Occurrence and molecular characterization of Escherichia coli strains isolated from black grouse (Lyrurus tetrix) from the Karkonosze National Park in Poland.

The purpose of this study was to characterize Escherichia coli (E. coli) strains isolated from wild black grouse (Lyrurus tetrix), carried out due to the crossing of hiking trails with wild bird habitats from the Karkonosze National Park. Twenty-seven E. coli isolates were obtained from fecal samples collected during the winter months of 2017 and 2018. The strains were assigned to their relevant phylo-groups and the prevalence of virulence genes characteristic of APEC strains (irp2, astA, iss, iucD, papC, tsh, vat, cva/cvi, stx2f) was checked using PCR analysis. In addition, the phenotypic and genotypic resistance to antibiotics was determined. The entire study provided a better understanding of the potential bacteriological threat to wild birds of the Karkonosze National Park. The results showed that 55.6% of the strains belonged to phylo-group B1 (15/27), 33.3% to group B2 (9/27) and 11.1% to group D (3/27). Among the virulence genes tested, irp2 was detected in 25.9% of isolates (7/27), vat in 22.2% (6/27) and iucD in 3.7% (1/27). The tested E. coli strains showed susceptibility to most antimicrobials, only 14 (51.9%) of them were intermediate resistant or resistant to sulfamethoxazole. The presence of none of the tested genes responsible for resistance to selected antibiotics was identified. Our research indicates a low level of transfer of antimicrobial substances to the natural environment and confirms the effectiveness of the Karkonosze National Park's activities to protect and restore black grouse habitats.

Genetic Evaluation of Bovine Papillomavirus Types Associated with Teat Papillomatosis in Polish Dairy Cattle with the Report of a New Putative Type.

Teat papillomatosis is reported to be one of the factors causing mastitis and milk losses in dairy cattle. Little is known about bovine papillomavirus (BPV) circulation in the European cattle population, and no reports can yet be found about its prevalence in Polish herds. In this study, 177 BPV-like lesions were collected from teats of 109 slaughtered cows. BPV was identified in 39 of the examined animals, using PCR amplification and Sanger dideoxy sequencing. In total, 10 BPV types were isolated, among which the most common were infections caused by types 8 and 7. Macroscopically, "rice-grain" type lesions dominated (76%) and were mainly found on one teat (57.4%). The diversity of BPV types causing teat papillomatosis in Polish cows seems to be large, with nine already known types isolated and a new putative type found. The spread of new types among the worldwide cattle population can be seen for the first time, as type 25 and so called isolates BPV42 and BPV43 were found in the European cattle population.

Isolation and characterization of multidrug resistant Gallibacterium anatis biovar haemolytica strains from Polish geese and hens.

Gallibacterium anatis biovar haemolytica is a bacterium that is frequently associated with infections of the reproductive tract and respiratory system in poultry. To assess the current prevalence and resistance profile of these bacteria in Poland, we collected and investigated 63 strains of Gallibacterium from diseased domestic poultry flocks including geese, laying hens, breeding hens and an ornamental hen. Detailed characterization of the isolates included the analysis of phenotypic antimicrobial resistance profiles and biofilm formation ability. Furthermore, the genetic background of 40 selected isolates regarding the presence of virulence and antimicrobial resistance genes and mobile genetic elements was determined. All investigated isolates were multidrug resistant, most prominently to ß-lactams, fluoroquinolones, sulfonamides and macrolides. A total of 48 different resistance profiles were detected. Of all isolates, 50.8% formed a strong biofilm, where strains isolated from geese appeared to be better at biofilm formation than strains isolated from laying and breeding hens. Single-nucleotide polymorphism genotyping revealed that G. anatis bv. haemolytica strains are restricted in host and geographical distribution, and the geese isolates showed greater phylogenetic similarity. Whole genome sequencing enabled identification of 25 different antimicrobial resistance determinants. The most common resistance genes were tetB, blaROB-1, and blaTEM-1 which may be located on mobile genetic elements. All isolates possessed the toxin gene gtxA, and the fimbrial gene flfA was identified in 95% of strains. Our results indicated that all G. anatis bv. haemolytica isolates showed multidrug resistant phenotypes. Strains isolated from geese were characterized by the highest percentage of isolates resistant to selected antimicrobials, probably reflecting host-related adaptations.

Quinolone Resistance in Gallibacterium anatis Determined by Mutations in Quinolone Resistance-Determining Region.

