Oxolane Ammonium Salts (Muscarine-Like)-Synthesis and Microbiological Activity.

Commercially available 2-deoxy-D-ribose was used to synthesize the appropriate oxolane derivative-(2R,3S)-2-(hydroxymethyl)oxolan-3-ol-by reduction and dehydration/cyclization in an acidic aqueous solution. Its monotosyl derivative, as a result of the quaternization reaction, allowed us to obtain eight new muscarine-type derivatives containing a quaternary nitrogen atom and a hydroxyl group linked to the oxolane ring. Their structure was fully confirmed by the results of NMR, MS and IR analyses. The crystal structure of the pyridinium derivative showed a high similarity of the conformation of the oxolane ring to previously published crystal structures of muscarine. Two reference strains of Gram-negative bacteria (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853), two reference strains of Gram-positive staphylococci (Staphylococcus aureus ATCC 25923 and Staphylococcus aureus ATCC 29213) and four reference strains of pathogenic yeasts of the genus Candida spp. (Candida albicans SC5314, Candida glabrata DSM 11226, Candida krusei DSM 6128 and Candida parapsilosis DSM 5784) were selected for the evaluation of the antimicrobial potential of the synthesized compounds. The derivative containing the longest (decyl) chain attached to the quaternary nitrogen atom turned out to be the most active.

In Silico Safety Assessment of Bacillus Isolated from Polish Bee Pollen and Bee Bread as Novel Probiotic Candidates.

Bacillus species isolated from Polish bee pollen (BP) and bee bread (BB) were characterized for in silico probiotic and safety attributes. A probiogenomics approach was used, and in-depth genomic analysis was performed using a wide array of bioinformatics tools to investigate the presence of virulence and antibiotic resistance properties, mobile genetic elements, and secondary metabolites. Functional annotation and Carbohydrate-Active enZYmes (CAZYme) profiling revealed the presence of genes and a repertoire of probiotics properties promoting enzymes. The isolates BB10.1, BP20.15 (isolated from bee bread), and PY2.3 (isolated from bee pollen) genome mining revealed the presence of several genes encoding acid, heat, cold, and other stress tolerance mechanisms, adhesion proteins required to survive and colonize harsh gastrointestinal environments, enzymes involved in the metabolism of dietary molecules, antioxidant activity, and genes associated with the synthesis of vitamins. In addition, genes responsible for the production of biogenic amines (BAs) and D-/L-lactate, hemolytic activity, and other toxic compounds were also analyzed. Pan-genome analyses were performed with 180 Bacillus subtilis and 204 Bacillus velezensis genomes to mine for any novel genes present in the genomes of our isolates. Moreover, all three isolates also consisted of gene clusters encoding secondary metabolites.

Probiotic potential of Bacillus Isolates from Polish Bee Pollen and Bee Bread.

The main goal of this study was the evaluation of the probiotic potential of 10 Bacillus spp. strains isolated from 5 bee bread and 3 bee pollen samples. The antagonistic interaction with Staphylococcus aureus and Escherichia coli was a primary criterion for the preliminary selection of the isolates. Three out of ten strains-PY2.3 (isolated from pollen), BP20.15 and BB10.1 (both isolated from bee bread)-were found to be possible probiotic strains. All these strains are safe for humans (exhibiting [Formula: see text]-hemolytic activity) and meet all essential requirements for probiotics in terms of viability in the presence of bile salts and acid conditions, hydrophobicity, auto-aggregation, and co-aggregation with the cells of important human pathogenic bacteria. They also assimilate more than 30% of cholesterol after 24 h of incubation. These three isolates are resistant to penicillin but sensitive (or exhibit moderate resistance) to the other nine antibiotics tested herein. On the basis of whole-genome sequencing, BP20.15 and BB10.1 were classified as B. subtilis and PY2.3 as B. velezensis. Moreover, genomic analyses revealed that all these isolates are potential producers of different antimicrobial compounds, including bacteriocins and secondary metabolites. The outcomes of this study have proven that some of the Bacillus strains isolated from bee pollen or bee bread are potential probiotics.

Whole-genome sequencing and antimicrobial potential of bacteria isolated from Polish honey.

