Minimum Bactericidal Concentration Assays Research Articles

Detection and Investigation of Eagle Effect Resistance to Vancomycin in Clostridium difficile With an ATP-Bioluminescence Assay.

Vancomycin was bactericidal against Clostridium difficile at eightfold the minimum inhibitory concentration (MIC) using a traditional minimum bactericidal concentration (MBC) assay. However, at higher concentrations up to 64 × MIC, vancomycin displayed a paradoxical “more-drug-kills-less” Eagle effect against C. difficile. To overcome challenges associated with performing the labor-intensive agar-based MBC method under anaerobic growth conditions, we investigated an alternative more convenient ATP-bioluminescence assay to assess the Eagle effect in C. difficile. The commercial BacTiter-GloTM assay is a homogenous method to determine bacterial viability based on quantification of bacterial ATP as a marker for metabolic activity. The ATP-bioluminescence assay was advantageous over the traditional MBC-type assay in detecting the Eagle effect because it reduced assay time and was simple to perform; measurement of viability could be performed in less than 10 min outside of the anaerobic chamber. Using this method, we found C. difficile survived clinically relevant, high concentrations of vancomycin (up to 2048 μg/mL). In contrast, C. difficile did not survive high concentrations of metronidazole or fidaxomicin. The Eagle effect was also detected for telavancin, but not for teicoplanin, dalbavancin, oritavancin, or ramoplanin. All four pathogenic strains of C. difficile tested consistently displayed Eagle effect resistance to vancomycin, but not metronidazole or fidaxomicin. These results suggest that Eagle effect resistance to vancomycin in C. difficile could be more prevalent than previously appreciated, with potential clinical implications. The ATP-Bioluminescence assay can thus be used as an alternative to the agar-based MBC assay to characterize the Eagle effect against a variety of antibiotics, at a wide-range of concentrations, with much greater throughput. This may facilitate improved understanding of Eagle effect resistance and promote further research to understand potential clinical relevance.

Antimicrobial Activity of Moringa oleifera, Aloe vera and Warbugia ugandensis on Multi-Drug Resistant Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus

Multi-drug resistant is a global public health concern. There has been an increase in infections caused by multidrug resistant micro-organisms in Sub Saharan Africa. This has led to extended illness, expensive health care and deaths. This experimental study was aimed to determine the anti-microbial activity of aqueous and methanol leaf extracts of Warbugia ugandensis, Moringa oleifera and Aloe vera on standard bacteria and multi-drug resistant clinical isolates of Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. Tetracycline drug was used as the reference drug. The bacteria were treated with extracts at different concentrations to determine the zones of inhibition through Agar Diffusion Assay, minimum inhibitory concentration and minimum bactericidal concentration assays. Raw data was analyzed using one-way and two-way analysis of variance followed by Tukey’s post hoc test. Zones of inhibition ranged from 6.5 mm to 9.98 mm on the multi-drug resistant isolates, while those of the standard bacteria ranged from 6.5 mm to 12.00 mm. Methanol extracts of W. ugandensis, M. oleifera and A. vera at the concentration of 400 mg/ml had higher zones of inhibition against multi-drug resistant S. aureus, P. aeruginosa and E. coli respectively. The antimicrobial activity of the extracts indicated a concentration-dependent response. The minimum bactericidal concentration values obtained were double the minimum inhibitory concentration values. Methanol extracts recorded lower minimum inhibitory and minimum bactericidal concentrations compared to aqueous extracts. Phytochemicals which were present, included alkaloids, cardenolide glycosides, phenols, flavonoids, coumarins, tannins, saponins and anthracin glycosides. These phytochemicals are associated with antimicrobial activities. This study showed potent antimicrobial activities of methanol and aqueous extracts of W. ugandensis, M. oleifera and A. vera against the multi-drug resistant and standard bacteria tested. The extracts, therefore, may be used to develop alternative therapeutics in the management of multi-drug resistant Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli.

In Vitro Evaluation of Antioxidant and Antimicrobial Activities of Melaleuca alternifolia Essential Oil.

