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Background In order to start to understand the function of individual members of gut microbiota, we cultured, sequenced and analysed bacterial anaerobes from chicken caecum. Results Altogether 204 isolates from chicken caecum were obtained in pure cultures using Wilkins-Chalgren anaerobe agar and anaerobic growth conditions. Genomes of all the isolates were determined using the NextSeq platform and subjected to bioinformatic analysis. Among 204 sequenced isolates we identified 133 different strains belonging to seven different phyla - Firmicutes , Bacteroidetes , Actinobacteria , Proteobacteria , Verrucomicrobia , Elusimicrobia and Synergistetes . Genome sizes ranged from 1.51 Mb in Elusimicrobium minutum to 6.70 Mb in Bacteroides ovatus . Clustering based on the presence of protein coding genes showed that isolates from phyla Proteobacteria , Verrucomicrobia , Elusimicrobia and Synergistetes did not cluster with the remaining isolates. Firmicutes split into families Lactobacillaceae , Enterococcaceae , Veillonellaceae and order Clostridiales from which the Clostridium perfringens isolates formed a distinct sub-cluster. All Bacteroidetes isolates formed a separate cluster showing similar genetic composition in all isolates but distinct from the rest of the gut anaerobes. The majority of Actinobacteria clustered closely together except for the representatives of genus Gordonibacter showing that the genome of this genus differs from the rest of Actinobacteria sequenced in this study. Representatives of Bacteroidetes commonly encoded proteins (collagenase, hemagglutinin, hemolysin, hyaluronidase, heparinases, chondroitinase, mucin-desulfating sulfatase or glutamate decarboxylase) that may enable them to interact with their host. Aerotolerance was recorded in Akkermansia and Cloacibacillus and was also common among representatives of Bacteroidetes . On the other hand, Elusimicrobium and the majority of Clostridiales were highly sensitive to air exposure despite their potential for spore formation. Conclusions Major gut microbiota members utilise different strategies for gut colonisation. High oxygen sensitivity of Firmicutes may explain their commonly reported decrease after oxidative burst during gut inflammation.

Chickens in commercial production are hatched in a clean hatchery environment in the absence of any contact with adult hens. However, Gallus gallus evolved to be hatched in a nest in contact with an adult hen which may act as a donor of gut microbiota. In this study, we therefore addressed the issue of microbiota development in newly hatched chickens with or without contact with an adult hen. We found that a mere 24-hour-long contact between a hen and newly hatched chickens was long enough for transfer of hen gut microbiota to chickens. Hens were efficient donors of Bacteroidetes and Actinobacteria. However, except for genus Faecalibacterium and bacterial species belonging to class Negativicutes, hens did not act as an important source of Gram-positive Firmicutes. Though common to the chicken intestinal tract, Lactobacilli and isolates from families Erysipelotrichaceae, Lachnospiraceae and Ruminococcaceae therefore originated from environmental sources instead of from the hens. These observation may have considerable consequences for the evidence-based design of the new generation of probiotics for poultry.

A series of new unique acetylene derivatives of 8-hydroxy- and 8-methoxyquinoline- 5-sulfonamide 3a–f and 6a–f were prepared by reactions of 8-hydroxy- and 8-methoxyquinoline- 5-sulfonyl chlorides with acetylene derivatives of amine. A series of new hybrid systems containing quinoline and 1,2,3-triazole systems 7a–h were obtained by reactions of acetylene derivatives of quinoline-5-sulfonamide 6a–d with organic azides. The structures of the obtained compounds were confirmed by 1H and 13C NMR spectroscopy and HR-MS spectrometry. The obtained quinoline derivatives 3a–f and 6a–f and 1,2,3-triazole derivatives 7a–h were tested for their anticancer and antimicrobial activity. Human amelanotic melanoma cells (C-32), human breast adenocarcinoma cells (MDA-MB-231), and human lung adenocarcinoma cells (A549) were selected as tested cancer lines, while cytotoxicity was investigated on normal human dermal fibroblasts (HFF-1). All the compounds were also tested against reference strains Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 and representatives of multidrug-resistant clinical isolates of methicillin-resistant S. aureus (MRSA) and vancomycin-resistant E. faecalis. Only the acetylene derivatives of 8-hydroxyquinoline-5-sulfonamide 3a–f were shown to be biologically active, and 8-hydroxy-N-methyl-N-(prop-2-yn-1-yl)quinoline-5-sulfonamide (3c) showed the highest activity against all three cancer lines and MRSA isolates. Its efficacies were comparable to those of cisplatin/doxorubicin and oxacillin/ciprofloxacin. In the non-cancer HFF-1 line, the compound showed no toxicity up to an IC50 of 100 µM. In additional tests, compound 3c decreased the expression of H3, increased the transcriptional activity of cell cycle regulators (P53 and P21 proteins), and altered the expression of BCL-2 and BAX genes in all cancer lines. The unsubstituted phenolic group at position 8 of the quinoline is the key structural fragment necessary for biological activity.

