Use of a Genus- and Species-Specific Multiplex PCR for Identification of Enterococci

Jackson, Charlene R.; Fedorka–Cray, Paula J.; Barrett, John B.

doi:10.1128/jcm.42.8.3558-3565.2004

Cited by 392 publications

(280 citation statements)

References 23 publications

(16 reference statements)

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“…Representative isolates were kept for further studies. Enterococcal species identification was performed by using conventional tests, including 0.04% telurite reduction, carbohydrate utilization, arginine dehydrolase activity, methyl-␣-D-glucopyranoside acidification, motility, pigmentation, and PCR using species-specific primers (8). E. faecium BM4147 and E. faecalis V583 were used as positive controls and were obtained from the Pasteur Institute, Paris.…”

Section: Methodsmentioning

confidence: 99%

Molecular Structure and Transferability of Tn 1546 -Like Elements in Enterococcus faecium Isolates from Clinical, Sewage, and Surface Water Samples in Iran

Talebi

Pourshafie

Katouli

et al. 2008

Appl Environ Microbiol

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The molecular structure and transferability of Tn1546 in 143 vancomycin-resistant Enterococcus faecium (VREF) isolates obtained from patients (n ‫؍‬ 49), surface water (n ‫؍‬ 28), and urban and hospital sewage (n ‫؍‬ 66) in Tehran, Iran, were investigated. Molecular characterization of Tn1546 elements in vanA VREF was performed using a combination of restriction fragment length polymorphism analysis and DNA sequencing of the internal PCR fragments of vanA transposons. Long-PCR amplification showed that the molecular size of Tn1546 elements varied from 10.8 to 12.8 kb. The molecular analysis of Tn1546 showed that 45 isolates (31.5%) harbored a deletion/mutation upstream from nucleotide 170. No horizontal transfer of Tn1546 was observed following filter-mating conjugation with these isolates. Nevertheless, the rates of transferability for other isolates were 10 ؊5 to 10 ؊6 per donor. Insertion sequences IS1216V and IS1542 were present in 103 (72%) and 138 (96.5%) of the isolates, respectively. The molecular analysis of Tn1546 elements resulted in three genomic organizations. The genomic organization lineage 1 was dominated by the isolates from clinical samples (3.4%), lineage 2 was dominated mostly by sewage isolates (24.5%), and lineage 3 contained isolates obtained from all sources (72.1%). The genetic diversity determined using pulsed-field gel electrophoresis (PFGE) revealed a single E. faecium clone, designated 44, which was common to the samples obtained from clinical specimens and hospital and municipal sewage. Furthermore, the results suggest that lineage 3 Tn1546 was highly disseminated among our enterococcal isolates in different PFGE patterns.

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Section: Methodsmentioning

confidence: 99%

Molecular Structure and Transferability of Tn 1546 -Like Elements in Enterococcus faecium Isolates from Clinical, Sewage, and Surface Water Samples in Iran

Talebi

Pourshafie

Katouli

et al. 2008

Appl Environ Microbiol

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“…In order to delineate isolates into different species of the genus Enterococcus, a PCR was performed as previously described by Jackson et al (2004) using amplification of the sodA gene, which has sequences specific for the following species: E. faecalis, E. faecium, E. hirae, E. durans and E. casseliflavus in monoplex PCR reactions. The Dream Taq PCR Master Mix (2Â) consisting of 4 mM MgCl 2 , 0.4 mM deoxynucleoside triphosphate mix and Taq polymerase enzyme (Thermo Scientific), and 1µl of 10 pM of each primer pair was added to constitute the reaction mixture in a PCR tube.…”

Section: Methodsmentioning

confidence: 99%

Macrolide, glycopeptide resistance and virulence genes in Enterococcus species isolates from dairy cattle

