutans were determined. Thirty one isolates of S. mutans were obtained from 47 carious teeth and antibiotic sensitivity testing was done using the disc diffusion assay for all isolates. The resistance pattern of each isolate against eight antibiotics was noted. Eight resistance patterns were identified, and 19 isolates showed resistance to at least 4 antibiotics; these were considered MDR. Eight MDR and 2 multi-drug sensitive isolates were then assayed against methanol, acetone, diethyl ether and aqueous extracts of E. undulata, E. divinorum and D. lycioides. The broth macro-dilution assay was used to determine the minimum inhibitory concentration (MIC) of the extracts. All E. divinorum extracts showed no antimicrobial activity. Stem methanol extracts of E. undulata and root and stem methanol extracts of D. lycioides showed anti S. mutans activity. The largest inhibition zones were exhibited by methanol extracts of E. undulata (60 mm) and D. lycioides (54 mm). MICs ranged from 0.385 to 11.22 mg/ml and minimum bactericidal concentration (MBCs) from 0.485 to 20.20 mg/ml. D. lycioides and E. undulata extracts have potential use as anticariogenic agents. Key words: Streptococcus mutans, dental caries, antimicrobial activity, multi-drug resistant

Phytochemical screening and antibacterial activity of different fractions of Operculina turpethum root and leaf

In Bangladesh, the herb Operculina turpethum is used for otitis media, suppurative sores, burns, conjunctivitis and skin diseases (eczema, abscesses, acne, scabies and warts) although the compounds responsible for the medicinal properties have not been identified. The present study has been undertaken for antibacterial activity of the ethanol, ether and chloroform extract of O. turpethum root and leaf. Antibacterial activity has been investigated against Shigella boydii, Shigella flese, Shigella dysenteriae, Escherichia coli, Proteus vulgaris, Salmonella typhi, Hafnia alvei, Staphylococcus epidermidis, Streptococcus pyogenes, Staphylococcus aureus, Enterococcus faecalis by disc diffusion and broth macro-dilution assay. The zone of inhibition has been observed with almost all bacteria with some exceptions. Minimum inhibitory concentrations (MIC) of the extracts were found to be significant.

In vitro susceptibility of European human Borrelia burgdorferi sensu stricto strains to antimicrobial agents

Broth microdilution and macrodilution assays were used to determine minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of six antimicrobial agents (ceftriaxone, cefuroxime sodium, azithromycin, amoxicillin, doxycycline and amikacin) for nine European human isolates of Borrelia burgdorferi sensu stricto (s.s.). Strains were obtained from patients diagnosed with Lyme borreliosis in Slovenia. Modified Kelly–Pettenkofer medium with a final inoculum of 105Borrelia cells/mL and incubation periods of 72h and of 3 weeks and 6 weeks were used in the determination of MICs and MBCs, respectively. Observed MICs indicated that all isolates were susceptible to all the tested antimicrobial agents with the exception of amikacin. Cefuroxime sodium (MIC90=0.063mg/L), azithromycin (MIC90=0.22mg/L) and ceftriaxone (MIC90=0.25mg/L) displayed the lowest MICs, followed by amoxicillin (MIC90=1mg/L) and doxycycline (MIC90=2mg/L); no strain was susceptible to amikacin (MIC90=256mg/L). MBCs after incubation for 3 weeks and 6 weeks were determined for amoxicillin (MBC90=32mg/L), doxycycline (MBC90=32mg/L) and amikacin (MBC90=1024mg/L) and were found to be high (but not defined) for azithromycin (MBC90>0.88mg/L), cefuroxime sodium (MBC90>4mg/L) and ceftriaxone (MBC90>4mg/L). In determination of borrelial susceptibility to antimicrobial agents, intrinsic low susceptibility or methodological factors could result in low in vitro susceptibility of individual strains. This study is the first report on the antibiotic susceptibility of a series of European human isolates of B. burgdorferi s.s.