The aim of this study was the whole-genome analysis and assessment of the antimicrobial potential of bacterial isolates from honey harvested in one geographical location-the north of Poland. In total, 132 strains were derived from three honey samples, and the antimicrobial activity of CFAM (cell-free after-culture medium) was used as a criterion for strain selection and detailed genomic investigation. Two of the tested isolates (SZA14 and SZA16) were classified as Bacillus paralicheniformis, and one isolate (SZB3) as Bacillus subtilis based on their ANI and phylogenetic analysis relatedness. The isolates SZA14 and SZA16 were harvested from the same honey sample with a nucleotide identity of 98.96%. All three isolates have been found to be potential producers of different antimicrobial compounds. The secondary metabolite genome mining pipeline (antiSMASH) identified 14 gene cluster coding for non-ribosomal peptide synthetases (NRPs), polyketide synthases (PKSs), and ribosomally synthesized and post-translationally modified peptides (RiPPs) that are potential sources of novel antibacterials. The BAGEL4 analysis revealed the presence of nine putative gene clusters of interest in the isolates SZA14 and SZA16 (including the presence of six similar clusters present in both isolates, coding for the production of enterocin Nkr-5-3B, haloduracin-alpha, sonorensin, bottromycin, comX2, and lasso peptide), and four in B. subtilis isolate SZB3 (competence factor, sporulation-killing factor, subtilosin A, and sactipeptides). The outcomes of this study confirm that honey-derived Bacillus spp. strains can be considered potential producers of a broad spectrum of antimicrobial agents. KEY POINTS: • Bacteria of the genus Bacillus are an important component of honey microbiota. • Honey-derived Bacillus spp. strains are potential producers of new antimicrobials.

Hydrazinolysis Products of Selected Sugar Lactones-Crystal Structure and Microbiological Activity.

Commercially available lactones, as well as those synthesized by us, turned out to be good substrates for the synthesis of sugar hydrazides. The exception was L-ascorbic acid, whose hydrazinolysis led to the formation of a hydrazinium salt, not the hydrazide as expected. The structure of all compounds was confirmed by NMR and X-ray analyses. The lower durability of hydrazinium L-ascorbate was additionally confirmed by thermogravimetric tests. All products were tested for biological activity against Gram-negative bacteria strains Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 and against Gram-positive Staphylococcus aureus ATCC 25923 and Staphylococcus aureus ATCC 29213. Their antifungal activity against Candida albicans SC5314, Candida glabrata DSM 11226 SM 11226, Candida krusei DSM 6128, and Candida parapsilosis DSM 5784 was also tested. The most interesting results of microbiological activity were obtained for D-gluconic acid hydrazide and hydrazinium L-ascorbate. The results of the latter encourage more extensive testing.

Synthesis, Antimicrobial and Mutagenic Activity of a New Class of d-Xylopyranosides.

Eight N-[2-(2',3',4'-tri-O-acetyl-α/ß-d-xylopyranosyloxy)ethyl]ammonium bromides, a new class of d-xylopyranosides containing a quaternary ammonium aglycone, were obtained. Their complete structure was confirmed using NMR spectroscopy (1H, 13C, COSY and HSQC) and high-resolution mass spectrometry (HRMS). An antimicrobial activity against fungi (Candida albicans, Candida glabrata) and bacteria (Staphylococcus aureus, Escherichia coli) and a mutagenic Ames test with Salmonella typhimurium TA 98 strain were performed for the obtained compounds. The greatest activity against the tested microorganisms was shown by glycosides with the longest (octyl) hydrocarbon chain in ammonium salt. None of the tested compounds exhibited mutagenic activity in the Ames test.

Virulence analysis and antibiotic resistance of Klebsiella pneumoniae isolates from hospitalised patients in Poland.

Klebsiella pneumoniae (KP) is a nosocomial pathogen causing difficult-to-treat infections. The presence of virulence genes and antibiotic resistance of 109 KP isolates from hospitalized patients were investigated. Among them, 68.8% were multi-drug resistant (MDR) and 59.6% produced extended-spectrum beta-lactamases (ESBLs). Metallo-ß-lactamases (MBLs) were produced by 22% of isolates (mainly from anus), including 16.5% of isolates producing New Delhi metallo-ß-lactamase (NDM-1). The genes encoding adhesins (fimH-91.7%, mrkD-96.3%), enterobactin (entB-100%) and yersiniabactin (irp-1-88%) were frequently identified. The genes encoding salmochelin (iroD-9.2%, iroN-7.3%) and colibactin (clbA, clbB-0.9%) were identified rarely. Iron acquisition system-related kfu gene and wcaG gene involved in capsule production were identified in 6.4% and 11% of isolates, respectively. The rmpA gene associated with hypermucoviscosity was present in 6.4% of isolates. In 19.2% of isolates magA gene was detected, specific for K1 capsule serotype, while 22.9% of isolates showed K2 capsule serotype. The rmpA, iroD or iroN genes being diagnostic biomarkers for hypervirulent KP (hvKP) were detected in 16.5% of isolates. We found that 55.5% of hvKP were MDR and produced ESBLs, thus hospital KP isolates pose a serious threat to the healthcare system.

