Microbial Biofilms at Meat-Processing Plant as Possible Places of Bacteria Survival.

Biofilm contamination in food production threatens food quality and safety, and causes bacterial infections. Study of food biofilms (BF) is of great importance. The taxonomic composition and structural organization of five foods BF taken in different workshops of a meat-processing plant (Moscow, RF) were studied. Samples were taken from the surface of technological equipment and premises. Metagenomic analysis showed both similarities in the presented microorganisms dominating in different samples, and unique families prevailing on certain objects were noted. The bacteria found belonged to 11 phyla (no archaea). The dominant ones were Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. The greatest diversity was in BFs taken from the cutting table of raw material. Biofilms' bacteria may be the cause of meat, fish and dairy products spoilage possible representatives include Pseudomonas, Flavobacterium, Arcobacter, Vagococcus, Chryseobacterium, Carnobacterium, etc.). Opportunistic human and animal pathogens (possible representatives include Arcobacter, Corynebacterium, Kocuria, etc.) were also found. Electron-microscopic studies of BF thin sections revealed the following: (1) the diversity of cell morphotypes specific to multispecies BFs; (2) morphological similarity of cells in BFs from different samples, micro-colonial growth; (3) age heterogeneity of cells within the same microcolony (vegetative and autolyzed cells, resting forms); (4) heterogeneity of the polymer matrix chemical nature according to ruthenium red staining.

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.

Evaluation of Antibiotic Resistance of Salmonella Serotypes and Whole-Genome Sequencing of Multiresistant Strains Isolated from Food Products in Russia.

Food products may be a source of Salmonella, one of the main causal agents of food poisoning, especially after the emergence of strains resistant to antimicrobial preparations. The present work dealt with investigation of the occurrence of resistance to antimicrobial preparations among S. enterica strains isolated from food. The isolates belonged to 11 serovars, among which Infantis (28%), Enteritidis (19%), and Typhimurium (13.4%) predominated. The isolates were most commonly resistant to trimethoprim/sulfamethoxazole (n = 19, 59.38%), cefazolin (n = 15, 46.86%), tetracycline (n = 13, 40.63%), and amikacin (n = 9, 28.13%). Most of the strains (68.75%) exhibited multiple resistance to commonly used antibiotics. High-throughput sequencing was used to analyse three multidrug-resistant strains (resistant to six or more antibiotics). Two of them (SZL 30 and SZL 31) belonged to S. Infantis, while one strain belonged to S. Typhimurium (SZL 38). Analysis of the genomes of the sequenced strains revealed the genes responsible for antibiotic resistance. In the genomes of strains SZL 30 and SZL 31 the genes of antibiotic resistance were shown to be localized mostly in integrons within plasmids, while most of the antibiotic resistance genes of strain SZL 38 were localized in a chromosomal island (17,949 nt). Genomes of the Salmonella strains SZL 30, SZL 31, and SZL 38 were shown to contain full-size pathogenicity islands: SPI-1, SPI-2, SPI-4, SPI-5, SPI-9, SPI-11, SPI-13, SPI-14, and CS54. Moreover, the genome of strain SZL 38 was also found to contain the full-size pathogenicity islands SPI-3, SPI-6, SPI-12, and SPI-16. The emergence of multidrug-resistant strains of various Salmonella serovars indicates that further research on the transmission pathways for these genetic determinants and monitoring of the distribution of these microorganisms are necessary.

Application of EvaGreen for the assessment of Listeria monocytogenes АТСС 13932 cell viability using flow cytometry.

Determination of eukaryotic cell viability using flow cytometry is widespread and based on the use of fluorescent dyes such as SYTO, DAPI, SYBR, PI, and SYTOX. For many years, traditional microbiological methods have been used to successfully analyze prokaryotic cells, but the application of flow cytometry should be considered because it provides an opportunity for quantitative assessment. A combination of SYTO 9 or SYBR green and PI has been used successfully. DNA-binding dyes such as SYTO 9, SYBR green, and EvaGreen are used in qPCR. The aim of this study was to assess the feasibility of EvaGreen to determine the viability of Listeria monocytogenes АТСС 13932 cells using flow cytometry. RNA from Escherichia coli ATCC 25922 was isolated using the MagNA Pure LC RNA Isolation Kit-High Performance (Roche, Germany) according to the kit instructions on MagNA Pure LC® 2.0 (Roche, Switzerland). Chicken DNA was isolated using the Sorb-GMO-B kit (Syntol CJSC, Russia) according to the kit instructions. RNA from E. coli ATCC 25922, chicken DNA, a positive control, and a negative control of L. monocytogenes АТСС 13932 were stained with EvaGreen and analyzed on the Guava EasyCyte flow cytometer (Merck Millipore, Germany). Chicken DNA demonstrated both green and red fluorescence, while E. coli RNA displayed only red fluorescence. While the positive L. monocytogenes АТСС 13932 control and chicken DNA demonstrated similar fluorescence properties, the negative control showed a localization similar to that observed with E. coli RNA. Degraded ssDNA and RNA stained with EvaGreen demonstrated red fluorescence. Although EvaGreen is a class III dye, we observed fluorescence of live L. monocytogenes АТСС 13932 cells in the positive control stained with EvaGreen. The observed phenomenon was linked to the solution composition. It is necessary to repeat this analysis with various solution compositions as well as a wide range of both Gram-positive and Gram-negative bacteria to determine the effects on cell envelope permeability of EvaGreen.