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Based on pharmacokinetic studies carried out according to the methodologies defined by the European Medicines Agency (EMA) using mass spectrometry analysis, a new formulation of a veterinary drug for the treatment of broiler chickens is proposed. Currently, the traditional trimethoprim–sulfamethoxazole drug used for broilers is applied in a 1:5 ratio, and the recommended dose is 45 mg kg−1 of live weight administered at 24 h intervals for 3 to 5 days. In this study, we propose a novel combination containing similar active substances in a newly established ratio of 1:4, with a recommended dosage of 20 mg kg−1 of live weight administered at 24 h intervals for 3 to 5 days. With this method, the currently recommended dose of the traditional trimethoprim–sulfamethoxazole drug used for broilers can be reduced by more than half. The efficacy of the newly designed formulation and dosage of the drug was verified in a bioassay for the treatment of broilers experimentally infected with an avian pathogenic strain of Escherichia coli. In the experiment, we compared the newly designed dosage with the traditional dosage in terms of efficacy and dosage. There were no statistically significant differences between the two drugs in efficacy regarding the survival of chickens after experimental infection or changes in their health status. The experimental results suggest that a significant reduction in the recommended daily dose of drugs containing trimethoprim and sulfamethoxazole for the treatment of bacterial infections in broilers is possible and can support the prudent use of antimicrobials, including the limitation of their overuse.

The aim of this work was to describe the pathotypes of Escherichia coli strains isolated from one-day-old chickens, as well as the occurrence of resistance and multidrug resistance (MDR) in these strains. A total of 429 mixed swabs from 4290 one-day-old chicks were examined between August 2021 and July 2023 (24 months) during routine point-of-destination inspections at 12 poultry farms in the Czech Republic. All samples were processed via cultivation methods using meat-peptone blood agar and Mc Conkey agar under aerobic conditions at 37 ± 1 °C for 18–24 h. The identification of the strains was performed using MALDI-TOF mass spectrometry. All confirmed strains of E. coli were screened via single or multiplex PCRs for the presence of genes encoding the virulence-associated factors iroN, cvaC, iss, felA, iutA, frz and tsh. Antimicrobial susceptibility tests were performed using the minimal inhibitory concentration (MIC) method, focusing on ampicillin, cefotaxime, tetracycline, doxycycline, enrofloxacin, florfenicol, amoxicillin with clavulanic acid and trimethoprim with sulfamethoxazole. A total of 321 E. coli strains (prevalence of 74.8%) were isolated, and 300 isolates were defined as avian pathogenic strains of E. coli (APEC) via multiplex PCR. Based on the defined virulence genes, the isolates were classified into 31 pathotypes. A total of 15.9% of the tested isolates were susceptible to all the tested antimicrobials. On the other hand, 20.5% of the isolates were identified as multidrug-resistant (8.7% of isolates were resistant to three antimicrobials, 7.3% to four antimicrobials, 3.6% to five antimicrobials and 0.9% to six antimicrobials). Monitoring pathogenic strains of E. coli in different animals and in the environment makes it possible to understand their spread in animal and human populations and, at the same time, reveal the sources of virulence and resistance genes.

Streptococcus uberis is one of the most important causative agents of mastitis and is a common reason for the use of antimicrobials in dairy cows. In this study, we assessed the antimicrobial susceptibility of 667 S. uberis isolates originating from 216 Czech dairy farms collected between 2019 and 2023 using the broth microdilution method. We tested 140 of the isolates for the presence of antimicrobial genes using whole-genome sequencing and evaluated their relationship with phenotypic resistance. Streptococcus uberis isolates showed high levels of resistance to tetracycline (59%), followed by streptomycin (38%) and clindamycin (29%). Although all of the isolates were susceptible to beta-lactams, a relatively high percentage of intermediately susceptible isolates was recorded for ampicillin (44%) and penicillin (18%). The isolates were mainly resistant to tetracycline alone (31.3%); the second most frequent occurrence of the phenotypic profile was simultaneous resistance to tetracycline, streptomycin, and clindamycin (16.6%). The occurrence of antibiotic resistance genes did not always match the phenotypic results; in total, 36.8% of isolates that possessed the ant(6)-Ia gene did not show phenotypic resistance to streptomycin. To a lesser extent, silent genes were also detected in clindamycin and tetracycline. This study confirmed the high susceptibility of S. uberis to penicillins used as first-line antimicrobials for S. uberis mastitis treatment.