Flavobacterium psychrophilum is currently one of the most important pathogens in aquaculture worldwide, causing high losses to farmed salmonids particularly during early growth stages with significant economic impact. Despite previous attempts, no effective vaccine has been developed, and protection against introduction into farms is difficult due to the ubiquitous occurrence of the pathogen. A better understanding of the mechanism of disease development is essential for targeted therapeutic and preventive measures in farms. Unfortunately, the pathogenesis of diseases caused by F. psychrophilum has not been elucidated yet. Previously, several putative virulence factors have been identified. Some appear to be essential for disease development, while others are probably dispensable. The importance of some factors has not yet been explored. This review focuses on the supposed virulence factors of F. psychrophilum and the current knowledge about their importance in the pathogenesis of the disease.

Background The salmonid pathogen Flavobacterium psychrophilum poses a significant economic threat to global aquaculture, yet our understanding of its genetic and phenotypic diversity remains incomplete across much of its geographic range. In this study, we characterise the genetic and phenotypic diversity of 70 isolates collected from rainbow trout ( Oncorhynchus mykiss ) and brown trout ( Salmo trutta m. fario ) from fish farms in the Czech Republic between 2012 and 2019 to compare their genomic content with all draft or complete genomes present in the NCBI database ( n  = 187). Results The Czech isolates underwent comprehensive evaluation, including multiplex PCR-based serotyping, genetic analysis, antimicrobial resistance testing, and assessment of selected virulence factors. Multiplex PCR serotyping revealed 43 isolates as Type 1, 23 as Type 2, with sporadic cases of Types 3 and 4. Multi-locus sequence typing unveiled 12 sequence types (ST), including seven newly described ones. Notably, 24 isolates were identified as ST329, a novel sequence type, while 22 were classified as the globally-distributed ST2. Phylogenetic analysis demonstrated clonal distribution of ST329 in the Czech Republic, with these isolates lacking a phage sequence in their genomes. Antimicrobial susceptibility testing revealed a high proportion of isolates classified as non-wild type with reduced susceptibility to oxolinic acid, oxytetracycline, flumequine, and enrofloxacin, while most isolates were classified as wild type for florfenicol, sulfamethoxazole-trimethoprim, and erythromycin. However, 31 isolates classified as wild type for florfenicol exhibited minimum inhibitory concentrations at the susceptibility breakpoint. Conclusion The prevalence of the Czech F. psychrophilum serotypes has evolved over time, likely influenced by the introduction of new isolates through international trade. Thus, it is crucial to monitor F. psychrophilum clones within and across countries using advanced methods such as MLST, serotyping, and genome sequencing. Given the open nature of the pan-genome, further sequencing of strains promises exciting discoveries in F. psychrophilum genomics.