The in vitro antioxidant and antimicrobial activity of the essential oil from Melaleuca alternifolia (M. alternifolia) was evaluated in this report. The antioxidant potential of the essential oil from M. alternifolia was evaluated by the DPPH (2,2-diphenyl-1-picrylhydrazyl) method, thiobarbituric acid reactive species (TBARS) assay, and the hydroxyl radical scavenging activity method. The essential oil from M. alternifolia was able to reduce DPPH with an EC50 (concentration for 50% of maximal effect) of 48.35 μg/ml, inhibit the lipid peroxidation with an IC50 (50% inhibitory concentration) of 135.9 μg/ml, and eliminate hydroxyl radicals with an EC50 of 43.71 μg/ml. Antimicrobial screening, minimum inhibitory concentration, and minimum bactericidal concentration assays showed that the essential oil from M. alternifolia inhibited strongly the growth of different types of microorganisms, including Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Penicillium italicum Wehmer, and Penicillium digitatum Sacc. Thus, the essential oil of M. alternifolia possesses antioxidant and antimicrobial activity and could be suitable for use as a natural preservative ingredient in food, agriculture, and pharmaceutical industries.

One pathogen two stones: are Australian tree frog antimicrobial peptides synergistic against human pathogens?

Antimicrobial peptides (AMPs) may act by targeting the lipid membranes and disrupting the bilayer structure. In this study, three AMPs from the skin of Australian tree frogs, aurein 1.2, maculatin 1.1 and caerin 1.1, were investigated against Gram-negative Escherichia coli, Gram-positive Staphylococcus aureus, and vesicles that mimic their lipid compositions. Furthermore, equimolar mixtures of the peptides were tested to identify any synergistic interactions in antimicrobial activity. Minimum inhibition concentration and minimum bactericidal concentration assays showed significant activity against S. aureus but not against E. coli. Aurein was the least active while maculatin was the most active peptide and some synergistic effects were observed against S. aureus. Circular dichroism experiments showed that, in the presence of phospholipid vesicles, the peptides transitioned from an unstructured to a predominantly helical conformation (>50%), with greater helicity for POPG/TOCL compared to POPE/POPG vesicles. The helical content, however, was less in the presence of live E. coli and S. aureus, 25 and 5%, respectively. Equimolar concentrations of the peptides did not appear to form greater supramolecular structures. Dye release assays showed that aurein required greater concentration than caerin and maculatin to disrupt the lipid bilayers, and mixtures of the peptides did not cooperate to enhance their lytic activity. Overall, aurein, maculatin, and caerin showed moderate synergy in antimicrobial activity against S. aureus without becoming more structured or enhancement of their membrane-disrupting activity in phospholipid vesicles.

Design, synthesis of novel chitosan derivatives bearing quaternary phosphonium salts and evaluation of antifungal activity

Two novel chitosan derivatives modified with quaternary phosphonium salts were successfully synthesized, including tricyclohexylphosphonium acetyl chitosan chloride (TCPACSC) and triphenylphosphonium acetyl chitosan chloride (TPPACSC), and characterized by FTIR, 1H NMR, and 13C NMR spectra. The degree of substitution was also calculated by elemental analysis results. Their antifungal activities against Colletotrichum lagenarium, Watermelon fusarium, and Fusarium oxysporum were investigated in vitro using the radial growth assay, minimal inhibitory concentration, and minimum bactericidal concentration assay. The fungicidal assessment revealed that the synthesized chitosan derivatives had superior antifungal activity compared with chitosan. Especially, TPPACSC exhibited the best antifungal property with inhibitory indices of over 75% at 1.0mg/mL. The results obviously showed that quaternary phosphonium groups could effectively enhance antifungal activity of the synthesized chitosan derivatives. Meanwhile, it was also found that their antifungal activity was influenced by electron-withdrawing ability of the quaternary phosphonium salts. The synthetic strategy described here could be utilized for the development of chitosan as antifungal biomaterials.

Effect of Bacoside A on growth and biofilm formation by Staphylococcus aureus and Pseudomonas aeruginosa.

