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 to optimise and compare two multiplex PCR assays for the detection of Listeria spp. and Listeria monocytogenes in biological samples including the liver, brain, and blood. Material and Methods: Three 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. Additionally, five other species of bacterium were used to evaluate the specificity of the tests. Results: Specific amplification products were obtained for both multiplex PCR assays, which confirmed the tested strains as Listeria spp. and L. monocytogenes, respectively. Isolates of other species did not yield PCR products. Conclusion: Both multiplex PCR assays proved to be significantly sensitive and highly-specific methods for the detection of Listeria strains.

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: Pasteurella multocida is an important pathogen in many species of domestic and wild mammals, as well as birds. It is also a commensal in the nasopharynx of clinically healthy animals. The aim of the study was to characterise and classify field strains of P. multocida isolated from rabbits in Poland. Material and Methods: A total of 115 field strains isolated from rabbits in 1999–2020 were used. The examined strains were evaluated morphologically and microscopically, and their ability to produce catalase and oxidase was determined. Identification of P. multocida strains was performed on basis of biochemical characteristics and serotype classification. Results: Based on the biochemical profile assessment using API 20E and the ID 32E (bioMérieux), the strains isolated from rabbits were identified as P. multocida . Among 17.4% were classified as P. multocida subsp. multocida , 80% as ornithine decarboxylase negative P. multocida subsp. multocida and 2.6% as P. multocida subsp. septica . The capsule of type A was found in 87.8% of strains, type D in 8.7%, and type F in 3.5%. The somatic antigen of serotype 12 was present in 64.3% of strains, and serotype 3 in 36.7% of strains. The capsular type A was predominant among strains from rabbits that were asymptomatic carriers, while the capsular type D and F were found mainly in strains isolated from sick animals. In turn, the somatic antigen of serotype 12 was present in strains from both healthy and diseased animals, while strains of serotype 3 were much more frequently isolated from diseased rabbits. Conclusion: The conducted research allowed to classify and obtain new in-depth data on the population structure of P. multocida strains isolated from rabbits in Poland.

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.