Our study describes the prevalence and spectrum of enterococci isolated from one-day-old chickens in the Czech Republic, their level of antimicrobial resistance, and the occurrence of multiresistance. Over a 24-month period from 1 August 2021 to 31 July 2023, a total of 464 mixed samples of one-day-old chicken organs were examined during routine inspections at 12 randomly selected poultry farms in the Czech Republic. The samples were processed via cultivation methods and suspected strains were confirmed using the MALDI–TOF Mass Spectrometry method. Antimicrobial susceptibility was determined using the MIC method for eight antimicrobials. A total of 128 isolates (prevalence of 27.6%) representing 4 species of enterococci were isolated, including Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, and Enterococcus hirae, with prevalence rates of 23.3%, 1.5%, 2.2%, and 0.6%, respectively. Susceptibility tests showed a high percentage of susceptible strains among E. faecalis, E. faecium, and E. gallinarum for penicillin-based antibiotics, sulfamethoxazole with trimethoprim, and florfenicol (80–100% susceptible strains). E. hirae was an exception, displaying complete resistance to enrofloxacin (0% susceptible strains) and a high degree of resistance to other tested antimicrobials (33.3% susceptible strains). Among the isolated strains, a total of 16 isolates (12.5%) showed resistance to 3 or more antimicrobials. Complete resistance to all eight antimicrobials simultaneously was observed in four isolates (3.1%). This research shows the possible sources of pathogenic enterococci and their virulence and resistance genes. The findings hold relevance for both veterinary and human medicine, contributing to a better understanding of enterococcal circulation in the human ecosystem and food chain, as well as the development of their resistance and multiresistance.

The results of the antimicrobial susceptibility testing of clinical isolates Streptococcus suis to amoxicillin and marbofloxacin obtained by the agar dilution method and broth microdilution method with the results obtained by the commercially available E-test were compared. Comparisons between the methods based on the determination of the minimal inhibitory concentration (MIC) of the antimicrobials were assessed based on the degree and frequency of the categorical agreement (Agar dilution method as a reference system) and the percentage of the categorical agreement and error rate. A statistical evaluation was determined using the Bland-Atman method. The presented MIC values, determined for the isolates in the E-test, were slightly different from the MIC values determined by the dilution tests, mainly due to the different defined testing concentrations. For the E-test as the test system and agar-dilution method as the reference system, no error of any class was detected (very major, major and minor error) and a complete categorical agreement was obtained between the evaluated methods for amoxicillin. For amoxicillin, the regression and correlation analysis show linear relationships between the E-test and the two dilution methods with significant coefficients of determination (0.62 and 0.75). The slopes of the equality and regression lines were not significantly different. However, the E-test tends to slightly overestimate the MIC values when compared to the microdilution. The reverse is true when compared with the agar dilution. There was good agreement between the E-test and the dilution methods with a low bias (0.001 3 and -0.005 0), all the experimental data were within the computed limits of agreement. For marbofloxacin, the same trends were observed with lower coefficients of determination (0.42 and 0.73) and a less favourable agreement. The E-test constantly underestimated the MIC values when compared to the two dilution methods. No significant difference between the microdilution and agar dilution was obtained.

Streptococcus suis is a serious pathogen in the pig industry with zoonotic potential. With respect to the current effort to reduce antibiotic use in animals, a prophylactic measure is needed to control the disease burden. Unfortunately, immunization against streptococcal pathogens is challenging due to nature of the interaction between the pathogen and the host immune system, but vaccines based on conjugates of capsular polysaccharide (CPS) and carrier protein were proved to be efficient. The main obstacle of these vaccines is manufacturing cost, limiting their use in animals. In this work, we tested an experimental vaccine against Streptococcus suis serotype 2 based on capsular polysaccharide conjugated to chicken ovalbumin (OVA) and compared its immunogenicity and protectivity with a vaccine based on CRM197 conjugate. Ovalbumin was selected as a cheap alternative to recombinant carrier proteins widely used in vaccines for human use. We found that the ovalbumin-based experimental vaccine successfully induced immune response in pigs, and the IgG antibody response was even higher than after immunization with capsular polysaccharide-CRM197 conjugate. Protectivity of vaccination against infection was evaluated in the challenge experiment and was found promising for both conjugates.

