Action Of Aminoglycoside Antibiotics Research Articles

The development of a formulation, the selection and harmonization of the nutrient medium for determining the activity of aminoglycoside antibiotics

The work is devoted to research on the selection of peptone for the optimization of the nutrient medium for the antimicrobial activity by the agar diffusion method of neomycin (framycetin), which have a similar chemical structure. The paper substantiates the relevance and importance of developing a formulation, selecting the optimal composition (ingredients), the quantitative ratio of ingredients, the pH of the medium, compliance of the proposed nutrient media with the State Pharmacopoeia of the Russian Federation and the European Pharmacopoeia for the standardization of antibiotics. The aim of the work was to study the components of the nutrient medium in comparison with the pharmacopoeial standards recommended for determining the effectiveness of antibiotics. It is shown that for carrying out microbiological tests in the standardization of antimicrobial drugs, the nutrient medium is the main one, and the correctness and accuracy of the interpretation of the research results depend on their quality. The experiment studied four brands of dry peptone and broth from different manufacturers - LLC NITSF and FBUN GNTs PMB (Obolensk) when working with the test strain of the spore culture Bacillus subtilis ATCC 6633. An optimal nutrient medium is proposed, the composition and conditions of its preparation are selected. To determine the antimicrobial activity of framycetin by the agar diffusion method, a developed medium for determining the effectiveness of neomycin when working with the spore-forming form of the test strain Baccillus subtillis ATCC 6633 is proposed.

Benzalkonium chloride antagonises aminoglycoside antibiotics and promotes evolution of resistance.

BackgroundBiocide disinfectants are essential tools in infection control, but their use can inadvertently contribute to emergence of antibiotic-resistant bacteria. In this study we systematically examine the effect of the biocide benzalkonium chloride, which is primarily used for surface disinfection but is also present as a preservative in many consumer products, on the activity of aminoglycoside antibiotics in Acinetobacter baumannii.MethodsThe effect of subinhibitory BAC on aminoglycoside treatment of A. baumannii ATCC17978 was investigated using time-to-kill assays, MIC determination, directed evolution experiments, fluctuation tests and labelled gentamicin accumulation assays. Further MIC determinations and directed evolution experiments were performed with additional Gram-negative ESKAPE pathogens.FindingsIn A. baumannii ATCC17978, BAC prevents gentamicin killing and drastically increases the frequency at which resistant mutants emerge, through reducing intracellular antibiotic accumulation. BAC also increases the MIC of multiple aminoglycoside antibiotics (kanamycin, tobramycin, streptomycin, gentamicin and amikacin). BAC promotes the emergence of mutants with reduced gentamicin susceptibility in other Gram-negative ESKAPE pathogens but does not always alter the MIC. These effects occur at BAC concentrations which are similar to residual levels in high-use environments, and just below the concentration range for BAC when used as a preservative in eye drops and ear drops.InterpretationOur results suggest that subinhibitory BAC has the potential to antagonise aminoglycoside activity and promote the emergence of bacterial mutants with reduced susceptibility. We suggest that the extremely widespread use of BAC in clinical and home settings and its long half-life mean there is potential for these interactions to occur in the environment, or in patients who use BAC-containing products while taking aminoglycosides to treat skin, eye or ear infections, although such co-exposure is likely to be rare. We suggest that biocide stewardship is needed to prevent the types of exposure that can contribute to antibiotic resistance.FundingThis work was funded by the National Health and Medical Research Council of Australia. The funders had no role in study design, interpretation or decision to publish.

Protective Mechanisms of Avocado Oil Extract Against Ototoxicity

Despite the excellent antimicrobial activity of aminoglycoside antibiotics, permanent inner ear damage associated with the use of these drugs has resulted in the need to develop strategies to address the ototoxic risk given their widespread use. In a previous study, we showed that avocado oil protects ear hair cells from damage caused by neomycin. However, the detailed mechanism by which this protection occurs is still unclear. Here, we investigated the auditory cell-protective mechanism of enhanced functional avocado oil extract (DKB122). RNA sequencing followed by pathway analysis revealed that DKB122 has the potential to enhance the expression of detoxification and antioxidant genes associated with glutathione metabolism (Hmox4, Gsta4, Mgst1, and Abcc3) in HEI-OC1 cells. Additionally, DKB122 effectively decreased ROS levels, resulting in the inhibition of apoptosis in HEI-OC1 cells. The expression of the inflammatory genes that encode chemokines and interleukins was also downregulated by DKB122 treatment. Consistent with these results, DKB122 significantly inhibited p65 nuclear migration induced by TNF-α or LPS in HEI-OC1 cells and THP-1 cells and the expression of inflammatory chemokine and interleukin genes induced by TNF-α was significantly reduced. Moreover, DKB122 treatment increased LC3-II and decreased p62 in HEI-OC1 cells, suggesting that DKB122 increases autophagic flux. These results suggest that DKB122 has otoprotective effects attributable to its antioxidant activity, induction of antioxidant gene expression, anti-inflammatory activity, and autophagy activation.

