Characterization and comparative analysis of the Escherichia marmotae M-12 isolate from bank vole (Myodes glareolus).

The Escherichia marmotae is a bacterium of the Enterobacterales order, which was first isolated from the Himalayan marmot (Marmota himalayana). Recently E. marmotae has been shown to cause severe infections in humans. Wild animals were suggested to be a natural reservoir of this bacterium. The present study describes the first case of E. marmotae isolation from an apparently healthy wild bank vole (Myodes glareolus). Phenotype, as well as genotype-based techniques, were applied to characterize E. marmotae M-12 isolate. E. marmotae M-12 had the capsule-positive phenotype, high adhesion to human erythrocytes and HEp-2 cells as well as a low invasion into HEp-2 cells. E. marmotae M-12 was avirulent in mice. The phylogenomic analyses of E. marmotae showed dispersed phylogenetic structure among isolates of different origins. Virulome analysis of M-12 isolate revealed the presence of the following factors: siderophores, heme uptake systems, capsule synthesis, curli and type I fimbriae, flagella proteins, OmpA porin, etc. Comparative virulome analysis among available E. marmotae genomes revealed the presence of capsule K1 genes mostly in pathogenic isolates and OmpA porin presence among all strains. We assume that the K1 capsule and OmpA porin play a key role in the virulence of E. marmotae. Pathogenesis of the latter might be similar to extraintestinal pathogenic E. coli.

The first detection of two Aeromonas strains in mice of the genus Apodemus.

Aeromonas spp. are gram-negative facultatively anaerobic bacilli recovered mainly from aquatic environments. Aeromonas spp. were reported to be associated with infections primarily in aquatic and to a lesser extent in terrestrial animals as well as in humans. Up-to-date little is known about aeromonads associated with wild animals, especially with rodents. This study reported the first isolation and characterization of two Aeromonas spp. from internal organs of apparently healthy wild rodents Apodemus uralensis and Apodemus flavicollis captured in the wild environment in the European part of Russia. Isolates were identified as A. hydrophila M-30 and A. encheleia M-2 using the multilocus sequence analysis (MLSA) approach. The isolation of the A. encheleia from rodents is the first described case. Both strains demonstrated beta-hemolytic activity towards human erythrocytes. Antimicrobial susceptibility testing showed that both Aeromonas strains were resistant and intermediate to carbapenems and piperacillin-tazobactam, which was caused by the expression of the genus-specific CphA carbapenemases. A. hydrophila M-30 also demonstrated trimethoprim resistant phenotype. This is usually caused by the carriage of the dfrA or dfrB genes in aeromonads which are frequently associated with integron class I. The latter however was absent in both isolates. Our results expand our understanding of possible aeromonad reservoirs and demonstrate the likelihood of the formation of natural foci of Aeromonas infection and a new link in the chain of the spread of antimicrobial resistance as well.

Genomic analysis of multidrug-resistant Delftia tsuruhatensis isolated from raw bovine milk.

Delftia tsuruhatensis is a gram-negative, aerobic bacterium mostly known as an organic pollutant degrading and growth-promoting microorganism. However, it recently emerged as an opportunistic human pathogen. To date, the source of D. tsuruhatensis infection is not clear. The majority of studies of D. tsuruhatensis have focused on environmental or clinical strains, while investigations of D. tsuruhatensis strains isolated from food sources are limited. In the present study, we report the case of D. tsuruhatensis isolation from raw bovine milk. Classical bacteriology approaches, as well as next-generation sequencing and comparative genomics, were used to characterize the features of the D. tsuruhatensis MR-6/3H strain. The MR-6/3H strain was resistant to 19 antimicrobials among 23 tested, including all aminoglycosides, phenicol, trimethoprim-sulfamethoxazole, and almost all ß-lactams. Phylogenetically, the MR-6/3H was close to clinical origin strains, including those previously isolated in Russia. Comparative genomics revealed the presence of putative antimicrobial resistance genes in the MR-6/3H isolate, mostly associated with efflux systems. Notably, genus-specific OXA-926-like ß-lactamase was also detected. In all, 27 putative virulence factors were predicted, the majority of which were associated with motility, adherence, stress survival, siderophore synthesis, and immunomodulation. In the MR-6/3H genome, the five prophage regions were identified, including two with intact levels. Integrons and CRISPR-Cas systems were not detected in the MR-6/3H isolate. Thus, our findings suggest that raw milk can be the potential source of and transmission route for the dissemination of multidrug-resistant D. tsuruhatensis.