The Development of Innovative Herbal Extract Oral Film for the Unpleasant Mouth Odour

The development of innovative herbal extract encapsulated in quick-dissolving films for the unpleasant mouth odor derived from the encapsulated Mangosteen extract nanoparticles incorporated into pullulan-alginate films and using solvent casting as film formation. The oral blend films were prepared from a mixture of pullulan and alginate. Encapsulated mangosteen extract nanoparticles (15 and 20% w/w), mint oil (0.2%, w/w) and glycerol (10%, w/w) were incorporated in the films to act as natural antimicrobial agent and plasticizer, respectively. The oral blend films exhibited antibacterial activity against S.mutan, S.sanguinis and P.gingivalis by using by macro broth dilution assay. Percent encapsulated mangosteen extract on antibacterial activity was investigated. The increasing in growth inhibition (%) was found when percent encapsulated mangosteen extract was increased. When 15% and 20% encapsulated mangosteen extract content its was shown that growth inhibition of more than 99 % were found for all (S.mutan S.sanguinis and P.gingivalis). Acceptance of innovative products and films suspended mouth odor herbal extract from a sample 50 found that overall satisfaction with the product. Prototype than product innovation in existing products on the market. In terms of convenience in use. Ingredients of products. Effectively suspended mouth odor, Nutrition, Innovation.

In vitro ethnotherapeutic potential of the acetone extract of the bark of Ziziphus mucronata Willd. subsp. mucronata Willd.: Antimicrobial and toxicity evaluations

The antimicrobial and the cytotoxicity activities of the crude acetone bark extract ofZiziphus mucronata were determined by agar diffusion, macrobroth dilution assays and brine shrimp lethality assay, respectively, to ascertain its relevance in ethnomedicine. The results show that both Gram negative and Gram positive bacteria tested were equally inhibited. At the highest concentration (10000 µg/ml) of the extract,the bacterial inhibition zones ranged between 16 and 21 ± 1.0 mm, the fungal inhibition zones ranged between 13 and 19 ± 1.0 mm while Fusarium sporotrichioides, Candida glabrata and Trichophyton mucoides were not affected. The minimum inhibitory concentrations (MICs) values of the extract were between 78.1 and 312.5 µg/ml for the bacteria while the minimum bactericidal concentrations (MBCs) were between 156.3 and 2500 µg/ml. For the fungi, the MICs were between 1250 and 10000 µg/ml, the minimum fungicidal concentrations (MFCs) were between 5000 and 20000 µg/ml and the LC50 of the brine shrimp lethality assay was 90.27 µg/ml indicating a low level of toxicity. The degree of the pharmacological activity of Z. mucronata shows the significant medicinal potential of this plant in the treatment of microbial infections and justifies its use in complementary and alternative medicines in South Africa. Key words: Ziziphus mucronata, antibacterial, cytotoxicity, fungicidal, ethanol extract, medicinal potential.

Evaluation of antifungal activity of essential oils against potentially mycotoxigenic Aspergillus flavus and Aspergillus parasiticus

The antifungal activity of essential oils of fennel (Foeniculum vulgare Mill., Apiaceae), ginger (Zingiber officinale Roscoe, Zingiberaceae), mint (Mentha piperita L., Lamiaceae) and thyme (Thymus vulgaris L., Lamiaceae) was evaluated against mycotoxin producers Aspergillus flavus and A. parasiticus. High Resolution Gas Chromatography was applied to analyze chemical constituents of essential oils. The effect of different concentrations of essential oils was determined by solid medium diffusion assay. Mycelial growth and sporulation were determined for each essential oil at the concentrations established by solid medium diffusion assay. At the fifth, seventh and ninth days the mycelial diameter (O mm) and spore production were also determined. FUN-1 staining was performed to assess cell viability after broth macrodilution assay. Trans-anethole, zingiberene, menthol and thymol are the major component of essential oils of fennel, ginger, mint and thyme, respectively. The effective concentrations for fennel, ginger, mint and thyme were 50, 80, 50 and 50% (oil/DMSO; v/v), respectively. The four essential oils analysed in this study showed antifungal effect. Additionally, FUN-1 staining showed to be a suitable method to evaluate cell viability of potential mycotoxigenic fungi A. flavus and A. parasiticus after treatment with essential oils.

Cryptococcus neoformans: The Model Organism for Yeast Antifungal Drug Susceptibility Testing

We describe an approach to antifungal susceptibility testing of the yeast Cryptococcus neoformans that shows promise for predicting the mycological response in patients to treatment. Quantitative cultures of the cerebrospinal fluid provide a direct measure of the patient's mycological response to treatment and have been used in multiple studies to identify the most promising antifungal drugs for subsequent testing in larger clinical studies. Using these quantitative measures of response, a modified macrobroth dilution assay system shows the potential for predicting the response of an individual patient to treatment with amphotericin B, fluconazole, or the combination of amphotericin B plus flucytosine. We describe this modified macrobroth dilution assay method, the statistical approach for assessing susceptibility, and the clinical decisions that can be guided by this in vitro antifungal drug susceptibility testing.