The ubiquitous occurrence and high heterogeneity of Streptococcus uberis strains cause difficulties in the development and implementation of effective control strategies in dairy herds. In this study, S. uberis strains from 74 farms, obtained predominantly from subclinical, acute, and chronic recurrent mastitis, as well as from udder surface swabs and milk from healthy udders, were analysed for their genetic diversity using multilocus sequence typing (MLST). Isolates were tested for the presence of the genes encoding the virulence factors using polymerase chain reaction. Antibiotic susceptibility testing was performed using a microdilution assay including 14 antimicrobials. The virulence profiles and antimicrobial (AMR) profiles of the isolates were assembled and the overall heterogeneity was evaluated. Among the 124 isolates, 89 MLST genotypes, 7 different virulence profiles, and 12 AMR profiles were identified. The large number of different MLST allelic profiles in this study points to the high heterogeneity of strains in dairy herds in the Czech Republic. Isolates of a certain MLST genotype may possess a different set of virulence factor genes. We detected up to three different resistance profiles within a single MLST genotype. The results of our study showed that fully susceptible isolates coexisted with resistant or even multiresistant isolates in the same herd. Multiple genotypes within a herd were detected on many farms (up to seven MLST genotypes and four AMR profiles in one herd). This heterogenic population structure might suggest that environmental transmission is the predominant route of infection in herds in the Czech Republic.

A determination of susceptibility/resistance to antimicrobials via serotype was carried out in 506 field isolates of Streptococcus suis, originating from pig farms in the Czech Republic in the period 2018–2022. A very high level of susceptibility of S. suis isolates was found to amoxicillin, in combination with clavulanic acid and sulfamethoxazole potentiated with trimethoprim. None of the tested isolates were resistant to these antimicrobial substances. Only two isolates were found to be intermediately resistant to enrofloxacin in 2020. With regard to ceftiofur, one isolate was intermediately resistant in 2020 and 2022, and two isolates were intermediately resistant in 2018 and 2021. A low level of resistance was detected to ampicillin (0.6% in 2021) and to florfenicol (1.15% in 2019; 1.3% in 2022). With regard to penicillin, a medium level of resistance was detected in 2018 (10.6%), but a low level of resistance was found in the following years (7.0% in 2019; 3.1% in 2020; 3.3% in 2021; 3.9% in 2022). On the contrary, a high or very high level of resistance was found to tetracycline (66.0% in 2018; 65.1% in 2019; 44.35% in 2020; 46.4% in 2021; 54.0% in 2022). Using molecular and serological methods, serotype 7 (16.4%) was determined to be predominant among S. suis isolates, followed by serotypes 1/2, 2, 9, 4, 3, 1, 29, 16, and 31 (10.7%; 8.5%; 5.7%; 5.5%; 4.5%; 4.3%; 3.6%; 3.4%; 3.4%, respectively). Other serotypes were identified among the investigated strains either rarely (up to 10 cases) or not at all. A relatively high percentage of isolates were detected as non-typeable (79 isolates; 15.6%). Dependence of resistance upon serotype assignment could not be proven in all but serotype 31, wherein all isolates (n = 17) were resistant or intermediately resistant to clindamycin, tilmycosin, tulathromycin, and tetracycline. The resistance to clindamycin and tetracycline may be related to the high consumption of these antibiotics on pig farms at present or in previous years. Macrolides (tilmicosin and tulathromycin) and tiamulin are not suitable for the treatment of streptococcal infections, but are used on pig farms to treat respiratory infections caused by gram-negative bacteria, so they were included in the study.

