New selective agent for isolation of Pseudomonas aeruginosa.

Results of minimal inhibitory concentration tests with a diversity of bacterial strains showed that 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan (C-390) inhibited the growth of all microorganisms tested (other than Pseudomonas aeruginosa) at 25 microgram/ml or less, whereas MICs obtained for P. aeruginosa ranged from to to greater than 100 microgram/ml. Therefore, C-390 was evaluated as a potential selective agent for P. aeruginosa in pseudomonas agar F. Recovery tests were conducted on this medium with 53 strains o P. aeruginosa, and the results were compared to those obtained in similar tests on commercially available selective media, i.e., pseudomonas isolation agar and Pseudosel agar. The results of these comparisons indicated that pseudomonas agar F with C-390 was significantly less inhibitory than Pseudosel agar and pseudomonas isolation agar and more selective than pseudomonas isolation agar. The incorporation of C-390 in pseudomonas agar F also provided a medium that was both selective and differential. Preliminary evidence also suggested that C-390 may be added to other basal media with comparable results.     

Synergistic antimicrobial effect of nisin whey permeate and lactic acid on microbes isolated from fish

The antimicrobial activity of a combination of lactic acid and whey permeate fermented by a nisin-producing Lactococcus lactis strain was tested by the agar diffusion method using bacteria isolated from fish as test organisms. Lactic acid inhibited all bacterial strains studied, but nisin whey permeate inhibited Gram-positive bacteria only. The combination was more effective than lactic acid alone against Pseudomonas fluorescens and Staphylococcus hominis isolated from fish, and Pseudomonas aeruginosa ATCC9721 and Micrococcus luteus ATCC9341.     

Studies on antimicrobial activity, in vitro, of Physalis angulata L. (Solanaceae) fraction and physalin B bringing out the importance of assay determination

Complex physalin metabolites present in the capsules of the fruit of Physalis angulata L. have been isolated and submitted to a series of assays of antimicrobial activity against Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, S. aureus ATCC 25923, S. aureus ATCC 6538P, Neisseria gonorrhoeae ATCC 49226, Escherichia coli ATCC 8739; E. coli ATCC 25922, Candida albicans ATCC 10231 applying different methodologies such as: bioautography, dilution broth, dilution agar, and agar diffusion techniques. A mixture of physalins (pool) containing physalins B, D, F, G inhibit S. aureus ATCC 29213, S. aureus ATCC 25923, S. aureus ATCC 6538P, and N. gonorrhoeae ATCC 49226 at a concentration of 200 mg/microl, using agar dilution assays. The mixture was inactive against P. aeruginosa ATCC27853, E. coli ATCC 8739; E. coli ATCC 25922, C. albicans ATCC 10231 when applying bioautography assays. Physalin B (200 microg/ml) by the agar diffusion assay inhibited S. aureus ATCC 6538P by +/- 85%; and may be considered responsible for the antimicrobial activity.     

Accuracy of the disk method for determining antimicrobic susceptibility of common Gram-negative bacilli

A standardized disk susceptibility test was evaluated by comparing results with minimal inhibitory concentrations obtained with agar dilution methods. The agar overlay method was used to test 152 Gram-negative bacilli against eight different antimicrobial agents. One to 3% of the isolates were resistant to an antimicrobic by the MIC method, but appeared to be susceptible by the disk method. Most very major discrepancies involved disk tests withProteus sp., a microorganism notoriously difficult to test reproducibly.Serratia sp. vs. the polymyxins andKlebsiella sp. vs. nitrofurantoin accounted for most other major discrepancies. With other microorganism-drug combinations, the disk test was a reasonably accurate technique for classifying bacteria into resistant or susceptible categories. Gentamicin disk tests were unsatisfactory, but when an intermediate zone category of 13–16 mm was applied, the false susceptible test results were reduced to 2.6%. Intermediate zone sizes were obtained with 6% of the disk tests; most of those isolates were resistant or susceptible but not intermediate in susceptibility. About 11% of the strains had intermediate MICs (5–20% with different drugs), but most of those strains were fully susceptible by the disk technique. *** DIRECT SUPPORT *** A01R4011 00007     

Standardization of antimicrobial disc susceptibility test for anaerobic bacteria.

Both agar diluiton and agar diffusion tests with 8 clinically useful or potentially useful antimicrobial agents were performed with 74 strains of Bacteroides fragilis. Correlation of results obtained by the two methods and applicability of the single disc test to the measurement of antimicrobial susceptibility of anaerobes were analyzed. Prediction of susceptibility, intermediate susceptibility, and resistance of anaerobic bacteria to antimicrobial agents, based on the measurement of inhibition zone diameter, appeared to be satisfactory generally.     

