Dr.T.V.Rao.

MD

Dr.T.V.Rao MD 1
 Introduction to Enterococci
 Enterococci are gram-positive

cocci which often occur in pairs
(diplococci)
 Two species are common
commensal organisms in the
intestines of humans:
E. faecalis and E. faecium
 Enterococci occur almost
everywhere, including soil, food,
water, plants, animals, birds, and
insects they inhabit in humans and
other animal's gastrointestinal
tract and the female genital tract

Dr.T.V.Rao MD 2
 Characters of Enterococci
 Gram(+) , Catalase(－)


Cocci
 Can grow in media :
6.5% sodium chloride
 E. faecalis and E. faecium
(90%)
 Part of the normal
bowel flora. the
prominent cause of
nosocomial infections.

Dr.T.V.Rao MD 3
Habitat of Enterococci

 Enterococci normally inhabit
the bowel. They are found in
the intestine of nearly all
animals, from cockroaches to
humans. Enterococci are
readily recovered outdoors
from vegetation and surface
water, probably because of
contamination by animal
excrement or untreated sewage
. In humans, typical
concentrations of enterococci in
stool are up to 108 CFU per
gram .

Dr.T.V.Rao MD 4
 Growing Importance of
Enterococci


 Enterococcus species are normal flora of the intestinal
tract. Enterococcus faecalis frequently causes infections
within the peritoneal cavity, especially following
penetrating trauma such as gunshot wounds, and
surgical wounds, urinary tract infections, prostate
infections, and infections of damaged or compromised
skin, such as diabetic or decubitus ulcers, burns, and
surgical wounds. Other opportunistic fecal streptococci
include E. faecium and E. durans.

Dr.T.V.Rao MD 5
 Prominent Cause of Nosocomial
Infections


 The enterococci have become
the second most common
bacterium isolated from
nosocomial urinary and
wound infections, and the
third most common cause of
nosocomial bacteremia.
Furthermore, the enterococci
are among the most
antibiotic resistant of all
bacteria, with some isolates
resistant to all known
antibiotics
Dr.T.V.Rao MD 6
 Clinical Manifestation


 Infections with VRE do not
differ from other enterococcal
infections other than in their
therapy.
 The most common sites of
infection :
The urinary tract and
bloodstream.
 In addition, enterococci may
cause endocarditis due to their
ability to adhere to heart
valves.
 They rarely cause respiratory
tract infections.
Dr.T.V.Rao MD 7
 Glycopeptides
Mechanism of Action


 Vancomycin and teicoplanin
inhibit cell wall synthesis by
forming complexes with
peptidyl-D-alanyl-D-alanine
termini
 vanA and vanB resistance
phenotypes are associated
with the acquisition of gene
clusters that lead to the
production of peptidoglycan
ending in D-alanyl-D-lactate

Dr.T.V.Rao MD 8
 Vancomycin Resistance Increases
Morbidity and Mortality


 Vancomycin-resistant enterococci (VRE), first reported in
Europe in 1988, are emerging as a global threat to public
health . The incidence of VRE infection and colonization
among hospitalized patients has increased rapidly in the
last 7 years. From 1989, the year VRE was first identified
in the United States, through 1993. Infection with VRE
may be associated with increased mortality , and no
effective antimicrobial therapy is available for many VRE
.

Dr.T.V.Rao MD 9
 VRE Epidemiology




Found world-wide, but rates vary greatly
Hospital outbreaks often involve clonal spread
 Also seen in nursing homes and long term care facilities
 In Europe, animals may be a source due to the use of the
glycopeptide, avoparcin
 First described in Europe
 Primarily a nosocomial pathogen
 Alarming increase from 1989 to 1993
 intensive care units
teaching hospitals
hospitals with more than 500 beds.

