Comparison of phenotypic and genotypic taxonomic methods for the identification of dairy enterococci (original) (raw)
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Quantitative analysis of enterococci isolated from dairy products
Zelena L., Tkachuk N., Okhmat O., Nevmyvaka S. (2022). Quantitative analysis of enterococci isolated from dairy products. BHT: Biota. Human. Technology, 2022. No 2, P. 22-29. https://doi.org/10.58407/bht.2.22.2, 2022
Purpose of the work. To evaluate the number of enterococci in the dairy products of industrial manufacturing. Methodology. Molecular-genetic methods were used to analyze enterococci in milk, kefir and sour cream samples. Metagenomic DNA was isolated from each product and used for amplification. PCR with specific primers to Enterococcus genus was carried out to amplify specific genome region and to detect enterococci in dairy products. Quantitative PCR (qPCR) with SYBR Green dye solution was performed to enumerate Enterococcus bacteria presented in three milk products. The melting curve analysis was used to define the specificity of amplification. The genome-equivalent values of enterococci in milk, kefir and sour cream were calculated using the standard curve analysis. Scientific novelty. In the present research the quantitative analysis of enterococci in the three milk products of industrial manufacturing was conducted using metagenomic DNA and qPCR-analysis. There were no significant differences of enterococci number between dairy products. Conclusions. The development of molecular-genetic methods and approaches gives a possibility to estimate the number of enterococci in dairy products by culture-independent way. The results suggested that similar amount of enterococci in all three dairy products can be due to their industrial production and using the same preservation agents. Although the genotyping and species identification of Enterococcus should be performed in the next researches. The fermented milk products have an important and in general beneficial effect on human health. Despite qualitative and quantitative variability of microorganisms comprising the composition in milk products, lactic acid bacteria represent the most numerous group. The ambiguous features characterize bacteria belonging to Enterococcus genus: they contribute to the specific flavor and taste of dairy products and can be used as a probiotic and adjunct starters but, at the same time, they can be the source of virulence factors and be an indicator of poor hygienic conditions during production. Thus, it is important to perform quantitative analysis and control of enterococci in dairy products.
Characterization and safety evaluation of enterococci isolated from Spanish goats' milk cheeses
International Journal of Food Microbiology, 2009
Enterococci are common constituents of dairy products such as cheese, in which it is believed that they contribute to the development of some of their organoleptic properties. This is particularly true in the Mediterranean region, where farmhouse cheeses are generally made from raw milk, which tends to harbour enterococci. In recent years, however, enterococci have emerged as serious pathogens in hospital environments, where vancomycin-resistant strains are on the increase. In this study we have characterized 95 enterococci isolated from 3 different goats' milk cheeses, belonging to the species Enterococcus devriesei, Enterococcus faecalis and E. malodoratus, among others. Genomic typification by RAPD and ERIC-PCR showed that the genotypes tended to be specific to particular cheeses; only two of the different cheeses contained two genotypes. E. malodoratus showed the highest intraspecific diversity. No more than 13% of the isolates demonstrated any antimicrobial activity, most of these belonging to E. devriesei. Screening for the most important enterocin structural genes by dot-blot hybridization proved negative for enterocins A, B and P. All the isolates belonging to E. faecalis, E. hirae and E. avium, on the other hand, hybridized with the enterocin EJ97 gene probe and all the E. faecalis strains hybridized with the AS-48 and MR10A structural gene probes. The genes gelE, esp, asa1, efaA and ace, all associated to virulence factors in enterococci, were detected at different levels in E. faecalis whilst they were not detected in the remaining species studied. No resistance to vancomycin was detected by PCR screening. The gene for tyrosine decarboxylase (tdc) was detected in all the E. faecalis and E. hirae isolates but in none of the others.
Occurrence of Enterococcus spp. isolated from the milk and milk products
Potravinarstvo, 2015
Enterococcus spp. is the most controversial group of lactic acid bacteria that have been for years ascribed with beneficial or detrimental role in food and feed. The aim of our study was to monitor the occurrence of Enterococcus spp. as the indicator of the contamination from collected samples of raw cow's milk, goat's colostrum and whey (n = 186). Cultures of enterococci were cultivated and purified and identified by the genus-specific and species-specific PCR method (n = 230). Among suspected isolates in total 222 isolates (96.5%) were identified as Enterococcus spp. The results were the same in all samples separately, more than 90% each of them were positive to Enterococcus spp. The results of counting the number of cultivated colonies showed that the largest number of enterococci is found in the samples of whey taken after the process of electrodialysis and the smallest in the native whey sample. From collected whey samples, 64 samples (90%) were PCR positive for enterococci species. Afterwards within the identification of several selected isolates that were identified, as Enterococcus spp. by the species-specific PCR method the most frequently presented in all of isolates was Enterococccus faecalis. Apparently the presence of enterococci was detected in all samples, but in amounts that aren't hazardous for human health. Although enterococci are oportunistic pathogens, it seems that they occur frequently in foods (especially fermented) in large numbers.