Effect of the essential oils from Piper sp. and blue led lights in the enhancement of the antibiotic activity of drugs against mdr bacterial strains

The indiscriminate use of antibiotics has made bacterial resistance an important public health problem, since many antibiotics have become ineffective. Phototherapy can be considered an alternative to reduce the abusive use of antimicrobials, thus impacting microbial resistance. The objective of this study was to determine the chemical profile and to evaluate the effect of blue LED lights on the antibacterial activity of essential oils from Piper species, as well as their aminoglycoside antibiotic activity modulation using the microdilution method to determine the Minimum Inhibitory Concentration (MIC). The antibiotic activity modulating effect of these oils was also determined using the broth microdilution method with 96-well plates which were exposed to LED light for 20 min. Chemical components were characterized by gas chromatography coupled to mass spectrometry, revealing β-copaen-4-α-ol, germacrene A and germacrene B as major essential oil constituents for Piper arboreum (OEPar), Piper aduncum (OEPad) and Piper gaudichaudianum (OEPg), respectively. OEPar obtained a MIC of 512 μg/mL against Staphylococcus aureus and a MIC ≥ 1024 μg/mL against Escherichia coli. OEPad and OEPg showed MIC values ≥ 1024 μg/mL against the utilized strains. The essential oils modulated the effect of the antibiotics amikacin and gentamicin, with this effect being potentiated when exposed to blue LED. The blue LED light in the absence of the essential oil also showed an ability to modulate aminoglycoside antibiotic activity in this study, presenting mostly synergistic effects. In conclusion, the results obtained in this study demonstrate that photodynamic therapy using blue LED light interferes with the antibacterial action of P. arboreum, P. aduncum and P. gaudichaudianum essential oils and aminoglycoside antibiotic activity.

Host metabolites stimulate the bacterial proton motive force to enhance the activity of aminoglycoside antibiotics

Antibiotic susceptibility of bacterial pathogens is typically evaluated using in vitro assays that do not consider the complex host microenvironment. This may help explaining a significant discrepancy between antibiotic efficacy in vitro and in vivo, with some antibiotics being effective in vitro but not in vivo or vice versa. Nevertheless, it is well-known that antibiotic susceptibility of bacteria is driven by environmental factors. Lung epithelial cells enhance the activity of aminoglycoside antibiotics against the opportunistic pathogen Pseudomonas aeruginosa, yet the mechanism behind is unknown. The present study addresses this gap and provides mechanistic understanding on how lung epithelial cells stimulate aminoglycoside activity. To investigate the influence of the local host microenvironment on antibiotic activity, an in vivo-like three-dimensional (3-D) lung epithelial cell model was used. We report that conditioned medium of 3-D lung cells, containing secreted but not cellular components, potentiated the bactericidal activity of aminoglycosides against P. aeruginosa, including resistant clinical isolates, and several other pathogens. In contrast, conditioned medium obtained from the same cell type, but grown as conventional (2-D) monolayers did not influence antibiotic efficacy. We found that 3-D lung cells secreted endogenous metabolites (including succinate and glutamate) that enhanced aminoglycoside activity, and provide evidence that bacterial pyruvate metabolism is linked to the observed potentiation of antimicrobial activity. Biochemical and phenotypic assays indicated that 3-D cell conditioned medium stimulated the proton motive force (PMF), resulting in increased bacterial intracellular pH. The latter stimulated antibiotic uptake, as determined using fluorescently labelled tobramycin in combination with flow cytometry analysis. Our findings reveal a cross-talk between host and bacterial metabolic pathways, that influence downstream activity of antibiotics. Understanding the underlying basis of the discrepancy between the activity of antibiotics in vitro and in vivo may lead to improved diagnostic approaches and pave the way towards novel means to stimulate antibiotic activity.

