Download Inclusion Criteria for HA-MRSA

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA
BANGALORE
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1.
Name of the Candidate
Address (in block letters)
2.
Name of the Institution
3.
Course of study and subject
4.
5.
Date of admission to course
Title of the topic:
Dr. PRIYANKA S
Second floor , PG Hostel ESICMC- Post
Graduate Institute Of Medical Science and
Research, Rajajinagar
Bangalore – 560010.
ESIC-Medical College & Post Graduate
Institute Of Medical Science and Research,
Rajajinagar, Bangalore – 560010.
M.D. (MICROBIOLOGY)
31st MAY 2011
Comparision of Biofilm Production in Methicillin Resistant Staphylococcus aureus and
Methicillin Sensitive Staphylococcus aureus as a marker of virulence along with their
antibiogram in clinical isolates at ESIMC- PGI MSR , Rajajinagar ,Bangalore.
6.
Brief resume of the intended work:
6.1 Need for the study
Staphylococcus aureus infection ranges from superficial infection to deep infection and has
emerged over the past several decades as a leading cause of hospital acquired and community
acquired infections. In recent years, many isolates of S. aureus have evolved resistance to
multiple antibiotics and becoming a potential epidemiological threat.(1)
Traditionally, Methicillin Resistant Staphylococcus aureus (MRSA) has been considered a
major nosocomial pathogen in healthcare facility, but in the past decade it has been observed
emerging in the community as well.(2)
Biofilm formation is the hallmark characteristic of S. aureus infection, which consists of
multiple layers of bacteria encased within an exopolysaccharide glycocalyx .Presence of
glycocalyx protects the enclosed bacteria from host defense and impedes delivery of
antibiotics.(3)
The identification of Biofilm in persistent infections may assist in deciding suitable
therapies.(4)
6.2 Review of Literature
Staphylococcus aureus is perhaps the pathogen of its greatest concern, because of its intrinsic
virulence its ability to cause a diverse array of life threatening infections and its capacity to
adapt to different environmental conditions. The mortality of S.aureus bacteremia remains
approximately 20-40% despite the availability of effective antimicrobials S.aureus is now the
leading cause of nosocomial infection and as more patients are treated outside the hospital
setting, is an increasing concern in the community. As rapidly as new antibiotics are
introduced, Staphylococci have developed efficient mechanisms to neutralize them.(5)
The mortality of patients with S aureus bacteremia in the pre antibiotic era exceeded 80%
and over 70% developed metastatic infections .The introduction of penicillin in the early
1940 dramatically improved, the prognosis of patients with staphylococcus infections.
However as early as 1942 , penicillin resistant staphylococci were recognized, first in
hospitals and subsequently in the community. Staphylococcal resistance to penicillin is
mediated by blaz, the gene that encodes Beta-lactamase. This extracellular enzyme,is
predominantly synthesized when staphylococci are exposed to Beta-lactam antibiotics,
hydrolyses the Beta-lactam ring, rendering the b-lactam inactive.(5)
Methicillin introduced in 1961 , was the first of the semi synthetic penicillinase resistant
penicillin. Its introduction was rapidly followed by reports of methicillin –resistant isolates
.For clinicians the spread of three methicillin-resistances has been a critical one. The
therapeutic outcome of infections that result from MRSA is worse than the outcome of those
that result from methicillin sensitive strain.(5)
Methicillin-resistance first detected in hospitals in the 1960, methicillin resistance is now
increasingly recognized in the community. While many of these infections occurred in
patients with some antecedent hospital experience, recently there has been an increasing
number of subjects with no prior hospital exposure. These community based infections have
been reported in patients from both rural and urban setting.
S.aureus especially involved in strong biofilm formation, bacteria embedded in biofilms, are
difficult to eradicate as it develops resistant to higher antibiotics by poor penetration of
antimicrobials, nutrient limitation, accumulation of toxic metabolites, decreased oxygen
tension and inherently resistant to host immune response and increases the virulence. As a
result treatment of many chronic S.aureus biofilm related infections are hindered.