Glaesserella (Haemophilus) parasuis is a part of the normal flora of the respiratory tract of pigs. However, under certain conditions it can also induce severe systemic disease with high morbidity and mortality leading to gross economic losses in the pig industry. The most prevalent serovars in pig herds in the Czech Republic are the virulent serovars 1, 4, 5 and 13. The currently available commercial vaccines are inactivated vaccines with certain limitations, such as no or poor cross-serovar protection. Therefore, the aim of the present study was to construct a subunit vaccine with a crude capsular polysaccharide extract (cCPS) isolated from G. parasuis CAPM 6475 (serovar 5) and evaluate its immunogenicity in a mouse model. Mice were immunised subcutaneously with two doses of the constructed vaccine in a 14-day interval and challenged intraperitoneally with various G. parasuis strains (serovars 1, 4, 5, 13) at 21 days after the second immunisation. The results of the ELISA test showed that the boost dose of the vaccine induced the production of IgG antibodies in high levels. On the basis of the death cases, the pathological findings and the bacterial isolation, the mice immunised with the cCPS were partially protected against the challenge with the homologous serovar 5 as well as with heterologous serovars 1, 4 and 13 of G. parasuis. The cross-reaction of the mixed serum from the immunised mice with the tested serovars was seen in the western-blotting also. Moreover, the most abundant protein found in the cCPS by mass spectrometry was catalase, a protein of molecular weight 55 kDa that may correspond to the strongest reaction seen in the western-blotting. Our findings indicated that the crude capsular polysaccharide extract may provide an effective immunogenicity in preventing a G. parasuis infection caused by the most prevalent serovars in the Czech Republic. However, the evaluation of the efficacy needs to be performed in pigs before any conclusions can be drawn.

The post-antibiotic effect is defined as the period of bacterial growth suppression that persists after a limited exposure of organisms to antimicrobials and knowledge of its duration is important in establishing and optimising current dosing schedules for the treatment of bacterial infections. The post-antibiotic effect of marbofloxacin, enrofloxacin and amoxicillin were evaluated in vitro for Actinobacillus pleuropneumoniae, Haemophilus parasuis and Pasteurella multocida strains which originated from clinical samples of diseased pigs and were confirmed as susceptible to all tested antimicrobials based on determination of minimal inhibitory concentrations. The post-antibiotic effect for individual antimicrobials was monitored at five and ten times minimum inhibitory concentrations for one and two hours. The duration of the post-antibiotic effect for tested antimicrobials was found to exhibit the following order for all tested pathogens: marbofloxacin > enrofloxacin > amoxicillin. The longest duration of post-antibiotic effect of all tested antimicrobials was found in A. pleuropneumoniae and the shortest post-antibiotic effect duration was detected in P. multocida. No statistical differences in post-antibiotic effect duration were found within marbofloxacin and enrofloxacin in A. pleuropneumoniae and H. parasuis strains. In P. multocida strains there was a statistically significant difference (P = 0.0189). On the other hand, the differences between amoxicillin and marbofloxacin or enrofloxacin were statistically significant in all cases (P-values ranged between 0.0058 and 0.008). The prolonged post-antibiotic effect of fluoroquinolones and amoxicillin on important Gram-negative swine pathogens was confirmed. The results can be used to clarify the effect and mechanism of action of antimicrobial drugs in veterinary medicine.

ABSTRACT The posttranslational Ca2+-dependent “clip-and-link” activity of large repeat-in-toxin (RTX) proteins starts by Ca2+-dependent structural rearrangement of a highly conserved self-processing module (SPM). Subsequently, an internal aspartate-proline (Asp-Pro) peptide bond at the N-terminal end of SPM breaks, and the liberated C-terminal aspartyl residue can react with a free ε-amino group of an adjacent lysine residue to form a new isopeptide bond. Here, we report a solution structure of the calcium-loaded SPM (Ca-SPM) derived from the FrpC protein of Neisseria meningitidis. The Ca-SPM structure defines a unique protein architecture and provides structural insight into the autocatalytic cleavage of the Asp-Pro peptide bond through a “twisted-amide” activation. Furthermore, in-frame deletion of the SPM domain from the ApxIVA protein of Actinobacillus pleuropneumoniae attenuated the virulence of this porcine pathogen in a pig respiratory challenge model. We hypothesize that the Ca2+-dependent clip-and-link activity represents an unconventional strategy for Gram-negative pathogens to adhere to the host target cell surface. IMPORTANCE The Ca2+-dependent clip-and-link activity of large repeat-in-toxin (RTX) proteins is an exceptional posttranslational process in which an internal domain called a self-processing module (SPM) mediates Ca2+-dependent processing of a highly specific aspartate-proline (Asp-Pro) peptide bond and covalent linkage of the released aspartyl to an adjacent lysine residue through an isopeptide bond. Here, we report the solution structures of the Ca2+-loaded SPM (Ca-SPM) defining the mechanism of the autocatalytic cleavage of the Asp414-Pro415 peptide bond of the Neisseria meningitidis FrpC exoprotein. Moreover, deletion of the SPM domain in the ApxIVA protein, the FrpC homolog of Actinobacillus pleuropneumoniae, resulted in attenuation of virulence of the bacterium in a pig infection model, indicating that the Ca2+-dependent clip-and-link activity plays a role in the virulence of Gram-negative pathogens.