The goal of this study was to evaluate the antibiofilm and antimicrobial activities of Bacoside A, a formulation of phytochemicals from Bacopa monnieri, against Staphylococcus aureus and Pseudomonas aeruginosa, which are known to form biofilms as one of their virulence traits. The antimicrobial effects of Bacoside A were tested using the minimum inhibitory concentration and minimum bactericidal concentration assays. A cell membrane disruption assay was performed to find its possible target site. MTT assay, crystal violet assay, and microscopic studies were performed to assess the antibiofilm activity. Bacoside A showed antimicrobial activity against both test organisms in their planktonic and biofilm states. At a subminimum inhibitory concentration of 200 μg·mL-1, Bacoside A significantly removed ∼88%-93% of bacterial biofilm developed on microtiter plates. Biochemical and microscopic studies suggested that the eradication of biofilm might be due to the loss of extracellular polymeric substances and to a change in cell membrane integrity of the selected bacterial strains treated with Bacoside A. These results indicate that Bacoside A might be considered as an antimicrobial having the ability to disrupt biofilms. Thus, either alone or in combination with other therapeutics, Bacoside A could be useful to treat biofilm-related infections caused by opportunistic bacterial pathogens.

Antibacterial constituents of Fructus Chebulae Immaturus and their mechanisms of action

BackgroundTo extract, purify, and identify the effective constituents of aqueous extract of Fructus Chebulae Immaturus, and analyze the bactericidal effects of total tannins.MethodsPreparative thin layer chromatography and semi-preparative high performance liquid chromatography were used to isolate and purify the total tannin fraction. 1H- and 13C- NMR spectroscopy were used to elucidate compound structures. The antibacterial activities of total tannins and ethyl gallate on Klebsiella pneumoniae (KP) and Staphylococcus aureus (SA) were determined through minimum inhibitory concentration and minimum bactericidal concentration assays. Their antibacterial mechanisms of action were explored by transmission electron microscopy and scanning electron microscopy.ResultsFive compounds were isolated: ellagic acid, ethyl gallate, arjugenin, β-sitosterol, and tri-n-butyl chebulate. Tri-n-butyl chebulate is a newly-reported compound. Total tannins and ethyl gallate both had favorable bactericidal effects against KP and SA.ConclusionIn vivo and in vitro pharmacodynamic experiment demonstrated that the effective components of Fructus Chebulae Immaturus possessed significant antibacterial effects, and were nontoxic and safe.Trial registrationNo results of a health care intervention on human participants

Antibacterial Activity of Selected Essential oils against Streptococcus sobrinus and Porphyromonas gingivalis

Plant essential oils have been used medicinally in history. The antibacterial screening of selected plant essential oils against Porphyromonas gingivalis and Streptococcus sobrinus was evaluated by MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) assay. Among them, thujopsis dolabrata (Asunaro) essential oil was selected as a fairly effective sample against P. gingivalis (MIC=1 mg/ml) and S. sobrinus (MIC=1 mg/ml) respectively. Moreover, Callitrisintratropica (Australian blue cypress; cypress) was also screened as a fairly effective sample only against S. sobrinus (MIC=0.5 mg/ml). Furthermore, five effective compounds, (+)-cuparene, (-)-thujopsene, (+)-cuparenol, (-)-guaiol and a novel sesquiterpenoid were isolated by a performance of a series of chromatography, and identified by nuclear magnetic resonance (NMR) and matrix assisted laser desorption/ionizationtime of flight mass spectrometry (MALDI-TOFMS). Among them, (+)-cuparenol was the most effective compound with a MIC of 0.125 mg/ml against P. gingivais. Additionally, the novel sesquiterpenoid (MIC=0.25 mg/ml) was identified as the first time isolation from C. intratropica.

Antioxidant, antibacterial activity, and phytochemical characterization of Melaleuca cajuputi extract.