Dr.T.V.Rao MD 10
 Vancomycin and Development
of VRE Strains


 Mechanism of action: Inhibits bacterial cell wall synthesis
 Spectrum of action: Gram positive organisms

Including: Listeria, Rhodococcus, Peptostreptococcus

Bacteriostatic against enterococcus
 Mechanism of resistance:

Enterococcus: Van A – E
 Peptidoglycan precursor has decreased affinity for
vancomycin – D-ala-D-ala replaced by D-ala-D-lac

Dr.T.V.Rao MD 11
 Vancomycin-Resistant
Enterococci


(VRE), are bacterial strains of the genus
Enterococcus that are resistant to the antibiotic
vancomycin. Enterococci are gram-positive
coccoid-shaped bacteria found in the digestive
tract of some humans. To become VRE,
vancomycin-sensitive enterococci typically
obtain new DNA in the form of plasmids or
transposons which encode genes that confer
vancomycin resistance.Dr.T.V.Rao MD 12
 vanA and vanB
Phenotypes
vanA vanB

Vancomycin MIC >64 4-1024

Teicoplanin MIC 16-512 ≤ 0.5

Usual species faecium, faecalis faecium, faecalis

Acquired Yes Yes

Transferable Yes Yes

 MOLECULAR BASIS OF
VANCOMYCIN RESISTNACE
vanC, vanD, and vanE Phenotypes
vanC vanD vanE

Vancomycin 2-32 128 16

MIC
Teicoplanin ≤ 0.5 4.0 0.5
MIC
Usual species gallinarum, faecium faecalis
casseliflavis,
flavescens
Acquired No Yes Yes

Transferable No No No
 Glycopeptide-Resistance
Transposons


 In E. faecium, vanA and
related genes are located on
a transposon (Tn1546)
which resides on a plasmid
 Resistance is associated
with a number of genes
(vanHAX gene cluster,
vanS, vanR, vanX and
vanZ)
 vanB phenotype is
associated with Tn5382

Dr.T.V.Rao MD 16
 vanA Genes and Their
Enzymatic Products (I)
vanA protein is a

ligase

produces D-ala-D-lac rather than D-ala-D-ala
(vanA alone does not lead to resistance)
vanH protein is a dehydrogenase

converts pyruvate to D-lactic acid
(the source of lactate for the above reaction)
vanX protein is D,D-dipeptidase

cleaves D-ala-D-ala
(Cetinkaya et al. 2000. Clin Micro Rev. 13: 686-707)
Dr.T.V.Rao MD 17
 vanA Genes and Their
Enzymatic Products (II)
vanS is a sensor



detects the presence of vancomycin or some effect of it
vanR is a regulator

may turn on vanHAX
vanY is a carboxypeptidase

cleaves terminal D-Ala
vanZ increases the MIC of teicoplanin

mechanism is unknown
Cetinkaya et al. 2000. Clin Micro Rev. 13: 686-707

Dr.T.V.Rao MD 18
 vanB Genes and Resistance
vanB is also a ligase


involved with D-ala-D-lac production
vanXB has dipeptidase activity

associated with high level vancomycin resistance
vanHB, vanYbB, vanSB and vanRB genes

similar to their vanA counterparts
Teicoplanin does not induce the synthesis of vanB
proteins and cells are still susceptible

Cetinkaya et al. 2000. Clin Micro Rev. 13: 686-707

Dr.T.V.Rao MD 19
 vanC, vanD and vanE
Resistance


 vanC ligases result in D-ala-D-ser

net result is reduced vancomycin binding
 vanD ligase

has some homology with other van ligases
 vanE ligase

rare

more closely related to vanC than the other van ligases

Dr.T.V.Rao MD 20
 Result
Spreading Resistance


 Enterococci that acquire
the vanA phenotype are
highly resistant to
vancomycin and to
teicoplanin
 Enterococci can pass the
vanA gene cluster to S.
aureus

E. faecalis rather tan E.
faecium (so far)

Dr.T.V.Rao MD 21
 Infections Caused by Vancomycin
Resistant Enterococci


 Urinary tract infection (most common)
 Intra-abdominal and pelvic infection (also common)
 Surgical wound infection
 Bacteremia—bacteria in the blood
 Endocarditis —infection of the inner surface of the heart
muscles and valves
 Neonatal sepsis —bacteria in the blood, occurring in
infants
 Meningitis —infection of the membranes that surround
the brain and spinal cord

