1. A PROPOSAL ON BIOFILM FORMATION AND
ANTIBIOTIC SUSCEPTIBILITY PATTERN OF
Staphylococcus aureus IN CLINICAL SAMPLES
Submitted By
Shreejana Mainali
September 2015

2. 1
1.INTRODUCTION
1.1 Background
Staphylococcus aureus is a significant human pathogen since it is one of
the most common cause of nosocomial as well as community acquired
infection (Rajbhandari, NJM 2003). The percentage of hospitals
isolating Methicillin Resistant Staphylococcus aureus (MRSA) in the
developed countries has increased from 2% in 70’s to 30% in 90’s.
MRSA emerged in 1961 as a cause of infection among patients exposed
to the bacteria in health care centers in UK (Jevons MP. J Br Med 1961).
Now, Staphylococcus aureus is a leading cause of hospital acquire
infection (HAI) and cause more than 50% of HAI that are more virulent
than methicillin sensitive strains. With the current emergence of multi-
drug resistant MRSA in hospitals on the one hand and the dramatically
increased incidence of hyper virulent community-associated MRSA on
the other hand, MRSA has been able to evolve rapidly and create new
clinical problems (Duckworth, BMJ 1993).
An aggregate of microbes with a distinct architecture, a biofilm is like a
tiny city in which microbial cells, each only a micrometers or two long,
form tower that can be hundreds of micrometers high. The “streets”
between the towers are really fluid filled channels that bring in nutrients,
oxygen and other necessities for live biofilm communities. CDC
(Centre for Disease Control and Prevention) estimate that over 65% of
nosocomial (hospital acquired) infections are caused by biofilms.
Microbial biofilms constitute a major reason for infections to occur as
well as to persist at various sites in the human body. Staphylococcus

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aureus being one of the major gram positive pathogen which causes
array of infections has the ability to colonize biofilms on damaged tissue
and/or implanted biomaterials (Donlan, 2001). These organisms can
persist in clinical settings and gain increased resistance to antimicrobial
agents through biofilm formation which appears to be a bacterial
survival strategy. (Donlan and Costerton, 2002; Hall-Stoodley et al;
2004). Therefore, MRSA biofilms become resistant to almost all
available antimicrobial agents used for its treatments (Goetz, 2002).
Production of biofilms can be a marker of virulence which can be
detected phenotypically (Jain and Agrawal, 2009). The ability of
microorganism to produce biofilm facilitates them to withstand the host
immune response and is recognized as one factor contributing to chronic
or persistent infections. It was demonstrated that the ica -encoded genes
lead to the biosynthesis of polysaccharide intracellular adhesion (PIA)
molecules, and may be involved in the accumulation phase of biofilm
formation. Different studies have shown the decisive role of the ica gene
as virulence factors in staphylococcal infections.
 Antibiotic susceptibility
The “susceptible” category implies that isolates are inhibited by the
usually achievable concentration of antimicrobial agent when the
recommended dosage is used for the site of infection. (CLSI definition).
The “resistant’’ category implies that isolates are not inhibited by the
usually achievable concentrations of the agent with normal dosage
schedules, and /or that demonstrate zone diameters that fall in the range
where specific microbial resistance mechanism (e.g.: beta-lactamases )
are likely, and clinical efficacy of the agent against the isolate has not
been reliably shown in treatment studies. (CLSI definition)

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The “intermediate’’ category includes isolates with antimicrobial
Minimum Inhibitory concentrations (MIC’s) that approach usually
attainable blood and tissue levels and for which response rates may be
lower than for susceptible isolates.
Bacteria growing in a biofilm are highly resistant antibiotics, up to 1000
times more resistant than the same bacteria not growing in a biofilm.
Standard antibiotic therapy is often useless and the only recourse may be
to remove the contaminated implant. MRSA are usually found to be
resistant to most commonly used antibiotics against Staphylococcal
isolate. In fact, many strains of MRSA exhibit resistant to both β-
lactams and aminoglycosides. These strains are seen processing elevated
resistant to a wide range of antibiotics, limiting the treatment options to
very few agent such as Vancomycin and teicoplanin. Hence, knowledge
of prevalence of MRSA and their antimicrobial profile becomes
necessary in the selection appropriate empirical treatment of these
infections and controlling nosocomial infection.

