Predominance of Acinetobacter pseudolwoffii among Acinetobacter species in domestic animals in the Czech Republic.

The aim of this study was to map the spectrum of microorganisms belonging to the genus Acinetobacter in domestic animals with a specific focus on the prevalence of Acinetobacter pseudolwoffii. Additionally, the susceptibility of isolates to antimicrobial agents was determined. In the period from January 1, 2014, to August 31, 2015, a total of 9 544 samples originating from gross lesions and pathological processes of animals exhibiting clinical symptoms of the disease were examined across 41 districts in the Czech Republic. The examinations were carried out using culture methods involving meat-peptone blood agar and Endo agar under aerobic conditions at a temperature of 37 ± 1 °C for 18-24 hours. Isolates were confirmed using molecular phenotypic method MALDI-TOF MS with the MBT Compass Library Revision L 2020 covering 3 239 species/entries (9 607 MSP) from Bruker Daltonics company. Out of the 108 isolates (prevalence 1.13%), 14 species of Acinetobacter spp. were identified, with 5 isolates remaining unclassified as species. A. pseudolwoffii was the predominant species isolated in 25 cases (prevalence 0.26%). Antimicrobial susceptibility was determined for 12 antimicrobials by the disc diffusion method, with A. pseudolwoffii isolates exhibiting the lowest susceptibility to ceftazidime (32%) and co-trimoxazole (60%).

Occurrence of virulence-associated genes in Streptococcus uberis and Streptococcus parauberis isolated from bovine mastitis.

Streptococcus uberis is one of the most important mastitis-causing pathogens. Although the pathogenesis and virulence factors required for the intramammary infection development are not yet well established, several putative virulence-associated genes have been described. This work aimed to investigate the presence of ten known and putative virulence-associated genes in S. uberis isolated from subclinical or clinical mastitis and its closely related species Streptococcus parauberis in 135 dairy farms in the Czech Republic. The PCR analysis detected that all the examined isolates possessed at least four virulence genes and most isolates carried eight out of ten virulence genes. All S. uberis isolates were positive for the oppF, gapC and sua genes. Among the most prevalent virulence-associated genes skc (98%) and pauA (97%) were also found. The hasA and hasB genes were always present together in 94% of the isolates. The genes cfu and lbp were detected in 6% and 2%, respectively. In the S. uberis isolates, 14 different virulence gene profiles were observed. The most frequent profile was hasA + hasB + sua + skc + pauA + gapC + oppF with variable hasC, observed in 86% of the tested isolates, occurring in 127 out of 135 farms. S. parauberis was identified very sporadically and, although it is closely related to S. uberis, only a rare occurrence of the examined virulence-associated genes was found.

Characterization of Gallibacterium anatis Isolated from Pathological Processes in Domestic Mammals and Birds in the Czech Republic.

Gallibacterium anatis, recognized as a resident and opportunistic pathogen primarily in poultry, underwent investigation in unwell domestic mammals and birds. The study encompassed the mapping and comparison of G. anatis isolates, evaluation of their genetic diversity, and determination of their susceptibility to antimicrobials. A total of 11,908 clinical samples were analyzed using cultivation methods and MALDI-TOF. Whole-genome sequencing was performed on seven calf isolates and six hen isolates. Among mammals, G. anatis was exclusively detected in 22 young dairy calves, while among domestic birds, it was found in 35 individuals belonging to four species. Pathological observations in calves were predominantly localized in the digestive tract, whereas in birds, multi-organ infections and respiratory system infections were most prevalent. Distinct groups of genes were identified solely in calf isolates, and conversely, those unique to hen isolates were also recognized. Novel alleles in the multilocus sequence typing scheme genes and previously unidentified sequence types were observed in both calf and hen isolates. Antimicrobial susceptibility exhibited variation between bird and calf isolates. Notably, G. anatis isolates from calves exhibited disparities in genotype and phenotype compared to those from hens. Despite these distinctions, G. anatis isolates demonstrated the capability to induce septicemia in both species.

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.

Genomic analysis of Paenibacillus larvae isolates from the Czech Republic and the neighbouring regions of Slovakia.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a devastating disease of honeybee larvae. In the Czech Republic, two large infested regions were recognised. This study aimed to analyse P. larvae strains occurring in the Czech Republic in the years 2016-2017 and to characterise the genetic structure of their population with the use of Enterobacterial Repetitive Intergenic Consensus genotyping (ERIC), multilocus sequence typing (MLST) and whole genome sequence (WGS) analysis. The results were complemented by the analysis of isolates collected in the year 2018 in areas of Slovakia located near the Czechia-Slovakia border. ERIC genotyping revealed that 78.9% of tested isolates belonged to the ERIC II genotype and 21.1% to ERIC I genotype. MLST showed six sequence types with ST10 and ST11 being the most frequent among isolates. Within six isolates we found discrepancies in correlations between MLST and ERIC genotypes. The use of MLST and WGS analysis of isolates revealed that each of the large infested geographic regions had its own dominating P. larvae strains. We assume that these strains represented primary sources of infection in the affected areas. In addition, the sporadic presence of strains identified by core genome analysis as genetically related was unveiled in geographically distant regions suggesting possible human-mediated transmission of AFB.

