Download RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
KARNATAKA, BANGALORE-41
REGISTRATION OF SUBJECT FOR DISSERTATION:
1.Name of Candidate and Address :
RUBY NANCY.W
Postgraduate (M.Sc.MLT)
Department of Microbiology,
St.John's Medical College Hospital,
Sarjapur road,
Bangalore-34
2. Name of the Institution :
St.John's Medical College Hospital,
Bangalore-34
3. Course of Study and Subject :
M.Sc.MLT(Microbiology)
4. Date of Admission to course :
01 Sept' 2010.
5. Title :
Speciation of Clinically Significant
Enterococci and their Antibiotic
Susceptibility Testing
6. BRIEF RESUME OF STUDY
6.1. NEED FOR STUDY :
Enterococci are the most common aerobic and facultative anaerobic, Gram positive cocci found in the
bowel flora of humans and other animals. In normal individuals, it causes urinary tract infection(UTI),
bacteremia/sepsis, endocarditis, intraabdominal and pelvic infections. In hospitalised patients, it causes
urinary tract infection(UTI), surgical wound infection and others. E.faecalis and E.faecium are the most
common species, both account for upto 90% of the clinical isolates. Presently there are 23 recognised
Enterococcus species. Nevertheless, the incidence of other species of Enterococci from clinical sources
shows an alarming increase with the properties of intrinsic resistance to several antibiotics. Thus proper
identification of Enterococci to species level is essential for management and prevention of these bacteria
in any health care facility.(1)
Antibiotic resistance in Enterococci can be intrinsic (inherent) or acquired.
The term intrinsic resistance is used to indicate resistance, which is the usual species characteristic present
in all or most of the strains of that species. Intrinsic resistance expressed by Enterococci are to(5,8,10)
1)Beta lactams
2)Cephalosporins
3)Low level of Aminoglycoside
4)Low level of Clindamycin, Trimethoprime, Sulfamethoxazole and Fluroquinolones.
Acquired resistance results from either a mutation in existing DNA or acquisation of new DNA(5).
Acquired resistance are to
1)High concentration of Beta lactams, through penicillin binding proteins or Beta lactamase.
2)High level Clindamycin, Aminoglycosides by means of 6-acetyl transferase-2phosphotransferase, glycopeptides(vancomycin) and lipopeptide(Teicoplanin) by lipase.
3)Penicillin by means of Penicillinase
Ampicillin was the drug of choice for enterococcal infection. Enterococci has become increasingly
resistant to Ampicillin making vancomycin the treatment of choice.Since 1988
resistance to vancomycin has emerged and increased worldwide.(9) In case of Endocarditis, combination
therapy of a B-lactam and an Aminoglycoside has been the treatment. Aminoglycosides are not routinely
used to treat other Enterococcal infections but in endocarditis the synergy between the two agents
provides a cure. Synergy doesnot occur if the organism has high level gentamicin resistance.(9) The
emergence of vancomycin resistant Enterococci (VRE) in addition to the increasing incidence of high level
aminoglycoside resistance(HLAR) presents a serious challenge to clinicians in treating the patients with
infection due to enterococci.(7)Antimicrobial susceptibility pattern to recommended drugs is very helpful
to the clinicians and with MIC determination which may prevent over utilisation or elimination of
unnecessary antibiotics.