International trade with ornamental fish is gradually recognized as an important source of a wide range of different antibiotic resistant bacteria. In this study we therefore characterized the prevalence of selected antibiotic resistance genes in the microbiota found in the carriage water of ornamental fish originating from 3 different continents. Real-time PCR quantification showed that the sul1 gene was present in 11 out of 100 bacteria. tet(A) was present in 6 out of 100 bacteria and strA, tet(G), sul2 and aadA were present in 1-2 copies per 100 bacteria. Class I integrons were quite common in carriage water microbiota, however, pyrosequencing showed that only 12 different antibiotic gene cassettes were present in class I integrons. The microbiota characterized by pyrosequencing of the V3/V4 variable region of 16S rRNA genes consisted of Proteobacteria (48%), Bacteroidetes (29.5%), Firmicutes (17.8%), Actinobacteria (2.1%) and Fusobacteria (1.6%). Correlation analysis between antibiotic resistance gene prevalence and microbiota composition verified by bacterial culture showed that major reservoirs of sul1 sul2, tet(A), tet(B) tet(G), cat, cml, bla, strA, aacA, aph and aadA could be found among Alpha-, Beta- and Gammaproteobacteria with representatives of Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae and Comamonadaceae being those most positively associated with the tested antibiotic resistance genes.

IntroductionAntimicrobial resistance (AMR) represents a growing One Health challenge at the human–animal–environment interface. Wildlife rescue centres may represent potential, underrecognized settings where resistant bacteria could emerge and disseminate due to close human–animal contact and antimicrobial use. We investigated AMR profiles and genomic features of Escherichia coli isolated from European and northern white-breasted hedgehogs (Erinaceus europaeus, Erinaceus roumanicus) admitted to a Czech wildlife rescue facility.Materials and methodsFaeces from 23 hedgehogs were collected during routine pen cleaning. E. coli isolates were obtained on MacConkey agar (MCA) and MCA with cefotaxime and confirmed by MALDI-TOF MS. Antimicrobial susceptibility to 13 antibiotics was assessed using the disc diffusion test. A subset of 26 isolates representing diverse resistance profiles was further characterised by whole-genome sequencing (WGS). Genomic analyses focused on sequence types, phylogroups, resistance genes, plasmid replicons, and virulence-associated genes.Results and discussionMore than half of the isolates (37/69; 54%) were multidrug-resistant, with resistance most frequently observed to ampicillin and nalidixic acid. No cefotaxime-resistant isolates or genes encoding extended-spectrum beta-lactamases or carbapenemases were detected. Whole-genome sequencing revealed substantial genetic diversity, including several sequence types that are commonly associated with human and animal infections, such as ST457, ST162, and ST624. Isolates carried a wide range of resistance determinants, including blaTEM-1 and qnrB2 genes, plasmid replicons, and virulence-associated genes, with phylogroup F showing the highest virulence gene content. Despite the modest sample size, our findings indicate that hedgehogs in rehabilitation settings can act as reservoirs of multidrug-resistant E. coli with diverse genomic backgrounds, contributing to the environmental dissemination of AMR. The presence of sequence types and resistance genes commonly associated with human and veterinary infections further supports the relevance of rehabilitated wildlife to the broader epidemiology of AMR. Enhancing biosafety practices and antimicrobial stewardship in wildlife rescue operations is therefore essential to mitigate zoonotic risks within a One Health framework.

