A simple colorimetric method for testing antimicrobial susceptibility of biofilmed bacteria

This study introduces a simple colorimetric method which can measure the antimicrobial susceptibility of bacteria in biofilms using trimethyl tetrazolium chloride (TTC) as an indicator of viable bacteria. The new method was utilized for the evaluation of antibiotic susceptibility of Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus biofilms.     

A new antimicrobial susceptibility testing method of Escherichia coli against ampicillin by MSPQC

A new antimicrobial susceptibility testing method by multi-channel series piezoelectric quartz crystal (MSPQC) was proposed. This method was used to test susceptibility of clinical Escherichia coli isolates against ampicillin. Both the minimum inhibitory concentrations (MICs) and interpretive categorization of clinical E. coli isolates were determined by proposed method. Comparing tests were run at the same time by the agar dilution method and the disk diffusion method. The experimental results showed that MSPQC method had a good agreement with the reference methods. Compared with those methods, the MSPQC method is simple, rapid, and convenient to perform. It can offer both a minimum inhibitory concentration (MIC) and an interpretive category result.     

Determination of proteins with fullerol by a resonance light scattering technique

Fullerol has been synthesized through the reaction of fullerene C60 with NaOH in aqueous solution by means of ultrasonic agitation and characterized by infrared and 1H-nuclear magnetic resonance spectroscopy. The fullerol obtained shows good solubility and excellent stability in water. A weak resonance light scattering (RLS) spectrum of fullerol was observed in aqueous solution. However, the intensity of the RLS signal could be enhanced in the presence of proteins, including bovine serum albumin (BSA), human serum albumin (HSA), pepsin (Pep), and lysozyme (Lys). Based on the enhancement of the RLS, a sensitive method for the determination of proteins has been established. The quantitative conditions were considered with regard to the effects of the pH, the ion strength, and the concentration of the fullerol. Under the optimum conditions, the intensity of the RLS was proportional to the concentration of proteins with the limits of detection of 9.7, 10.9, 57.4, and 8.5 ng mL(-1) for BSA, HSA, Pep, and Lys, respectively. Almost no interference can be observed from some amino acids, nucleic acids, and most of the metal ions. The model samples and human serum samples were determined satisfactorily with the proposed method.     

A new gel tube method for the direct detection, identification and susceptibility testing of bacteria in clinical samples

We recently developed a simple new method which is designed to separate and concentrate bacteria from a sample by centrifugation in a gel system. Bacterial enzyme activity is then detected inside the gel without further manipulation using a colorimetric or fluorogenic substrate. The method provides a rapid, direct means of detecting bacteria in clinical samples, dispensing with the 24-h period normally required to isolate colonies on agar. Various applications of the method are described below, e.g. screening of negative urine samples, identification of Escherichia coli in urine samples, identification of Staphylococcus aureus in blood culture broths and detection of oxacillin-resistant S. aureus in blood culture broths. The advantages of the gel system and other applications are discussed.     

Use of the Etest method for antimicrobial susceptibility testing of obligate anaerobes]

The aim of this study was to evaluate Etest usefulness for antimicrobial susceptibility testing of obligate anaerobes and to compare the activity of five antibacterial drugs against clinical strains of anaerobes. One hundred strains of obligate anaerobes were tested: 2 reference strains (B. fragilis ATCC 25285 and B. thetaiotaomicron ATCC 29741) and 98 clinical strains isolated from patients of the Infant Jesus Clinical Hospital--Center for Trauma Treatment in Warsaw during the last three years (1997-1999). Strains of seven genera of obligate nonsporeforming anaerobes (Bacteroides, Prevotella, Porphyromonas, Fusobacterium, Peptostreptococcus, Propionibacterium and Actinomyces) and strains of two sporeforming species (C. perfringens and C. difficile) were examined. The MIC values were determined by the gradient diffusion method Etest (AB BIODISK, Sweden). Wilkins-Chalgren solid medium supplemented with 5% of sheep blood was used. Test plates were incubated at 35 degrees C for 48 hours in glove-box (85% N2, 10% H2, 5% CO2). The MIC values for each strain and antimicrobial agent, and the MIC ranges for bacteria of the same species were established. Ten strains resistant to clindamycin, ten resistant to piperacillin, and ten resistant to imipenem were detected. Seven strains were resistant to metronidazole and two strains to piperacillin combined with tazobactam. Tazobactam restored the susceptibility of eight strains to piperacillin. Obtained results confirm that Etest method is useful for antimicrobial susceptibility testing of obligate anaerobes. Older (clindamycin and metronidazole) and newer (piperacillin, piperacillin/tazobactam and imipenem) antimicrobial agents revealed high and comparable activity against clinical strains of obligate anaerobes. The percentage of strains susceptible to tested antimicrobials was > or = 90. These antimicrobials may be still useful in the empiric treatment of infections caused by medically important anaerobes.     