BackgroundThe threat posed by drug-resistant pathogens has resulted in the increasing momentum in research and development for effective alternative medications. The antioxidant and antibacterial properties of phytochemical extracts makes them attractive alternative complementary medicines. Therefore, this study evaluated the phytochemical constituents of Melaleuca cajuputi flower and leaf (GF and GL, respectively) extracts and their antioxidant and antibacterial activities.MethodsRadical scavenging capacity of the extracts was estimated using 2,2-diphenyl-2-picrylhydrazyl and Fe2+-chelating activity. Total antioxidant activity was determined using ferric reducing antioxidant power assay. Well diffusion, minimum inhibitory concentration, and minimum bactericidal concentration assays were used to determine antibacterial activity against eight pathogens, namely Staphylococcus aureus, Escherichia coli, Bacillus cereus, Staphylococcus epidermidis, Salmonella typhimurium, Klebsiella pneumonia, Streptococcus pneumoniae, and Pasteurella multocida. We identified and quantified the phytochemical constituents in methanol extracts using liquid chromatography/mass spectrometry (LC/MS) and gas chromatography (GC)/MS.ResultsThis study reports the antioxidant and radical scavenging activity of M. cajuputi methanolic extracts. The GF extract showed better efficacy than that of the GL extract. The total phenolic contents were higher in the flower extract than they were in the leaf extract (0.55 ± 0.05 and 0.37 ± 0.05 gallic acid equivalent per mg extract dry weight, respectively). As expected, the percentage radical inhibition by GF was higher than that by the GL extract (81 and 75 %, respectively). A similar trend was observed in Fe2+-chelating activity and β-carotene bleaching tests. The antibacterial assay of the extracts revealed no inhibition zones with the Gram-negative bacteria tested. However, the extracts demonstrated activity against B. cereus, S. aureus, and S. epidermidis.ConclusionsIn this study, we found that M. cajuputi extracts possess antioxidant and antibacterial activities. The results revealed that both extracts had significant antioxidant and free radical-scavenging activity. Both extracts had antibacterial activity against S. aureus, S. epidermidis, and B. cereus. The antioxidant and antimicrobial activities could be attributed to high flavonoid and phenolic contents identified using GC/MS and LC/MS. Therefore, M. cajuputi could be an excellent source for natural antioxidant and antibacterial agents for medical and nutraceutical applications.

Turmeric: Alternative Therapy Against MDR Staphylococcus aureus, Preservative, Shelf-life the Miced Meat

Aqeous and alcoholic extract of turmeric (Curcuma longa) were evaluated for its antimicrobial action against the food borne and food spoilage pathogen, Staphylococcus aureus, that resistant concentrations (Minimum inhibition concentration (MIC) and Minimum bactericidal concentration (MBC)) of C.longa against S.aureus were found using MIC and MBC assay,miced meat was taken as the standard sample to test for the turmeric upon MDR S. aureusduring 6 days storage and compared with sodium nitrate.Turmeric extract exhibited anti-MDR S.aureus activity with all concentration-dependent relationship.the alcohol extracts were the most efficient,then aqueous extracts and both of them with high significant differences compared with sodium nitrate(preservative). The value of MIC and MBC of turmeric was 2.5 ml and was the efficient when used in miced meat storage assay for 6 days with high significant differences compared with sodium nitrate.

Antimicrobial efficacy and wound-healing property of a topical ointment containing nitric-oxide-loaded zeolites.

Topical delivery of nitric oxide (NO) through a wound dressing has the potential to reduce wound infections and improve healing of acute and chronic wounds. This study characterized the antibacterial efficacy of an ointment containing NO-loaded, zinc-exchanged zeolite A that releases NO upon contact with water. The release rate of NO from the ointment was measured using a chemiluminescence detection system. Minimum bactericidal concentration assays were performed using five common wound pathogens, including Gram-negative bacteria (Escherichia coli and Acinetobacter baumannii), Gram-positive bacteria (Staphylococcus epidermidis and meticillin-resistant Staphylococcus aureus) and a fungus (Candida albicans). The time dependence of antimicrobial activity was characterized by performing log-reduction assays at four time points after 1-8 h ointment exposure. The cytotoxicity of the ointment after 24 h was assessed using cultured 3T3 fibroblast cells. Minimum microbicidal concentrations (MMCs) for bacterial organisms (5×10(7) c.f.u.) ranged from 50 to 100 mg ointment (ml media)(-1); the MMC for C. albicans (5×10(4) c.f.u.) was 50 mg ointment (ml media)(-1). Five to eight log reductions in bacterial viability and three log reductions in fungal viability were observed after 8 h exposure to NO-zeolite ointment compared with untreated organisms. Fibroblasts remained viable after 24 h exposure to the same concentration of NO-zeolite ointment as was used in antimicrobial tests. In parallel studies, full-thickness cutaneous wounds on Zucker obese rats healed faster than wounds treated with a control ointment. These data indicate that ointment containing NO-loaded zeolites could potentially be used as a broad-spectrum antimicrobial wound-healing dressing.