Role of Antibiofilm-Antimicrobial Agents in Controlling Device-Related Infections

To assess the effects of N-acetylcysteine (NAC) on organism viability in planktonic and biofilm phases, biofilm thickness, and extracellular polysaccharide content. We performed time-kill curves and broth macrodilution assays of bacterial and fungal clinical isolates with varying concentrations of NAC. We also created in vitro bacterial biofilms, incubated them with NAC or control, and then stained with propidium iodide and FITC-labeled concanavalin A. We measured biofilm thickness, number of non-viable cells, and fluorescent intensity as a marker of extracellular matrix via a confocal laser scanning microscope. All experiments were conducted in triplicate. Tested organisms included methicillin-sensitive and -resistant Staphylococcus aureus (MSSA, MRSA), S. epidermidis, vancomycin-resistant Enterococcus faecalis (VRE), Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae, Candida albicans and C. krusei. NAC 80 mg/ml was uniformly bactericidal (>99.9% reduction) against all tested bacteria with no recoverable organisms after 30 minutes of incubation, but was fungistatic against candida species. Minimum inhibitory and bactericidal concentrations of NAC ranged from 5-10 mg/ml. Biofilm thickness was significantly decreased in NAC-treated biofilms for all organisms except VRE. The number of non-viable cells in NAC-treated Gram-positive biofilms was increased (p<0.05 for MRSA and VRE). NAC-treated Gram-negative biofilms had scant cellularity and lacked complex 3-dimensional structures that were characteristic of controls. Fluorescent intensity was similar in the experimental and control arms. NAC is bactericidal against clinically relevant and drug-resistant bacteria and also leads to biofilm disruption. NAC has the potential for use as a novel agent for prevention or treatment of biofilm-related infections.

Survey of the Antimicrobial Activity of Commercially Available Australian Tea Tree (Melaleuca alternifolia) Essential Oil ProductsIn Vitro

The aim of this study was to investigate the antimicrobial activity of a range of commercially available tea tree oil (TTO) products and to evaluate whether formulation plays a significant part in their antiseptic activity. The antimicrobial activity of the purchased products and control TTO solutions was assessed against Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa, and Candida albicans using well diffusion, broth microdilution, and broth macrodilution assays. Zone sizes obtained by the agar well diffusion assay ranged from 0 to 49.8 mm, with the more viscous and lipophilic products producing the smallest zones. Micro- and macrodilution methods showed that eight products had minimum inhibitory concentrations that were lower than the nonformulated TTO control. The remaining three products showed activity equivalent to the TTO control. In general, the commercially available antiseptic TTO products showed antimicrobial activity that was equivalent to, or greater than the nonformulated TTO control. This suggests that the TTO within these products has retained its antimicrobial activity. Furthermore, the enhanced activity of the products may be attributed to other antimicrobial excipients within the products such as preservatives, or to synergistic antimicrobial interactions between the TTO and other product excipients. The observation that the commercially available antiseptic TTO products tested in this study retained adequate antimicrobial activity emphasizes the importance of considering how product bases and excipients may interact with the active compound during formulation to ensure efficacy of the final product. Finally, the current data suggest that these TTO products may also be active in vivo. However, this can only be determined through further studies and in clinical trials.

Effect of Plant Oils on Candida albicans

Candida species, notably Candida albicans, is the major fungal pathogen in humans. It is a dimorphic fungus capable of causing superficial mucosal infections, as well as systemic infections, in immunocompromised individuals. The factors responsible for its pathogenesis are still not fully understood and increasing resistance to commonly used antifungal agents necessitates the search for new formulations. The inhibitory effect of 30 different plant oils on Candida albicans isolated from clinical samples was evaluated. The antifungal agent fluconazole was used as a positive control. Plant oils were tested at concentrations from 0.03% to 3% (v/v) to determine the minimum inhibitory concentration and minimum fungicidal concentration (MFC) using agar dilution and macro broth dilution assays. Of the 30 plant oils tested, 18 were found to be effective and 12 were ineffective. Based on their MFCs, effective oils were placed into three categories: most effective, moderately effective and least effective. Eucalyptus and peppermint oils were most effective, with MFC values of 0.12% and 0.15% (v/v), respectively. The significant antifungal activity of these oils suggests that they could serve as a source of compounds with therapeutic potential against Candida-related infections.