Chemical Characterization and Cytoprotective Effect of the Hydroethanol Extract from Annona coriacea Mart. (Araticum).

Introduction:Annona coriacea Mart. (araticum) is a widely distributed tree in the cerrado. Its value is attributed principally to the consumption of its fruit which possesses a large nutritive potential. The objective was to identify the chemical profile and evaluate the antimicrobial and cytoprotective activity of the hydroethanol extract of A. coriacea Mart. (HEAC) leaves against the toxicity of mercury chloride.Materials and Methods:The characterization of components was carried out using high-performance liquid chromatography (HPLC). The minimum inhibitory concentration (MIC) was determined by microdilution method in broth with strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. For evaluation of the modulatory and cytoprotective activity of aminoglycoside antibiotics (gentamicin and amikacin) and mercury chloride (HgCl2), the substances were associated with the HEAC at subinhibitory concentrations (MIC/8).Results and Discussion:The HPLC analysis revealed the presence of flavonoids such as Luteolin (1.84%) and Quercetin (1.19%) in elevated concentrations. The HEAC presented an MIC ≥512 μg/mL and significant antagonistic action in aminoglycosides modulation, and it also showed cytoprotective activity to S. aureus (significance P < 0.0001) and E. coli (significance P < 0.05) bacteria against the mercury chloride heavy metal with significance, this action being attributed to the chelating properties of the flavonoids found in the chemical identification.Conclusions:The results acquired in this study show that the HEAC presents cytoprotective activity over the tested strains in vitro and can also present antagonistic effect when associated with aminoglycosides, reinforcing the necessity of taking caution when combining natural and pharmaceutical products.SUMMARY The hydroalcoholic extract of A. coriacea Mart. presents in vitro cytoprotective activity against the toxic effect of Hg. Abbreviations Used: HPLC-DAD: High-performance liquid chromatography with a diode array detector; MIC: Minimum inhibitory concentration; DMSO: Dimethyl sulfoxide

In vitro Potentiation of the Aminoglycoside Antibiotic Activity by Croton campestris A. Against Multiresistant Bacteria

Croton campestris A., popularly known as “Velame do campo” (Euphorbiaceae), is a shrub originating from Brazil, mainly present in the Southeast and Northeast regions. The objective of this study was to evaluate the antibacterial activity of the ethanol extract (EECC), hexane (HFEECC) and dichloromethane fractions (DFEECC), obtained from the ethanol extract of Croton campestris A. leaves. Antibacterial and modulating activity (on bacterial resistance) was determined by micro dilution method to identify the MIC (Minimum Inhibitory Concentration). In the antibacterial activity tests the fractions showed a MIC of ≥ 1024 µg/mL (where ≤ 128 µg/mL for MIC is considered clinically relevant). In regards to modulation of bacterial resistance, all products showed synergism when combined with antibiotic against bacterial strains. It was observed that all products potentiated the antibiotic action of all antibiotics assayed against all bacterial strains. The results indicate that the extracts and fractions obtained from Croton campestris leaves could represent an alternative source of natural products capable of modifying and interfering with bacterial resistance to aminoglycosides.

Essential oil of Alpinia zerumbet (Pers.) B.L. Burtt. & R.M. Sm. (Zingiberaceae): chemical composition and modulation of the activity of aminoglycoside antibiotics

Essential oils are new natural medicines, which have been studied extensively in recent years. In this paper, the chemical composition of the essential oil from Alpinia zerumbet leaves and its ability to modulate the activity of aminoglycoside antibiotics is reported. The essential oil was extracted by hydrodistillation and analyzed by gas chromatography (GC) and GC–mass spectrometry (GC/MS). The antibacterial activity of the essential oil alone or in association with aminoglycosides was determined by a microdilution test against twelve strains (standards and multi-resistant clinical isolates obtained from sputum and wounds), seven Gram negative and five Gram positive. The major components identified by GC/MS were 4-terpineol (32.9 ± 4.0%), 1,8-cineole (21.4 ± 1.2%), γ-terpinolene (10.0 ± 1.8%) and sabinene (7.1 ± 4.6%), with a strong predominance of monoterpenes. The results indicated that the multi-resistant clinical isolates Staphylococcus aureus 358 and Escherichia coli 27 were more sensitive to the essential oil, exhibiting minimum inhibitory concentrations (MICs) of 32 and 128 μg/mL, respectively. The essential oil was capable of enhancing the antimicrobial activity of aminoglycosides against all strains tested, except for S. aureus 358. Very significant reductions in the MICs of the antibiotics were found, with a 94% decrease in MIC of neomycin against Klebsiella pneumoniae ATCC 10031. The results obtained indicate that the A. zerumbet essential oil has a significant synergistic antibiotic effect when combined with aminoglycosides, demonstrating that its constituents may act as potentiators of the antibiotic activity of aminoglycosides.