Bacterial biofilms are complex, monopolymicrobian communities adhering to biotic/abiotic
surfaces. This adaptation has been implicated as a survival strategy. The formations enhance
the virulence of the pathogen and have their potential role in various infections.(6)
O Objectives of the Study
1. 1. To differentiate MRSA into Hospital acquired (HA) MRSA and Community Associated (CA)
MRSA
2. 2. To know the antimicrobial susceptibility pattern of MRSA and Methicillin Sensitive
Staphylococcus Aureus (MSSA)
3. 3. To detect the biofilm production in MRSA and MSSA
4. 4. To correlate the biofilm production in MRSA and MSSA and its contribution as a virulent
factor.
7.
Material and methods
S 7.1 Source of data
F From clinical samples of In patients and out patients of various department of ESIMC and
PGIMSR.
Inclusion criteria for CA- MRSA (7)
1.If it occurs in outpatient setting/if a positive culture for MRSA is identified within 48hrs after
admission to the hospital.
2. If there is no medical history in the past year of hospitalization , admission to a nursing
home, skilled nursing facility, or hospice, dialysis, or surgery.
3. If there are no permanent indwelling catheters or medical devices that pass through the
skin into the body
Exclusion Criteria for CA-MRSA
1. If I occurs >48hrs after admission to the hospital.
2. If there is a history in the past year of hospitalization, admission to a nursing home, skilled
nursing facility or hospice, dialysis or surgery.
3. If there are any indwelling catheters or medical devices that pass through the skin in to the
body.
Inclusion Criteria for HA-MRSA
1. If it occurs >48hrs after admission to hospital.
2. If there is a history in the past year of hospitalization admission to the nursing home,
skilled nursing facility, or hospice, dialysis or surgery.
3. If there are any indwelling catheters or medical devices that pass through the skin into the
body.
Exclusion Criteria for HA-MRSA
1. If it occurs in the outpatient setting/if a positive culture for MRSA is identified within 48
hrs after admission to the hospital.
2. If there is no medical history in the past year of hospitalization, admission to the nursing
home, skilled nursing facility, or hospice, dialysis or surgery.
3. If there are no permanent indwelling catheters or medical devices that pass through the
skin into the body.
7.2. Method of collection of data :
S .aureus isolated from clinical samples like pus/wound, blood, sputum, central venous
catheter tips, indwelling urinary catheter and urine for a period of 1 year will be included for
the study. The isolate will be confirmed as S.aureus by standard isolation and identification
methods.(8)
Antibiotic sensitivity test will be performed by Kirby-Baure’s disc diffusion method.(8)
1. To differentiate MRSA into HA-MRSA and CA-MRSA
Using 0.5 Mc Farland standard, MHA (Muller Hinton Agar) plate supplemented with Nacl will
be inoculated with be strain to the screened and cefoxitin 30ug disc will be placed on MHA
plate, along with control strain of MRSA.The plate will be incubated at 30’ C and observed
after 18-24 hrs. Depending on zone of inhibition if the strains are resistant to above drug they
are considered as MRSA and if the strain sensitive to cefoxitin will be considered as MSSA.(9)
2. Antibiogram of MRSA and MSSA will be carried out using the following
panel of antibiotics as per CLSI (Clinical and Laboratory Standard
Institute) 2010.
Vancomycin (30 mcg), gentamicin (10 mcg), co trimoxazole (1.25/23.5 mcg), tetracycline (30
mcg), ciprofloxacin (5 mcg), linezolid (30 mcg), clindamycin (2 mcg), amoxyclav(30 mcg),
piperacillin-tazobactum (100/10 mcg), cefazolin (30 mcg), cephalexin (100 mcg),
penicillin(10 units), cefoxitin (30 mcg).
