Identification and Characterization of Enterococcus spp. in Local Surface Waters

Team Microbiology

Advisor: Dr. June MiddletonAssistant: Alex Kohl

Neha Bansal, Laura Decker, Kevin Huang, Ashwinee Ragam, Angela Sekerke, Reema Shah, Sarah Song, Pallavi Yerramilli, Eddie Zhang, Jeremy Zornow

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

Characteristics• Gram-positive cocci

• Group D glycerol teichoic acids

• 6.5% NaCl

• Esculin Esculin test

Medical Significance• E. faecium and E. faecalis

• Nosocomial infections– Urinary tract infection– Wound infection– Bacterial endocarditis E. faecium

E. faecalis

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

Antibiotic Resistance

the potential of a microorganism to resist and endure the effects of an antibiotic

Vancomycin-Resistant Enterococcus spp.

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.

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.

Sample Collection

Burnham Pond, Drew Pond, Loantaka Brook, and Whippany River

Culture Isolation

Whippany River1 mL

Whippany River5 mL

Whippany River25 mL

Drew Pond1 mL

Drew Pond5 mL

Drew Pond10 mL

Speciation

Yellow = Fermentation(Positive)

Red = Negative

Sugars Tested:arabinosemannitolmethyl α-D-glucopyranoside (MGP)ribosesorbosesorbitol

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)

β hemolysis

α hemolysisVirulence Factors

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

Counts

3150

1100 880

240

Comparison of Total Enterococci Levels in Local Water Sources

1

10

100

1000

10000

Whippany River (W) Loantaka Brook (LB) Drew Pond (DREW) Burnham Pond (BP)

Water Source

log

Co

lon

ies

per

100

mL

Relative Abundance

Relative Abundance of Enterococcal Species in Local Water Sources

0

10

20

30

40

50

60

Whippany River (n=46) Drew Pond (n=48) Loantaka Brook (n=41) Burnham Pond (n=33)

Water Source

Perc

en

t o

f T

ota

l Is

ola

tes

E. faecium

E. faecalis

E. durans/hirae

E. gallinarum

E. casseliflavus

E. mundtii

Relative Abundance con’t

• Low concentration of Enterococcus faecalis

• High concentration of Enterococcus casseliflavus

• Varying concentrations of Enterococcus faecium

Antibiotic ResistanceRelative Antibiotic Resistance of Total Enterococci in Local Water Sources

0

20

40

60

80

100

Whippany River (n=46) Burnham Pond (n=33) Drew Pond (n=48) Loantaka Brook (n=41)

Water Source

Perc

en T

otal

Isol

ates

Penicillin

Cephalothin

Tetracycline

Streptomycin

Gentamycin

Ciprofloxacin

Vancomycin

Antibiotic Resistance con’t

• High resistance to Penicillin, Cephalothin, Streptomycin

• Moderate resistance to Gentamycin and Vancomycin

• Low resistance to Tetracycline and Ciprofloxacin

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

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

Conclusions con’t• E. faecalis and E. faecium are

nosocomial pathogens (causes of secondary infections in hospitals)

• Only pathogenic species of Enterococcus

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

Conclusions con’t• E. faecalis and E. faecium were found

to have similar resistance patterns

• One main exception – E. faecium displayed susceptibility to vancomycin

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

Paul Quinn the Bacterial Contamination!!!!!

Fin