Iweriebor

Chikwelu

Okoh

2016

Journal of Medical Microbiology

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The genus Enterococcus is known to possess the capacity to acquire and disseminate antimicrobial resistant determinants alongside the ability to produce various virulence genes that enables it to establish infections. We assessed the prevalence and antibiogram profiles of Enterococcus spp. in faecal samples of dairy cattle. Faecal swab samples were collected from 400 dairy cattle from two commercial cattle farms in two rural communities in the Eastern Cape, South Africa. Confirmation of enterococci isolates was carried out by PCR targeting of the tuf gene. Species delineation was by species-specific primers targeting the superoxide dismutase (sodA) gene in a multiplex PCR assay. Isolates were screened for the presence of the following virulence genes (ace, gelE, esp, efaA, cylA and hylE) and antimicrobial resistance determinants to erythromycin, vancomycin and streptomycin were evaluated molecularly. A total of 340 isolates were confirmed as belonging to the genus Enterococcus. Species distribution among the isolates consisted of Enterococcus faecium (52.94 %) and Enterococcus durans (23.53 %) in preponderance compared to the three other species, namely Enterococcus faecalis (8.8 %), Enterococcus hirae (8.6 %) and Enterococcus casseliflavus (5.9 %). All were resistant to vancomycin, while 99 % showed resistance to aminoglycoside and 94 % to macrolide. Three virulence genes (ace, gelE and esp) were detected in almost all the confirmed isolates. The resistance determinants vanB (19.7 %), vanC1 (25 %), vanC2/3 (26.3 %) ermB (40.29 %) and strA (50.88 %) were detected among the isolates. A high prevalence of multidrug-resistant enterococci isolates was detected in this study and the genetic repertoire to survive in the presence of antimicrobial agents was present in these organisms. INTRODUCTIONMembers of the genus Enterococcus are known to be abundantly present in the intestinal tracts of humans and warmblooded animals, as well as in water and soil environments. These bacteria have become notorious recently because of their ability to cause nosocomial infections, especially among both immunocompromised patients and elderly people. Pathogenic enterococci have been reported to harbour a pathogenicity island that encodes for aggregation substance (AS), gelatinase, extracellular surface proteins (esp), cytolysin, hyaluronidase and other proteins (Giridhara Upadhyaya et al., 2009;Shankar et al., 2002). These virulence factors enable enterococci to cause tissue damage and inflammation.Besides their possession of several virulence factors, members of the genus Enterococcus also have inherent capacity to accumulate and disseminate antimicrobial resistance determinants. Enterococci, like other bacteria are capable of developing strategies to enhance their survival in adverse situations (Garcia-Migura et al., 2005). They have emerged as an important opportunistic pathogen causing life-threatening infections in patients, alongside the fact that they have developed remarkable resistance to a battery of antimicrobial agents belo...

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“…The sequences were compared by a BLAST search in GenBank/EMBL/DDBJ database and on EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al, 2012). The multiplex PCR assay based on sodA gene reported by Jackson, Fedorka-Cray, and Barrett (2004) was applied to confirm species identity. Finally, pheS partial sequence was obtained for strain WFE31 as previously reported (Naser et al, 2005) and compared in GenBank/EMBL/DDBJ database.…”

Section: Identification Of Enterococci At Species Level and Strain DImentioning

confidence: 99%

Production, stability, gene sequencing and in situ anti-Listeria activity of mundticin KS expressed by three Enterococcus mundtii strains

Settanni¹,

Guarcello²,

Gaglio³

et al. 2014

Food Control

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a b s t r a c tThree enterococci (WFE3, WFE20 and WFE31) selected as presumptive bacteriocin producers were found to be active against Listeria monocytogenes. In this study, due to their potential industrial/food applications, the three bacterial isolates were extensively characterized. Identification was performed by means of a combined 16S rRNA gene sequencing and multiplex PCR approach, and was confirmed with the sequencing of a partial region of a protein-encoding gene, namely pheS. The three isolates belonged unequivocally to the species Enterococcus mundtii. The randomly amplified polymorphic DNA (RAPD) analysis recognized three distinct strains. The supernatants were mainly active against Listeria spp., but some lactic acid bacteria were also inhibited. The proteinaceous nature of the three supernatants was detected after treatment with proteinase K, protease B and trypsin. The bacteriocins were found to be heat resistant, stable in a large pH range and in presence of ethanol. The bacteriocins were not adsorbed onto the surface of the producer cells and their effect was bactericidal. The production of bacteriocins was higher at neutral pHs and temperatures in the range 30e37 C. The active supernatants did not show cytotoxicity against human erythrocytes and the three strains were susceptible to the action of common antibiotics. The genetic characterization of the bacteriocin genes showed that all three strains produced mundticin KS. They produced it in five food model systems, sterilized by thermal treatment or filtration, prepared from fresh vegetables, cereals, cheeses, meats and fishes. The in situ anti-listerial activities of the strains WFE3, WFE20 and WFE31 were quantitatively different.

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Use of a Genus- and Species-Specific Multiplex PCR for Identification of Enterococci

Cited by 392 publications

References 23 publications

Molecular Structure and Transferability of Tn 1546 -Like Elements in Enterococcus faecium Isolates from Clinical, Sewage, and Surface Water Samples in Iran

Molecular Structure and Transferability of Tn 1546 -Like Elements in Enterococcus faecium Isolates from Clinical, Sewage, and Surface Water Samples in Iran

Macrolide, glycopeptide resistance and virulence genes in Enterococcus species isolates from dairy cattle

Production, stability, gene sequencing and in situ anti-Listeria activity of mundticin KS expressed by three Enterococcus mundtii strains

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