In vitro antifungal and antibacterial activities of extracts of Galium tricornutum subsp. longipedunculatum

The aim of this study was to evaluate the antimicrobial activity of crude ethanolic extracts and fractions of the ariel parts and the fruits of Galium tricornutum subsp. longipedunculatum, traditionally used in northern areas of Pakistan for treating microbial infections of skin. Extracts and their fractions were tested against six bacteria and six fungal strains using the hole diffusion method and macrodilution method. All extracts and fractions possessed significant antimicrobial effect. Four fungal strains, Candida albicans, Trichophyton longifusus, Fusarium.solani and Candida glabrata, showed interesting susceptibility profiles when evaluated using the extracts and fractions with MICs ranging from 0.18 to 200 mg/mL. In case of bacterial strains, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi were significantly susceptible to the extracts and fractions with MICs ranging from 0.12 to 200 mg/mL. Comparative results were carried out using imepenem, miconazole and amphotericin B as standard antibiotics.     

Synergistic Antimicrobial Activity of Camellia sinensis and Juglans regia against Multidrug-Resistant Bacteria

Synergistic combinations of antimicrobial agents with different mechanisms of action have been introduced as more successful strategies to combat infections involving multidrug resistant (MDR) bacteria. In this study, we investigated synergistic antimicrobial activity of Camellia sinensis and Juglans regia which are commonly used plants with different antimicrobial agents. Antimicrobial susceptibility of 350 Gram-positive and Gram-negative strains belonging to 10 different bacterial species, was tested against Camellia sinensis and Juglans regia extracts. Minimum inhibitory concentrations (MICs) were determined by agar dilution and microbroth dilution assays. Plant extracts were tested for synergistic antimicrobial activity with different antimicrobial agents by checkerboard titration, Etest/agar incorporation assays, and time kill kinetics. Extract treated and untreated bacteria were subjected to transmission electron microscopy to see the effect on bacterial cell morphology. Camellia sinensis extract showed higher antibacterial activity against MDR S. Typhi, alone and in combination with nalidixic acid, than to susceptible isolates.” We further explore anti-staphylococcal activity of Juglans regia that lead to the changes in bacterial cell morphology indicating the cell wall of Gram-positive bacteria as possible target of action. The synergistic combination of Juglans regia and oxacillin reverted oxacillin resistance of methicillin resistant Staphylococcus aureus (MRSA) strains in vitro. This study provides novel information about antimicrobial and synergistic activity of Camellia sinensis and Juglans regia against MDR pathogens     

糠秕马拉色菌药敏试验新方法的探讨

目的 建立一种新的糠秕马拉色菌药敏试验方法.方法 在ATBF2半固体培养基中添加不同种类和含量的脂质,用ATB Fungus 3药敏板条对ATCC14521和临床分离的糠秕马拉色菌进行了MIC测试.结果 孵育时间为72 h时对照孔中糠秕马拉色菌生长充分,吐温40浓度为1％时糠秕马拉色菌生长充分,且对药敏结果的影响最小.脂质的种类和含量对实验结果有影响.结论 用改良ATB Fungus 3药敏试验方法对糠秕马拉色菌进行抗真菌药敏试验,方法操作简便、结果易观察、试验结果重复性好.临床分离菌株与糠秕马拉色菌ATCC14521在ATB Fungus 3对照孔中的生长状况相同,结果易于判断.     

Drug resistance and pulsed-field gel electrophoresis patterns of Lactococcus garvieae isolates from cultured Seriola (yellowtail, amberjack and kingfish) in Japan

AIMS: To investigate the existing antimicrobial susceptibility and genetic characteristics of Lactococcus garvieae isolates from cultured Seriola in Japan. METHODS AND RESULTS: Minimum inhibitory concentrations (MICs) of 14 antimicrobial agents for 170 isolates were determined using the agar dilution method. Seventy-five isolates (44.1%) were simultaneously resistant to erythromycin (EM) (MIC>or=2 microg ml-1), lincomycin (LCM) (MIC>or=128 microg ml-1) and oxytetracycline (OTC) (MIC>or=4 microg ml-1). Resistance to EM was grouped as intermediate- and high-level resistant by MIC values. All resistant isolates possessed ermB and tet(S) genes. The number of different bands between pulsed-field gel electrophoresis patterns of 25 isolates and two ATCC strains (isolated in 1974), determined using two enzymes (ApaI and SmaI), did not exceed 3. CONCLUSIONS: The present resistance pattern observed with ermB and tet(S) is similar to that observed in previous reports. Moreover, the genetic characteristics of L. garvieae isolates from a wide area in Japan in 2002 and ATCC strains were closely related. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that EM-, LCM- and OTC-resistant isolates have been present for 15 years and that L. garvieae strains with same origin have spread among Seriola spp. in Japan since 1974.     