The aim of this study was to describe the prevalence of different Staphylococcus species isolated from pathological processes and lesions in domestic animals in the Czech Republic and to detect and describe oxacillin (methicillin)-resistant strains (MRS). During the years 2019–2020, a total of 5218 veterinary clinical samples from the Czech Republic were tested. Testing was performed by culture methods and typing by molecular phenotypic methods MALDI-TOF MS and PCR. Antimicrobial susceptibility testing of the strains was performed by the disk diffusion method. A total of 854 staphylococci strains were identified (16.37% prevalence), out of which 43 strains of 6 species of staphylococci were MRS (n = 43; 0.82% prevalence). Of the MRS strains, the most prevalent species were Staphylococcus pseudintermedius (n = 24; 0.46% prevalence) and Staphylococcus aureus (n = 7; 0.13% prevalence). Susceptibility testing showed resistance to beta-lactam antibiotics and, depending on the species, also to trimethoprim/sulfamethoxazole, gentamicin, tetracycline, erythromycin, clindamycin, and enrofloxacin. For further characterization of MRS, PCR assay for virulence factor genes was performed. Seven of the 14 target genes were observed only in S. aureus, except for the eno gene encoding laminin-binding protein, which was also detected in other staphylococci. It is necessary to emphasize the issue of correct using of antimicrobials in practice and antibiotic policy in university teaching and to create stricter legislation that would prevent the widespread use of antimicrobials in veterinary medicine, especially in livestock to reduce the emergence and spread of antimicrobial resistance.

Streptococcus uberis is one of the primary causative agents of mastitis, a clinically and economically significant disease that affects dairy cattle worldwide. In this study, we analyzed 140 S. uberis strains isolated from mastitis milk samples collected from 74 cow herds in the Czech Republic. We employed whole-genome sequencing to screen for the presence of antimicrobial resistance (AMR) genes and genes encoding virulence factors, and to assess their genetic relationships. Our analysis revealed the presence of 88 different sequence types (STs), with 41% of the isolates assigned to global clonal complexes (GCCs), the majority of which were affiliated with GCC5. The STs identified were distributed across the major phylogenetic branches of all currently known STs. We identified fifty-one putative virulence factor genes, and the majority of isolates carried between 27 and 29 of these genes. A tendency of virulence factors and AMR genes to cluster with specific STs was observed, although such clustering was not evident within GCCs. Principal component analysis did not reveal significant diversity among isolates when grouped by GCC or ST prevalence. The substantial genomic diversity and the wide array of virulence factors found in S. uberis strains present a challenge for the implementation of effective anti-mastitis measures.

As in other countries, in the Czech Republic, Streptococcus suis infection in pigs is considered an economically significant disease for the pig industry, though little is known about its population structure. We collected S. suis isolates from 144 farms in the years 2018–2022. All samples were taken from animals suffering from symptoms indicating possible S. suis infection. Serotyping revealed the presence of 23 different serotypes, and 18.94% were non-typable strains. The most common was S7 (14.96%), while other serotypes had frequencies of less than 10%. Sequence typing identified 56 different sequence types, including 31 newly assigned sequence types together with 41 new alleles in genes in the MLST schema. A large portion of isolates (25.70%) were of unknown sequence type. The most common sequence types were ST29 (14.77%) and ST28 (10.04%); the other sequence types had frequencies of less than 10%. In total, 100 different combinations of serotypes and sequence types were identified. Among them, S7ST29 was found in 72 isolates, representing 13.63% of all isolates, and was significantly associated with the central nervous system. Many other isolates of particular serotype and sequence type combinations were found in a few cases, and a number of isolates were non-typable.