Molecular identification and diversity of enterococci isolated from Slovak Bryndza cheese
The Journal of General and Applied Microbiology, 2006
Three hundred and eight presumed enterococcal isolates were recovered from Bryndza, a soft sheep milk cheese. The cheese samples were obtained from five different commercial distributors in Slovakia and were taken at three different seasonal intervals. All isolates were identified to the species level using genotypic tools. Species-specific PCR using ddl genes highlighted the predominance of Enterococcus faecium (176 isolates) and assigned 50 isolates to the species Enterococcus faecalis. The remaining 82 isolates were classified using repetitive element sequence-based polymerase chain reaction (PCR) with primer (GTG) 5-(GTG) 5-PCR, in combination with phenylalanyl-tRNA synthase gene (pheS) sequence analysis and by whole-cell protein analysis (SDS-PAGE). These strains were identified as Enterococcus durans (59 strains), Enterococcus italicus (8 strains), Enterococcus casseliflavus (3 strains), Enterococcus gallinarum (3 strains), Enterococcus hirae (1 strain), and 8 strains were members of the species Lactococcus lactis. Of the seven enterococcal species isolated, three of them, E. durans, E. faecalis and E. faecium were present in all samples studied, with E. faecium as the predominant one. The precise identification of enterococci in Bryndza cheese is an essential step in the process of evaluation of their functional properties which will be further studied and assessed.
Enterococcus lactis sp. nov., from Italian raw milk cheeses
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 2012
Ten atypical Enterococcus strains were isolated from Italian raw milk cheeses. The 16S rRNA gene, phenylalanyl-tRNA synthase alpha subunit (pheS), RNA polymerase alpha subunit (rpoA) and the 16S-23S rRNA intergenic transcribed spacer (ITS) sequences, randomly amplified polymorphic DNA (RAPD) PCR and the phenotypic properties revealed that the isolates represent a novel enterococcal species. On the basis of 16S rRNA gene sequence analysis, the isolates were closely related to Enterococcus hirae ATCC 8043 T , Enterococcus durans CECT 411 T and Enterococcus faecium ATCC 19434 T , with 98.8, 98.9 and 99.4 % sequence similarity, respectively. On the basis of sequence analysis of the housekeeping gene pheS, the reference strain, BT159 T , occupied a position separate from E. faecium LMG 16198. The group of isolates could be easily differentiated from recognized species of the genus Enterococcus by 16S-23S rRNA ITS analysis, RAPD-PCR and phenotypic characteristics. The name Enterococcus lactis sp. nov. is proposed, with BT159 T (5DSM 23655 T 5LMG 25958 T ) as the type strain.