Accurate assessment of antibiotic susceptibility and screening resistant strains of a bacterial population by linear gradient plate

The dynamics of a bacterial population exposed to the minimum inhibitory concentration (MIC) of an antibiotic is an important issue in pharmacological research. Therefore, a novel antibiotic susceptibility test is urgently needed that can both precisely determine the MIC and accurately select antibiotic-resistant strains from clinical bacterial populations. For this purpose, we developed a method based on Fick's laws of diffusion using agar plates containing a linear gradient of antibiotic. The gradient plate contained two layers. The bottom layer consisted of 15 mL agar containing the appropriate concentration of enrofloxacin and allowed to harden in the form of a wedge with the plate slanted such that the entire bottom was just covered. The upper layer consisted of 15 mL plain nutrient agar added with the plate held in the horizontal position. After allowing vertical diffusion of the drug from the bottom agar layer for 12 h, the enrofloxacin concentration was diluted in proportion to the ratio of the agar layer thicknesses. The uniform linear concentration gradient was verified by measuring the enrofloxacin concentration on the agar surface. When heavy bacterial suspensions were spread on the agar surface and incubated for more than 12 h, only resistant cells were able to form colonies beyond the boundary of confluent growth of susceptible cells. In this way, the true MIC of enrofloxacin was determined. The MICs obtained using this linear gradient plate were consistent with those obtained using conventional antibiotic susceptibility tests. Discrete colonies were then spread onto a gradient plate with higher antibiotic concentrations; the boundary line increased significantly, and gene mutations conferring resistance were identified. This new method enables the rapid identification of resistant strains in the bacterial population. Use of the linear gradient plate can easily identify the precise MIC and reveal the dynamic differentiation of bacteria near the MIC. This method allows the study of genetic and physiological characteristics of individual strains, and may be useful for early warning of antibiotic resistance that may occur after use of certain antimicrobial agents, and guide clinical treatment.     

The influence of AMPs from HaCaT keratinocytes on some bacteria strains derived from skin changes

The aim of this study was to evaluate antimicrobial activity of protein extracts from HaCaT cell line against bacterial strains, isolated from clinical materials, obtained from patients with clinical symptoms of acne (Propionibacterium acnes) and gas gangrene (Clostridium perfringens and Sterptococcus pyogenes). Reference strain of Staphylococcus aureus ATCC 25923 also was used. Protein extracts from cultured HaCaT cells were obtained by 3-fold freezing/defreezing cells in dry ice following by centrifugation and incubated with appropriate bacterial suspension (0.5 McFarland scale) during 6 and 24 hours. We observed time-depending and strain-depending activity of HaCaT--protein extract. Interestingly, high activity was demonstrated against strains of S. pyogenes and C. perfringens. Because of increasing bacterial resistance to antibiotics further studies in the field of antimicrobial peptides are required.     

Susceptibility of Brazilian Staphylococcal Strains to Glycopeptides Evaluated by Different Testing Methods

Reports of staphylococci with reduced susceptibility to glycopeptides are cause for concern. This study evaluated the susceptibility of 84 staphylococci clinical isolates to glycopeptides by the disk diffusion, agar dilution, E-test, and BHIA screening methods. Vancomycin agar dilution showed all strains presented minimum inhibitory concentration (MIC) ranging from 0.5 to 2 μg/ml, and the E-test showed similar results. Teicoplanin agar dilution test showed MICs ranging from ≤ 0.5 to 2 μg/ml for Staphylococcus aureus and MICs ranging from <0.25 to 32 μg/ml for coagulase-negative Staphylococcus (CNS). Ten CNS isolates presented MICs ranging from 8 to 32 μg/ml for agar dilution and/or E-test. All the staphylococci were susceptible to vancomycin by the disk diffusion test (DDT), but two CNS isolates presented intermediate resistance to teicoplanin by the DDT and MICs of susceptibility, with two other CNS strains, teicoplanin-susceptible by the DDT, presented MICs of intermediate resistance. On the vancomycin-containing agar, 20 CNS isolates were able to grow, but no S. aureus strain. All these isolates showed MICs to teicoplanin (4–32 μg/ml) higher than those isolates that did not grow on the agar screen plate. PFGE of chromosomal SmaI digests showed a wide diversity of these CNS strains, without any predominance of a single PFGE pattern. Received: 25 May 2001 / Accepted: 9 July 2001     

