Identification of Clinically Relevant Streptococcus and Enterococcus Species Based on Biochemical Methods and 16S rRNA, sodA, tuf, rpoB, and recA Gene Sequencing

Kosecka-Strojek, Maja; Wolska, Mariola; Żabicka, Dorota; Sadowy, Ewa; Międzobrodzki, Jacek

doi:10.3390/pathogens9110939

Cited by 6 publications

(6 citation statements)

References 68 publications

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“…The rpoB gene has been previously reported for its high identification potential over other genes, such as tuf , recA , and sodA etc. [ 43 ].…”

Section: Resultsmentioning

confidence: 99%

“…To design the specific primers, species-specific genes with highly conserved sequence regions for E. faecalis were chosen. The genes encoding tuf (elongation factor Tu) [ 40 ], rpo A, and rpo B (RNA polymerase subunits A and B respectively) [ 41 , 42 , 43 ] have previously reported to be suitable targets for enterococci identification. Therefore, primers encoding these conserved regions were prepared.…”

Section: Methodsmentioning

confidence: 99%

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Development of A Rapid, Low-Cost Portable Detection Assay for Enterococci in Wastewater and Environmental Waters

Batra

Cottam²,

Lepesteur³

et al. 2023

Microorganisms

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Waterborne diseases are known as a leading cause of illness and death in both developing and developed countries. Several pathogens can be present in contaminated water, particularly waters containing faecal material; however, routine monitoring of all pathogens is not currently possible. Enterococcus faecalis, which is present in the microflora of human and animals has been used as a faecal indicator in water due to its abundance in surface water and soil. Accurate and fast detection methods are critical for the effective monitoring of E. faecalis in the environment. Although conventional and current molecular detection techniques provide sufficient sensitivity, specificity and throughput, their use is hampered by the long waiting period (1–6 days) to obtain results, the need for expensive laboratory equipment, skilled personnel, and cold-chain storage. Therefore, this study aimed to develop a detection system for E. faecalis that would be simple, rapid, and low-cost, using an isothermal DNA amplification assay called recombinase polymerase amplification (RPA), integrated with a lateral flow assay (LFA). The assay was found to be 100% selective for E. faecalis and capable of detecting rates as low as 2.8 × 103 cells per 100 mL from water and wastewater, and 2.8 × 104 cells per 100 mL from saline water. The assay was completed in approximately 30 min using one constant temperature (38 °C). In addition, this study demonstrated the quantitation of E. faecalis using a lateral flow strip reader for the first time, enhancing the potential use of RPA assay for the enumeration of E. faecalis in wastewater and heavily contaminated environmental waters, surface water, and wastewater. However, the sensitivity of the RPA-LFA assay for the detection of E. faecalis in tap water, saline water and in wastewater was 10–1000 times lower than that of the Enterolert-E test, depending on the water quality. Nevertheless, with further improvements, this low-cost RPA-LFA may be suitable to be used at the point-of-need (PON) if conjugated with a rapid field-deployable DNA extraction method.

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“…The rpoB gene has been previously reported for its high identification potential over other genes, such as tuf , recA , and sodA etc. [ 43 ].…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Development of A Rapid, Low-Cost Portable Detection Assay for Enterococci in Wastewater and Environmental Waters

Batra

Cottam²,

Lepesteur³

et al. 2023

Microorganisms

View full text Add to dashboard Cite

show abstract

“…A lower number of taxa for tuf , an amplicon known for a high Firmicutes resolution especially for Staphylococcus , Streptococcus , and Enterococcus species (32–34), were observed. In the tuf dataset, the Moraxella genus was by far the most abundant taxon, specifically an unclassified member of the genus Moraxella (Figure 3B).…”

Section: Resultsmentioning

confidence: 99%

“…In piglets the development of the nasal microbiome has implications for health and disease, but longitudinal development is understudied, and most microbiome studies lack taxonomic resolution We obtained longitudinal data of the porcine nasal microbiota from birth to day 70 of life, from three farms across three different countries, using a combination of 16S rRNA and tuf gene amplicon sequencing achieving phylogenetic resolution up to species level. Using tuf sequencing the species resolution of Firmicutes (34, 35) in the piglet nasal microbiome improved. This allowed us to describe CAGs of bacterial species that are commonly found together and consistently present in piglets in the studied countries.…”

Section: Discussionmentioning

confidence: 99%

The developing pig respiratory microbiome harbours strains antagonistic to common respiratory pathogens

