Enterococcus
Pathogenesis: enterococci
25% mobile genetic elements drug resistance virulence factors Virulence: E. faecalis Drug resistant: E. faecium
Pathogenesis: E. faecium
3 genes implicated in virulence: ESP (Enterococcal Surface Protein): adhesion ACM (virulence protein): adhesion of collagen Ebp (Endocarditis and biofilm associated pili):
Clinical Significance: enterococcal infections
80-90% E. faecalis 10-15% E. faecium <5% other
Pathogenesis: virulence factors
Cytolysins (lysis): cell membranes RBCs/ erythrocytes other Gram-pos Aggregation substance: pheromone-responsive plasmids transfer ag.-sub. biofilms Superoxide production: extracellular damage tissue Resistance to antimicrobial agents: acquires/exchanges genes that encode resistance
Laboratory Diagnosis: Table Enterococci Differentiation
Differentiate: E. faecalis from E. faecium tellurite (inorganic substance, oxide mineral)
General Metabolism: Enterococcus genus
Facultative anaerobe: chemoorganotrophic (fermentative + some respiratory) aerobic conditions (hemin) fermentation product (lactic acid ) catalase negative oxidase negative PYR positive (pyrrolidonyl-beta-napthylamide) LAP positive (Leucine aminopeptidase) acid production (starch) growth: 40% bile high salt concentrations wide Temp broad pH
General Characteristics: Enterococcus genus
Gram-positive cocci/ovoid (0.6-2.5µm) pairs/short chains sometimes motile (scanty flagella) no spores facultative anaerobe G:C 37-45%
Laboratory Diagnosis: microscopy
Gram-stain positive: cocci young culture (CV) single/pairs
Laboratory Diagnosis: Identification Table Enterococci
Positive for: Voges-Proskaeur (detects acetoin) Beta-glucosidase LAP (Leucine amino peptidase) Hydrolysis: PYR Esculin Resistance: 40% Bile Acid production: NAG (n-acetyl glucomsamine) Sugars metabolized: ribose xylulose Negative for: Urease (can't hydrolyze Urea)
Laboratory Diagnosis: PYR test
Pyrrolidonyl-beta-napthylamide test rapid biochemical identifies enterococci colonies (minutes) tests hydrolysis PYR appears dk/pink
Pathogenesis: E. faecalis
aggregation substance: encoded by plasmid gene adheres to eukaryotic cells aggregates conjugation lipoteichoic acids (adhesion): polyglycerol ESP (Enterococcal Surface Protein): colonization/persistence urogenital tract ACE (virulence protein): adhesion of collagen Ebp (Endocarditis and biofilm associated pili): Extracellular superoxide production Gelatinase (gellatin, collagen, fibrogen, hemoglobin) Hyaluronidase (permeabilize host-cm) Cytolysin
Treatment, Prevention and Control: UTI isolates
ampicillin nitrofurantoin tetracycline quinolones
Laboratory Diagnosis: Intergenic ribosomal PCR
another method to genome/DNA sequencing amplifies noncoding region (between 16S & 23S rRNAgenes) discriminates enterococcal strains to species level
Treatment, Prevention and Control: resistance
antimicrobial agents: cephalosporins aminoglycosides vancomycin (specifically E. faecium) vancomycin resistance mediated by: modified antibiotic target terminal D-ala-D-ala converted to Dala-Dlal (lactine) penicillin resistance mediated by: modified PBP (penicillin binding protein)
Laboratory Diagnosis: Commercial Biochemical Systems
automated ID system kits
Treatment, Prevention and Control: systemic life-threatening infections
cell wall active agents: penicillin ampicillin vancomycin in combination with aminoglycoside: gentamycin streptomycin newer agents: linezdid daptomycin multi-drug resistant strains: chloramphenicol (last resort) (side affects)
General Characteristics: Enterococcus cell wall
cell-wall associated streptococcal antigen: Polyglycerol lipoteichoic acid
Clinical Significance: enterococcal endocarditis
children rarely in infants risk factors: genitourinary & biliary portals heart disease underlying
Laboratory Diagnosis: Bile-Esculin test
differentiate enterococci and GDS glycoside esculin hydrolyzed = esculetin + dextrose esculetin + ferric citrate =dark phenolic iron complex
Natural Habitats: transmission
horizontal transmission fomites
Clinical Significance: nosocomial infections
hospital horizontal and fomite transfer 2nd to MRSA as cause of nosocomial infections
Natural Habitats: E. faecalis, E faecium
human GI tract female urogenital tract outside (fecal pollutants) E. faecium: fermentation products (cheese/sausage) some strains, probiotic
Laboratory Diagnosis: cultivation Enterococci
isolated from pathologocal specimen nonselective blood agar/ blood agar supplemented with: colistin (polymyxtin) nalidixic acid oxolonic acid BAP appearance: grey colonies non-pigmented colonies (other 17 spp.) hemolysis: beta (E. faecalis) alpha (E. faecium) 40%bile 6.5% NaCl pH 9.6 10ºC/45ºC/ if 60ºC, 30 min. (wide range kills other G. sp.)
Natural Habitats: E. mundtii, E. casseliflavus
plant associated yellow pigmented
Laboratory Diagnosis: molecular techniques for typing enterococcal isolates
robust molecular techniques generates web-based repository both techniques are: reproducible, portable, discriminatory, unambiguous MLST more rapid/cheaper MLST (multilocus sequence typing): identify alleles via sequencing of 7 internal fragments of housekeeping genes (always expressed for survival) Multiple locus VNTR (variable number tandem repeat) analysis: different numbers of the same repeat based on differences in VNTR in multiple loci dispersed over enterococcal genome
Natural Habitats: Enterococci
soil food water normal flora (animals, humans, birds)
Laboratory Diagnosis: Multiplex PCR
species-specific PCR assay: 4 primers E. faecium (Vancomycin resistant genes) encoding: D-ala D-alanine ligases ddl genes tuf (translation elongation factor) genes: cpn60 & cpn10 genes (GroEL & GROES) (molecular chaperons protein folding)
Clinical Significance: enterococcal bacteremia
typical source: UTI other sources: intra-abdominen and pelvis spontaneous peritonitis (cirrhotics (liver), nephrotics (kidneys)) chronic peritonitis (chronic ambulatory peritoneal) dialysis peripartum mothers: endometritis abscess following cesarean section infants bone intravascular catheters central nervous system: meningitis underlying disorder long-term illness invasive procedure CNS prior antibiotic therapy