Inhibition of the ANT(2″)-Ia resistance enzyme and rescue of aminoglycoside antibiotic activity by synthetic α-hydroxytropolones

Aminoglycoside-2″-O-nucleotidyltransferase ANT(2″)-Ia is an aminoglycoside resistance enzyme prevalent among Gram-negative bacteria, and is one of the most common determinants of enzyme-dependant aminoglycoside-resistance. The following report outlines the use of our recently described oxidopyrylium cycloaddition/ring-opening strategy in the synthesis and profiling of a library of synthetic α-hydroxytropolones against ANT(2″)-Ia. In addition, we show that two of these synthetic constructs are capable of rescuing gentamicin activity against ANT-(2″)-Ia-expressing bacteria.

Engineering persister-specific antibiotics with synergistic antimicrobial functions.

Most antibiotics target growth processes and are ineffective against persister bacterial cells, which tolerate antibiotics due to their reduced metabolic activity. These persisters act as a genetic reservoir for resistant mutants and constitute a root cause of antibiotic resistance, a worldwide problem in human health. We re-engineer antibiotics specifically for persisters using tobramycin, an aminoglycoside antibiotic that targets bacterial ribosomes but is ineffective against persisters with low metabolic and cellular transport activity. By giving tobramycin the ability to induce nanoscopic negative Gaussian membrane curvature via addition of 12 amino acids, we transform tobramycin itself into a transporter sequence. The resulting molecule spontaneously permeates membranes, retains the high antibiotic activity of aminoglycosides, kills E. coli and S. aureus persisters 4–6 logs better than tobramycin, but remains noncytotoxic to eukaryotes. These results suggest a promising paradigm to renovate traditional antibiotics.

Structure-guided optimization of protein kinase inhibitors reverses aminoglycoside antibiotic resistance

Activity of the aminoglycoside phosphotransferase APH(3')-Ia leads to resistance to aminoglycoside antibiotics in pathogenic Gram-negative bacteria, and contributes to the clinical obsolescence of this class of antibiotics. One strategy to rescue compromised antibiotics such as aminoglycosides is targeting the enzymes that confer resistance with small molecules. We demonstrated previously that ePK (eukaryotic protein kinase) inhibitors could inhibit APH enzymes, owing to the structural similarity between these two enzyme families. However, limited structural information of enzyme-inhibitor complexes hindered interpretation of the results. In addition, cross-reactivity of compounds between APHs and ePKs represents an obstacle to their use as aminoglycoside adjuvants to rescue aminoglycoside antibiotic activity. In the present study, we structurally and functionally characterize inhibition of APH(3')-Ia by three diverse chemical scaffolds, anthrapyrazolone, 4-anilinoquinazoline and PP (pyrazolopyrimidine), and reveal distinctions in the binding mode of anthrapyrazolone and PP compounds to APH(3')-Ia compared with ePKs. Using this observation, we identify PP derivatives that select against ePKs, attenuate APH(3')-Ia activity and rescue aminoglycoside antibiotic activity against a resistant Escherichia coli strain. The structures described in the present paper and the inhibition studies provide an important opportunity for structure-based design of compounds to target aminoglycoside phosphotransferases for inhibition, potentially overcoming this form of antibiotic resistance.