Control of the important pathogen, Gallibacterium anatis, which causes salpingitis and peritonitis in poultry, relies on treatment using antimicrobial compounds. Among these, quinolones and fluoroquinolones have been used extensively, leading to a rise in the prevalence of resistant strains. The molecular mechanisms leading to quinolone resistance, however, have not previously been described for G. anatis, which is the aim of this study. The present study combines phenotypic antimicrobial resistance data with genomic sequence data from a collection of G. anatis strains isolated from avian hosts between 1979 and 2020. Minimum inhibitory concentrations were determined for nalidixic acid, as well as for enrofloxacin for each included strain. In silico analyses included genome-wide queries for genes known to convey resistance towards quinolones, identification of variable positions in the primary structure of quinolone protein targets and structural prediction models. No resistance genes known to confer resistance to quinolones were identified. Yet, a total of nine positions in the quinolone target protein subunits (GyrA, GyrB, ParC and ParE) displayed substantial variation and were further analyzed. By combining variation patterns with observed resistance patterns, positions 83 and 87 in GyrA, as well as position 88 in ParC, appeared to be linked to increased resistance towards both quinolones included. As no notable differences in tertiary structure were observed between subunits of resistant and sensitive strains, the mechanism behind the observed resistance is likely due to subtle shifts in amino acid side chain properties.

Bacteria Single-Cell and Photosensitizer Interaction Revealed by Quantitative Phase Imaging.

Quantifying changes in bacteria cells in the presence of antibacterial treatment is one of the main challenges facing contemporary medicine; it is a challenge that is relevant for tackling issues pertaining to bacterial biofilm formation that substantially decreases susceptibility to biocidal agents. Three-dimensional label-free imaging and quantitative analysis of bacteria-photosensitizer interactions, crucial for antimicrobial photodynamic therapy, is still limited due to the use of conventional imaging techniques. We present a new method for investigating the alterations in living cells and quantitatively analyzing the process of bacteria photodynamic inactivation. Digital holographic tomography (DHT) was used for in situ examination of the response of Escherichia coli and Staphylococcus aureus to the accumulation of the photosensitizers immobilized in the copolymer revealed by the changes in the 3D refractive index distributions of single cells. Obtained results were confirmed by confocal microscopy and statistical analysis. We demonstrated that DHT enables real-time characterization of the subcellular structures, the biophysical processes, and the induced local changes of the intracellular density in a label-free manner and at sub-micrometer spatial resolution.

Phenotypic and genotypic characterization of mcr-1-positive multidrug-resistant Escherichia coli ST93, ST117, ST156, ST10, and ST744 isolated from poultry in Poland.

BACKGROUND: A plasmid-mediated mechanism of bacterial resistance to polymyxin is a serious threat to public health worldwide. The present study aimed to determine the occurrence of plasmid-mediated colistin resistance genes and to conduct the molecular characterization of mcr-positive Escherichia coli strains isolated from Polish poultry. METHODS: In this study, 318 E. coli strains were characterized by the prevalence of mcr1-mcr5 genes, antimicrobial susceptibility testing by minimal inhibitory concentration method, the presence of antimicrobial resistance genes was screened by PCR, and the biofilm formation ability was tested using the crystal violet staining method. Genetic relatedness of mcr-1-positive E. coli strains was evaluated by multilocus sequence typing method. RESULTS: Among the 318 E. coli isolates, 17 (5.35%) harbored the mcr-1 gene. High antimicrobial resistance rates were observed for ampicillin (100%), tetracycline (88.24%), and chloramphenicol (82.35%). All mcr-1-positive E. coli strains were multidrug-resistant, and as many as 88.24% of the isolates contained the blaTEM gene, tetracycline (tetA and tetB), and sulfonamide (sul1, sul2, and sul3) resistance genes. Additionally, 41.18% of multidrug-resistant, mcr-1-positive E. coli isolates were moderate biofilm producers, while the rest of the strains showed weak biofilm production. Nine different sequence types were identified, and the dominant ST was ST93 (29.41%), followed by ST117 (17.65%), ST156 (11.76%), ST 8979 (11.76%), ST744 (5.88%), and ST10 (5.88%). Moreover, the new ST was identified in this study. CONCLUSIONS: Our results showed a low occurrence of mcr-1-positive E. coli strains isolated from Polish poultry; however, all the isolated strains were resistant to multiple antimicrobial agents and were able to form biofilms at low or medium level.

Myricetin as an Antivirulence Compound Interfering with a Morphological Transformation into Coccoid Forms and Potentiating Activity of Antibiotics against Helicobacter pylori.