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.

Screening of Antibiotic and Virulence Genes from Whole Genome Sequenced Cronobacter sakazakii Isolated from Food and Milk-Producing Environments.

The objective of this study was to use whole-genome sequencing (WGS) to screen for genes encoding for antibiotic resistance, fitness and virulence in Cronobacter sakazakii strains that had been isolated from food and powdered-milk-producing environments. Virulence (VGs) and antibiotic-resistance genes (ARGs) were detected with the Comprehensive Antibiotic Resistance Database (CARD) platform, ResFinder and PlasmidFinder tools. Susceptibility testing was performed using disk diffusion. Fifteen presumptive strains of Cronobacter spp. were identified by MALDI-TOF MS and ribosomal-MLST. Nine C. sakazakii strains were found in the meningitic pathovar ST4: two were ST83 and one was ST1. The C. sakazakii ST4 strains were further distinguished using core genome MLST based on 3678 loci. Almost all (93%) strains were resistant to cephalotin and 33% were resistant to ampicillin. In addition, 20 ARGs, mainly involved in regulatory and efflux antibiotics, were detected. Ninety-nine VGs were detected that encoded for OmpA, siderophores and genes involved in metabolism and stress. The IncFIB (pCTU3) plasmid was detected, and the prevalent mobile genetic elements (MGEs) were ISEsa1, ISEc52 and ISEhe3. The C. sakazakii isolates analyzed in this study harbored ARGs and VGs, which could have contributed to their persistence in powdered-milk-producing environments, and increase the risk of infection in susceptible population groups.

Gordonia species as a rare pathogen isolated from milk of dairy cows with mastitis.

While Gordonia species have long been known to cause severe inflammation in humans, the pathogenic effects of Gordonia species in veterinary medicine have rarely been described. Between 2010 and 2019, we collected microorganisms of the genus Gordonia isolated from milk samples from dairy cows with mastitis. We describe the growth properties of these microorganisms and their prevalence, virulence factors and susceptibility to antimicrobial agents. From 31,534 quarter milk samples processed by standard culture methods, 27 isolates of Gordonia species (0.086% prevalence) were identified by a molecular phenotyping method. The isolates originated from 17 farms in 12 districts of the Czech Republic. Twenty-one isolates were tested for susceptibility to 7 antimicrobials by the disc diffusion method. Notably, 100% of these isolates were susceptible to streptomycin and neomycin, 85.7% to cefovecin and tetracycline, 76.2% to penicillin G, 47.6% to trimethoprim/sulfamethoxazole and 0% to clindamycin. The species was determined to be Gordonia paraffinivorans by whole genome sequencing for 9 isolates (from 8 farms in 7 districts). These isolates showed the highest similarity to two reference strains from the environment. In all these isolates, we identified genes encoding virulence factors that are very similar to genes encoding virulence factors expressed in Mycobacterium tuberculosis and Mycobacterium smegmatis. However, genome analysis revealed 61 unique genes in all 9 sequenced isolates.

[Direct detection of the brucellosis-causing pathogen--a case report]. / Primý prukaz puvodce maltské horecky--kazuistika.

Bacterial zoonoses still represent a serious medical problem. One of the less frequent but severe zoonoses is brucellosis caused by the bacterium Brucella melitensis. The presented case report describes relapsing imported brucellosis in a young male. In addition to four serological tests, the diagnosis was confirmed by direct detection of the pathogen in blood culture. The isolate of Brucella melitensis was identified using the MALDI-TOF BioTyper method and subsequently also by PCR.

Corrigendum: American Foulbrood in the Czech Republic: ERIC II Genotype of Paenibacillus Larvae Is Prevalent.

[This corrects the article DOI: 10.3389/fvets.2021.698976.].

Oxacillin (Methicillin) Resistant Staphylococci in Domestic Animals in the Czech Republic.

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.

American Foulbrood in the Czech Republic: ERIC II Genotype of Paenibacillus Larvae Is Prevalent.