Vlasblom,

Duim,

Patel

et al. 2023

Preprint

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In the global efforts to combat antimicrobial resistance and reduce antimicrobial use in pig production, there is a continuous search for methods to prevent and/or treat infections. Within this scope, we explored the relationship between the developing piglet nasal microbiome and (zoonotic) bacterial pathogens from birth until ten weeks of life. The nasal microbiome of 54 pigs was longitudinally studied over 16 time-points on nine farms in three European countries (Germany, Ireland, and the Netherlands) using amplicon sequencing targeting the V3-V4 16S rRNA region as well as thetufgene for itsStaphylococcaldiscrimination power. The piglets’ age, the farm, and the litter affected the nasal microbiome, with piglets’ age explaining 19% of the variation in microbial composition between samples. Stabilization of the microbiome occurred around two weeks post-weaning. Notably, while opportunistic pathogens were ubiquitously present, they did not cause disease. The piglet nasal microbiome often carried species associated with gut, skin, or vagina, which suggests that contact with the vaginal and faecal microbiomes shape the piglet nasal microbiome. We identified bacterial Co-Abundance Groups (CAGs) of species that were present in the nasal microbiomes in all three countries over time. Anticorrelation between these species and known bacterial pathogens identified strains that might be exploited for pathogen reduction. Further experimental evidence is required to confirm these findings. Overall, this study advances our understanding of the longitudinal development and factors influencing the piglet nasal microbiome, providing insights into its role in health and disease.ImportanceOur study on longitudinal analysis of the developing nasal microbiota of piglets in farms in three European countries showed consistent microbiome compositions and that colonization of porcine pathogens occurred in relation with anticorrelating species. These findings enhance our knowledge of co-colonizing species in the nasal cavity, and the identified microbial interactions can be explored for the development of interventions to control pathogens in porcine husbandry.

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“…Streptococci and enterococci are also significant opportunistic pathogens in epidemiology and infectious medicine. In addition, the most challenging aspects of species identification include the high genetic and taxonomic similarities and several reclassifications within a genus [38].…”

Section: Discussionmentioning

confidence: 99%

Biochemical and molecular identification of Gram-positive isolates with β-hemolysis activity isolated from the nasal swab of pigs during the human meningitis outbreak in Badung Regency, Bali-Indonesia

et al. 2022

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Background and Aim: The nasal cavity of a pig serves as an entry point and a habitat for the colonization of commensal microbes and pathogenic bacteria. Based on biochemical and serological tests, Streptococcus β-hemolytic Group C was identified as the Gram-positive bacteria, which resulted in the 1994 outbreak and death of thousands of pigs in Bali. Furthermore, this agent is zoonotic and frequently results in the development of meningitis lesions in the infected pig. Recently, a meningitis outbreak in humans was also reported after the consumption of pig-derived foods at Sibang Kaja, Badung-Bali. This study aimed to identify and characterize Gram-positive β-hemolytic organisms collected from nasal swab of pigs from the outbreak area, as well as to compare API Kit and 16S rRNA gene analysis methods. Materials and Methods: This study commenced with the cultivation of two isolates, Punggul Swab Nasal (PSN) 2 and PSN 19, which were characterized by β-hemolysis activity. These samples were then conventionally and molecularly identified using Kit API 20 Strep and 16S ribosomal RNA (rRNA) gene primers, respectively. Results: Using the Kit API 20 Strep, both isolates were identified as Enterococcus faecium, which was previously classified as Group D Streptococci. Based on the 16S rRNA gene sequencing, PSN 2 and PSN 19 were molecularly confirmed to have 99 and 98.1% similarities with E. faecium (NR042054), respectively. Furthermore, both isolates share the same clade in the phylogenetic tree analysis. Conclusion: Using Kit API 20 Strep and 16S rRNA gene analysis, the PSN 2 and PSN 9 Gram-positive isolates with β-hemolysis activity from pig nasal swabs were identified as E. faecium.

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Identification of Clinically Relevant Streptococcus and Enterococcus Species Based on Biochemical Methods and 16S rRNA, sodA, tuf, rpoB, and recA Gene Sequencing

Cited by 6 publications

References 68 publications

Development of A Rapid, Low-Cost Portable Detection Assay for Enterococci in Wastewater and Environmental Waters

Development of A Rapid, Low-Cost Portable Detection Assay for Enterococci in Wastewater and Environmental Waters

The developing pig respiratory microbiome harbours strains antagonistic to common respiratory pathogens

Biochemical and molecular identification of Gram-positive isolates with β-hemolysis activity isolated from the nasal swab of pigs during the human meningitis outbreak in Badung Regency, Bali-Indonesia

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