Use of sodium lauroyl sarcosinate in a high-sensitivity protein assay by resonance light scattering technique

A simple and high-sensitivity method has been developed for the determination of proteins in aqueous solutions by resonance light scattering (RLS) technique. At pH 3.4 and ionic strength 1.2 x 10(-3), the weak RLS intensity of sodium lauroyl sarcosinate was greatly enhanced by the addition of proteins with the maximum peak located at 391 nm. Under the optimum conditions, the enhanced RLS intensities were in proportion to the concentrations of proteins in the range of 0.04 to 2.1 microg/mL for lysozyme, 0.0025 to 1.2 microg/mL for bovine serum albumin, 0.0075 to 0.9 microg/mL for human serum albumin, 0.02 to 1.4 microg/mL for gamma-globulin, 0.02 to 0.8 microg/mL for egg albumin, and 0.01 to 0.6 microg/mL for hemoglobin. Low detection limits ranging from 0.8 ng/mL to 4.3 ng/mL depending on the kind of proteins that have been achieved. The protein concentrations in synthetic samples and real biochemical samples were determined with satisfactory results. This method presented here is not only sensitive and simple but also reliable and suitable for practical bioassay applications.     

A new technique for visualizing proanthocyanidins by light microscopy

We describe a new technique for visualizing proanthocyanidin-containing elements in plant tissues. Our innovation is the fixation of condensed tannins with an exogenous protein prior to alcohol dehydration. In this way, tannins do not undergo partial solubilization during the dehydration sequence and appear as sharply contoured globules of various diameters.     

Evaluation of a new method for antifungal susceptibility testing for azoles

The interpretation of the end points in azole antifungal drug susceptibility testing is challenging, in part due to incomplete growth inhibition of Candida species. Since the reference Clinical and Laboratory Standards Institute (CLSI) broth microdilution method have limitation with azoles, a new modification of the CLSI microdilution protocol was evaluated. We measure the decrease in growth rate (μ) of exponentially growing cultures in accordance to different azole concentrations at time intervals up to 10 h. Using 15 different Candida strains, an overall agreement within ± 2 dilutions by the CLSI method at 24 h in RPMI and the μ-dependent method for three antifungal agents (fluconazole- itraconazole and voriconazole) was achieved. MIC measurement by the new method was less sensitive to the medium used or the inoculum size applied. The presented data suggested that, measuring the in vitro inhibition kinetics at the logarithmic phase could have advantages for addressing susceptibility testing toward azoles.     

A new technique for mapping epitope specificities of monoclonal antibodies using quasi-elastic light scattering spectroscopy

Quasi-elastic light scattering (QLS) was used to determine relative epitope specificities of a group of anti-bovine serum albumin (BSA) monoclonal antibodies (MAb). QLS is a non-invasive technique which can determine the mean size and size distributions of macromolecular scatterers by analysis of the fluctuations in the intensity of laser light scattering. When two MAbs are mixed together with antigen, QLS detects the complex formation which results from the Ag-Ab reaction, and can easily distinguish between the large complexes formed by interaction of non-competitive MAbs and the smaller complexes formed by competitive MAbs. In this report, the competitive or non-competitive behavior of six anti-BSA MAbs were assessed by radioimmunoassay (RIA) and QLS analysis. The results obtained by QLS analysis confirmed the RIA findings indicating that the six MAbs examined can be categorized into three distinct, non-interacting groups. QLS analysis represents a simple, and extremely rapid technique for epitope mapping studies.     

Selective determination of cysteine by resonance light scattering technique based on self-assembly of gold nanoparticles

The interaction between cysteine and gold nanoparticles was studied. Through the covalent combination with the -SH group and the electrostatic binding with the -NH3+ group of cysteine, gold nanoparticles can self-assemble to form a network structure, which results in greatly enhanced resonance light scattering (RLS). The experimental results demonstrate that the RLS technique offers a sensitive tool for investigations of self-assembly of nanoparticles. On the other hand, the RLS method can be applied to selectively determine cysteine with high sensitivity and simple operation. The linear range of determination of cysteine is from 0.01 to 0.25 microg/mL with the detection limit of 2.0 ng/mL (16.5 nM, 3sigma). None of the amino acids found in proteins interferes with the determination.     

