Identification and Characterization of Enterococcus spp. in Local Surface Waters

1. Identification and Characterization of Enterococcus spp. in Local Surface Waters Team Microbiology Advisor: Dr. June Middleton Assistant: Alex Kohl Neha Bansal, Laura Decker, Kevin Huang, Ashwinee Ragam, Angela Sekerke, Reema Shah, Sarah Song, Pallavi Yerramilli, Eddie Zhang, Jeremy Zornow

2. Enterococcus spp. History • Closely related to Streptococcus • Separate genus formed in 1984 • 16 different species • Located in intestines of warm-blooded animals, plants, soil, and water

3. Characteristics • Gram-positive cocci • Group D glycerol teichoic acids • 6.5% NaCl • Esculin Esculin test

4. Medical Significance • E. faecium and E. faecalis • Nosocomial infections • Urinary tract infection • Wound infection • Bacterial endocarditis E. faecium E. faecalis

5. Antibiotics: antimicrobial compounds that terminate or impede the proliferation of bacteria derived from certain microorganisms Selective Toxicity: the ability of an antibiotic to target bacteria without harming the host cells

6. Antibiotic Resistance the potential of a microorganism to resist and endure the effects of an antibiotic Vancomycin-Resistant Enterococcus spp.

8. Virulence Factors • The products of any genes that help bacteria invade and colonize a host. • Virulence factors work synergistically to help the cell invade a host. • The more virulence factors a bacterium expresses, the more dangerous it is to our health.

9. Virulence Factors • Hemolysins • Compounds that destroy red blood cells. • Bacteriolysins • Compounds that kill “rival” bacteria, leading to the dominance of an area by one strain or species. • Gelatinase • Disassembles gelatins and collagen – the bacterial cell will then recycle the peptide segments for use among the colonizing bacteria.

10. Sample Collection Burnham Pond, Drew Pond, Loantaka Brook, and Whippany River

11. Culture Isolation Whippany River 1 mL Whippany River 5 mL Whippany River 25 mL Drew Pond 1 mL Drew Pond 5 mL Drew Pond 10 mL

12. Speciation Yellow = Fermentation (Positive) Red = Negative Sugars Tested: • arabinose • mannitol • methyl α-D-glucopyranoside (MGP) • ribose • sorbose • sorbitol

13. Antibiotic Resistance Antibiotics Tested: • cephalothin (30µg/ml) • chloramphenicol (30µg/ml) • ciprofloxacin (5µg/ml) • erythromycin (15µg/ml) • gentamycin (10µg/ml) • nitrofurantoin (300µg/ml) • penicillin (10µg/ml) • streptomycin (10µg/ml) • tetracycline (30µg/ml) • vancomycin (30µg/ml)

14. Virulence Factors α hemolysis β hemolysis Virulence Factors Tested: • Gelatinase • Hemolysins • Bacteriocins~ Tested against: • Streptococcus mitis PI 519 • Enterococcus faecalis ATCC 29212 • Enterococcus mundtii O • Enterococcus mundtii P • Enterococcus mundtii R

15. Counts 3150 1100 880 240

16. Relative Abundance

17. Relative Abundance con’t • Low concentration of Enterococcus faecalis • High concentration of Enterococcus casseliflavus • Varying concentrations of Enterococcus faecium

18. Antibiotic Resistance

19. Antibiotic Resistance con’t • High resistance to Penicillin, Cephalothin, Streptomycin • Moderate resistance to Gentamycin and Vancomycin • Low resistance to Tetracycline and Ciprofloxacin

20. Virulence • Majority of Enterococcus from Whippany River lysed all five of the bacteria • Majority of Enterococcus from Drew Pond and Loantaka Brook lysed none of the bacteria • Burnham Pond possessed no colonies of virulent bacteria

21. Conclusions • Maximum level of Enterococcus allowed for drinking water is 7 isolates per 100 mL and for bathing water is 100 isolates per 100 mL • All of the water sources we tested were unsuitable for drinking or bathing

22. Conclusions con’t • E. faecalis and E. faecium are nosocomial pathogens (causes of secondary infections in hospitals) • Only pathogenic species of Enterococcus

23. Conclusions con’t • Overuse of common antibiotics has led to high bacterial resistance • Three antibiotics virtually ineffective in curbing Enterococcus spp. growth • Penicillin • Cephalothin • Streptomycin • Three antibiotics displayed 100% effectiveness against Enterococcus spp. • Chloramphenicol • Erythromycin • Nitrofurantoin

24. Conclusions con’t • E. faecalis and E. faecium were found to have similar resistance patterns • One main exception – E. faecium displayed susceptibility to vancomycin

25. Conclusions con’t • Whippany River - large concentration of E. faecalis and E. faecium • Enterococcus found in Drew Pond and Loantaka Brook possess lower virulence capability • Burnham Pond –no isolates of E. faecalis or E. faecium found in pond

26. Fin Paul Quinn the Bacterial Contamination!!!!!