Nanofibers as carrier systems for antimicrobial microemulsions. II. Release characteristics and antimicrobial activity

AbstractAntimicrobial nanofibers were prepared by electrospinning microemulsions composed of the essential oil component eugenol solubilized in an aqueous nonionic micellar surfactant solution (Surfynol®465) with poly(vinyl alcohol) (PVA). Nanofibers contained microemulsions composed of 0.75–1.5 wt% eugenol and 5–10 wt % Surfynol. Scanning electron microscopy revealed substantial difference in fiber morphology depending on microemulsion composition with fiber diameters increasing as the concentration of either surfactant or essential oil component in the fibers increased. Release studies suggested a burst release of the encapsulated eugenol, potentially due to the hydrophilicity of the polymeric carrier resulting in rapid dissolution of the carrier matrix and high‐fiber porosity. The eugenol release rate depended on the amount of eugenol and surfactant incorporated within the fibers. The antimicrobial activity of nanofibers carrying eugenol was evaluated against two strains of Salmonella typhimurium (2476 and 2576) and Listeria monocytogenes (Scott A and 101) using a macrobroth dilution assay. Presence of nanofibers in bacterial suspensions successfully suppressed growth of foodborne pathogens and in some cases decreased initial cell numbers. Generally, nanofibers were more efficient against Gram‒negative than Gram‒positive bacterial strains. Results suggest that addition of microemulsions carrying lipophilic components to polymer solutions subjected to electrospinning offers a novel means to further enhance the functionality of nanofibers. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010

Evaluation of Combined Antibacterial Effects of Eugenol, Cinnamaldehyde, Thymol, and Carvacrol against E. coli with an Improved Method

In this study, the antibacterial activities of eugenol, cinnamaldehyde, thymol, carvacrol, and their combinations against E. coli were investigated separately. First, broth macro-dilution assay was adopted to determine the minimal inhibitory concentration (MIC) of the 4 components. Second, the combination testing was performed using chequerboard method. Finally, the combined effects were evaluated with an improved method, which was based on the indices of fractional inhibitory concentration (FIC) and Effect of the Combination (EC) jointly. The results indicated that MICs of the 4 components were 1600, 400, 400, and 400 mg/L, respectively; treatments with cinnamaldehyde/eugenol, thymol/eugenol, carvacrol/eugenol, and thymol/carvacrol revealed synergistic effects according to the 2 indices. By means of combination, MICs of eugenol, cinnamaldehyde, thymol, and carvacrol decreased to 400, 100, 100, and 100 mg/L, respectively. Consequently, the negative impacts of unpleasant smell of these 4 components could be minimized, making it possible to add them to foods as preservatives. In addition, this improved evaluation method provided a more accurate and comprehensive way to evaluate combined effects.

Caracterização fenotípica e análise plasmidial de cepas de Klebsiella pneumoniae em pacientes iranianos

É importante os conhecimentos locais de susceptibilidade antimicrobiana para Klebsiella pneumoniae a fim de que haja uma implementação efetiva de política hospitalar em relação aos anti- bacterianos. Foram isoladas 100 culturas para K. pneumoniae coletadas a partir de 3 diferentes hospitais no Irã durante o ano de 2004; para a susceptibilidade foram selecionados treze antibióticos diferentes, utilizando o método de difusão em disco e ensaio em caldo de diluição. Os isolados foram então submetidos à análise de endonucleases restritas ao DNA plasmidial. Todos os isolados foram sensíveis ao imipenem. As taxas de resistência a outros antibióticos foram na seguinte ordem: amicacina (10%), piperacilina-tazobactam (2%), ciprofloxacina (20%), ceftizoxima (14%), cefexime (31%), ceftazidima (28%) , cefotaxima (33%), ácido nalidíxico (32%), cefalexina (32%), gentamicina (30%), nitrofurantoína (31%) e piperacilina (66%). A produção de betalactamase de espectro estendido (ESBL) que hidrolisa a ceftazidima e a cefotaxima foi detectada em 54% dos isolados. Das 100 amostras testadas, 67 portavam plasmídeos e o restante faltava qualquer plasmidial. Embora a prevalência do fenótipo ESBL no Irã seja superior a países ocidentais, os números reportados são restritos de uma região. As evidências de focos com certos isolados de K. pneumoniae foram encontradas pela análise das endonucleases restritas de DNA plasmidial. Esta técnica também mostrou a persistência de infecções do trato urinário de vários pacientes.

