Survey of Genotype Diversity, Virulence, and Antimicrobial Resistance Genes in Mastitis-Causing Streptococcus uberis in Dairy Herds Using Whole-Genome Sequencing.

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.

Pathotypes and Phenotypic Resistance to Antimicrobials of Escherichia coli Isolates from One-Day-Old Chickens.

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.

Enterococci Isolated from One-Day-Old Chickens and Their Phenotypic Susceptibility to Antimicrobials in the Czech Republic.

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.

Antimicrobial Susceptibility and Resistance Genes in Streptococcus uberis Isolated from Bovine Mastitis in the Czech Republic.

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.

Dosage Optimisation of Trimethoprim and Sulfamethoxazole for the Treatment of an Avian Pathogenic Strain of Escherichia coli in Broiler Chickens.

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.

Vaccine against Streptococcus suis Infection in Pig Based on Alternative Carrier Protein Conjugate.

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.

Multilocus Sequence Genotype Heterogeneity in Streptococcus uberis Isolated from Bovine Mastitis in the Czech Republic.

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.

Resistance of Streptococcus suis Isolates from the Czech Republic during 2018-2022.

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.

Characterisation of Streptococcus suis Isolates in the Czech Republic Collected from Diseased Pigs in the Years 2018-2022.

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.

The Role of Antibodies Against the Crude Capsular Extract in the Immune Response of Porcine Alveolar Macrophages to In Vitro Infection of Various Serovars of Glaesserella (Haemophilus) parasuis.

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.

Mass spectrometric identification and quantification of the antibiotic clavulanic acid in broiler chicken plasma and meat as a necessary analytical tool in finding ways to increase the effectiveness of currently used antibiotics in the treatment of broiler chickens.

Clavulanic acid is a molecule with antimicrobial effect used in several livestock species treatment. Its inclusion in the treatment of infectious diseases of broilers requires determination of pharmacokinetic and pharmacodynamic parameters in order to determine the appropriate dosage for broilers and ensure safety of chicken products for human health. The present study describes the optimisation of analytical LC-MS/MS method for identification and quantification of clavulanic acid in broiler chicken plasma and meat. The limit of detection and the limit of quantification for the developed method were 3.09 µg·L-1 and 10.21 µg·L-1 for plasma and 2.57 µg·kg-1 and 8.47 µg·kg-1 for meat. The recoveries of the developed plasma and tissue extraction procedure were > 105.7% and > 95.6%, respectively. The achieved coefficient of variation of within-run precision ranged from 2.8 to 10.9% for plasma and from 6.5 to 8.5% for meat. The pharmacokinetic experiment was performed in 112 Ross broiler chickens assigned into time interval groups ranging from 10 min to 24 h in accredited animal facilities. Administered dose of clavulanic acid was 2.5 mg·kg-1 according to the manufacturer's recommendations. The pharmacokinetic parameters obtained from the experiment are as follows: Cmax = 1.82 ± 0.91 mg·L-1, Tmax = 0.25 h, T1/2 = 0.87 h, Kel = 0.80 ± 0.04 h-1, AUC0-∞ = 2.17 mg·h ·L-1.

Resolution of Streptococcus suis Serotypes 1/2 versus 2 and 1 versus 14 by PCR-Restriction Fragment Length Polymorphism Method.

Streptococcus suis is an important pathogen of pigs but is also transmissible to humans, with potentially fatal consequences. Among 29 serotypes currently recognized, some are clinically and epidemiologically more important than others. This is particularly true for serotypes 2 and 14, which have a large impact on pig production and also on human health. Conventional PCR-based serotyping cannot distinguish between serotype 1/2 and serotype 2 or between serotype 1 and serotype 14. Although serotype 1/2 and serotype 2 have a very similar cps locus, they differ in a single-nucleotide substitution at nucleotide position 483 of the cpsK gene. Similarly, serotypes 1 and 14 have a very similar cps locus but also differ in the same nucleotide substitution of the cpsK gene. Fortunately, this cpsK 483G→C/T substitution can be detected by BstNI restriction endonuclease. A PCR-restriction fragment length polymorphism (RFLP) detection method amplifying a fragment of the cpsK gene digested by BstNI restriction endonuclease was developed and tested in reference strains of these serotypes and also in field isolates.

Effect of cysteine peptidase inhibitor of Eudiplozoon nipponicum (Monogenea) on cytokine expression of macrophages in vitro.

The gills of the common carp, whose mucosal surface belongs to the key defence mechanisms of piscine immunity, can be infested with both the larval and adult stage of Eudiplozoon nipponicum (Monogenea). Although on their own, monogeneans do not considerably compromise their hosts' health status, fish with epithelial barriers damaged in parasite feeding and attachment sites are at an increased risk of bacterial challenge with possible harmful consequences. Several studies suggest that helminth parasites of teleost fish evade and manipulate host immune system via their excretory-secretory products, but our knowledge of these processes in the monogeneans is limited. Cysteine peptidase inhibitors (CPI), which are found in the secretions of numerous parasites, often induce immunosuppression by subverting Th1 mechanisms and drawing the immune system towards a Th2/Treg response. We employed the qPCR to test the effect of recently characterised CPI of E. nipponicum (rEnStef) on the mRNA expression of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10 produced by porcine macrophages in vitro. After an initial preincubation with rEnStef, we stimulated the macrophages using LPS. By inducing a Th1 pro-inflammatory response, we imitated the immune reaction during a bacterial challenge in tissue damaged by the feeding and attachment of E. nipponicum. We observed a significant dose-dependent downregulation of the expression of TNF-α and IL-10 cytokines. The observed suppression of TNF-alpha expression by rEnStef could result in decreased pathogen control, which might in turn lead to increased rates of secondary bacterial infections in fish infected by E. nipponicum.

Concurrent infection of monocyte-derived macrophages with porcine reproductive and respiratory syndrome virus and Haemophilus parasuis: A role of IFNα in pathogenesis of co-infections.

Porcine reproductive and respiratory syndrome virus (PRRSV) predisposes pigs to secondary bacterial infection caused by Haemophilus parasuis. The aim of the present study was to analyse the immune response of monocyte-derived macrophages (MDMs), serving as a model of macrophages accumulating at the site of inflammation. The second part of the study was focused on the role of IFNα in the production of inflammatory cytokines in co-infected MDMs. Concurrent infection with PRRSV and H. parasuis decreased gene expression of pro-inflammatory cytokines (IL-1ß, IL-8) in MDMs in comparison with MDMs infected with PRRSV or H. parasuis alone. Our data showed that MDMs express IFNα after PRRSV infection. Thereafter, we exposed cells to the experimental addition of IFNα and a subsequent infection with H. parasuis, and detected a decreased expression/production of pro-inflammatory cytokines. Thus, we assume that IFNα, produced after PRRSV infection, could affect the immune response of monocyte-derived macrophages. Down-regulation of pro-inflammatory cytokine expression in inflammatory macrophages may allow the development of secondary bacterial infections in pigs.

Concurrent infection with porcine reproductive and respiratory syndrome virus and Haemophilus parasuis in two types of porcine macrophages: apoptosis, production of ROS and formation of multinucleated giant cells.

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.