Correction of spectra for studying dye aggregates by resonance light scattering

Resonance light scattering (RLS), a phenomenon of abrupt enhancement of Rayleigh light scattering in close proximity to an absorption band, is easily detectable in solutions of strongly absorbing chromophores, which form large aggregates with strong π-electronic coupling among the chromophores. RLS spectra need to be corrected for the sensitivity of the spectrofluorimeter as well as for the effects of internal light filter. A method for correcting the measured RLS is described. It was shown by the method that addition of KCl induces formation of extended supramolecular aggregates (probably of H-type) of the anionic dye merocyanine 540 in water. The RLS spectra of a photosensitizer m-tetra(hydroxyphenyl)chlorin (Foscan®) indicate formation of J-aggregates of this dye in aqueous medium.     

Identification and antimicrobial susceptibility testing of clinical isolates of nonfermenting gram-negative bacteria by the Phoenix Automated Microbiology System

The Phoenix Automated Microbiology System (Becton Dickinson, Sparks, MD) was evaluated for its ability to identify nonfermenting gram-negative pathogens and measure their drug susceptibility. Isolates producing rare extended-spectrum beta-lactamases (PER-1, IMP-2, VIM-1, and VIM-2) were included in the study. Species identification was compared to that given by the ATB System (bio-Mérieux, Marcy l'Etoile, France), whereas susceptibility results were compared to those produced by a reference broth microdilution test (panels manufactured by Pasco Laboratories, Becton Dickinson). The Phoenix system consistently identified all isolates of Pseudomonas aeruginosa (n = 55) and Stenotrophomonas maltophilia (n = 28), while in other cases species agreement was obtained for 47/53 isolates (Acinetobacter baumannii, 29/31; Pseudomonas putida, 10/11; Burkholderia cepacia, 6/7; and Pseudomonas fluorescens, 2/4). Overall, the Phoenix and ATB systems gave equal results in 130/136 cases (95.6%). For two isolates, consistent identification was obtained at the genus level, thus bringing the cumulative agreement to 97.1%. MIC values (interpreted according to NCCLS guidelines) gave essential and categorical agreement in 94.2% and 93.1% of cases, respectively. Minor and major errors were 5.1% and 5.2%, respectively. No very major errors were produced. The mean time to results (TTR) for the Phoenix system was 14.8 +/- 1.6 h (mean +/- SD), with the shortest TTR being observedfor A. baumannii (13.0 +/- 1.8 h) and the longest one for P. aeruginosa (15.6 +/- 1.2 h). In conclusion, the Phoenix system performed rapidly and correctly in the identification of clinical isolates of important opportunistic pathogens and in measuring their susceptibility to antipseudomonal drugs.     

Detection of Bifidobacteriumspecies by enzymatic methods and antimicrobial susceptibility testing

Twenty four strains of bifidobacteria and 16 strains of lactobacilli were examined for activities of fructose-6-phosphate phosphoketolase, a-galactosidase, and a-glucosidase and the resistance to metronidazole and mupirocin over 24 h. The detection of a-galactosidase, a-glucosidase, and metronidazole susceptibility gave unclear results. The mupirocin susceptibility using disc diffusion method (200 mg mupirocin per disc) had the same validity as the detection of fructose-6-phosphate phosphoketolase – the key enzyme of the bifidobacteria carbohydrate metabolism. While 24 bifidobacteria strains tested were mupirocin-resistant, 16 lactobacilli strains were susceptible. Hence, the resistance to mupirocin may serve as a simple criterion distinquishing Bifidobacterium spp. from Lactobacillus spp.     

Evaluation of a scanner-assisted colorimetric MIC method for susceptibility testing of gram-negative fermentative bacteria

We describe the ScanMIC method, a colorimetric MIC method for susceptibility testing of gram-negative fermentative bacteria. The method is a slight modification of the National Committee for Clinical Laboratory Standards (NCCLS) recommended broth microdilution method that uses a redox indicator 2,3,5-triphenyltetrazolium chloride (TTC) to enhance the estimate of bacterial growth inhibition in a microplate and a flatbed scanner to capture the microplate image. In-house software was developed to transform the microplate image into numerical values based on the amount of bacterial growth and to generate the MICs automatically. The choice of indicator was based on its low toxicity and ease of reading by scanner. We compared the ScanMIC method to the NCCLS recommended broth microdilution method with 197 coliform strains against seven antibacterial agents. The interpretative categorical agreement was obtained in 92.4% of the assays, and the agreement for MIC differences (within +/-1 log(2) dilution) was obtained in 96% for ScanMIC versus broth microdilution and 97% for a two-step incubation colorimetric broth microdilution versus the broth microdilution method. The method was found to be labor-saving, not to require any initial investment, and to show reliable results. Thus, the ScanMIC method could be useful for epidemiological surveys that include susceptibility testing of bacteria.     

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.