The increasing prevalence of antimicrobial resistance in livestock, particularly the dissemination of extended-spectrum beta-lactamase-producing , poses a significant zoonotic and public health risk. This study investigates the genomic characteristics of cefotaxime-resistant isolates from dairy calves across 23 Czech farms and their caretakers. Bacteriological cultivation on McConkey agar with cefotaxime was used for their isolation, susceptibility to selected antibiotics was determined by disc diffusion method, production of extended-spectrum beta-lactamases (ESBL) was demonstrated by double disc synergy test. The PCR was applied to confirm the presence of selected genes encoding resistance to some beta-lactams and genes encoding resistance to quinolones carried on plasmids. Using whole-genome sequencing, we evaluated resistance genotypes, sequence types, serotypes, plasmid replicons, and virulence genes. Among 266 rectal samples obtained from the calves, 128 (48%) harbored cefotaxime-resistant . Whole-genome analysis revealed genes in 91% (116/128) of isolates, with (44%) and (34%) being the dominant variants. Other beta-lactamase gene was found in 40% (51/128) of isolates. Notably, no cephamycin resistance genes have been identified. The plasmid-mediated quinolone resistance (PMQR) gene was present at 21% (27/128) of isolates. The colistin resistance gene was found in a single ST2325 isolate. Sequence typing revealed significant clonal diversity, with 21 different STs detected among 68 sequenced isolates. ST10 was the most prevalent (27%), followed by ST69 (12%), ST29 (7%) and others. The phylogenetic distribution showed a predominance of commensal groups A (54%) and B1 (21%). The most common serotypes included O101:H9 (21%), O15:H18 (12%), H12, and O70:H11 (7%). Analysis of plasmid content revealed a complex distribution of 18 distinct plasmid replicon types, especially IncF, followed by Col-type and IncI1-type plasmids. Cross-species transmission was indicated by the detection of clonal strains shared between calves and caretakers, notably ST10-O101:H9 and ST34-O68:H30. The prevalence of high-risk clones and the presence of mobile resistance elements underscore the urgent need for stringent monitoring, antimicrobial stewardship, and improved biosecurity measures in livestock environments like increased caution and personal hygiene of animal handlers to mitigate the spread of resistant between animals and humans.

This study evaluated the carriage of AmpC and extended-spectrum beta-lactamase (ESBL) genes and associated plasmids in faecal bacteria of Canadian corvids. Faecal samples from 449 birds in five roosting sites across Canada were analyzed using selective media, screening for AmpC and ESBL genes by PCR, and sequencing. Genomic relatedness was determined by PFGE and MLST. Plasmid mobility was studied by conjugation and transformation experiments, followed by plasmid typing. In total, 96 (21%, n = 449) cefotaxime-resistant Escherichia coli and three (0.7%) Klebsiella pneumoniae isolates were identified. ESBL genes blaCTX-M-1 (n = 3), blaCTX-M-14 (n = 2), blaCTX-M-32 (n = 2) and blaCTX-M-124 (n = 1) were detected in eight E. coli isolates, whereas blaSHV-2 (2) was found in two K. pneumoniae. E. coli isolates contained blaCMY-2 (n = 83) and blaCMY-42 (n = 1). The high genetic diversity of the isolates and presence of clinically important E. coli ST69 (n = 1), ST117 (n = 7) and ST131 (n = 1) was revealed. AmpC genes were predominantly carried by plasmids of incompatibility groups I1 (45 plasmids), A/C (10) and K (7). The plasmid IncI1/ST12 was most common and found in diverse E. coli STs in all sites. Highly diverse E. coli isolates containing AmpC and ESBL genes, including clinically important clones and emerging plasmids, are in circulation throughout Canadian wildlife.

Epidemiological data show that the composition of gut microbiota influences host health, disease status, and even behaviour. However, to confirm these epidemiological observations in controlled experiments, pure cultures of gut anaerobes must be obtained. Since the culture of gut anaerobes is not a simple task due to the large number of bacterial species colonising the intestinal tract, in this study we inoculated 174 different culture media with caecal content from adult hens, and compared the microbiota composition in the original caecal samples and in bacterial masses growing in vitro by 16S rRNA sequencing. In total, 42% of gut microbiota members could be grown in vitro and since there were some species which were not cultured but for which the culture conditions are known, it is likely that more than half of chicken gut microbiota can be grown in vitro. However, there were two lineages of Clostridiales and a single lineage of Bacteroidetes which were common in chicken caecal microbiota but resistant to culture. Of the most selective culture conditions, nutrient broths supplemented with mono- or di-saccharides, including those present in fruits, positively selected for Lactobacillaceae. The addition of bile salts selected for Veillonellaceae and YCFA (yeast casitone fatty acid agar) enriched for Desulfovibrionaceae. In addition, Erysipelotrichaceae were positively selected by colistin, trimethoprim, streptomycin and nalidixic acid. Culture conditions tested in this study can be used for the selective enrichment of desired bacterial species but also point towards the specific functions of individual gut microbiota members.