Fatty acids as molecular carriers in cleavable antifungal conjugates.

Conjugates composed of C2-18 fatty acid (FA) residues as a molecular carrier and 5-fluorocytosine (5-FC) as an active agent, released upon the action of intracellular esterases on the ester bond between FA and "trimethyl lock" intramolecular linker, demonstrate good in vitro activity against human pathogenic yeasts of Candida spp. The minimal inhibitory concentrations (MIC) values for the most active conjugates containing caprylic (C8), capric (C10), lauric (C12), or myristic (C14) acid residues were in the 2-64 µg mL-1 range, except for these against the least susceptible Candida krusei. The least active conjugates containing C2, C16, or C18 FA were slowly hydrolyzed by esterase and probably poorly taken up by Candida cells, as found for their analogs containing a fluorescent label, Nap-NH2 instead of 5-FC.

New Peptide Based Fluconazole Conjugates with Expanded Molecular Targets.

Infections of Candida spp. etiology are frequently treated with azole drugs. Among azoles, the most widely used in the clinical scenario remains fluconazole (FLC). Promising results in treatment of dangerous, systemic Candida infections demonstrate the advantages of combined therapies carried out with combinations of at least two different antifungal agents. Here, we report five conjugates composed of covalently linked FLC and cell penetrating or antimicrobial peptide: TP10-7-NH2, TP10-NH2, LFcinB(2-11)-NH2, LFcinB[Nle1,11]-NH2, and HLopt2-NH2, with aspects of design, chemical synthesis and their biological activities. Two of these compounds, namely FLCpOH-TP10-NH2 and FLCpOH-TP10-7-NH2, exhibit high activity against reference strains and fluconazole-resistant clinical isolates of C. albicans, including strains overproducing drug transporters. Moreover, both of them demonstrate higher fungicidal effects compared to fluconazole. Analysis performed with fluorescence and scanning electron microscopy as well as flow cytometry indicated the cell membrane as a molecular target of synthesized conjugates. An important advantage of FLCpOH-TP10-NH2 and FLCpOH-TP10-7-NH2 is their low cytotoxicity. The IC90 value for the human cells after 72 h treatment was comparable to the MIC50 value after 24 h treatment for most strains of C. albicans. In reported conjugates, FLC was linked to the peptide by its hydroxyl group. It is worth noting that conjugation of FLC by the nitrogen atom of the triazole ring led to practically inactive compounds. Two compounds produced by us and reported herein appear to be potential candidates for novel antifungal agents.

Antimicrobial, Cytotoxic and Mutagenic Activity of Gemini QAS Derivatives of 1,4:3,6-Dianhydro-l-iditol.

A series of quaternary diammonium salts derivatives of 1,4:3,6-dianhydro-l-iditol were synthesized, using isommanide (1,4:3,6-dianhydro-d-mannitol) as a starting material. Both aromatic (pyridine, 4-(N,N-dimethylamino)pyridine (DMAP), (3-carboxamide)pyridine; N-methylimidazole) and aliphatic (trimethylamine, N,N-dimethylhexylamine, N,N-dimethyloctylamine, N,N-dimethyldecylamine) amines were used, giving eight gemini quaternary ammonium salts (QAS). All salts were tested for their antimicrobial activity against yeasts, Candida albicans and Candida glabrata, as well as bacterial Staphylococcus aureus and Escherichia coli reference strains. Moreover, antibacterial activity against 20 isolates of S. aureus collected from patients with skin and soft tissue infections (n = 8) and strains derived from subclinical bovine mastitis milk samples (n = 12) were evaluated. Two QAS with octyl and decyl residues exhibited antimicrobial activity, whereas those with two decyl residues proved to be the most active against the tested pathogens, with MIC of 16-32, 32, and 8 µg/mL for yeast, E. coli, and S. aureus reference and clinical strains, respectively. Only QAS with decyl residues proved to be cytotoxic in MTT assay against human keratinocytes (HaCaT), IC50 12.8 ± 1.2 µg/mL. Ames test was used to assess the mutagenic potential of QAS, and none of them showed mutagenic activity in the concentration range 4-2000 µg/plate.