Identification of a Serine Proteinase Homolog (Sp-SPH) Involved in Immune Defense in the Mud Crab Scylla paramamosain

Clip domain serine proteinase homologs are involved in many biological processes including immune response. To identify the immune function of a serine proteinase homolog (Sp-SPH), originally isolated from hemocytes of the mud crab, Scylla paramamosain, the Sp-SPH was expressed recombinantly and purified for further studies. It was found that the Sp-SPH protein could bind to a number of bacteria (including Aeromonas hydrophila, Escherichia coli, Staphylococcus aureus, Vibrio fluvialis, Vibrio harveyi and Vibrio parahemolyticus), bacterial cell wall components such as lipopolysaccharide or peptidoglycan (PGN), and β-1, 3-glucan of fungus. But no direct antibacterial activity of Sp-SPH protein was shown by using minimum inhibitory concentration or minimum bactericidal concentration assays. Nevertheless, the Sp-SPH protein was found to significantly enhance the crab hemocyte adhesion activity (paired t-test, P<0.05), and increase phenoloxidase activity if triggered by PGN in vitro (paired t-test, P<0.05). Importantly, the Sp-SPH protein was demonstrated to promote the survival rate of the animals after challenge with A. hydrophila or V. parahemolyticus which were both recognized by Sp-SPH protein, if pre-incubated with Sp-SPH protein, respectively. Whereas, the crabs died much faster when challenged with Vibrio alginolyiicus, a pathogenic bacterium not recognized by Sp-SPH protein, compared to those of crabs challenged with A. hydrophila or V. parahemolyticus when pre-coated with Sp-SPH protein. Taken together, these data suggested that Sp-SPH molecule might play an important role in immune defense against bacterial infection in the mud crab S. paramamosain.

Synthesis, optimization, and characterization of silver nanoparticles from Acinetobacter calcoaceticus and their enhanced antibacterial activity when combined with antibiotics

BackgroundThe development of nontoxic methods of synthesizing nanoparticles is a major step in nanotechnology to allow their application in nanomedicine. The present study aims to biosynthesize silver nanoparticles (AgNPs) using a cell-free extract of Acinetobacter spp. and evaluate their antibacterial activity.MethodsEighteen strains of Acinetobacter were screened for AgNP synthesis. AgNPs were characterized using various techniques. Reaction parameters were optimized, and their effect on the morphology of AgNPs was studied. The synergistic potential of AgNPs on 14 antibiotics against seven pathogens was determined by disc-diffusion, broth-microdilution, and minimum bactericidal concentration assays. The efficacy of AgNPs was evaluated as per the minimum inhibitory concentration (MIC) breakpoints of the Clinical and Laboratory Standards Institute (CLSI) guidelines.ResultsOnly A. calcoaceticus LRVP54 produced AgNPs within 24 hours. Monodisperse spherical nanoparticles of 8–12 nm were obtained with 0.7 mM silver nitrate at 70°C. During optimization, a blue-shift in ultraviolet-visible spectra was seen. X-ray diffraction data and lattice fringes (d =0.23 nm) observed under high-resolution transmission electron microscope confirmed the crystallinity of AgNPs. These AgNPs were found to be more effective against Gram-negative compared with Gram-positive microorganisms. Overall, AgNPs showed the highest synergy with vancomycin in the disc-diffusion assay. For Enterobacter aerogenes, a 3.8-fold increase in inhibition zone area was observed after the addition of AgNPs with vancomycin. Reduction in MIC and minimum bactericidal concentration was observed on exposure of AgNPs with antibiotics. Interestingly, multidrug-resistant A. baumannii was highly sensitized in the presence of AgNPs and became susceptible to antibiotics except cephalosporins. Similarly, the vancomycin-resistant strain of Streptococcus mutans was also found to be susceptible to antibiotic treatment when AgNPs were added. These biogenic AgNPs showed significant synergistic activity on the β-lactam class of antibiotics.ConclusionThis is the first report of synthesis of AgNPs using A. calcoaceticus LRVP54 and their significant synergistic activity with antibiotics resulting in increased susceptibility of multidrug-resistant bacteria evaluated as per MIC breakpoints of the CLSI standard.

