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Cycle 39 Organism 3 P.O. Box 131375, Bryanston, 2074 Ground Floor, Block 5 Bryanston Gate, 170 Curzon Road Bryanston, Johannesburg, South Africa www.thistle.co.za Tel: +27 (011) 463 3260 Fax: +27 (011) 463 3036 Fax to Email: + 27 (0) 86-538-4484 e-mail : [email protected] Please read this section first The HPCSA and the Med Tech Society have confirmed that this clinical case study, plus your routine review of your EQA reports from Thistle QA, should be documented as a “Journal Club” activity. This means that you must record those attending for CEU purposes. Thistle will not issue a certificate to cover these activities, nor send out “correct” answers to the CEU questions at the end of this case study. The Thistle QA CEU No is: MT- 16/009. Each attendee should claim THREE CEU points for completing this Quality Control Journal Club exercise, and retain a copy of the relevant Thistle QA Participation Certificate as proof of registration on a Thistle QA EQA. MICROBIOLOGY LEGEND CYCLE 39 ORGANISM 3 ENTEROCOCCUS FAECALIS Enterococci are part of the normal intestinal flora of humans and animals. They have been long recognized as important human pathogens and are becoming increasingly so. The genus Enterococcus includes more than 17 species, although only a few cause clinical infections in humans. Since the beginning of the antibiotic era, they have posed major therapeutic challenges, including the need for synergistic combinations of antibiotics to successfully treat enterococcal infective endocarditis (IE). Enterococcus species are facultative anaerobic organisms that can survive temperatures of 60°C for short periods and that grow in high salt concentrations. In the laboratory, enterococci are distinguished by their morphologic appearance on Gram stain and culture (gram-positive cocci that grow in chains) and their ability to (1) hydrolyze esculin in the presence of bile, (2) their growth in 6.5% sodium chloride, (3) their hydrolysis of pyrrolidonyl arylamidase and leucine aminopeptidase, and (4) their reaction with group D antiserum. Before they were assigned their own genus, Enterococci were members of the genus Streptococcus and were classified as group D Streptococci. Enterococcus faecalis and Enterococcus faecium are the most prevalent species cultured from humans, accounting for more than 90% of clinical isolates. Other enterococcal species known to cause human infection include Enterococcus avium, Enterococcus gallinarum, Enterococcus casseliflavus, Enterococcus durans, Enterococcus raffinosus, and Enterococcus mundtii. E faecium is responsible for most vancomycin-resistant enterococci (VRE) infections. Enterococcus faecalis Prior to 1984, E. faecalis was known as Streptococcus faecalis and formerly classified as part of the group D Streptococcus system. E. faecalis is a Gram-positive, commensal bacterium inhabiting the gastrointestinal tracts of humans and other mammals. Like other species in the genus Enterococcus, E. faecalis can cause life- threatening infections in humans, especially in the nosocomial (hospital) environment, where the naturally high levels of antibiotic resistance found in E. faecalis contribute to its pathogenicity. E. faecalis has been frequently found in root canal-treated teeth in prevalence values ranging from 30% to 90% of the cases. Root canal-treated teeth are about nine times more likely to harbor E. faecalis than cases of primary infections. Physiology E. faecalis is a nonmotile microbe; it ferments glucose without gas production, and does not produce a catalase reaction with hydrogen peroxide. It can produce a pseudo-catalase reaction if grown on blood agar. The reaction Page 1 of 3 P.O. Box 131375, Bryanston, 2074 Ground Floor, Block 5 Bryanston Gate, 170 Curzon Road Bryanston, Johannesburg, South Africa www.thistle.co.za Tel: +27 (011) 463 3260 Fax: +27 (011) 463 3036 Fax to Email: + 27 (0) 86-538-4484 e-mail : [email protected] is usually weak. It produces a reduction of litmus milk, but does not liquefy gelatin. It shows consistent growth throughout nutrient broth which is consistent with being an aerotolerant anaerobe. They catabolize a variety of energy sources including glycerol, lactate, malate, citrate, arginine, agmatine, and many keto acids. Enterococci survive very harsh environments including extremely alkaline pH (9.6) and salt concentrations. They resist bile salts, detergents, heavy metals, ethanol, azide, and desiccation. They can grow in the range of 10 to 45°C and survive at temperatures of 60°C for 30 min. Enterococcus faecalis basic characteristics Gram-positive cocci Non-motile Non-spore-forming Catalase: negative (a pseudo catalase is sometimes produced and a weak effervescence is observed in the catalase test) Oxidase: Negative Facultatively anaerobic bacteria Enterococcus faecalis tests for identification Grows in broth containing 6.5% NACL Hydrolyse esculin in the presence of bile salts (bile-esculin medium) Hydrolyse pyrrolidonyl-β-naphthylamide (PYR test - The PYR test determines the activity of pyrrolidonyl aminopeptidase, an enzyme produced by Enterococcus faecalis and many other Enterococcus species. L- pyrrolidonyl-β-naphthylamide impregnated into the strip serves as the substrate for the pyrrolidonyl arylamidase. Catabolism of the substrate produces β-naphthylamide which reacts with the PYR reagent (0.01% cinnamaldehyde reagent) to form a bright red color). Produce a cell-wall associated glycerol teichoic acid antigen that is identified as the streptococcal group D antigen Enterococcus faecalis - PYR test Enterococcus faecalis bile esculin agar Pathogenesis E. faecalis can cause endocarditis and septicaemia, urinary tract infections, meningitis, and other infections in humans. Several virulence factors are thought to contribute to E. faecalis infections. A plasmid-encoded hemolysin, called the cytolysin, is important for pathogenesis in animal models of infection, and the cytolysin in combination with high-level gentamicin resistance is associated with a five-fold increase in risk of death in human bacteraemia patients. A plasmid-encoded factor called "aggregation substance" is also important for virulence in animal models of infection. Page 2 of 3 P.O. Box 131375, Bryanston, 2074 Ground Floor, Block 5 Bryanston Gate, 170 Curzon Road Bryanston, Johannesburg, South Africa www.thistle.co.za Tel: +27 (011) 463 3260 Fax: +27 (011) 463 3036 Fax to Email: + 27 (0) 86-538-4484 e-mail : [email protected] Antibacterial resistance E. faecalis is resistant to many commonly used antimicrobial agents (aminoglycosides, aztreonam, cephalosporins, clindamycin, the semisynthetic penicillins (nafcillin and oxacillin) and trimethoprim- sulfamethoxazole). Resistance to vancomycin in E. faecalis is becoming more common. Treatment options for vancomycin-resistant E. faecalis include nitrofurantoin (in the case of uncomplicated UTIs), linezolid, and daptomycin, although ampicillin is preferred if the bacteria are susceptible. Quinupristin/dalfopristin can be used to treat Enterococcus faecium but not E. faecalis. In root canal treatments, NaOCl and chlorhexidine (CHX) are used to fight E. faecalis before isolating the canal. However, recent studies determined that NaOCl or CHX showed low ability to eliminate E. faecalis. References 1. http://emedicine.medscape.com/article/216993-clinical 2. https://en.wikipedia.org/wiki/Enterococcus_faecalis 3. http://www.microbiologyinpictures.com/bacteria-photos/enterococcus-faecalis-images.html Questions 1. Discuss the morphological characteristics of E. faecalis. 2. Discuss the role of E. faecalis in disease. 3. Discuss the tests used in the identification of E. faecalis. Page 3 of 3 .
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