Quest for the Molecular Basis of Improved Selective Toxicity of All-Trans Isomers of Aromatic Heptaene Macrolide Antifungal Antibiotics.

Three aromatic heptaene macrolide antifungal antibiotics, Candicidin D, Partricin A (Gedamycin) and Partricin B (Vacidin) were subjected to controlled cis-trans→ all trans photochemical isomerization. The obtained all-trans isomers demonstrated substantially improved in vitro selective toxicity in the Candida albicans cells: human erythrocytes model. This effect was mainly due to the diminished hemotoxicity. The molecular modeling studies on interactions between original antibiotics and their photoisomers with ergosterol and cholesterol revealed some difference in free energy profiles of formation of binary antibiotic/sterol complexes in respective membrane environments. Moreover, different geometries of heptaene: sterol complexes and variations in polyene macrolide molecule alignment in cholesterol-and ergosterol-containing membranes were found. None of these effects are of the crucial importance for the observed improvement of selective toxicity of aromatic heptaene antifungals but each seems to provide a partial contribution.

Bee Pollen and Bee Bread as a Source of Bacteria Producing Antimicrobials.

The principal objective of the study was the isolation and identification of bacteria that are present in mature bee bread (BB) and dried (ready for selling and consumption) bee pollen (BP). Obtained isolates were screened for their potential to inhibit select human pathogenic bacteria and their ability to produce enzymes of particular industrial importance. Four and five samples of BP and BB, respectively, were used for the study. In total, 81 strains of bacteria were isolated, and 34 (42%) of them exhibited antagonistic interactions with at least one reference strain of pathogenic bacteria, namely Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis 12228, Pseudomonas aeruginosa ATCC 27857, and Escherichia coli ATCC 25922. The sequencing of the 16S rRNA gene revealed that all strains producing antimicrobials belong to the genus Bacillus spp., and among them, five species were identified: B. pumilus (n = 17), B. altitudinis (n = 9), B. licheniformis (n = 4), B. subtilis (n = 2), and B. safensis (n = 1). Furthermore, 69, 54, 39, and 29 of the strains exhibited lipolytic, proteolytic, cellulolytic, and esterolytic activity, respectively. Alpha amylase and beta galactosidase activity were rarely observed, and none of the strains produced laccase. The outcomes of the study revealed that BP and BB can be considered potential sources of bacteria producing antimicrobial agents and/or enzymes of particular industrial importance. Of course, additional research is required to verify this hypothesis, but the results of preliminary studies are promising.

Antibiotic Resistance of Uropathogens Isolated from Patients Hospitalized in District Hospital in Central Poland in 2020.

The aim of this study was to determine antibiotic resistance patterns and the prevalence of uropathogenes causing urinary tract infections (UTIs) in patients hospitalized in January-June 2020 in central Poland. Antimicrobial susceptibility testing was performed using the disk-diffusion method. Escherichia coli (52.2%), Klebsiella pneumoniae (13.7%), Enterococcus faecalis (9.3%), E. faecium (6.2%), and Proteus mirabilis (4,3%) were most commonly isolated from urine samples. E. coli was significantly more frequent in women (58.6%) (p = 0.0089) and in the age group 0-18, while K. pneumoniae was more frequent in men (24.4%) (p = 0.0119) and in individuals aged 40-60 and >60. Gram-negative species showed resistance to ampicillin. K. pneumoniae were resistant to amoxicillin plus clavulanic acid (75.0%), piperacillin plus tazobactam (76.2%), cefotaxime (76.2%), cefuroxime (81.0%), ciprofloxacin (81.0%), and trimethoprim plus sulphamethoxazole (81.0%). Carbapenems were effective against all E. coli and P. mirabilis. Some K. pneumoniae (13.6%) produced metallo-ß-lactamases (MBLs). E. coli (22.6%), K. pneumoniae (81.8%), and all E. faecium were multidrug-resistant (MDR). Some E. coli (26.2%), K. pneumoniae (63.6%), and P. mirabilis (14.3%) isolates produced extended-spectrum beta-lactamases (ESBL). Vancomycin-resistant E. faecium was also found. This study showed that the possibilities of UTIs therapy using available antibiotics become limited due to the increasing number of antibiotic-resistant uropathogens.