International Journal of …, 2003
Enterococci are part of the dominant microbiota of several dairy products. They are also present in the gut of humans and animals. Their presence in traditional raw milk cheeses is probably due to faecal contamination of milk during milking. Due to their importance as a cause of nosocomial infections, enterococci are acquiring increased significance. Such infections are becoming more and more difficult to treat as resistance to antibiotics increases. The aim of this investigation was to compare the potential virulence of Enterococcus faecium isolated from different ecological habitats and to establish if strains isolated from dairy products should really be considered as potential pathogens. In the present work, the antibiotic resistance pattern of 40 E. faecium strains isolated from dairy products, 26 E. faecium isolated from ewes' faeces and 28 clinical isolates of the same species was studied, and checks were made to see if known virulence determinants were present. Resistance to 12 different antibiotics commonly used in the treatment of human infections was tested using the broth microdilution method as described by the NCCLS. In addition, polymerase chain reaction (PCR) tests were carried out to see if genes for vancomycin resistance were present. The presence of the aggregation substance (AS) gene, the surface protein gene esp, the accessory colonisation factor ace, the Enterococcus faecalis endocarditis antigen efaA and the gelatinase gelE gene, which are involved in the virulence of enterococci, were also tested by PCR. The results of this study clearly indicate that E. faecium strains isolated from both cheese and sheep faeces are less pathogenic than those isolated from clinical samples. A similar pattern of resistance to antibiotics was observed in both dairy and animal strains. It was also found that there was difference in the kind of virulence determinants present in dairy and clinical isolates, while no virulence traits were found in sheep faeces strains. The results of this study suggest that E. faecium from traditional Sardinian raw milk cheeses should not be considered to be the main source of untreatable nosocomial enterococcal infections in humans in the island of Sardinia. D
International Journal of Food Microbiology, 2003
Enterococci are part of the dominant microbiota of several dairy products. They are also present in the gut of humans and animals. Their presence in traditional raw milk cheeses is probably due to faecal contamination of milk during milking. Due to their importance as a cause of nosocomial infections, enterococci are acquiring increased significance. Such infections are becoming more and more difficult to treat as resistance to antibiotics increases. The aim of this investigation was to compare the potential virulence of Enterococcus faecium isolated from different ecological habitats and to establish if strains isolated from dairy products should really be considered as potential pathogens. In the present work, the antibiotic resistance pattern of 40 E. faecium strains isolated from dairy products, 26 E. faecium isolated from ewes' faeces and 28 clinical isolates of the same species was studied, and checks were made to see if known virulence determinants were present. Resistance to 12 different antibiotics commonly used in the treatment of human infections was tested using the broth microdilution method as described by the NCCLS. In addition, polymerase chain reaction (PCR) tests were carried out to see if genes for vancomycin resistance were present. The presence of the aggregation substance (AS) gene, the surface protein gene esp, the accessory colonisation factor ace, the Enterococcus faecalis endocarditis antigen efaA and the gelatinase gelE gene, which are involved in the virulence of enterococci, were also tested by PCR. The results of this study clearly indicate that E. faecium strains isolated from both cheese and sheep faeces are less pathogenic than those isolated from clinical samples. A similar pattern of resistance to antibiotics was observed in both dairy and animal strains. It was also found that there was difference in the kind of virulence determinants present in dairy and clinical isolates, while no virulence traits were found in sheep faeces strains. The results of this study suggest that E. faecium from traditional Sardinian raw milk cheeses should not be considered to be the main source of untreatable nosocomial enterococcal infections in humans in the island of Sardinia. D
A survey of the enterococci isolated from an artisanal Italian goat's cheese (semicotto caprino)
Journal of Applied Microbiology, 2000
Enterococci were isolated from semicotto caprino cheese, a traditional cheese produced in Southern Italy: they were a signi®cant part of the microbial population of this cheese, con®rming the importance of the presence of these micro-organisms during cheese-making and ripening. They were also identi®ed and studied for their phenotypic and genotypic characteristics: Enterococcus faecalis and Ent. faecium were the most frequently isolated species, followed by Ent. durans, Ent. hirae and Ent. gallinarum. None of the isolates showed lipolytic activity, whereas they were characterized by a relevant proteolytic activity as well as an antagonistic activity towards Listeria innocua. One strain of Ent. gallinarum showed a low-level resistance to vancomycin, while six out of the 79 Ent. faecalis strains possessed b-haemolysis reaction. The highest acidifying potential in skim milk was obtained by Ent. faecalis isolates. Thirty enterococcal strains representative of the different species at different ripening times were analysed by means of RAPD-PCR, and revealed species-speci®c pro®les for all the considered species.
OCCURANCE OF ENTEROCOCCI IN MILK WITH SPECIAL REFERANCE TO ENTEROCOCCUS FAECALIS
A total of 115 unheated milk samples (61 street peddlers samples and54 farmers milk samples), were collected from various localities in Kafr El-Sheikh Governorate (Egypt). The samples were examined bacteriologically for enterococci with percentages of (100% of street peddlers samples and 62.96% of farmers milk samples) while The percentage of Enterococcus strains in positive samples were [ (100%) for Ent. faecalis, (21.31%) for Ent. faecium and (16.39%) for Ent. durans] in street peddlers samples and [(100%) for Ent. faecalis, (23.53%) for Ent. faecium and (5.88%) for Ent. durans] in farmers milk samples. Amplification of 185 bp fragment specific for the groES gene of Enterococcus faecalis used for confirmation of its identification.