Decreased Susceptibility to Azithromycin and Doxycycline in Clinical Isolates of Chlamydia trachomatis Obtained from Recurrently Infected Female Patients in India

Background: Recurrent genital Chlamydia trachomatis infection often results in serious sequelae and has a major impact on reproductive health. Materials and Methods: Recurrent infections were determined in symptomatic female patients. In vitro susceptibility assay was performed for azithromycin and doxycycline using the cell culture technique against 21 clinical isolates obtained from C. trachomatis-positive patients including those who were recurrently infected. Results: Thirteen isolates (61.9%) were found to be susceptible to azithromycin and doxycycline with minimum inhibitory concentration (MIC) values ≤0.125 and ≤0.25 µg/ml, respectively. Eight isolates (38%) were found to be less susceptible to the drugs. Two of them had MICs of 8 µg/ml for both the drugs and could not be completely eradicated as observed by minimum bactericidal concentration assay. Conclusions: Decreased antibiotic susceptibility to the current first-line drugs (azithromycin and doxycycline) for chlamydial infection treatment was observed in isolates obtained from recurrently infected patients.

Aggregation of Staphylococcus aureus following treatment with the antibacterial flavonol galangin

The flavonol galangin, an antimicrobial constituent of the traditional medicines propolis and Helichrysum aureonitens, is being assessed as part of an ongoing investigation into the antibacterial activity of flavonoids. The present study sought to establish whether galangin has any aggregatory effect on bacterial cells. In preparatory time-kill assays, 50 microg ml(-1) of galangin was found to reduce colony counts of c. 5 x 10(7) CFU ml(-1)Staphylococcus aureus NCTC 6571 by approximately 15 000-fold during 60 min of incubation. Subsequent light microscopy studies demonstrated significant increases in the number of large clusters of bacterial cells in populations treated with the flavonol. Data presented here show that galangin causes aggregation of bacterial cells. The finding that galangin causes bacterial cells to clump together may implicate the cytoplasmic membrane as a target site for this compound's activity. More importantly, this observation indicates that decreases in CFU numbers detected in time-kill and minimum bactericidal concentration (MBC) assays in previous investigations were at least partially attributable to this aggregatory effect. This raises the possibility that galangin is not genuinely bactericidal in action, and calls into question the suitability of time-kill and MBC assays for determining the nature of activity of naturally occurring flavonoids.

Comparison of Four Methods for Determining Lysostaphin Susceptibility of Various Strains of Staphylococcus aureus

Lysostaphin is an endopeptidase that cleaves the pentaglycine cross-bridges of the staphylococcal cell wall rapidly lysing the bacteria. Recently, lysostaphin has been examined for its potential to treat infections and to clear Staphylococcus aureus nasal colonization, requiring a reliable method for determining the lysostaphin susceptibility of strains of S. aureus. We compared four methods for determining the lysostaphin susceptibility of 57 strains of methicillin-sensitive S. aureus, methicillin-resistant S. aureus, vancomycin intermediately susceptible S. aureus (VISA), mupirocin-resistant S. aureus, and various defined genetic mutants of S. aureus. Three reference lysostaphin-resistant S. aureus variants were also included in the assays as negative controls. The assays examined included turbidity, MIC, minimum bactericidal concentration (MBC), and disk diffusion assays. All of the strains of S. aureus tested, including a VISA strain which had previously been reported to be lysostaphin resistant, were susceptible to lysostaphin by all four methods. The three reference lysostaphin-resistant variants were resistant by all four methods. The disk diffusion assay was the simplest method to differentiate lysostaphin-susceptible S. aureus strains from lysostaphin-resistant variants, while the MBC assay could be used as a follow-up assay if required. In the disk diffusion assay, all strains of S. aureus tested revealed zones of inhibition of >/=11 mm using a 50-microg lysostaphin disk, while the three reference lysostaphin-resistant S. aureus variants had no zones of inhibition. In MBC assays, concentrations of lysostaphin ranging from 0.16 microg/ml to 2.5 microg/ml were found to cause a 3 log or greater drop from the initial CFU of S. aureus within 30 min for all strains tested.