Chemical composition of the essential oils of Lantana camara L. and Lantana montevidensis Briq. and their synergistic antibiotic effects on aminoglycosides

Recently, several plants have been evaluated not only for antimicrobial activity but also for resistance-modifying action. In this work, the chemical composition and antibacterial and antibiotic-modulatory activities of the essential oils from Lantana camara L. and Lantana montevidensis Briq. were analyzed. The essential oils extracted from the leaves by hydrodistillation and analyzed by gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS) were characterized by a high percentage of sesquiterpene hydrocarbons. Among the 34 constituents identified, bicyclogermacrene (19.4%), isocaryophyllene (16.7%), valencene (12.9%) and germacrene D (12.3%) were the main constituents of the oil from L. camara, while in the oil from L. montevidensis, β-caryophyllene (31.5%), germacrene D (27.5%) and bicyclogermacrene (13.9%) predominated. The essential oils were examined for antibiotic activities alone and in combination with aminoglycosides by a microdilution assay utilizing five bacterial strains. They exhibited significant antibacterial activities, mainly against Proteus vulgaris (MIC 64 μg/mL, L. camara; MIC 128 μg/mL, L. montevidensis). Both oils also showed a synergistic effect on the activity of aminoglycoside antibiotics. Thus, the essential oils of L. camara and L. montevidensis could be used as a source of plant-derived natural products with resistance-modifying antibacterial activity.

Phytochemical Prospection and Modulation of Aminoglycoside Antibiotic Activity by Croton campestris A.

Background:Escherichia coli is known to produce enterotoxins whose properties and role in diarrheal disease have been extensively investigated; besides, this bacterium is related to several extra-intestinal problems in the intensive care unit and in surgical wound infections. Some species of Staphylococcus are recognized as etiological agents of opportunistic infections in animals and humans. This study is the first test on the modulation of antibiotic activity by Croton campestris A. against multiresistant strains of E. coli and Staphylococcus aureus. Methods: In this study, the hexane and methanol extract of C. campestris A. was tested for antibacterial activity alone and in combination with aminoglycosides against bacterial strains. The synergy of the methanol and hexane extract was verified by microdilution method. Results: A synergistic effect of both extracts combined with the aminoglycosides was demonstrated. Conclusions: It is suggested that the extracts from C. campestris A. could be used as a source of natural product derived from this plant with resistance-modifying antibacterial activity, providing a new weapon against the problem of bacterial resistance to antibiotics.

Potentiation of aminoglycoside antibiotic activity using the body fat from the snake Boa constrictor

Boa constrictor is widely used in traditional communities in many different folk remedies and products derived from it are sold in public markets throughout northeastern Brazil and as its body fat has many different therapeutic indications as a folk remedy. The present work evaluates the antibacterial activity of the body fat from the snake Boa constrictor when employed either alone or in combination with antibiotics and discusses the ecological implications of the use of this traditional remedy. Oil (OBC) was extracted from body fat located in the ventral region of B. constrictor using hexane as a solvent. The antibacterial activity of OBC was tested against standard as well as multi-resistant lines, either alone and in combination with antibiotics. OBC did not demonstrate any relevant antibacterial activity against standard or multidrug-resistant bacterial strains. OBC showed synergistic activity when combined with the aminoglycoside antibiotics. Our results indicate that the body fat of Boa constrictor does not possess bactericidal activity, from the clinical point of view, but when combined with an antibiotic, the fat demonstrated a significant synergistic activity.

In VitroAntibacterial, Phototoxic, and Synergistic Activity of Ethanol Extracts fromCostuscf.arabicusL.

In this study, ethanol extracts of stems and leaves of Costus cf. arabicus L. were evaluated for antibacterial activity against multidrug-resistant strains of Escherichia coli and Staphylococcus aureus. The antibacterial and modulatory activities of the extracts were assayed by microdilution. The light-enhanced antibacterial activity was assayed by a light regimen. The growth of the bacteria tested was not inhibited by the extracts. The minimum inhibitory concentration values were ≥1,024 μg/mL. However, the antibiotic activity of aminoglycosides was synergistically enhanced when these extracts at subinhibitory concentrations were combined with the antibiotics. Also, both extracts showed activity against the wild-type bacterial strains, but the leaf extract was the more active extract, being active against both S. aureus and E. coli. Therefore, we conclude that the ethanol extracts of stems and leaves of C. cf. arabicus L. have potential light-induced antibacterial activity and synergistic antibiotic activity. This study showed that these extracts may be a promising source of antibacterial and modulatory agents.