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1.2 Historical Background
In 1959, Methicillin, the first beta-lactamase resistant penicillin, was
licensed in England and was first MRSA isolates was identified in 1960.
During 1968-mid1990, MRSA gradually recognized as an endemic
pathogen in hospitals, especially in large urban university hospitals.
Moreover, in the period of 1998-2008, rates of CA-MRSA
(Community-associated MRSA) increased while rates of HA-MRSA
(Health care-associated MRSA) infections remained stable. Similarly
MRSA infections decreased among patients with health care associated
infections that began in community and also decreased among those
with hospital- onset invasive diseases in 2010. *Infection Control and
Hospital Epidemiology-
published study in 2012 showed that MRSA infections doubled at
academic medical centers in the U.S. The CDC study looked only at the
cases of invasive MRSA- infections found in blood, spinal fluid, or deep
tissues: it excluded infections of the skin.
In context of Nepal, history of MRSA has not been reported yet as
well as data on nosocomial infection by MRSA is also not available.

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2.ProblemStatement
Methicillin Resistant Staphylococcus aureus has been identified as one
of the major causative agents of nosocomial infection, most prevalent in
skin infections followed by lower respiratory tract infection causing
significant morbidity and mortality. The biofilms formed by MRSA
have now been known to be resistant to most available antimicrobial
agents like methicillin, ampicillin, ofloxacin, tetracycline, ciprofloxacin,
cotrimoxazole, etc. as they are fast grower and can persist in clinical
settings. Various studies on biofilm production and nosocomial
infections by MRSA shows that biofilm formation attributes to the
resistance as well as major contributor of nosocomial infections. Biofilm
impairs the action of both host immune system and antimicrobial
activities and its production is an important virulence factor of S. aureus
(Dhanawade et al., 2009). The formation of biofilms is recognized
method of MRSA’s ability to establish and maintain certain infections
and a way which increases its persistence and boosts its level of
antimicrobial resistance (Monre, 2007). Disease such as endocarditis,
osteomyelitis and medical-device related infections are caused by S.
aureus biofilms and are not readily treatable with antibiotics.
Studies shows that the mainstay of treatment of MRSA infection is
antibiotics Vancomycin. However, Multi-Drug Resistant Staphylococcus
aureus with Intermediate resistance to Vancomycin have been isolated
from patients in Scotland, U.K. (Hood, et al. 2000). This resistance
severely limits therapeutic options and increases the high rate of
morbidity and mortality in patients. To compound this problem further,
Staphylococcus aureus shows the ability to colonize and form biofilm in
implanted biomaterials. These biofilm structures are resistant to
antibiotics and difficult to eradicate from the infected hosts as they can
display resistance towards antimicrobial agents 10-1000 times more than

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equivalent population of free floating planktonic cells (Potera,1999 ;
Donlan, 2001;Gilbert et al. 2002; Parsek and Fuqua, 2004).
The prevalence of resistance in S. aureus also is increasing globally and
the infection caused by biofilm forming MRSA in Nepal is under
reported. Most of the studies on biofilm formation by MRSA are limited
to the developed countries only and there are very few reports from
Nepal and the whole South East Asian Region.

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3.Justificationof Study
This research justifies the problem caused by prevalence of biofilm
producing MRSA infections persisted at various sites in human body
causing an array of infections. In case of Nepal, no extensive and
effective research works have been conducted on biofilm producing
MRSA which is gaining resistance to most beta-lactam antibiotics,
hence the major purpose of our study is to detect the biofilm producing
MRSA and the antibiotic susceptibility pattern of the organism prevalent
in the area of our survey. The identification of possible associations
between biofilm production and pathogenesis as well as antibiotics
susceptibility profiles of Methicillin Resistant Staphylococcus aureus
will provide better control measures particularly among immune
compromised individuals in the hospitals of our survey. Hence our study
will determine the prevalence of biofilm formation among
Staphylococcus aureus isolates from clinical samples.

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4.Limitationof the study
This study is primarily limited by short period of time for survey and its
evaluation. In addition to this, detailed and extensive researches on
biofilm producing MRSA have not been carried out in Nepal which
caused difficulties during the literature reviews of the study. Moreover,
the research would give accurate and authentic data if the laboratory
facilities and practices that is provided, is under the guidelines of
Clinical and Laboratory Standard Institute (CLSI). The methodology
would be more accurate if there is provision of ELISA auto reader in
Tissue Culture Plate method for Biofilm Detection Method. A greater
depth of information will be obtained from this research if sufficient
funds is given from respective institution.