Editorial for the Special Issue: "Honey Bee Products as an Alternative or Complement to Classical Antibiotics".

Based on World Health Organization reports, the resistance of bacteria to well-known antibiotics is becoming a major global health challenge [...].

Bee Bread Exhibits Higher Antimicrobial Potential Compared to Bee Pollen.

This study aimed at investigation of the antimicrobial potential of ethanolic extracts of bee bread (BB) and bee pollen (BP) and suspensions of these products in MHB (Mueller Hinton Broth). We covered 30 samples of BP and 19 samples of BB harvested in Polish apiaries. Slightly lower activity was observed against Gram-negative bacteria compared to Gram-positive staphylococci. BB extracts exhibited higher inhibitory potential with minimum inhibitory concentration (MIC) values in the range from 2.5 to 10% (v/v) against Staphylococcus aureus ATCC 25923 and ATCC 29213. Most active BB extracts, namely, BB6, BB11 and BB19, effectively inhibited growth of clinical isolates of S. aureus (n = 9), including MRSA (methicillin resistant Staphylococcus aureus) strains (n = 3) at concentrations ranging from 2.5 to 5.0% (v/v). Minimal bactericidal concentration (MBC) values were in the same range of concentrations; however, a shift from 2.5 to 5.0% (v/v) was observed for some products. The most active BP extracts inhibited the growth of reference strains of S. aureus at a concentration of 5% (v/v). Up to the concentration of 20% (v/v) three and seven BP extracts were not able to inhibit the growth of S. aureus ATCC 29213 and S. aureus ATCC 25923 respectively. The growth of staphylococci was also importantly inhibited in suspensions of the products in MHB. No correlation between phenolic content and antimicrobial activity was observed.

Synergistic Effects of Propolis Combined with 2-Phenoxyethanol and Antipyretics on the Growth of Staphylococcus aureus.

The present investigation aimed to assess the combinational effect of commonly used antipyretics and antiseptics with ethanolic extracts of propolis (EEPs) on the growth inhibition of Staphylococcus aureus. The broth microdilution checkerboard assay revealed synergistic interactions between all investigated antipyretics, namely acetylsalicylic acid, ibuprofen, and acetaminophen, with EEPs samples. The values of the fractional inhibitory concentration (ΣFIC) index for all these combinations were <0.5. While, in the case of considered antiseptics, namely chlorhexidine, octenidine dihydrochloride, and 2-phenoxyethanol, the positive interaction was confirmed only for the last one (values of ΣFIC in the range 0.0625-0.25). Combinations of two other agents with all four samples of EEPs resulted in an important antagonistic effect (values of ΣFIC ≥ 4.5). Propolis is mostly dedicated to the treatment of skin/wound infections; thus, these findings are of particular practical importance. The outcomes of the study also support the hypothesis that the propolis's antimicrobial effect is due to the combined (synergistic) action of several ingredients rather than the presence of one component of high antibacterial activity. The composition of 13 ingredients of EEPs (at a concentration below the MIC (minimum inhibitory concentration) of the most active agent) exhibited considerably high anti-staphylococcal efficiency with MIC = 128 µg/mL.

Effect of Ethanol Extracts of Propolis (EEPs) against Staphylococcal Biofilm-Microscopic Studies.

Staphylococci growing in the form of biofilm exhibit high resistance to a plethora of antibiotics. The aim of the study was to assess the influence of ethanolic extract of propolis (EEPs) on S. epidermidis ATCC 35984 biofilm using fluorescent microscopy. Propidium iodide (PI) and SYTO 9 were used for differentiation of live and dead cells, and calcofluor white was used to stain the extracellular matrix, the self-produced extracellular polymeric substances (EPS). The outcomes of the research confirm the promising potential of EEPs for eradication of staphylococcal biofilm. However, its activity cannot be classified as fully satisfactory, either in terms of the effectiveness of elimination of bacterial cells or disturbing the EPS structure. A two or even four times higher concentration of EEPs compared to MIC (Minimum Inhibitory Concentration) against planktonic cells (128 µg/mL) was necessary for effective (estimated for 90%) elimination of living cells from the biofilm structure. Unfortunately, even at that concentration of EEPs, the extracellular matrix was only partially disturbed and effectively protected the residual population of living cells of S. epidermidis ATCC 35984. In our opinion, a combination of EEPs with agents disrupting components of EPS, e.g., proteases, lysines, or enzymes degrading extracellular DNA or PIA (polysaccharide intercellular adhesin).