3. To detect biofilm production (10)
Biofilm formation will be detected by inoculating the isolates into Trypticase-soy broth (TSB)
with 0.5 % glucose and incubated at 370C. After overnight incubation, the culture will be
diluted 1:40 in fresh TSB-0.5% glucose.200ul liters of the diluted solution will be added to
plate bottomed polystyrene microtitre well and incubated for 48 hrs at 370C. Wells will be
gently washed 3 times with distilled water.After drying the plate in an inverted position at
room temperature for 1hr,the adherent biofilm will be stained with 0.1 % safranin and
allowed to stand for 20 mins at room temperature .Adsorbance of biofilm on the bottom
surface of each well of the dried plates will be determined at 490nm in an ELISA reader.
Follow up of the patients will be carried out from whom MRSA and MSSA were isolated and a
conclusion will be done about the severity of the disease and the presence or absence of
biofim production.
7.3 Does the study require any investigation or interventions to be
conducted on patients or other humans or animals? If so, please describe
briefly.
A)Culture and sensitivity of various samples will be done.
B)Detection of biofilm production.
7.4 Has ethical clearance been obtained from your institution in case of 7.3
YES
8.
List of References
1. Malachy CV, Damian CO, Emmanuel CI, Charles OE. Antibiotic resistance pattern of
staphylococcus aureus isolated from nostril of healthy human subjects in a south eastern
Nigeria locality. Maced J Med Sci, 2009; 2 (4) :294-300.
2. Hsin H ,Neil MF, Jeff HK, Caroline M, Margaret M, Stuart HC. Comparisons of community
associated methicillin resistant staphylococcus aureus and hospital associated methicillin
resistant staphylococcus aureus infection in Sacramento, California. J Clin Microbial, 2006; 44
(7) :2423-2427.
3.Stewart PS. Mechanisms of antibiotic resistance in bacterial biofilm. Ind J Med Microbial,
2002; 29: 107-113.
4. Donlan RM, Costerton JW. Biofilms survival mechanism of clinically relevant
microorganisms. Clin Microbial, 2002; 15: 167-193.
5.Franklin DL. Staphylococcus aureus infections Engl J Med, 1988; 339: 520-532
6.Rajesh S, Vandana B.Practioner’s section, Bacterial biofilm formation, pathogenecity,
diagnostics and control an overview, Ind J Med, 2009; 63:313
7.Centres for disease control and Prevention 2005, Feb 3, community associated methicillin
resistant staphylococcus aureus and hospital associated methicillin resistant staphylococcus
aureus information for clinician retrieved Dec 20, 2007, sitedoct 1 2011 from
http://www.cdc.gov/ncidod/dhqp/av_MRSA_ca.clinician.html.
8. Collee JG, Miles RS, Watt B. Tests for the identifications of bacteria. In: Mackie and Mc
Cartney. Practical medical microbiology.14th ed. Collee JG, Frans AG, Marmion BP, Simmons A
(eds ), 2008; 131-150.
9.Skov R , Smith R, Laren A R, Frimodt M N, Kohimeter G. Evaluation of cefoxitin for the
detection of methicillin resistance in staphylococci. Journal of antimicrobial chemotherapy,
2005; 55 (2): 157-161.
10.Giridhara UPM, Umapathy BL, Ravikumar KL. Comparative study for the presence of
Enterococcal virulence factors gelatinase, hemolysin and biofilm among clinical and
commensal
isolates
of
Enterococcus
faecalis.
Journal
of
Laboratory
physicians,2010;2(2):100-104.
9.
Signature of the candidate
10. Remarks of the Guide
11
Identification of Biofilm production in HAMRSA and CA – MRSA helps to find out
whether Biofilm production has any
contributory effect on virulence of HA –
MRSA as compare to CA – MRSA and also
MSSA
Name & Designation of (in block letters)
11.1 Guide
DR. B.L. UMAPATHY
PROFESSOR,
DEPARTMENT OF MICROBIOLOGY,
11.2 Signature
11.3 Co-Guide
11.4 Signature
11.5 Head of department
11.6 Signature
12
Remarks of the
Dean
Signature
DR. B.V. NAVANEETH
PROFESSOR AND HEAD,
DEPARTMENT OF MICROBIOLOGY,