Helicobacter pylori, a gastric pathogen associated with a broad range of stomach diseases, has a high tendency to become resistant to antibiotics. One of the most important factors related to therapeutic failures is its ability to change from a spiral to a coccoid form. Therefore, the main aim of our original article was to determine the influence of myricetin, a natural compound with an antivirulence action, on the morphological transformation of H. pylori and check the potential of myricetin to increase the activity of antibiotics against this pathogen. We observed that sub-minimal inhibitory concentrations (sub-MICs) of this compound have the ability to slow down the process of transformation into coccoid forms and reduce biofilm formation of this bacterium. Using checkerboard assays, we noticed that the exposure of H. pylori to sub-MICs of myricetin enabled a 4-16-fold reduction in MICs of all classically used antibiotics (amoxicillin, clarithromycin, tetracycline, metronidazole, and levofloxacin). Additionally, RT-qPCR studies of genes related to the H. pylori morphogenesis showed a decrease in their expression during exposure to myricetin. This inhibitory effect was more strongly seen for genes involved in the muropeptide monomers shortening (csd3, csd6, csd4, and amiA), suggesting their significant participation in the spiral-to-coccoid transition. To our knowledge, this is the first research showing the ability of any compound to synergistically interact with all five antibiotics against H. pylori and the first one showing the capacity of a natural substance to interfere with the morphological transition of H. pylori from spiral to coccoid forms.

Photolon Nanoporous Photoactive Material with Antibacterial Activity and Label-Free Noncontact Method for Free Radical Detection.

The worldwide increase in bacterial resistance and healthcare-associated bacterial infections pose a serious threat to human health. The antimicrobial photodynamic method reveals the opportunity for a new therapeutic approach that is based on the limited delivery of photosensitizer from the material surface. Nanoporous inorganic-organic composites were obtained by entrapment of photosensitizer Photolon in polysiloxanes that was prepared by the sol-gel method. The material was characterized by its porosity, optical properties (fluorescence and absorbance), and laser-induced antimicrobial activity against Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. The permanent encapsulation of Photolon in the silica coating and the antimicrobial efficiency was confirmed by confocal microscope and digital holotomography. The generation of free radicals from nanoporous surfaces was proved by scanning Kelvin probe microscopy. For the first time, it was confirmed that Kelvin probe microscopy can be a label-free, noncontact alternative to other conventional methods based on fluorescence or chemiluminescence probes, etc. It was confirmed that the proposed photoactive coating enables the antibacterial photodynamic effect based on free radicals released from the surface of the coating. The highest bactericidal efficiency of the proposed coating was 87.16%. This coating can selectively limit the multiplication of bacterial cells, while protecting the environment and reducing the risk of surface contamination.

On the application of multi-parametric optical phenotyping of bacterial colonies for multipurpose microbiological diagnostics.

The development of new diagnostics techniques and modalities is critical for early detection of microbial contamination. In this study, the novel integrated system for multi-parametric optical phenotyping and characterization of bacterial colonies, is presented. The system combines Mach-Zehnder interferometer with a spectral imaging system for capturing multispectral diffraction patterns and multispectral two-dimensional transmission maps of bacterial colonies, along with the simultaneous interferometric profilometry. The herein presented investigation was carried out on five representative bacteria species and nearly 3000 registered multispectral optical signatures. The interferograms were analyzed by four-step phase shift algorithm to reconstruct the colony profile to enable the obtaining of the comparable optical signatures. The dedicated image processing algorithms were used for extraction of quantitative features of these signatures. The random forest algorithm was applied for selection of the most predictive set of features, which were used in classification model based on Support-Vector Machine. Obtained results have shown that the use of multiple multispectral optical signatures provide a multi-parametric bacteria identification at an exceptionally high accuracy (99.4-100%), significantly better than in case of classification based on each of these signatures (multispectral diffraction patterns, two-dimensional transmission coefficient maps), separately. Obtained results revealed that analysis of multispectral signatures can also be applied for characterisation of physical, physicochemical and chemical properties of the bacterial colonies in the presence of the antimicrobial factors. Therefore, the proposed label-free, non-destructive optical technique has perspectives to be exploited in the multipurpose diagnostics and it can be used as a pre-screening tool in microbiological laboratories.

Development of the Correction Algorithm to Limit the Deformation of Bacterial Colonies Diffraction Patterns Caused by Misalignment and Its Impact on the Bacteria Identification in the Proposed Optical Biosensor.

Recently proposed methods of bacteria identification in optical biosensors based on the phenomenon of light diffraction on macro-colonies offer over 98% classification accuracy. However, such high accuracy relies on the comparable and repeatable spatial intensity distribution of diffraction patterns. Therefore, it is essential to eliminate all non-species/strain-dependent factors affecting the diffraction patterns. In this study, the impact of the bacterial colony and illuminating beam misalignment on the variation of classification features extracted from diffraction patterns was examined. It was demonstrated that misalignment introduced by the scanning module significantly affected diffraction patterns and extracted classification features used for bacteria identification. Therefore, it is a crucial system-dependent factor limiting the identification accuracy. The acceptable misalignment level, when the accuracy and quality of the classification features are not affected, was determined as no greater than 50 µm. Obtained results led to development of image-processing algorithms for determination of the direction of misalignment and concurrent alignment of the bacterial colonies' diffraction patterns. The proposed algorithms enable the rigorous monitoring and controlling of the measurement's conditions in order to preserve the high accuracy of bacteria identification.