RESUMEN
Gut microbiota (GM) plays many key functions and helps maintain the host's health. Consequently, the development of GM cultivation under in vitro stimulating physiological conditions has gained extreme interest in different fields. In this study, we evaluated the impact of four culture media: Gut Microbiota Medium (GMM), Schaedler Broth (SM), Fermentation Medium (FM), and Carbohydrate Free Basal Medium (CFBM) on preserving the biodiversity and metabolic activity of human GM in batch in vitro cultures using PMA treatment coupled with 16S rDNA sequencing (PMA-seq) and LC-HR-MS/MS untargeted metabolomics supplemented with GC-MS SCFA profiling. Before the experiments, we determined the possibility of using the pooled faecal samples (MIX) from healthy donors (n = 15) as inoculum to reduce the number of variables and ensure the reproducibility of in vitro cultivation tests. Results showed the suitability of pooling faecal samples for in vitro cultivation study. Non-cultured MIX inoculum was characterized by higher α-diversity (Shannon effective count, and Effective microbial richness) compared to inocula from individual donors. After 24 h of cultivation, a significant effect of culture media composition on GM taxonomic and metabolomic profiles was observed. The SM and GMM had the highest α-diversity (Shannon effective count). The highest number of core ASVs (125) shared with non-cultured MIX inoculum and total SCFAs production was observed in the SM. These results might contribute to the development of standardized protocols for human GM in vitro cultivation by preventing methodological bias in the data.
Asunto(s)
Microbioma Gastrointestinal , Humanos , ADN Ribosómico , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem , Heces , Medios de Cultivo/farmacología , ARN Ribosómico 16S/genética , MetabolómicaRESUMEN
This study aimed to evaluate the effectiveness of the phage cocktail to improve the microbiological quality of five different mixed-leaf salads: rucola, mixed-leaf salad with carrot, mixed-leaf salad with beetroot, washed and unwashed spinach, during storage in refrigerated conditions. Enterobacterales rods constituted a significant group of bacteria in the tested products. Selected bacteria were tested for antibiotic resistance profiles and then used to search for specific bacteriophages. Forty-three phages targeting bacteria dominant in mixed-leaf salads were isolated from sewage. Their titer was determined, and lytic activity was assessed using the Bioscreen C Pro automated growth analyzer. Two methods of phage cocktail application including spraying, and an absorption pad were effective for rucola, mixed leaf salad with carrot, and mixed leaf salad with beetroot. The maximum reduction level after 48 h of incubation reached 99.9% compared to the control sample. In washed and unwashed spinach, attempts to reduce the number of microorganisms did not bring the desired effect. The decrease in bacteria count in the lettuce mixes depended on the composition of the autochthonous saprophytic bacteria species. Both phage cocktail application methods effectively improved the microbiological quality of minimally processed products. Whole-spectral phage cocktail application may constitute an alternative food microbiological quality improvement method without affecting food properties.
Asunto(s)
Bacteriófagos , Bacterias , Carga Bacteriana , LactucaRESUMEN
Salmonella is one of the most important foodborne pathogens. Fifty-three strains of Salmonella deposited in the Culture Collection of Industrial Microorganisms-Microbiological Resources Center (IAFB) were identified using molecular and proteomic analyses. Moreover, the genetic similarity of the tested strains was determined using the PFGE method. Main virulence genes were identified, and phenotypical antibiotic susceptibility profiles and prevalence of resistance genes were analyzed. Subsequently, the occurrence of the main mechanisms of ß-lactam resistance was determined. Virulence genes, invA, fimA, and stn were identified in all tested strains. Phenotypic tests, including 28 antibiotics, showed that 50.9% of the strains were MDR. The tet genes associated with tetracyclines resistance were the most frequently identified genes. Concerning the genes associated with ESBL-producing Salmonella, no resistance to the TEM and CTX-M type was identified, and only two strains (KKP 1597 and KKP 1610) showed resistance to SHV. No strains exhibited AmpC-type resistance but for six Salmonella strains, the efflux-related resistance of PSE-1 was presented. The high number of resistant strains in combination with multiple ARGs in Salmonella indicates the possible overuse of antibiotics. Our results showed that it is necessary to monitor antimicrobial resistance profiles in all food chain links constantly and to implement a policy of proper antibiotic stewardship to contain or at least significantly limit the further acquisition of antibiotic resistance among Salmonella strains.
RESUMEN
The food industry is still searching for novel solutions to effectively ensure the microbiological safety of food, especially fresh and minimally processed food products. Nowadays, the use of bacteriophages as potential biological control agents in microbiological food safety and preservation is a promising strategy. The aim of the study was the isolation and comprehensive characterization of novel bacteriophages with lytic activity against saprophytic bacterial microflora of minimally processed plant-based food products, such as mixed leaf salads. From 43 phages isolated from municipal sewage, four phages, namely Enterobacter phage KKP 3263, Citrobacter phage KKP 3664, Enterobacter phage KKP 3262, and Serratia phage KKP 3264 have lytic activity against Enterobacter ludwigii KKP 3083, Citrobacter freundii KKP 3655, Enterobacter cloacae KKP 3082, and Serratia fonticola KKP 3084 bacterial strains, respectively. Transmission electron microscopy (TEM) and whole-genome sequencing (WGS) identified Enterobacter phage KKP 3263 as an Autographiviridae, and Citrobacter phage KKP 3664, Enterobacter phage KKP 3262, and Serratia phage KKP 3264 as members of the Myoviridae family. Genome sequencing revealed that these phages have linear double-stranded DNA (dsDNA) with sizes of 39,418 bp (KKP 3263), 61,608 bp (KKP 3664), 84,075 bp (KKP 3262), and 148,182 bp (KKP 3264). No antibiotic resistance genes, virulence factors, integrase, recombinase, or repressors, which are the main markers of lysogenic viruses, were annotated in phage genomes. Serratia phage KKP 3264 showed the greatest growth inhibition of Serratia fonticola KKP 3084 strain. The use of MOI 1.0 caused an almost 5-fold decrease in the value of the specific growth rate coefficient. The phages retained their lytic activity in a wide range of temperatures (from -20 °C to 50 °C) and active acidity values (pH from 4 to 11). All phages retained at least 70% of lytic activity at 60 °C. At 80 °C, no lytic activity against tested bacterial strains was observed. Serratia phage KKP 3264 was the most resistant to chemical factors, by maintaining high lytic activity across a broader range of pH from 3 to 11. The results indicated that these phages could be a potential biological control agent against saprophytic bacterial microflora of minimally processed plant-based food products.