Susceptibility of compound 48/80-sensitized Pseudomonas aeruginosa to the hydrophobic biocide triclosan

Pseudomonas aeruginosa is intrinsically resistant to the hydrophobic biocide triclosan, and yet it can be sensitized to low concentrations by permeabilization of the outer membrane using compound 48/80. A selective plating assay revealed that compound 48/80-permeabilized YM64, a triclosan-recognizing efflux pump-deficient variant, was unable to initiate growth on a medium containing triclosan. Macrobroth dilution assay data revealed that treatment with compound 48/80 synergistically decreased minimal inhibitory concentrations of the hydrophobic antibacterial agents rifamycin SV and chloramphenicol for all cell envelope variant strains examined. A low concentration of triclosan exerted a transient bactericidal effect on permeabilized wild-type strain PAO1, after which exponential growth resumed within 4 h. Permeabilized strain YM64 was unable to overcome the inhibition; yet, both strains remained susceptible to chloramphenicol for as long as 6 h, thereby suggesting that the outer membrane remained permeable to nonpolar compounds. These data support the notion that the transitory nature of compound 48/80 sensitization to triclosan in P. aeruginosa does not involve obviation of the hydrophobic diffusion pathway through the outer membrane. The inability of strain YM64 to overcome the synergistic effect of compound 48/80 and triclosan strongly suggests that triclosan-recognizing efflux pumps are involved in maintaining viability in wild-type cells whose outer membranes are otherwise compromised.

Prevalence of Aminoglycoside-Modifying Enzymes Genes Among Isolates ofEnterococcus faecalisandEnterococcus faeciumin Iran

Disks containing 120 microg of gentamicin were used to detect high-level gentamicin-resistant phenotype (HLGR) among isolates of Enterococcus faecalis (n = 79) and E. faecium (n = 35). These isolates were collected from three hospitals in Tehran during 2002-2004. The macrobroth dilution assay was then used to determine the minimum inhibitory concentration (MIC) of gentamicin. The susceptibility of isolates against amikacin, netilmicin, tobramycin, and kanamycin were also determined by Kirby-Bauer method. All isolates were subjected to polymerase chain reaction (PCR) assays targeting aminoglycoside modifying enzyme (AMEs) genes including aac(6 ')-aph(2 "), aph(2 ")-Ib, aph(2 ")-Ic, aph(2 ")-Ia, aph(2 ")-Id, aph(3 ')-IIIa, and ant(4 ')-Ia. Fifty-nine isolates (52%) showed HLGR phenotype. All isolates with HLGR phenotype and those showing 64 < MIC < 500 microg/ml contained aac(6 ')-aph(2 "). The aph(3 ')-IIIa was found in 61% of the isolates with HLGR phenotypes and in 65% of isolates with MIC < 500. Coexistence of aac(6 ')-aph(2 ") and aph(3 ')-IIIa gene among HLGR isolates of E. faecalis and E. faecium were 60% and 65%, respectively. The gene aph(2 ")-Ic was amplified in two isolates of E. faecium. The results of PCR for aph(2 ")-Id, ant(4 ')-Ia and aph(2 ")-Ib genes were negative. The aac(6 ')-aph(2 ") was the most frequent gene encoding resistance to gentamicin and other aminoglycosides followed by aph(3 ')-IIIa. Isolates lacking these genes were susceptible to all aminoglyocosides tested in this study.

Effect of outer membrane permeabilisation on intrinsic resistance to low triclosan levels in Pseudomonas aeruginosa

The present study was undertaken to investigate the possibility that outer cell envelope impermeability might be involved in the intrinsic resistance of Pseudomonas aeruginosa to low levels of the hydrophobic biocide triclosan. Macrobroth dilution and batch cultural turbidimetric assays were employed to assess the ability of compounds that render the Gram-negative outer membrane permeable to non-polar molecules to sensitise cell envelope variants to triclosan. Pseudomonas aeruginosa strains possessing highly refractory (PAO1) and atypically permeable (Z61) outer cell envelopes as well as a PAO1 derivative lacking four multidrug efflux pumps (YM64) were examined. Whilst the triclosan minimal inhibitory concentrations (MICs) differed dramatically for both PAO1 and Z61, significant decreases were seen for both strains in the presence of the outer membrane permeabiliser polymyxin B-nonapeptide. Strain YM64 was as resistant to triclosan as strain PAO1. Turbidimetric assessments of batch cultural growth kinetics revealed that the three chemically unrelated outer membrane permeabilisers polymyxin B-nonapeptide, compound 48/80 and ethylenediaminetetraacetate (EDTA) sensitised all strains to a sub-MIC concentration of triclosan (2.0 μg/mL). These data support the notion that the outer membrane exclusionary properties of P. aeruginosa for non-polar molecules confer intrinsic resistance to low concentrations of triclosan such as might be expected to occur in environmental residues. Moreover, a role for outer cell envelope impermeability is suggested for resistance to high triclosan concentrations in vitro.

