European multi-centre study to establish MIC and zone diameter epidemiological cut-off values for Bacillusanthracis.

OBJECTIVES: Bacillus anthracis clinical breakpoints, representing a systematic approach to guide clinicians in selecting the most appropriate antimicrobial treatments, are not part of the guidance from the European Committee on Antimicrobial Susceptibility Testing (EUCAST). This is because defined distributions of MIC values and of epidemiological cut-off values (ECOFFs) have been lacking. In this study, a Europe-wide network of laboratories in collaboration with EUCAST, aimed at establishing standardized antimicrobial susceptibility testing methods, wild-type MIC distributions, and ECOFFs for ten therapeutically relevant antimicrobials. METHODS: About 335 B. anthracis isolates were tested by broth microdilution and disc diffusion methodologies. MIC and inhibition zone diameters were curated according to EUCAST SOP 10.2 and the results were submitted to EUCAST for ECOFFs and clinical breakpoint determination. RESULTS: Broth microdilution and disc diffusion data distributions revealed putative wild-type distributions for the tested agents. For each antimicrobial agent, ECOFFs were defined. Three highly resistant strains with MIC values of 32 mg/L benzylpenicillin were found. MIC values slightly above the defined ECOFFs were observed in a few isolates, indicating the presence of resistance mechanisms to doxycycline, tetracycline, and amoxicillin. DISCUSSION: B. anthracis antimicrobial susceptibility testing results were used by EUCAST to determine ECOFFs for ten antimicrobial agents. The MIC distributions were used in the process of determining clinical breakpoints. The ECOFFs can be used for the sensitive detection of isolates with resistance mechanisms, and for monitoring resistance development. Genetic changes causing phenotypic shifts in isolates displaying slightly elevated MICs remain to be investigated.

Adaptation of Brucella melitensis Antimicrobial Susceptibility Testing to the ISO 20776 Standard and Validation of the Method.

Brucellosis, mainly caused by Brucella (B.) melitensis, is associated with a risk of chronification and relapses. Antimicrobial susceptibility testing (AST) standards for B. melitensis are not available, and the agent is not yet listed in the EUCAST breakpoint tables. CLSI recommendations for B. melitensis exist, but they do not fulfill the requirements of the ISO 20776 standard regarding the culture medium and the incubation conditions. Under the third EU Health Programme, laboratories specializing in the diagnostics of highly pathogenic bacteria in their respective countries formed a working group within a Joint Action aiming to develop a suitable method for the AST of B. melitensis. Under the supervision of EUCAST representatives, this working group adapted the CLSI M45 document to the ISO 20776 standard after testing and validation. These adaptations included the comparison of various culture media, culture conditions and AST methods. A Standard Operation Procedure was derived and an interlaboratory validation was performed in order to evaluate the method. The results showed pros and cons for both of the two methods but also indicate that it is not necessary to abandon Mueller-Hinton without additives for the AST of B. melitensis.

Evaluation of Real-time PCR Based on SYBR Green I Fluorescent Dye for Detection of Bacillus Anthracis Strains in Biological Samples.

INTRODUCTION: The aim of the study was the application and evaluation of real-time PCRs based on the fluorescence of SYBR Green I intercalating dye for the detection of three Bacillus anthracis genes in contaminated liver and blood samples. The goals for detection were rpoB gene as a chromosomal marker, pag gene located on plasmid pXO1, and capC gene located on plasmid pXO2. MATERIAL AND METHODS: Five B. anthracis strains were used for the experiments. Additionally, single strains of other species of the genus Bacillus, i.e. B. cereus, B. brevis, B. subtilis, and B. megaterium, and strains of six other species were used for evaluation of the specificity of the tests. Three SYBR Green I real-time PCRs were conducted allowing confirmation of B. anthracis in the biological samples. RESULTS: The observation of amplification curves in real-time PCRs enabled the detection of the chromosomally encoded rpoB gene, pag gene, and capC gene of B. anthracis. The specificity of the tests was confirmed by estimation of the melting temperature of the PCR products. The sensitivity and linearity of the reactions were determined using regression coefficients. Strains of other microbial species did not reveal real-time PCR products. CONCLUSION: All real-time PCRs for the detection of B. anthracis in biological samples demonstrated a significant sensitivity and high specificity.

Detection of Listeria Spp. and Listeria Monocytogenes in Biological Samples by SYBR Green I and TaqMan Probe-based Real-time PCRs.

INTRODUCTION: The aim of the study was the application and comparison of real-time PCR methods based on the fluorescence of SYBR Green I intercalating dye and TaqMan probes for the detection of the 23S rDNA gene of Listeria spp. and the hlyA gene of Listeria monocytogenes in biological samples of the liver, brain, and blood. MATERIAL AND METHODS: Five strains of L. monocytogenes and single strains of each species L. ivanovii, L. innocua,L. grayi, L. welshimeri, and L. seeligeri were used for the experiments. Additionally, five strains of other species of bacteria were used for evaluation of the specificity of tests. In the first stage of the study SYBR Green I real-time PCRs, one allowing detection of the 23S rDNA gene and two based on the amplification the hlyA gene, were performed. In the next part, three TaqMan probe-based real-time PCRs allowing confirmation of belonging to Listeria spp. and L. monocytogenes were conducted. RESULTS: The observation of amplification curves in real-time PCRs enabled the detection of both genes. A high regression coefficient of 0.99 was found for all reactions. Specific amplification products were obtained for the 23S rDNA and hlyA genes which confirm their belonging to Listeria spp. and L. monocytogenes, respectively. Other microbial species did not reveal real-time PCR products. CONCLUSION: Both real-time PCR methods for the detection of Listeria spp. and L. monocytogenes in biological samples demonstrated a significant sensitivity and high specificity.