Termite usage associated with antibiotic therapy: enhancement of aminoglycoside antibiotic activity by natural products of Nasutitermes corniger (Motschulsky 1855)

BackgroundSeveral species from Insecta are used as remedies. Among these species, the termite Nasutitermes corniger is commonly used in traditional medicine in Northeast Brazil. The present work tests the modifying antibiotic activity of Nasutitermes corniger, a termite used in folk medicine in Northeastern region of Brazil.MethodsChlorpromazine and decocts of N. corniger were collected from two different plant species used in the traditional medicine were tested for their antimicrobial activity against strains of Escherichia coli resistant to aminoglycosides. The growth of two bacterial strains of E. coli was tested using decocts and chlorpromazine alone or associeted with aminogycosides.ResultsThe MIC and MBC values were ≥1024 μg/ml for both strains of E. coli assayed. A significant synergism was observed between both decocts and chlorpromazine when assyed with neomycin. This synergism with neomycin indicates the involvement of an efflux system in the resistance to this aminoglycoside.ConclusionTherefore it is suggested that natural products from N. corniger could be used as a source of zoo-derived natural products with modifying antibiotic activity to aminoglycosides, being a new weapon against the bacterial resistance to antibiotics.

Broad-Spectrum Peptide Inhibitors of Aminoglycoside Antibiotic Resistance Enzymes

The action of aminoglycoside antibiotics is inhibited by chemical modification catalyzed by aminoglycoside inactivating enzymes, which bind these cationic saccharides with active site pockets that contain a preponderance of negatively charged residues. In this study, it was observed that several cationic antimicrobial peptides, representing different structural classes, could serve as inhibitors of such aminoglycoside resistance enzymes. The bovine antimicrobial peptide indolicidin and synthetic analogs appeared to be especially effective against a range of resistance enzymes, inhibiting enzymes belonging to both aminoglycoside phosphotransferase and aminoglycoside acetyltransferase classes, where the mode of action was dependent on the class of antibiotic resistance enzyme. These peptides represent the first example of broad-spectrum inhibitors of aminoglycoside resistance enzymes.

Antibiotic sensitivity of the ribosomes of the ultra-thermophilic archaebacterium Pyrococcus woesei

1. Summary The effect of several antibiotics on polypeptide synthesis and translational accuracy by ribosomes from the ultra-thermophilic archaebacterium Pyrococcus woesei was investigated. Pyrococcus was found to be unique among the sulfur-dependent extreme thermophiles in being sensitive to the miscoding-inducing action of aminoglycoside antibiotics (except streptomycin). On the whole, the antibiotic sensitivity patterns indicate phylogenetic closeness of Pyrococcus to the methanogenic-halophilic branch of the archaebacterial tree.

Blockage of the transduction channels of hair cells in the bullfrog's sacculus by aminoglycoside antibiotics

The action of aminoglycoside antibiotics on transduction by hair cells was investigated in isolated preparations of the bullfrog's sacculus. Bath application of aminoglycosides produced a reversible blockage of extracellularly recorded responses to displacements of the otolithic membrane. The half-blocking concentrations for various drugs were in the range 2–95 μM. The effect of dihydrostreptomycin on the receptor currents of individual hair cells was studied under two-electrode, voltage-clamp conditions. Iontophoretic application of drug to the apical cellular surface caused a reduction of the receptor current within 20 ms; the reduction was reversible within 1 s. The effect was most striking at holding potentials more negative than −60 mV and was relieved by depolarization. The effect of intracellular aminoglycosides was investigated in cells voltage-clamped with the tight-seal, whole-cell technique. Gentamicin and dihydrostreptomycin, at concentrations near 100 μM, did not block transduction under these conditions. The acute, reversible blocking effect of aminoglycosides therefore occurs from the extracellular membrane surface. The results are consistent with aminoglycosides' plugging the poorly ion-selective transduction channels of hair cells.

Interaction of aminoglycosides and ionophores in the killing of Crithidia fasciculata.

Crithidia fasciculata was utilized as a prescreen to determine the antiprotozoal action of aminoglycoside antibiotics alone and in combination with surface-altering agents. Paromomycin was tested with the carrier ionophores nigericin and valinomycin, the channel ionophore gramicidin and the polyene antibiotics amphotericin B and nystatin. After exposure to the drugs in suspension, organisms were plated out to determine the survival of C. fasciculata. Killing was time dependent for both the antibiotic and the ionophore. Paromomycin action was found to be potentiated by all the surface altering agents. The aminoglycosides kanamycin, gentamycin and streptomycin were studied alone and in combination with nigericin. Synergistic effects were demonstrated both with kanamycin and gentamycin in combination with nigericin. Streptomycin was ineffective both alone and with surface-altering agents.