Antimicrobial Resistance and Comparative Genomic Analysis of Elizabethkingia anophelis subsp. endophytica Isolated from Raw Milk.

Elizabethkingia anophelis is an emerging multidrug-resistant pathogen that causes severe nosocomial and community-acquired infections worldwide. We report the first case of E. anophelis isolation in Russia and the first isolation from raw cow's milk. The ML-44 demonstrated resistance to 28 antimicrobials of 33 tested in the disk-diffusion test. Whole genome-based phylogeny showed ML-44 strain clustered together with the F3201 strain isolated from a human patient in Kuwait in 1982. Both strains were a part of the "endophytica" clade. Another clade was formed by subsp. anophelis strains. Each of the E. anophelis compared genomes carried 18 to 21 antibiotic resistance determinants. The ML-44 chromosome harbored nine efflux system genes and three beta-lactamase genes, along with six other antimicrobial resistance genes. In total, 72 virulence genes were revealed. The set of virulence factors was quite similar between different E. anophelis strains and included LPS and capsule encoded genes, type IV pili, oxidative stress response genes, and genes encoding TIVSS and TVISS effectors. The particular interest caused the mip and zmp1 gene homologs, which can be essential for intracellular survival. In sum, our findings suggest that raw milk might be a source of E. anophelis harboring a set of virulence factors and a broad resistance to generally used antimicrobials.

Diversity of Listeria monocytogenes Strains Isolated from Food Products in the Central European Part of Russia in 2000-2005 and 2019-2020.

Totally, 45 L. monocytogenes strains isolated from meat, poultry, dairy, and fish products in the Central European part of Russia in 2001-2005 and 2019-2020 were typed using a combined MLST and internalin profile (IP) scheme. Strains belonged to 14 clonal complexes (CCs) of the phylogenetic lineages I and II. Almost half of the strains (20 of 45) belonged to six CCs previously recognized as epidemic clones (ECs). ECI and ECV strains were isolated during both studied periods, and ECII, ECIV, ECVI, and ECVII strains were isolated in 2001-2005, but not in 2019-2020. ECI, ECIV, ECV, and ECVII strains were isolated from products of animal origin. ECII and ECVI were isolated from fish. Testing of invasion efficiencies of 10 strains isolated in different years and from different sources and belonging to distinct CCs revealed a statistically significant difference between phylogenetic lineage I and II strains but not between ECs and non-EC CCs or strains differing by year and source of isolation. Strains isolated in 2001-2005 were characterized by higher phylogenetic diversity and greater presentation of ECs and CCs non-typical for natural and anthropogenic environments of the European part of Russia comparatively to isolates obtained in 2019-2020.Closing of the Russian market in 2019-2020 for imported food might be responsible for these differences.

Antimicrobial Resistance of Listeria monocytogenes Strains Isolated from Humans, Animals, and Food Products in Russia in 1950-1980, 2000-2005, and 2018-2021.

Susceptibility of 117 L. monocytogenes strains isolated during three time periods (1950-1980; 2000-2005, and 2018-2021) to 23 antibiotics was tested by the disk diffusion method. All strains were sensitive to aminoglycosides (gentamicin, kanamycin, neomycin, streptomycin), glycopeptides (vancomycin and teicoplanin), clarithromycin, levofloxacin, amoxicillin/clavulanic acid, and trimethoprim/sulfamethoxazole. Resistance to clindamycin was observed in 35.5% of strains. Resistance to carbapenems, imipenem and meropenem was found in 4% and 5% of strains, respectively. Resistance to erythromycin, penicillin G, trimethoprim, and ciprofloxacin was found in 4%, 3%, 3%, and 2.5% of strains, respectively. Resistance to tylosin, ampicillin, enrofloxacin, linezolid, chloramphenicol, and tetracycline was found in less than 2%. Three strains with multiple antibiotic resistance and 12 strains with resistance to two antibiotics were revealed. Comparison of strains isolated in different time periods showed that the percentage of resistant strains was the lowest among strains isolated before 1980, and no strains with multiple antibiotic resistance were found among them. Statistical analysis demonstrated that the temporal evolution of resistance in L. monocytogenes has an antibiotic-specific character. While resistance to some antibiotics such as ampicillin and penicillin G has gradually decreased in the population, resistance to other antibiotics acquired by particular strains in recent years has not been accompanied by changes in resistance of other strains.