American foulbrood (AFB) is a dangerous disease of honeybees (Apis mellifera) caused by the spore-forming bacterium Paenibacillus larvae. According to the ERIC (enterobacterial repetitive intergenic consensus) classification, five genotypes are distinguished, i.e., I, II, III, IV, and V, which differ in their virulence and prevalence in colonies. In the Czech Republic, AFB prevalence is monitored by the State Veterinary Administration; however, the occurrence of specific P. larvae genotypes within the country remains unknown. In this study, our aim was to genotype field P. larvae strains collected in the Czech Republic according to the ERIC classification. In total, 102 field isolates from colonies with AFB clinical symptoms were collected from various locations in the Czech Republic, and the PCR genotypization was performed using ERIC primers. We confirmed the presence of both ERIC I and II genotypes, while ERIC III, IV, and V were not detected. The majority of samples (n = 82, 80.4%) were identified as ERIC II, while the ERIC I genotype was confirmed only in 20 samples (19.6%). In contrast to other European countries, the ERIC II genotype is predominant in Czech honeybee colonies. The ERIC I genotype was mostly detected in border regions close to Poland, Slovakia, and Austria.

Melissococcus plutonius Can Be Effectively and Economically Detected Using Hive Debris and Conventional PCR.

European foulbrood (EFB) is an infectious disease of honey bees caused by the bacterium Melissococcus plutonius. A method for DNA isolation and conventional PCR diagnosis was developed using hive debris, which was non-invasively collected on paper sheets placed on the bottom boards of hives. Field trials utilized 23 honey bee colonies with clinically positive symptoms and 21 colonies without symptoms. Bayes statistics were applied to calculate the comparable parameters for EFB diagnostics when using honey, hive debris, or samples of adult bees. The reliability of the conventional PCR was 100% at 6.7 × 103 Colony Forming Unit of M. plutonius in 1 g of debris. The sensitivity of the method for the sampled honey, hive debris, and adult bees was 0.867, 0.714, and 1.000, respectively. The specificity for the tested matrices was 0.842, 0.800, and 0.833. The predictive values for the positive tests from selected populations with 52% prevalence were 0.813, 0.833, and 0.842, and the real accuracies were 0.853, 0.750, and 0.912, for the honey, hive debris, and adult bees, respectively. It was concluded that hive debris can effectively be utilized to non-invasively monitor EFB in honey bee colonies.

New sequence type ST3756 of Staphylococcus aureus subspecies anaerobius as the causative agent of abscessing lymphadenitis in sheep.

Morel's disease is a form of abscessing lymphadenitis of sheep and goats caused by Staphylococcus aureus subspecies anaerobius. In Europe and Africa, the disease is linked to S. aureus of multilocus sequence type 1464. In an outbreak recorded in 2015 in a flock of 530 animals in the district of Nymburk, Czech Republic, Europe, the causative agent was cultured and subsequently confirmed by Maldi-TOF. Neither antibiotic therapy nor surgical interventions met any success, although the strain isolated was found to be sensitive to antibiotics used. Vaccination and revaccination with inactivated autogenous vaccine administered subcutaneously was relatively successful. Subsequent multilocus sequence typing revealed the presence of new S. aureus sequence type 3756, different from 1464 in three out of seven genes typed. The isolate thus represents a new sequence type of Staphylococcus aureus ssp. anaerobius which should be considered as a causative agent of Morel's disease.

Revising the taxonomy of the Acinetobacter lwoffii group: The description of Acinetobacter pseudolwoffii sp. nov. and emended description of Acinetobacter lwoffii.

In 1986, Bouvet and Grimont delineated two related taxa of the genus Acinetobacter termed genospecies (GS) 8 and 9. They proposed the name Acinetobacter lwoffii for GS8, which included the supposed type strain (CIP 64.10). As the authenticity of CIP 64.10 was later questioned, this study aimed at reassessing the taxonomy of these genospecies. We investigated 52 strains of GS8 or GS9, including CIP 64.10 and the genuine type strain of A. lwoffii (NCTC 5866T). All strains were subjected to the genus-wide comparative analyses of MALDI-TOF whole-cell mass spectra, rpoB gene sequences and metabolic traits while whole-genome sequences were analysed for 16 strains. The strains were classified into two distinct groups corresponding to GS8 (n=15) and GS9 (n=37). CIP 64.10 fell within GS8 whereas NCTC 5866T belonged to GS9. Intraspecies ANIb values for the genomes of GS8 (n=6) and GS9 (n=10) were ≥96.1% and ≥95.4%, respectively, whereas the ANIb values between them were 86.8-88.6%. Based on core genome phylogeny, GS8 and GS9 formed a distinct clade within the genus, with two respective, strongly supported subclades. GS8 and GS9 were similar in physiological and catabolic properties but were separable by MALDI-TOF MS. We conclude that the name A. lwoffii pertains to GS9 and not to GS8 as originally assumed and that these groups represent two species. We propose the name Acinetobacter pseudolwoffii sp. nov. for GS8, with ANC 5044T (=CCM 8638T=CCUG 67963T=CIP 111642T) as the type strain, and provide the emended description of A. lwoffii.