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.

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.

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.

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. Five strains of L. monocytogenes and single strains of each of the 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 the tests. QuantiTect SYBR Green PCR and QuantiTect Probe PCR kits were selected for the study. In the first stage of the study, SYBR Green I real-time PCRs were performed under several methods, the first one allowing detection of the 23S rDNA gene and the remainder based on the amplification of the hlyA gene. In the next part, three varied in method TaqMan probe-based real-time PCRs allowing confirmation of strains belonging to Listeria spp. and L. monocytogenes were conducted. The observation of amplification curves in real-time PCR methods enabled the detection of both genes, and these methods demonstrated a significant sensitivity and high specificity. 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 confirmed the tested strains as Listeria spp. and L. monocytogenes respectively. Isolates of other microbial species did not yield real-time PCR products.

Pasteurella multocida is a pathogen of numerous mammal and bird species. Based on capsular antigens, five capsular types of P. multocida (A, B, D, E, and F) are distinguished. The aim of this study was to evaluate the usefulness of multiplex PCR and MALDI-TOF MS for the identification and capsular typing of P. multocida strains isolated from rabbits. A total of 115 field strains previously classified as P. multocida, isolated in Poland between 1999 and 2020, were analysed. Multiplex PCR was applied for simultaneous species identification and determination of capsular types. Most strains belonged to capsular type A (87.8%), while capsular types D (8.7%) and F (3.5%) were detected less frequently. The examined strains were subsequently identified by MALDI-TOF MS, which correctly assigned all strains to the species P. multocida. The results demonstrate that multiplex PCR is a rapid and reliable alternative to conventional species identification and serological capsular typing of P. multocida. In addition, MALDI-TOF MS proved to be a valuable tool for accurate species-level identification. The application of these methods in routine clinical microbiology laboratories may significantly improve the speed and reliability of P. multocida identification.

Introduction: Bacillus anthracis causes an infectious disease called anthrax. Herbivores are more susceptible to the disease than omnivores, carnivores and humans. Grazing animals are the highest-risk group, and among them, anthrax outbreaks have extremely high fatality rates and impose heavy costs, besides posing a grave zoonotic risk. The aim of the study was the application and evaluation of simultaneous use of multiplex PCR and PCR-restriction fragment length polymorphism (PCR-RFLP) allowing the differentiation of strains of the B. anthracis species from other species of the Bacillus genus. Material and Methods: The experiment involved 21 strains of B. anthracis . Strains of other species of the Bacillus genus were also included in the experiment. In the first part of the studies, two genes responsible for virulence – pag and cap , located on plasmids pXO1 and pXO2 – and the chromosomal sequence Ba813 were used for a multiplex PCR. In the next stage, PCR-RFLP, in which restriction analysis of the SG-749 sequence using the Alu I enzyme was performed. Results: The multiplex PCR allowed the identification of virulent B. anthracis strains, as well as the detection of the presence of the chromosomal sequence Ba813. Then, PCR-RFLP showed the restriction pattern characteristic of B. anthracis strains. Conclusion: The simultaneous use of multiplex PCR and PCR-RFLP enables the distinction of B. anthracis strains with and without plasmids from other strains of the Bacillus genus, including those with the Ba813 chromosomal sequence.