In Glässer’s disease outbreaks, Glaesserella (Haemophilus) parasuis has to overcome the non-specific immune system in the lower respiratory tract, the alveolar macrophages. Here we showed that porcine alveolar macrophages (PAMs) were able to recognize and phagocyte G. parasuis with strain-to-strain variability despite the presence of the capsule in virulent (serovar 1, 5, 12) as well in avirulent strains (serovar 6 and 9). The capsule, outer membrane proteins, virulence-associated autotransporters, cytolethal distending toxins and many other proteins have been identified as virulence factors of this bacterium. Therefore, we immunized pigs with the crude capsular extract (cCE) from the virulent G. parasuis CAPM 6475 strain (serovar 5) and evaluated the role of the anti-cCE/post-vaccinal IgG in the immune response of PAMs to in vitro infection with various G. parasuis strains. We demonstrated the specific binding of the antibodies to the cCE by Western-blotting assay and immunoprecipitation as well as the specific binding to the strain CAPM 6475 in transmission electron microscopy. In the cCE, we identified several virulence-associated proteins that were immunoreactive with IgG isolated from sera of immunized pigs. Opsonization of G. parasuis strains by post-vaccinal IgG led to enhanced phagocytosis of G. parasuis by PAMs at the first two hours of infection. Moreover, opsonization increased the oxidative burst and expression/production of both pro- and anti-inflammatory cytokines. The neutralizing effects of these antibodies on the antioxidant mechanisms of G. parasuis may lead to attenuation of its virulence and pathogenicity in vivo. Together with opsonization of bacteria by these antibodies, the host may eliminate G. parasuis in the infection site more efficiently. Based on these results, the crude capsular extract is a vaccine candidate with immunogenic properties.

Streptococcus suis represents a primary health problem (such as meningitis, septicemia and arthritis in piglets and fatteners) in the swine industry worldwide and also an emerging zoonotic pathogen. In the Czech Republic, many pig farms repopulated their herds over the past decades to reduce morbidity and minimize treatment. The study analysed serotypes, sequence types and antimicrobial susceptibility in 39 S. suis isolates obtained from organs of diseased pigs from selected 16 repopulated farms with a history of S. suis-associated diseases and routine antimicrobial treatment with tulathromycin and/or amoxicillin. The analysis revealed diversity of collected isolates with regular occurrence of more than three serotypes per farm. The serotypes identified were 1/2 and 7, each in six isolates, followed by serotype 2 and 3 found in five isolates each, other serotypes were less frequent. Seven isolates were not typable by multiplex PCR and we also found sequence type of unknown type in thirteen isolates. The majority of S. suis isolates were resistant to clindamycin (n = 31), tetracycline (n = 29) and tilmicosin and tulathromycin (n = 28). On the other hand, with the exception of two isolates that were intermediately susceptible to penicillin and ampicillin, all isolates were susceptible to all three tested subgroups of beta-lactam antibiotics.

Abstract Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant and economically important infectious diseases affecting swine worldwide and can predispose pigs to secondary bacterial infections caused by, e.g. Haemophilus parasuis. The aim of the presented study was to compare susceptibility of two different types of macrophages which could be in contact with both pathogens during infection with PRRS virus (PRRSV) and in co-infection with H. parasuis. Alveolar macrophages (PAMs) as resident cells provide one of the first lines of defence against microbes invading lung tissue. On the other hand, monocyte derived macrophages (MDMs) represent inflammatory cells accumulating at the site of inflammation. While PAMs were relatively resistant to cytopathogenic effect caused by PRRSV, MDMs were much more sensitive to PRRSV infection. MDMs infected with PRRSV increased expression of pro-apoptotic Bad, Bax and p53 mRNA. Increased mortality of MDMs may be also related to a higher intensity of ROS production after infection with PRRSV. In addition, MDMs (but not PAMs) infected with H. parasuis alone formed multinucleated giant cells (MGC); these cells were not observed in MDMs infected with both pathogens. Higher sensitivity of MDMs to PRRSV infection, which is associated with limited MDMs survival and restriction of MGC formation, could contribute to the development of multifactorial respiratory disease of swine.