In vitro antifungal and antibacterial activities of extracts of Galium tricornutum subsp. longipedunculatum

The aim of this study was to evaluate the antimicrobial activity of crude ethanolic extracts and fractions of the ariel parts and the fruits of Galium tricornutum subsp. longipedunculatum, traditionally used in northern areas of Pakistan for treating microbial infections of skin. Extracts and their fractions were tested against six bacteria and six fungal strains using the hole diffusion method and macrodilution method. All extracts and fractions possessed significant antimicrobial effect. Four fungal strains, Candida albicans, Trichophyton longifusus, Fusarium.solani and Candida glabrata, showed interesting susceptibility profiles when evaluated using the extracts and fractions with MICs ranging from 0.18 to 200 mg/mL. In case of bacterial strains, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi were significantly susceptible to the extracts and fractions with MICs ranging from 0.12 to 200 mg/mL. Comparative results were carried out using imepenem, miconazole and amphotericin B as standard antibiotics.     

Media- and method-dependent variations in minimal inhibitory concentrations of antiplaque agents on oral bacteria

AIMS: To determine minimal inhibitory concentrations (MICs) and the percentage of nonsusceptible bacteria-- those still cultivable above a threshold concentration--in human supragingival dental plaque and saliva for antiplaque/antimicrobial agents including triclosan (TCS) and trichlorocarbanilide (TCC), and a new potential antimicrobial, 2-t-butyl-5-(4-t-butylphenyl)-phenol (DTBBP). METHODS AND RESULTS: Broth and agar dilution-based MIC tests were performed using 28 oral and nonoral bacterial strains representing 17 species. MICs for TCS were lowest and more than 100-fold lower than DTBBP (P < 0.0005) by both methods. MICs for TCS were lower in broth-based tests compared with TCC. The additions of defibrinated blood to agar and horse serum to broth increased MICs--in the case of TCS, 10- to 15-fold. Significantly higher proportions of nonsusceptible plaque and salivary bacteria were recovered from agar media containing DTBBP or TCC compared with TCS (P < 0.05). CONCLUSIONS: TCS is a more effective antimicrobial agent than either TCC or DTBBP as determined by in vitro testing. SIGNIFICANCE AND IMPACT OF THE STUDY: The utility of in vitro testing for antiplaque agents as a predictor of in vivo efficacy is affected by the methods used.     

Survey of antimicrobial resistance in lactic streptococci

A total of 26 strains of Streptococcus cremoris and 12 strains of Streptococcus lactis were challenged with 18 antimicrobial agents and with nisin in the Bauer-Kirby disk susceptibility test. All strains were susceptible to ampicillin, bacitracin, cephalothin, chloramphenicol, chlortetracycline, erythromycin, penicillin G, tetracycline, and vancomycin. All strains were resistant to trimethoprim, and almost all strains were resistant to sulfathiazole. Variability in resistance to gentamicin, kanamycin, lincomycin, nafcillin, neomycin, nisin, rifampin, and streptomycin was observed. MICs of these substances for the less susceptible strains were determined, and high-level resistance factors could not be detected, except in the case of nisin. S. lactis ATCC 7962 was resistant to at least 40-fold-higher concentrations of nisin (greater than 64 micrograms/ml) than most other strains tested. This strain was a potent nisin producer.     

Survey of antimicrobial resistance in lactic streptococci.

A total of 26 strains of Streptococcus cremoris and 12 strains of Streptococcus lactis were challenged with 18 antimicrobial agents and with nisin in the Bauer-Kirby disk susceptibility test. All strains were susceptible to ampicillin, bacitracin, cephalothin, chloramphenicol, chlortetracycline, erythromycin, penicillin G, tetracycline, and vancomycin. All strains were resistant to trimethoprim, and almost all strains were resistant to sulfathiazole. Variability in resistance to gentamicin, kanamycin, lincomycin, nafcillin, neomycin, nisin, rifampin, and streptomycin was observed. MICs of these substances for the less susceptible strains were determined, and high-level resistance factors could not be detected, except in the case of nisin. S. lactis ATCC 7962 was resistant to at least 40-fold-higher concentrations of nisin (greater than 64 micrograms/ml) than most other strains tested. This strain was a potent nisin producer.     