Dr.T.V.Rao MD 22
Dr.T.V.Rao MD 23
 Diagnosis of VRE
Specimens can proceeded as per clinical
Needs


Diagnosis requires culturing the organism. VRE
is easily grown on culture plates in a laboratory.
To get material to culture, a sample of the
infected tissue is taken. For a wound infection, a
swab is usually rubbed over the surface to get
infected material. Blood is drawn and cultured to
detect sepsis or endocarditis. Urine samples are
taken to identify urinary infections .

Dr.T.V.Rao MD 24
 Enterococci are Bile Esculin
Test Positive
 Enterococci are able to grow


in the presence of bile and
hydrolyze the esculin; the
liberated
diphydroxycourmarin
complexes with ferric citrate
present in the media to form
a dark brown/black soluble
compound.

Dr.T.V.Rao MD 25
Bile Esculin Test Positive for group D
streptococci and enterococci

Dr.T.V.Rao MD 26
 Detection of Vancomycin
Resistance


 Susceptibility to vancomycin
was performed by Kirby-
Bauer Disc Diffusion
Method on Mueller Hinton
Agar by using 30µg
vancomycin disc .
Vancomycin resistance was
also determined by
Vancomycin agar screen
method using 6µg/ml of
vancomycin incorporated in
Brain Heart Infusion (BHI)
agar.

Dr.T.V.Rao MD 27
 Detection of Vancomycin
Resistance


 Minimum Inhibitory
Concentration (MIC) of all
the isolates were done by
Macro broth dilution
method, using dilutions
of vancomycin ranging
from 2 µg/ml to 512
µg/ml.

Dr.T.V.Rao MD 28
Drug Resistance can be
Established by E-Test

Dr.T.V.Rao MD 29
 *Chromogenic Methods in
Diagnosis of VRE


Chromogenic medium
for the detection of
Vancomycin Resistant
Enterococcus (VRE) E.
faecalis and E. faecium
 * Colorex™ Prepared
Chromogenic Media by BioMed
Diagnostics

Dr.T.V.Rao MD 30
 Genotypic Detection of
VRE


 Rapid detection of
vancomycin resistance
by polymerase chain
reaction (PCR). useful
in epidemiologic
studies
 PCR cant be performed
directly on clinical
specimens.

Dr.T.V.Rao MD 31
 Control and Prevention
Limiting the use of certain broad spectrum

antibiotics may also lead to a decrease in the rates
of VRE colonization and infection.
One study suggested that reduction of third-
generation cephalosporins with the substitution of
piperacillin/tazobactam could reduce the
incidence of VRE in an intensive care unit setting

Hospital Infection Control Practices Advisory Committee (HICPAC). Recommendations for preventing the spread of
vancomycin resistance. Infect Control Hosp EpidemiolDr.T.V.Rao
1995; 16:105
MD 32
 Control and Prevention

preventing the spread of
The CDC has recently published
recommendations for
vancomycin resistance
 Prudent use of vancomycin
 Education of hospital staff regarding the problem
 Rapid and accurate identification of VRE in the
microbiology laboratory
 Aggressive infection control measures utilizing contact
isolation and cohorting where necessary to prevent
person-to-person transmission

Hospital Infection Control Practices Advisory Committee (HICPAC). Recommendations for preventing the spread of vancomycin
resistance. Infect Control Hosp Epidemiol 1995; 16:105 Dr.T.V.Rao MD 33
Hand Washing can Reduce the
Spread of VRE

Dr.T.V.Rao MD 34


Created by Dr.T.V.Rao MD for ‘ e ‘
Learning Resources for Medical and
Paramedical Professionals in
Developing World
Email
doctortvrao@gmail.com

Dr.T.V.Rao MD 35