Antifungal effect of Hevea brasiliensis latex with various fungi. Its synergistic action with amphotericin B against Candida albicans.

Antifungal activity of latex from Hevea brasiliensis was observed with various fungi in macrobroth dilution assays. The strongest antifungal effect was obtained with Trichosporon cutaneum (MIC 80% = 40.615 microg protein ml(-1), Kaff = 0.075 microg(-1) protein ml) and Cryptococcus neoformans (MIC 80% = 56.078 microg protein ml(-1), Kaff = 0.059 microg(-1) protein ml). Amphotericin B was synergized with all H. brasiliensis latex concentrations tested. The rates of synergy were about 50, 44 and 55% with 15, 30 and 60 microg protein ml(-1) latex, respectively. The putative role of glycosidase activities measured in crude latex, especially alpha-d-mannosidase and N-acetyl-beta-d-glucosaminidase activities which are potentially able to split off intraparietal linkages between glycosidic residues, is discussed. The possible role of another antifungal factor associated with rubber particles of latex is suggested.

Melanization of Cryptococcus neoformans and Histoplasma capsulatum reduces their susceptibilities to amphotericin B and caspofungin.

The fungal pathogens Cryptococcus neoformans and Histoplasma capsulatum produce melanin-like pigments in the presence of L-dopa in vitro and during mammalian infection. We investigated whether melanization affected the susceptibilities of the fungi to amphotericin B, caspofungin, fluconazole, itraconazole, or flucytosine (5FC). Using the standard macrodilution MIC protocol (the M27A protocol) of the National Committee for Clinical Laboratory Standards for yeast, we found no difference in the susceptibilities of melanized and nonmelanized C. neoformans and H. capsulatum isolates. Killing assays demonstrated that melanization reduced the susceptibilities of both fungi to amphotericin B and caspofungin. Laccase-deficient C. neoformans cells grown with L-dopa were significantly more susceptible than congenic melanin-producing yeast to killing by amphotericin B or caspofungin. Preincubation of amphotericin B or caspofungin with melanins decreased their antifungal activities. Elemental analysis of melanins incubated with amphotericin B or caspofungin revealed an alteration in the C:N ratios of the melanins, which indicated binding of these drugs by the melanins. In contrast, incubation of fluconazole, itraconazole, or 5FC with melanins did not significantly affect the antifungal efficacies of the drugs or the chemical composition of the melanins. The results suggest a potential explanation for the inefficacy of caspofungin against C. neoformans in vivo, despite activity in vitro. Furthermore, the results indicate that fungal melanins protect C. neoformans and H. capsulatum from the activities of amphotericin B and caspofungin and that this protection is not demonstrable by standard broth macrodilution assays.

In vitro susceptibility of Microsporum canis and other dermatophyte isolates from veterinary infections during therapy with terbinafine or griseofulvin.

We investigated the in vitro activity of terbinafine against fresh veterinary isolates of Microsporum canis and the potential of this organism to develop resistance in vivo during oral therapy. Dermatophyte cultures (n = 300) were obtained from naturally infected cats and dogs undergoing oral therapy with terbinafine or griseofulvin. M. canis comprised 92% of isolates; other species included Microsporum gypseum and Trichophyton mentagrophytes. Minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of terbinafine and griseofulvin were determined by broth macrodilution assay. Terbinafine was highly active against all three species with MIC90< or =0.03 microg ml(-1), in agreement with published data. However, terbinafine exhibited primary cidal activity against 66% of Microsporum isolates (n = 275) in contrast to the almost complete cidal effect in Trichophyton (n = 18). Griseofulvin was significantly less active than terbinafine (MIC90 = 4 microg ml(-1)) but had a primary cidal action on about 40% of the isolates. The data were analysed for changes in MIC and MFC during the course of therapy, which could be indicative for development of acquired resistance. Oral treatment of 37 animals with terbinafine for up to 39 weeks caused no increase in MIC or MFC of terbinafine, either in individual patients or in the whole group.