VITEK－IMSGNS系列药敏卡对~（162）株绿脓杆菌的药敏分析

本文就临床标本中分离的１６２株绿脓杆菌,用ＶＩＴＥＫ－ＩＭＳ全自动微生物系统的ＧＮＳ－ＰＡ等四种药敏卡,共测定２３种抗生素药敏试验．该菌对亚胺硫霉素的敏感度（敏感和中度敏感）最高９７．７８％,其次是哌拉西林、替卡西林、丁胺卡那霉素、妥布霉素、环丙氟哌酸、头孢他啶和头孢哌酮,复方新诺明等八种抗生素耐药率均在９０％以上。分析该菌对１０种抗生素药敏结果发现,几种常用抗绿脓杆菌药物的最小抑菌浓度（ＭＩＣ）值普遍增高,并结合绿脓杆菌生物微膜形成和该菌对β－内酰胺类抗生素抗性机制作了讨论。     

Antimicrobial activity of chlorhexidine diacetate and benzalkonium chloride against Pseudomonas aeruginosa and its response to biocide residues

AIMS: The aims of this study were to evaluate the antimicrobial activity of chlorhexidine diacetate (CHX) and benzalkonium chloride (BZK) for strains of Pseudomonas aeruginosa exhibiting increased minimum inhibitory concentrations (MIC) for CHX, and to determine whether residues of chlorhexidine digluconate (CHG) and Hibiscrub (Hib, a formulation containing CHG) affect the susceptibility of P. aeruginosa to these biocides and a number of antibiotics. METHODS AND RESULTS: The bactericidal activity of CHX and BZK was evaluated for strains of P. aeruginosa exhibiting increased MIC for CHX with established suspension and surface disinfection tests. None of the strains of P. aeruginosa exhibiting raised MIC for CHX was less sensitive than the parent strain to CHX or BZK in either method. A test was designed to investigate the effects of dried CHG and Hib residues on P. aeruginosa cells. Exposure of P. aeruginosa to dried residues of CHG or Hib did not result in the organism becoming less sensitive to either biocide or a number of antibiotics. CONCLUSIONS: Pseudomonas aeruginosa strains with raised MIC to CHX were no less sensitive than the parent strain to CHX and BZK in bactericidal investigations. Exposure to dried residues of CHG and Hib did not render P. aeruginosa less sensitive to either of these agents or a number of antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: An increase in the MIC for a biocide in a micro-organism does not necessarily result in a failure of the biocide to effectively kill the organism. The residue that remains after the use of an antimicrobial agent can be at a far lower concentration than that initially applied and this study highlights the necessity for further investigations into the effect of residues, at low concentration, on bacterial populations and their role, if any, in the continued problem of antibiotic resistance.     

Antifungal and antibacterial activities of Taxus wallichiana Zucc

Current study was undertaken to evaluate the in vitro antifungal and antibacterial potential of methanol extract and subsequent fractions obtained after partitioning in organic solvents with variable polarity of the aerial parts of the tree Taxus wallichiana Zucc. Traditionally, this plant is often used in folk medicines in Pakistan for treating microbial infections. In order to rationalize the traditional use, methanol extracts of leaf, bark, and heartwood of Taxus wallichiana Zucc. were tested against six bacteria and six fungal strains using the Hole diffusion and macro-dilution methods. All extracts and fractions displayed significant antimicrobial effect. Only three fungal strains, Trichophyton longifusus, Microspoum canis, and Fusarium solani were susceptible to the extracts and fractions with MICs ranging from 0.08 to 200 mg/mL. In case of bacterial strains, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi were susceptible to the extracts and fractions with MICs ranging from 0.08 to 200 mg/mL. Comparison results were carried out using imipinem, miconazole and amphotericin B as standard antibiotics.     

Modulation of antibiotic resistance in bacterial pathogens by oleanolic acid and ursolic acid

Antibiotic resistance among bacterial pathogens is a serious problem for human and veterinary medicine, which necessitates the development of novel therapeutics and antimicrobial strategies. Some plant-derived compounds, e.g. pentacyclic triterpenoids such as oleanolic acid (OA) and ursolic acid (UA), have potential as a new class of antibacterial agents as they are active against many bacterial species, both Gram-positive and Gram-negative, and specifically target the cell envelope. The aim of the present study was to investigate the influence of OA and UA on the susceptibility of four bacterial pathogens (Pseudomonas aeruginosa, Listeria monocytogenes, Staphylococcus aureus and Staphylococcus epidermidis) to the β-lactam antibiotics ampicillin (Ap) and oxacillin (Ox). Antimicrobial assays were conducted with bacteria growing in liquid suspension cultures (planktonic cells) or as biofilms. Using FICI value estimation and the time-kill method it was demonstrated that in some combinations, the tested compounds acted in synergy to lower the susceptibility of S. aureus, S. epidermidis and L. monocytogenes to ampicillin and oxacillin, but no synergy was observed for P. aeruginosa. These results indicate that OA and UA may be useful when administered in combination with β-lactam antibiotics to combat bacterial infections caused by some Gram-positive pathogens.