Draft genome sequence of antimicrobial producing Paenibacillus alvei strain MP1 reveals putative novel antimicrobials.

OBJECTIVE: A Paenibacillus strain isolated in previous research exhibited antimicrobial activity against relevant human pathogens including Staphylococcus aureus and Listeria monocytogenes. In this study, the genome of the aforementioned strain, designated as MP1, was shotgun sequenced. The draft genome of strain MP1 was subject to multiple genomic analyses to taxonomically characterize it and identify the genes potentially responsible for its antimicrobial activity. RESULTS: Here we report the draft genome sequence of an antimicrobial producing Paenibacillus strain, MP1. Average Nucleotide Identity (ANI) analysis established strain MP1 as a new strain of the previously characterized Paenibacillus alvei. The genomic analysis identified several putative secondary metabolite clusters including seven Nonribosomal Peptide Synthetase clusters (NRPS) (> 10,000 nt), one bacteriocin or other unspecified Ribosomally Synthesized and Post-Translationally modified Peptide Product (RiPP), one lanthipeptide, and six hybrid clusters (NRPS-Type I Polyketide synthase (T1PKS) and NRPS-trans Amino Transferase Polyketide Synthase (AT-PKS)).

Paenibacillus alvei MP1 as a Producer of the Proteinaceous Compound with Activity against Important Human Pathogens, Including Staphylococcus aureus and Listeria monocytogenes.

An emerging need for new classes of antibiotics is, on the one hand, evident as antimicrobial resistance continues to rise. On the other hand, the awareness of the pros and cons of chemically synthesized compounds' extensive use leads to a search for new metabolites in already known reservoirs. Previous research showed that Paenibacillus strain (P. alvei MP1) recovered from a buckwheat honey sample presented a wide spectrum of antimicrobial activity against both Gram-positive and Gram-negative pathogens. Recent investigation has confirmed that P. alvei MP1 (deposited at DDBJ/ENA/GenBank under the accession WSQB00000000) produces a proteinaceous, heat-stable compound(s) with the maximum antimicrobial production obtained after 18 hours of P. alvei MP1 growth in LB medium at 37 °C with continuous shaking at 200 RPM. The highest activity was found in the 40% ammonium sulfate precipitate, with high activity also remaining in the 50% and 60% ammonium sulfate precipitates. Moderate to high antimicrobial activity that is insensitive to proteases or heat treatment, was confirmed against pathogenic bacteria that included L. monocytogenes FSL - X1-0001 (strain 10403S), S. aureus L1 - 0030 and E. coli O157: H7. Further studies, including de novo sequencing of peptides by mass spectrometry, are in progress.

New Approaches for Escherichia coli Genotyping.

Easy-to-perform, fast, and inexpensive methods of differentiation of Escherichia coli strains beyond the species level are highly required. Herein two new, original tools for genotyping of E. coli isolates are proposed. The first of the developed method, a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) test uses a highly variable fliC gene, encoding the H antigen as a molecular target. The designing of universal pair of primers and selection of the optimal restriction enzyme RsaI was preceded by in silico comparative analysis of the sequences of the genes coding for 53 different serotypes of H-antigen (E. coli flagellin). The target fragments of E. coli genomes for MLST method were selected on the basis of bioinformatics analysis of complete sequences of 16 genomes of E. coli. Initially, seven molecular targets were proposed (seven pairs of primers) and five of them were found useful for effective genotyping of E. coli strains. Both developed methods revealed high differentiation power, and a high genetic diversity of the strains tested was observed. Within the group of 71 strains tested, 29 and 47 clusters were revealed with fliC RFLP-PCR and MLST methods, respectively. Differentiation of the strains with the reference BOX-PCR method revealed 31 different genotypes. The in silico analysis revealed that the discriminatory power of the new MLST method is comparable to the Pasteur and Achtman schemes and is higher than the discriminatory power of the method developed by Clermont. From the epidemiology point of view, the outcomes of our investigation revealed that in most cases, the patients were infected with unique strains, probably from environmental sources. However, some strains isolated from different patients of the wards of pediatrics, internal medicine, and neurology were classified to the same genotype when the results of all three methods were taken into account. It could suggest that they were transferred between the patients.