6.2. REVIEW OF LITERATURE
INTRODUCTION
The name Enterococci was proposed by Kalina in 1970 and confirmed by Schleifer and kilpper - Balz
in 1984 because they showed biochemical and other differences from other streptococci. The organism
now classified in the genus Enterococcus were formerly classified as faecal Streptococci. Enterococci
contain a C-carbohydrate that reacts with group D antisera. Therefore in the past, they were considered
group D streptococci. Today DNA analysis and other properties have placed them in genus
Enterococcus(11,12)
CLASSIFICATION
Enterococcus is classified into five groups based on acid Formation from mannitol, sorbitol and sorbose
broth and hydrolysis of arginine.(11,12)
Group 1 E.avium
Group11 E.faecalis,E.faecium,E.gallinarum
Group 111 E.durans
Group 1V E.sulfurous, E.cecorum
Group V Variants of E.faecalis, E.gallinarum
EPIDEMIOLOGY
Enterococci are part of normal fecal flora. However, they can also colonize oral mucous membranes and
skin especially in hospital settings.These organisms are highly resistant to environment and chemical
agents, and can persist on fomites(11,12).Most infection occur in hospitals.In India several studies are
suggested an increase in nosocomial infection rates for Enterococci in recent years, national nosocomial
infection surveillance system data show little change in the percentage of Enterococcal infections over
the past 2 decades(1).In U.S the percentage of nosocomial infections caused by Enterococci has increased
more than 20 fold between 1989 and 1993,indicating rapid dissemination.(2,6)
DISEASES
Enterococci seldom cause disease in normal, healthy individuals. However, under conditions
where host resistance is lowered, or where the integrity of the gastrointestinal or genitourinary tract has
been disrupted. It causes urinary tract infection (UTI) in Hospitalised patients bacteremia /sepsis, subacute
bacterial endocarditis and intra abdominal abcesses.(11,12).
LABORATORY DIAGNOSIS
Laboratory diagnosis of Enterococcus involves culture from different clinical samples like
urine, blood, sterile fluids like Ascitic, peritoneal fluid and fluid from other sterile sites.
Routine media
1.Macconkey agar
2.Blood agar
Gram staining :
Gram positive spherical or oval cocci arranged singly, pairs or short chains
Genus identification by
1. Bile-esculin test
2. Salt tolerance ( 6.5% Nacl) test
3. Heat tolerance test
Species identification by
1.Arginine dehydrolase test
2.Voges proskauer test
3.Potassium tellurite reduction
4.Motility by hanging drop method
5.Pigment production on tryptic soya agar
6..Sugar fermentation test (13)
In addition several molecular methods are available in the speciation of Enterococci including PCR.
Antibiotic sensitivity is usually determined by modified Kirby Baurer disc diffusion method and MIC is
obtained by Agar dilution method or broth dilution. The resistance genes could also be identified using
molecular methods like Multiplex PCR which may be useful to predict resistance in vivo(15).
6.3. AIMS AND OBJECTIVES
1.Speciation of clinically significant Enterococci
2.Antibiotic susceptibility testing by modified kirby-bauer disc diffusion method
3.MIC to Vancomycin and High Level Gentamicin
7. MATERIALS AND METHODS
7.1 SOURCE OF DATA
50 consecutive samples of
1.Urine
2.Blood
3. Sterile fluids [Ascitic fluid, peritoneal fluid, CSF and fluids from other sterile sites ]
4.Catheter tip [central venous catheter, Artery line when it is associated with blood culture]
5.Pus [only if it is predominant organism]
These samples will be collected from diagnostic laboratory of microbiology,
SJMCH Bangalore.
STUDY PERIOD - Jan 2011 – Dec 2011
7.2 INCLUSION CRITERIA
Enterococci isolated from urine, blood, sterile fluids [ascitic fluid, peritoneal fluid,
CSF and fluids from other sterile sites],catheter tip, pus [which are Enterococci predominant] will
be included in the study.
7.3 EXCLUSION CRITERIA
Enterococci from samples which are contaminated with other commensals sputum,
stool,GI tract, female genital tract, foley’s catheter tip will be excluded from the study .
7.4 METHODS
Suspected Enterococcal isolates
Characteristic colony morphology
Gram’s stain
Catalase test
Confirmation as genus Enterococcus
Bile -esculin test
Salt tolerance test
Heat tolerance test
Enterococcus sp. Identification
1.Arginine dehydrolase test
2.Voges Proskauer test
3.Potassium tellurite reduction
4.Motility by hanging drop method
5.Pigment production on tryptic soya agar
6.Sugar fermentation test
(Glucose, lactose, sucrose, Mannitol, sorbitol, arabinose)
Antibiotic susceptibility testing
1.Modified Kirby Baurer disc diffusion method
a) Ampicillin 10mg
b) Penicillin
10U
c) Ciprofloxacin 5mg
d)Vancomycin 30mg
e)Teicoplanin 30mg
f) Linezolid
30mg
g)High level Gentamicin 120mg
MIC by Agar dilution method
1) Vancomycin
Range 1-128 mg/ml,Resistance >32mg/ml
2) High level Gentamicin
Range 250-2000mg/ml,Resistance>500mg/ml
7.5. Does the study require any investigation of intervention to be conduced on patients or other than
human beings or animals ? If so, describe briefly.