Anti-Salmonella activity of medicinal plants from Cameroon.

To evaluate the effects of herbal extracts derived from plants commonly prescribed by traditional practitioners for the treatment of typhoid fever. A cross sectional study. Departments of Life Sciences and Chemistry, University of Buea, Cameroon. Methanol extracts of plant parts commonly used in Cameroon for the treatment of typhoid fever. Antimicrobial activity was tested using the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) assays. Methanol extracts of plant parts commonly used in Cameroon for the treatment of typhoid fever were tested for antibacterial activity against Salmonella typhi, S. paratyphi and S. typhimurium. The formulations used were: 1) Formulation A comprising Cymbogogon citratus leaves, Carica papaya leaves, and Zea mays silk. 2) Formulation B comprising C. papaya roots, Mangifera indica leaves, Citrus limon fruit and C. citratus leaves. 3) C. papaya leaves. 4) Emilia coccinea whole plant. 5) Comelina bengalensis leaves. 6) Telfaria occidentalis leaves. 7) Gossypium arboreum whole plant. Antimicrobial activity was tested using the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) assays. Generally, Formulation A elicited inhibitory activity at a lower range of 0.02 to 0.06 mg/ml. Similarly, Formulation B elicited bacterial activity at the lowest range of 0.06 to 0.25 mg/ml. C. bengalensis leaves on the other hand, showed the lowest activity with a concentration range of 0.132 to 2.0 mg/ml and 1 to 4 mg/ml in MIC and MBC assays respectively. S. paratyphi was most sensitive to the formulations (concentration range of 0.02 to 1 mg/ml in both MIC and MBC assays) while S. typhimurium was the least sensitive and concentrations of up to 4 mg/ml were required to be bactericidal. It is concluded that plant extracts with low MIC and MBC values (1 mg/ml and lower) may contain compounds with therapeutic activity.

Highly reproducible bactericidal activity test results by using a modified National Committee for Clinical Laboratory Standards broth macrodilution technique.

Bactericidal testing historically has exhibited variable reproducibility, even when prior standardized methods were employed. Several modifications to the National Committee for Clinical Laboratory Standards (NCCLS) broth macrodilution method are proposed to improve reproducibility. Recommended changes from the approved NCCLS guidelines (M21-A and M26-A) include omitting serum supplementation of Mueller-Hinton broth, incubating tubes at 35 degrees C for 24 h with no agitation until they are sampled, running all tests in duplicate with six dilutions instead of nine, reincubating the test for an additional 24 h to resolve discrepant bactericidal activity test results, using a single 0.1-ml sample from each clear tube for subculture, and adopting an alternate method for calculating endpoint determination. In order to test these recommendations in a clinical laboratory setting, we used the modified methodology on 224 separate tests for bactericidal activity. There were 102 serum bactericidal titer (SBT) and 122 minimum bactericidal concentration (MBC) assays performed. By defining reproducibility as agreement between duplicate tests +/- 1 dilution, we found 207 of 224 tests (92%) were reproducible at the 24-h subculture point (94% for the SBT assay and 91% for the MBC assay). When the 17 assays with discrepant results were incubated an additional 24 h for a second subculture, only 1 of 224 tests (0.4%) remained discrepant. The method used is practical for a clinical laboratory that chooses to perform bactericidal activity testing and assures a high level of reproducibility between duplicate assays. The total cost of a test was approximately $25.00.