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5.Objectives of the study
 To investigate the prevalence of MRSA and their rate of resistance
to different anti-Staphylococcal antibiotics.
 To collect information about ongoing researches on biofilm
forming MRSA in Nepal.
 To determine infections caused by MRSA and its control
measures, antibiotic prophylaxis and treatment of MRSA isolated
from clinical samples.
 To identify possible associations between biofilm production and
pathogenesis of MRSA.
 To determine the relative risks of cross-transmission of the
possible infections caused by biofilm producing MRSA to other
(high-risk groups) patients in the hospital.
 To compare the results of biofilm forming MRSA obtained from
this research with past research projects.
 Data on nosocomial infection by MRSA is not available so present
study is performed to find out the prevalence of nosocomial
infection by MRSA.
 The recalcitrance and biofilm mediated infections has an advanced
effect on patient health, hence the main objective of this study was
to investigate the capacity of clinical isolates of Staphylococcus
aureus to form biofilm.

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6.ConceptualFramework
Identification of S.
aureus by coagulase
test
Culture in suitable
media
Screening of all
samples for MRSA by
Kirby Bauer Disc
Diffusion method
Detection of
Methicillin- Resistant
Staphylococcus
aureus
Detection of Biofilm
forming MRSA by
Tissue Culture Plate
Method and Tube
Method
Hospital
Associated
Sample

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7.LiteratureReviews
 Joshi N.P. and Murugan S. , Department of Biotechnology,
school of Biotechnology and Health Sciences, Karunya
University, Coimbatore, Tamil Nadu,641 114, India (2014)
conducted a report on “Biofilm Formation by Methicillin
Resistant Staphylococcus aureus and their Antibiotic
Susceptibility Pattern : An in vitro Study” reported that out of
259 S. aureus isolates 209 (80.69%) were confirmed as MRSA
and only 35 (16.76%) of the MRSA isolates were found to be
strong biofilm producers by Tissue Culture Plate on the basis of
optical density values. The antibiotic resistance pattern of
biofilm producing MRSA isolates were found to be highly
variable with maximum resistance (100%) to Ampicillin,
ampicillin/sulfbactam, ofloxacin, tetracycline, ciprofloxacin,
cotrimoxazole and 100% sensitivity observed towards
Vancomycin. Hence, Vancomycin was the effective drug of
choice for the treatment of biofilm producing MRSA infections
and TCP is the reliable method for detecting the biofilm
formation. The research concluded that frequent monitoring of
pathogens in the hospital and community settings should be
made mandatory and unambiguous antibiotics policy should
also be formulated to decrease the spread of pathogens.1
 Pandey S., Raza M.S., Bhatta C.P., Department of
Microbiology, Nobel Medical College, Teaching Hospital,

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Biratnagar and Kathmandu Medical College, Teaching Hospital,
Kathmandu (2012) piloted a research on “Prevalence and
Antibiotic Sensitivity Pattern of Methicillin Resistant-
Staphylococcus aureus in Kathmandu Medical College-
Teaching Hospital” and reported that of 111 S. aureus isolates,
29 (26.12%) were identified to be MRSA. The rate of multi
drug resistance was 75.86% for MRSA and 6.09% for MSSA.
All the staphylococcal isolates were resistant to penicillin.
However, all strains were sensitive to Vancomycin. They
concluded that Vancomycin, a glycopeptides seems to be only
antimicrobial agent which showed 100% sensitivity through all
parts of Nepal and may be used as a drug of choice for treating
multidrug resistant MRSA infections.2
 R.K. Sanjana, Shah R., Chaudhary N., Singh Y.L., Department
of Microbiology, College of Medical Sciences- Teaching
Hospital, Bharatpur, Chitwan district, Nepal conducted a
research on “Prevalence and Antimicrobial Susceptibility
Pattern of MRSA in CMS- Teaching Hospital: a preliminary
report” and reported that out of total of 348 S. aureus strains
isolated from various clinical samples, 138 (39.6%) were found
to be Methicillin Resistant. Among MRSA isolates, 86(62.3%)
were from different in patient department, whereas, 52 (37.7%)
of the isolates were from out patients. All MRSA were resistant
to penicillin. More than 70% of MRSA strains were resistant to
cephalexin, ciprofloxacin, cloxacillin, while less than 10% of
them were resistant to azithromycin, amikacin and tetracycline.
Many MRSA strains were multidrug resistance. However, no
strains were resistant to Vancomycin. The preliminary report
showed a high prevalence of MRSA in their hospital and to
reduce the prevalence of MRSA, regular surveillance of

14. 13
hospital acquired infection and isolation was the need of the
hour.3
 Ando E., Monden K., Mitsuhata R., Kariyama R., Kumon H.,
Department of Urology, Okayama University, Graduated
School of Medicine and Dentistry Okayama, Japan conducted a
research on “Biofilm formation among MRSA isolates from
patients with Urinary Tract Infection” and reported that 109
MRSA isolates were collected from patients (one isolated per
patient) with UTI at the urology ward of Okayama University
Hospital. Upon review of associated medical records, concluded
that the Biofilm- forming capacities of MRSA isolates from
catheter-related cases were significantly greater than those from
catheter- unrelated cases.4
 Tambekar D. H.,Dhanohkar D.V.,Gulhane S.R.,and Dudhane
M.N.;P.G. Department of Microbiology,S.G.B. Amravati
University,Amravati-44462(India)(2006) conducted a research
on “ Prevalence and antimicrobial susceptibility pattern of
MRSA from Health Care and Community associated sources “
which was carried out in Amravati,Maharastra stat taking
sample of 150 healthcare associated sources (HA) (doctors
mobile phone and wound/pus swabs) and 160 community
associated (CA) sources(hand swab). The samples were
screened for MRSA and their antibiotic resistance pattern was
performed. Out of 41 isolated strains of S aureus, 77%from
H.A. and 50% C.A.samples were found to be methicillin
resistant. There were high prevalence of MRSA doctor’s mobile
phone (83%), wound pus(71%) (HA sources) than the hand

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swab. Almost all HA and CA MRSA strains were resistant to
penicillin and penicillin V (100%) followed by cloxacillin and
cephalexin, co-trimoxazole. About 56-67% HA and CA- MRSA
strains were resistant to erythromycin, ceftazidime, lincomycin,
cephalexin, erythromycin and tetracycline indicating high
degree of multi-resistance MRSA prevalent in the region.
However, 67% strains of CA and 56% strains of HA were
sensitive to Vancomycin. The study showed high prevalence of
MRSA in hospital setting indicating need of good control
measure such as proper hand hygiene, avoiding mobile phone
while wound dressing and treating patient, survilliance cultures
and monitoring of susceptibility pattern of MRSA may also
help in arresting the spread of infections in this part of India.5
 Shrestha B., Pokhrel B. and Mahapatra T. of Tribhuvan
University, IOM, Department of Microbiology and Banaras
Hindu University, IOMs, Department of Microbiology
conducted a research on “Study of Nosocomial isolates of S.
aureus with special reference to Methicillin Resistant
Staphylococcus aureus in tertiary care hospital in Nepal” (2009)
and reported that of 149 S. aureus isolates, skin infection
isolates contributed a major part (72.5%) making nosocomial
infection by S. aureus most prevalent in skin infection followed
by lower respiratory tract infection 11.41% and UTI 8.7%.
Overall MRSA prevalence was 45.0%. MRSA isolation was
high from Lower respiratory tract of patients admitted in ICU,
coronary care unit, sub- acute ICU, Intermediate coronary care
unit, Neurology wart and Post- operative ward. They also
reported that data on nosocomial infection by S. aureus and
MRSA in Nepal was not available and the major objective of

16. 15
their study was to find out prevalence of nosocomial infection
by S. aureus with special focus on MRSA. The research led to
the conclusion that due to ever increasing MRSA infection in
hospitals and its spread in other patients as well as community
via the health care workers, every hospital or health care
settings should survey the MRSA prevalence and follow
universal precautions, the most important one being washing
hands with alcohol based soap after each patient care.6
 Sah P., Rijal K.R., Shakya B., Tiwari B.R. and Ghimire P.,
Central Department of Microbiology, TU, conducted a research
on “Nasal carriage Rate of S. aureus in Hospital Personnel of
Nepal Medical College and Teaching Hospital and their
Antibiotics Susceptibility Pattern” and stated that out of 54
nasal swabs taken from hospital staffs, nasal carriage rate of S.
aureus was found to be 20.37%. All nasal S. aureus were
sensitive to Amikacin and Vancomycin and MRSA rate was
found to be 45.5%. The research led to conclusions that the
health care personnel require awareness regarding the
nosocomial infection and should know their status of nasal
carriage fo MRSA and accordingly take necessary measures to
prevent possible transmissions.7
 Kumari N., Mahopatra T., Singh Y.I., Department of
Microbiology, B. P. Koirala Institute of Health Sciences,
Dharan, Nepal (2008) conducted research on “Prevalence of
Methicillin Resistant Staphylococcus aureus (MRSA) in
Tertiary Care Hospital in Eastern Nepal which was carried out

17. 16
in various clinical samples like pus, sputum, genital specimens (
vaginal swab, semen, urethral discharge), urine, blood, body
fluids (cerebrospinal fluid, ascitic fluid, plural fluid, synovial
fluid etc.), devices (urinary catheter, endotracheal tube, CVP
catheter, tracheostomy tube, PD catheter, drain, suction
catheter) and tissues. Outof a total of 750
Staphylococcus aureus strains isolated from various
clinical samples, 196 (26.14%) were found to be Methicillin-
resistant. Seventy percent isolates of MRSA were from inpatient
departments and amongst them only 10% of the isolates were
from intensive care units (ICU). More than 65% of MRSA were
found to be resistant to Penicillin, Cephalosporins,
Ciprofloxacin, Gentamicin Erythromycin and Tetracycline,
while 47.96% of them were resistant to Amikacin. Many
MRSA strains were multidrug-resistant. However, no strains
were resistant to Vancomycin. The study concluded that to
reduce the prevalence of MRSA, the surveillance of hospital
acquired infection, isolation nursing of patients who carry
MRSA, monitoring of antimicrobial susceptibility pattern and
formulation of a definite antibiotic policy might be helpful.8
 Majumder S., Wolffs P.F.G., Hoebe J.P.A. and Rahmatollah M.,
Department of Microbiology and Serology, NH Health City,
Banglore, Faculty of Health, Medicine and Life Sciences,
Department of Medical Microbiology, Maastricht Infection
Center (MINC), School of Public Health and Primary Care
(CAPHRI), Maastricht University Medical Centre (MUMC+),
Department of Pharmacy, University of Development
Alternative, Dhaka Bangladesh conducted a report on “Rapid

18. 17
Detection of Methicillin Resistance and Biofilm Formation in
Staphylococcus spp.” and reported that out of 242 bacterial
isolates, 162 Methicillin- Resistant S. aureus and 80 Methicillin
resistant coagulase negative S. aureus from clinical samples was
included. The study resulted that 96 (40%) strains were detected
which indicates the strains to be MRSA with biofilm producing
ability and 66 isolates were positive which identified them to be
MR-CoNS with no biofilm producing ability. The study led to
conclusion that it is important to diagnose to give prophylactic
antibiotics just before and during the surgical procedure to
eliminate planktonic bacteria before they can form biofilm.9
 Tyagi A., Kapil A. and Singh P., Former Research Fellow, Civil
Engineering Department, Indian Institute of Technology;
Department of Microbiology, All India Instititute of Medical
Science; Head Department of Microbiology, Gurukul Kangri
University conducted a research on “ Incidence of Methicillin
Resistant Staphylococcus aureus (MRSA) in pus samples at a
Tertiary Care Hospital, AIIMS, New Delhi” and reported that
out of 2,080 pus samples, MRSA prevalence rate was 915.2
(44%) of all S. aureus isolates. All isolates were sensitive to
Vancomycin, rifampicin and teichoplanin.10
 Vidya Pal, Venkatakrishna I Rao and Sunil P Rao conducted a
research on “Prevalence and Antimicrobial Susceptibility
Pattern of Methicillin-Resistant Staphylococcus aureus
(MRSA) isolates at Tertiary Care Hospital in Mangalore, South
India” and reported that out of 237 isolates that were obtained

19. 18
from various clinical samples like pus, sputum, urine, blood and
body fluids, the prevalence rate of MRSA was found to be 29%
which is in accordance with investigators from India (32.8%)
and Nepal (26.4%). Multidrug resistant among MRSA strains
was higher than those that were sensitive to Methicillin.
Ciprofloxacin was proposed to be an alternate therapy for
MRSA infection. The research reported that although rapidly
developing resistance to Ciprofloxacin has been reported,
(68.2%) in their hospital which might be due to the differential
clonal expansion and drug pressure in the community. The
research concluded that the higher price of Vancomycin, its
unavailability in many parts of the country, and also the
possibility of emergence of resistance to the drug should at least
make the clinicians look into the alternatives. Therefore, regular
surveillance of hospital- associated infections including
antimicrobial susceptibility pattern of MRSA and formulation
of a definite antibiotic policy may be helpful in reducing the
burden of MRSA infections in the hospital.11
 Ornskov D. et al. Department of Clinical Microbiology and
Department of Clinical Biochemistry, Vejle Hospital, Vejle,
Denmark (2007) conducted a research on “Screening for
Methicillin-Resistant Staphylococcus aureus in clinical swabs
using a high-throughput real- time PCR- based method” and
reported that presence of MRSA in hospitals and the community
was a serious problem which was implemented in the country of
Vejle, Denmark to identify colonized and intended individuals
and to control the spread of MRSA. Since 2005, all patients
involving analysis of 300-400 samples daily in 6 month. To deal

20. 19
with this number of samples a PCR based method customized
for high throughout analysis. It concluded that in 6 month
period from July to December 2006. 42112 assays were
performed to 24687 individuals with 393 assays for 114
individuals being MRSA positive.12
 Girou E., Pujade G., Legrand P., Cizeu F. and Brun-Buisson C.,
United Hygiene et Prevention de I’Infection, Service de
Reanimation Medicale and Service de Bacteriologie Virologie-
Hygiene Hospital Henri Mondor, Creteil, France conducted a
research on “Selective Screening of Carriers for Control of
Methicillin-Resistant Staphylococcus aureus (MRSA) in High-
Risk Hospital Areas with a High Level of Endemic MRSA” and
reported that out of 3,686 patients admitted during a 4 year
period 44% screened, MRSA was recovered from 293 patients
(8%). There were 150 imported cases and 143 ICU-acquired
cases of which 51% and 45% respectively were first identified
through screening. Nasal swab cultures identified 84% of
MRSA carriers. The research concluded that selective screening
for nasal carriage during admission to high risk areas may
contribute to identification of a substantial proportion of cases
of MRSA and to early implementation of effective control
measures.13
 Huong S.S. et al (2002) piloted a research on “Risk of
Methicillin-Resistant Staphylococcus aureus Infection after
Previous Infection or Colonization” and determined the 18-
month risk of MRSA infection among 209 adult patients newly

21. 20
identified as harboring MRSA. Twenty nine percent of patients
(60) developed subsequent MRSA infections (90) which were
often severe. Twenty eight percent of infections involved
bacteremia and fifty six percent involved pneumonia, soft tissue
infection, osteomyelitis or septic arthritis. Eighty percent of
patients (48 of 60) with subsequent MRSA infection developed
the infection at a new site and forty nine percent of new MRSA
infections (44 of 90) first became manifest after discharge from
the hospital. The study led to findings that the respiratory tract
was the most common source of newly identified MRSA
isolates for cases of both colonization and infection and
concluded that high risk of infection increases the attractiveness
of eradicating MRSA carriage, particularly in view of the recent
emergence of strains of S. aureus fully resistant to Vancomycin.
Eradication measures may include treatment with topical
mupirocin or with a combination of active systemic agents such
as rifampin, doxycycline, or trimethoprim-sulfamethoxazole
have not been widely studied or implemented in the United
States. Even if instituted only at the time of hospital discharge,
an eradication measure with 50% efficacy over several months
could reduce the overall burden of subsequent MRSA infection
by 20%. 14

22. 21
 Khanal L.K. and Jha B.K., Department of Microbiology, Nepal
Medical College, Jorpati, Kathmandu, College of Medical
Sciences, Bharatpur, Chitwan, Nepal presented a paper on
“Prevalence of Methicillin resistant Staphylococcus aureus
(MRSA) among skin infection cases at a hospital in Chitwan,
Nepal” (2010) and reported that a total of 600 S. aureus isolates
isolated from 1,334 specimens from skin and soft tissue
infection, prevalence of MRSA was 68.0% (408/600) and the
rate was significantly higher among males (75.0%) than females
(63.4%) (p<0.05). MRSA isolation rate was higher from wound
(76.9%), followed by purulent exudates (67.7%) and abscesses
(64.1%). The high prevalence of MRSA found in this study
might be due to hospital based specimen. Report of this research
showed B-lactam antibiotics like penicillin and cephalexin are
ineffective drugs against S. aureus. Penicillin resistance was >
97.0% and quinolone - ciprofloxacin was also found to be
resistant (70.0%). All clinical isolates of this study have shown
that only 78.0% of S. aureus were susceptible to Vancomycin
whereas sensitivity to other drugs was also poor. This indicates
that Vancomycin resistance is also getting spread day by day,
both in community as well as hospital settings. Therefore it
requires continuous isolation and identification of S. aureus
from carriers, patients and health care workers, so that regular
monitoring and routine testing of other newer glycopeptides like
teicoplanin should be carried out against it.15
 Mukhiya R.K., Shrestha A., Rai S.K., Pant K., Rai G. and
Singh R. of St. Xavier’s College, Nat’l Institute of Tropical
Medicine and Public Health Research, Central Department of

23. 22
Microbiology, Tribhuvan University, Kirtipur, Nepal Police
Hospital , conducted a research on “Methicillin-resistant
Staphylococcus aureus in Clinical Samples of hospital located
in Kathmandu Valley, Nepal” and reported out of 630
specimens isolated from pus and urine, prevalence of MRSA
was 62.0% (13/21). MRSA infection was higher in outpatients
(69.2%) than in inpatients (30.7%). The maximum isolates was
isolated from pus (64.7%). Isolates showed high resistance
towards the co-trimoxazole and methicillin but 100.0% sensitive
towards vancomycin. The high prevalence of MRSA found in
this study might be due to hospital based specimen. Research
revealed that isolates were resistant to commonly used antibiotic
except 100.0% sensitive towards Vancomycin. The research
concluded that there is a need for surveillance of MRSA and
definite antimicrobial policy may be helpful for reducing the
incidence of these infections. In addition, regularly carrier
pattern of health care workers should be conducted because they
are directly responsible for causing nosocomial infections.16
 Mishra S.K., Basukala P., Basukala O., Parajuli K., Mani
Pokhrel B. and Rijal B.P. conducted a research on the topic
“Detection of Biofilm Production and Antibiotic Resistace
Pattern in Clinical Isolates from Indwelling Medical Devices”
and reported that out of 67 clinical isolates from IMDS, Tissue
culture Plate detected 31 (46.3%) biofilm producers and 36
(53.7%) biofilm non-producers. Klebsiella pneumoniae,
Pseudomonas aeruginosa and Burkholderia ceacia complex
were found to be the most frequent biofilm producers. The
research also reported that higher antibiotic resistance was
observed in biofilm producers than the biofilm non-producers
and the most effective antibiotics for biofilm producing Gram-

24. 23
positive isolates were Vancomycin and that for biofilm
producing Gram negative isolates were Polymyxin-B, Colistin
Sulphate and Tigecycline. The research led to conclusion that
biofilm pose a great threat for patients requiring nosocomial
infections and hence it is crucial to follow appropriate method
for the detection of biofilms.17

25. 24
8. Methodology
8.1 Study Area: The study area of research includes Tribhuvan
University Teaching Hospital located at Maharajgunj, Kathmandu. It has
the largest number of medical specialties compared to any hospital in
Nepal. The study will be based on retrospective data of samples
collected from different wards and OPDs of TU-Teaching Hospital,
Kathmandu.
8.2 Study Period: This research culminates in a formal report which is
designed to be accomplished within three months (November 2015-
January 2016).
8.3 Sample Size: A total of ≤150 consecutive and non-duplicate clinical
samples such a pus, blood, urine and sputum would be collected from
the TU-Teaching Hospital which would later be transported immediately
to the laboratory for further processing.
 Sample Processing and Bacterial Identification:
The samples would be cultured in selective media, Mannitol Salt Agar
(MSA) and the organism could be identified on the basis of colonial
morphology, microscopic observations (Gram staining), and catalase and
coagulase tests.
 Antibiotic Susceptibility Testing:
Antibiogram of MRSA would be based on the susceptibility patterns for
selected antibiotics representing various classes of antimicrobial agents.
The antibiotic susceptibility pattern can be determined by Kirby- Bauer
Disc Diffusion method as recommended by CLSI guidelines. The entire
surface of Mueller-Hinton Agar (MHA) plate with 2% NaCl is covered
with inoculums of isolated S. aureus and turbidity is matched with 0.5

26. 25
McFarland standards by a sterile cotton swab stick and the antibiotic
discs is laid on the surface of the plate.
 Detection of Biofilm Formation:
All the samples are subjected for biofilm formation. The methods that
would be used for biofilm detection are tube method tissue culture plate
method. In tube method adherence can be observed by ring formation on
the inside walls of the test tube when stained with crystal violet. Optical
densities (OD) of stained adherent bacteria in tissue culture plate method
would be determined with a micro ELISA auto reader.
 Statistical analysis:
Chi- square test would be used for analysis of categorical data and
for comparison of different variables and the correlation between
all the tests to be performed. A p value of < 0.001 is considered to
be statistically significant.
 Laboratory facilities:
The analysis of sample collected would be done in microbiology
laboratory, Department of Microbiology, Amrit Campus under the
guidance of the research supervisor. The lab has containment level 1
(bio-safety level 1) to be able to work safely with pathogens. The lab
space is partitioned into two separate containment. Materials and
equipment required for experimental analysis include:
 Laminar flow hood
 Incubator
 Autoclave
 Hot air oven
 Refrigerator
 Light microscope
 Petri plates, Media, etc.

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9. Expected Outcomes
MRSA is associated with high morbidity and mortality rates because of
the development of multi-drug resistance and their ability to form
biofilms which is difficult to eradicate and can thus, cause an array of
infections. To overcome this problem, regular surveillance and
monitoring of hospital associated infections caused by MRSA at regular
time intervals to minimize the risks of spread of MRSA is very essential.
Most of the researches on biofilm formation by MRSA have been
extensively studied by developed countries only but there are only very
few reports on the topic in Nepal. Hence, this research is expected to
conduct detailed study on the antibiotic susceptibility pattern of
Methicillin-resistant Staphylococcus aureus as well as their capacity to
form biofilms. Moreover, this document has proposed to identify the
possible associations between biofilm formation and pathogenesis of
MRSA as well as their antibiotic susceptibility. Thus, the results of the
study further expects to provide better control measures of the
nosocomial infections associated with biofilm forming MRSA as well
as facilitate epidemiologists to understand the nature of MRSA in
Nepal.

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10. Conclusion
This report is especially focused on detection of prevalence of biofilm
forming MRSA in hospital. Surveillance of MRSA and its antimicrobial
profile is a need for treatment regarding nosocomial infections. MRSA
from patient can contaminate the environment, whereas MRSA from
environment might be potentially transmitted to patients via health care
workers under unsatisfactory infection control. So, the hospital infection
control policy and guidelines that already exist should be strictly
implemented and followed so as to enable the clinicians to deliver better
and proper health care to the patients.

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11. Recommendations
The prevalence of resistance in S. aureus is increasing globally and the
infection caused by biofilm forming MRSA in Nepal is under reported
and of the studies on biofilm formation by MRSA are limited to the
developed countries only. Hence, screening of biofilm forming MRSA
should be made an essential protocol to access the carrier transmitted
drug resistant strains of Staphylococci from community to the hospital
settings and hospital settings to community. Proper hand hygiene,
sterilized tools and equipment should be used by doctors and health
workers to arrest the spread of infections in patient. Definite
antimicrobial policy may be helpful for reducing the incidence of
nosocomial infections, which serves as a potential health hazard in
Nepal. Therefore, every hospitals or health care settings should survey
the MRSA prevalence and follow the universal precautions, the most
important one being washing hand with alcohol based soap after each
patient care. In addition to these, further studies can be carried out in
order to identify the genetic features contributing to biofilm formation
among clinical isolates for better management of S, aureus infections
among the patients in the hospitals of Nepal to reduce the high levels of
morbidity and mortality.

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12. Estimatedcosts
We have estimated a cost of around Rs.50, 000 to undertake the study
described above. The majority of funds will go towards materials and
laboratory equipment required for assessment of the clinical samples
collected from TU-Teaching Hospital. This also includes compensation
for the supervisor/mentor of the research. Hence, the estimated direct
expenses for the project is Rs. 30,000 and include the costs of supplies
as well as printing charges and other petty charges. The estimated travel
costs of the research is of Rs.2, 000. No any external sources of funding
from NGOs/INGOs and from the respective authorities has been
provided for the research.

31. 30

32. 31
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