No
7.6 Has ethical clearance been obtained from your institution incase of 7.5.
Not Applicable
7.7 STATISTICAL ANALYSIS:
This is a descriptive study and results will be expressed in terms of
Percentages.
8. REFERENCE
1.Prakash VP, Rao SR, Parija SC .Emergence of unusual species of Enterococci causing infections,
South India. BMC Infectious Diseases 2005;5(14):106-110
2. Huyele MM, Sahm DF, Gilmore MS. Mutiple drug resistant Enterococci,nature of the
problem and an agenda for the future. Journal of Emerging Infectious Diseases 1998:11058.
3. L.D. Wells, A. Von Graevenitz .Clinical significance of Enterococci in blood cultures
from adult patients. Infection 8(1980).
4. Richard A. Horvitz and Alexander Von Graevenitz.A clinical study of the role
Enterococci as sole agents of wound and tissue infection.The yale journal of biology and
medicine 50(1977),391-395
5. Moellering RC. Emergence of Enterococci as a significant pathogen. J Clin
Infect Dis 1992; 14:1173-1178
6. Gordon S, Swenson JM, Hill BC, Pigott NE, Facklam RR, Cooksey RC et al,
Antimicrobial susceptibility pattern of common and unusual species of
Enterococci causing infections in the United States. J Clin Microbiology 1992
Sept; 30(9): 2373-2378.
7. Patterson JE, Masecar BL, Kauffman CA, Sachaberg DR. Hicrholzer WJ. et
al. Gentamicin resistance plasmids of Enterococci from diverse geographic
areas are heterogenous. The J Infect Dis 1988 July; 158(1): 212-216.
8. Cetinkaya Y, Falk P, Mayhall CG. Vancomycin resistant Enterococci. Clin
Microbiol Rev 2000 Oct; 13(4): 686-707.
9.Ruoff K.L, Maza Ldela, Murtagh MJ,Pango JDS and Furaro MJ.
Antimicrobial resistance in Enterococcus species in Australian surveillance 2007.Commun Dis
Intill,2007;(6-22).
10. Mundy LM, Sahm DF, Gilmore M. Relationship between Enterococcal
virulence and antimicrobial resistance. Clin Microbiol Rev 2000 Oct; 13(4):
513-522.
11. Anathanarayan R and Paniker CK. Text book of microbiology 7th Ed.
Chennai, India; Orient Longman publications: 2005.
12. Baveja CP. Text Book of microbiology, 2nd Ed. New Delhi; Arya publications:
2005.
13. Ross PW, Streptococcus and Enterococcus. In Collee JG, Fraser BP, Marmion
BP, Simmons A, editors, Mackei and McCartney practical Medical Microbiology, 14th
Ed. USA; Churchill Living stone:1966.
14.Jackson R Charlene, Cray Fedorka J Paula,Barrett B John,and Ladely R,Scott.Genetic
Relatedness of High-level Aminoglycoside resistant Enterococci. Avian diseases;2004
Jan ;48.(102-104)
15.Sergei B.Vakulenko,Susan M.Donabedian, Anatoliy M.Voskresenskiy,Marcus
J.Zervos,Stephen a.Lerner,and Joseph W.Chow Multiplex PCR for Detection of
Aminoglycoside Resistance Genes in Enterococci. Antimicro. Agents Chemother 2003
April;47(4):1423-1426.
9
Signature of Candidate:
10
Remarks of the Guide:
11
Name and Designation of:
11.1 Guide : Dr.Mary Dias,M D (Microbiologist)
Assistant Professor
St.John's Medical College
Bangalore
11.2 Signature:
11.3 Co-guide:
(If any)
11.4 Signature:
11.5 Head of the Department: Dr.Muralidharan.S MD(Microbiologist)
St.John's Medical College
Bangalore
11.6 Signature:
12
12.1 Remarks of the Chairman & Principal:
12.2 Signature: