ABSTRACT
Staphylococcus aureus is a versatile human pathogen possessing numerous virulence factors and is also the major cause of important infections in community and hospital settings. This study was undertaken to determine the prevalence of multidrug resistant S. aureus from wound infection patients in Federal Medical Center Umuahia, Abia State and also to characterize the S. aureus. A total of 300 wound swab samples from diabetic leg ulcers, accident wound, burn wounds and surgical site wounds were collected from different wards and cultured on Blood agar and Mannitol salt agar. Colonies with typical morphologies were selected and Gram stained, standard biochemical test and molecular analysis was done to identify the isolates. Antibiotic susceptibility test was performed using the disc diffusion method following the CLSI guidelines. Biofilm forming potential, beta lactamase production and methicillin resistance of the isolates were done using Congo red agar medium, rapid penicillinase paper strip test and cefoxitin disk diffusion method respectively. Plasmid analysis was done using the QIAprep Spin Plasmid kit according to the manufacturer’s instructions and curing of the plasmid were both done. The data was analyzed using IBM SPPS version 20. Staphylococcus aureus isolates from the 300 processed wound swabs were 50 (16.7%), of these, 14 (15%) were from diabetic leg ulcer, 11 (14.6%) from accident wound, 16 (23.1%) from burn wound and 9 (14.2%) from surgical site wound. There were no significant difference (P>0.05) in the different wards and sites of collection and between age and those with highly resistant S.aureus. Antimicrobial resistance profile of the isolates in the study from the various wound types showed increased resistance to norfloxacin (82%), ampiclox (80%), amoxicillin (76%), chloramphenicol (62%) and rifampicin (52%) among others. The overall prevalence of methicillin resistance S. aureus was 14.0%, while 66% of the S.aureus was multidrug resistant. 62% showed biofilm forming potential while 66% produced beta lactamase. Results of the study showed the presence high multidrug resistance. Therefore, antibiotic susceptibility testing should be performed prior to treatment; also adequate measures should be taken to avoid the transfer of multidrug resistant S.aureus among patients and health care workers
TABLE OF CONTENTS
Title i
Declaration ii
Dedication iii
Certification iv
Acknowledgement v
Table of content vi
List of tables ix
List of figures x
Abstract xi
CHAPTER 1: INTRODUCTION
1.1 Background
of the Study 1
1.2 Emergence
of Drug Resistance among S. aureus
strain 2
1.2.1 Mechanism
of methicillin resistance 4
1.2.2 Phenotypes
of MRSA 5
1.2.3 Resistance
to other antibiotics 5
1.3 Justification
of the study 6
1.3.1 Aims
6
1.3.2 Specific
objectives 7
CHAPTER 2: LITERATURE REVIEW
2.1 General
Features of S. aureus 8
2.2 Clinical Manifestations
of S. aureus 9
2.2.1 Bacteremia
and endocarditis 10
2.2.2 Wound
infections 11
2.2.3 Burn
Wound Infection 11
2.2.4 Surgical
site infection 12
2.2.5 Diabetic
foot infections 13
2.2.6 Catheter
associated Infections 13
2.2.7 Soft
tissue and skin infections 14
2.2.8 Infection
of the central nervous system 14
2.2.9 Eyes
Diseases 15
2.2.10 Osteomyelitis
and other infections 15
2.2.11 Infections
of the respiratory tract 16
2.2.12 infection
of urinary tract 16
2.2.13 Toxin-mediated
syndromes 17
2.2.14 Food
Poisoning 17
2.3 Virulence
Factors of S. aureus 18
2.3.1 Toxin
19
2.3.2 S. aureus Enzymes 22
2.4 Occurrence
of Drug Resistance among S. aureus
strains 24
2.5 protocol
of Antimicrobial Resistance to Antibiotic and
their
Genetic Basis 26
2.5.1 Methicillin resistance 28
2.5.2 Glycopeptide
Resistance (Vancomycin Resistance) 31
2.5.3 Aminoglycoside
resistance 32
2.5.4 Resistance
to Quinolones 32
2.6 Epidemiology
of Methicillin-Resistant S. aureus
(MRSA) 33
2.7 Genetic
Relationship and Molecular Study of MRSA and MSSA 36
2.8 Plasmid
Profiling 39
2.9 Laboratory
Diagnosis and Molecular Study of S.
aureus 40
2.10 Prevention
and Control of S. aureus 45
CHAPTER 3: MATERIAL AND METHODS
3.1 Study
Area 48
3.2 Study
Design 48
3.3 Study
Size 48
3.4 Sample
Collection 49
3.5 Sample
Processing 49
3.5.1 Media
Preparation 50
3.5.2 Isolation
and Identification of Bacteria 50
3.5.3 Gram
Staining Technique 50
3.5.4 Biochemical
Tests 51
3.5.4.1 Catalase Production 51
3.5.4.2 Coagulase Test 51
3.6 Molecular Analysis 51
3.6.1 DNA extraction 52
3.6.2 Gel electrophoresis 53
3.6.3 PCR reaction: amplification
of DNA 53
3.6.4 DNA Sequencing 54
3.7 Antibiotic
Susceptibility test 54
3.8 Determination
of Biofilm Forming Potential of the Isolates 55
3.9 Detection
of Beta Lactamase Production. 55
3.9.1 Preparation
of paper strip. 55
3.10 Protocol
for Methicillin Detection. 56
3.10.1 Multi
drug resistance 56
3.10.2 Calculation
for Multiple antibiotic Resistance Index (MARI) 56
3.11 Plasmid
Analysis 57
3.11.1 Plasmid curing 57
3.12 Ethical
Consideration 58
3.13 Statistical Analysis 58
CHAPTER 4: RESULT AND DISCUSSION
4.1 Result 59
4.2 Discussion 84
CHAPTER 5: CONCLUSION AND RECOMMENDATION
5.1 Conclusion 90
5.2 Recommendation 91
LIST OF TABLES
TABLE TITLE
PAGE
4.1 Demographic
distribution of S. aureus from
different units and sites
of sample collection.
61
4.2 Antibiotic resistance profile of the test
organism 63
4.3a Sensitivity and resistance of antibiotics
by sex and age 64
4.3b Sensitivity and resistance of antibiotics
in wards and wound types 65
4.4 Multiple antibiotics resistance index
(MARI) of the isolates 66
4.5 Percentage distribution of S.aureus with potential for biofilm
production 67
4.6 Prevalence of beta lactamase production
of the test isolate 68
4.7 Prevalence of methicillin resistance of
the test isolate 69
4.8 Plasmid Profile of Isolated organism 71
4.9 Resistance patterns before curing and
after curing 73
LIST OF FIGURES
Fig 1 Types of SSCmec components of Staphylococcus areus 30
Fig 2: Prevalence of multidrug resistant S. aureus 62
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
Staphylococcus aureus is a Gram-positive and spherical coccus, it measures 1µm –1.3µm in
diameter. It appears in groups like lots of grapes on microscopic examination.
Some of the strains produce toxins while growing in food. The toxins produced
can cause gastrointestinal disease which is generally referred to as Staphylococcal
food poisoning. The enterotoxin produced is a heat-stable protein that resists
heating at 100°C
for 30–70 min. Food borne infections can also be seen to be caused by S. aureus. (Garcia-Alvarez et al., 2011).
Various disease conditions caused by this
organism are: toxic shock syndrome, septicaemia and wound infections. Some of
the consequence of S. aureus beside
skin pustules, impetigo, osteomyelitis, renal abscess, pneumonia, endocarditis,
meningitis, gastroenteritis may also include serious conditions in patients undergoing
hemodialysis, diabetic mellitus etc. (Lewis
and Jorgensen, 2005).
Identification of S.aureus can be done by several methods which include methods such
as Gram’s staining, cell morphology, production of catalase and coagulase
enzymes, pigment production, susceptibility to lysostaphin and lysozyme, and
anaerobic production of acid from glucose (Paul et al., 2009). Several other commercially available systems that
allow strains to be biochemically characterized, have also been developed.
Other classes of Staphylococcus types are also implicated in similar disease
conditions. For example, S. epidermidis
is involved in bacterial endocarditis, prosthetic heart valve endocarditis,
bacteremia, surgical wound contagions, infections of catheter within the blood
vessels, postoperative endophthalmitis, conjunctivitis and keratitis. Other
species of Staphylococcus such as S.
saprophyticus, S. hyicus and S.
intermedius may sometimes be involved but these can be differentiated from S. aureus. The coagulase negative
Staphylococci (CoNS) species have been implicated at low incidence in a variety
of infections. For example, S.saprophyticus
is has often been observed to be more important an adaptable pathogen compared
to S.epidermidis in human urinary
tract infections (UTIs), especially in female youth who are sexually active.
Other staphylococcal species frequently found as contaminants of blood cultures
are S. hominis, S. haemolyticus, and S.
lugdunensis. These organisms could also be closely associated with a range
of infections (Martineau et al.,
2001).
S. aureus, is
found almost everywhere it is widely distributed, particularly on the outer
membranes of humans and animals, it is the most pathogenic species of
Staphylococci (Mathanraj et al., 2009).
It has been estimated that about 20% of the human populations are carriers and
about 60% of the population colonized by S.
aureus. The nose is most favorable place of colonization (Zorgani et al., 2009) but the organism can be
seen to survive also on the membrane and in the environment for a extended
time. S. aureus that are methicillin
resistant (MRSA) also colonizes
additional locations not only the nose e.g. throat, armpit, appendix, perineum (Eveillard
et al., 2006) which might be involved
in a major function during the spread of infection. Recently, MRSA has been primarily considered as acquired in
hospital sites (commonly known as nosocomial infection) mainly affecting
healthcare workers (Zorgani et al.,2009).
1.2 OCCURRENCE OF
DRUG RESISTANCE AMONG S. aureus
STRAIN
Treatment of some conditions has become very
tough to treat (conditions such as wound infections, gonorrhoea, tuberculosis,
pneumonia, diabetic foot infection and childhood ear infections) with the
commercially available antibiotics because of the increase in resistance in
bacteria against these antibiotics. MRSA
has emerged as a major epidemiological problem in hospitals around the world
and requires continuous attention. Those strains often times can be called
superbug (Ahmed et al., 2010). The two kinds of MRSA
are:
i.
Hospital acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) and
ii.
Community acquired methicillin-resistant Staphylococcus aureus (CA-MRSA).
The
latter is bacteriologically, clinically and epidemiologically, distinct from
the former (CDC, 1999), and continues to be prominently involved in nosocomial
infections. The major reservoir host of S.
aureus is colonized hospital workers, carriers at risk for emerging
endogenous infection or spreading infection to healthcare workers and patients.
Carriage of the organisms on the hands of health care workers has been the main
mechanism for patient to patient transmission (Dar et al., 2006). Animals can also be seen as reservoirs of MRSA and serve as source (Milk) for further
transmission (Weese et al., 2010,
Garcia-Alvarez et al., 2011). Cows
were the first source of isolation of clinical MRSA
(Devriese et al., 1972).
There is evidence that MRSA can infect domestic animals which may be a
great challenge to vet doctors and to the general public at large (Khanna et al., 2008). Different animals have
since been reported to be a zoonotic source of MRSA,
which can infect their owners or attendants subsequently (Baptiste et al., 2005). These animals could thus,
serve as a basis (source) of zoonotic MRSA
and people in close contact with these animals could contract the MRSA infection. It has also been noted that humans
are also associated in the transfer of MRSA
to their close animals; few reports suggest the likelihood of transmission
(Seguin et al., 1999). Infections as a result of CA-MRSA in
particular, are seen now to be a major challenge to the health of the public
(Martin and Henry, 2008). In emerging countries, infections initiated by Staphylococcus aureus are not seen as
serious in relations to illness and death as compared to other diseases such as
malaria, tuberculosis, and HIV infection which are seen as highly infectious.
This organism is a pathogen of developing country. In South-East Asia, diseases
caused by Staphylococcus are prevalent in low-income and lower-middle income
countries based on several studies (Nickerson, 2009). The association of Staphylococcus aureus in several
clinical conditions is not connected to methicillin resistance.
Genetic Basis of
CA-MRSA
CA-MRSA strains appear to be resistant to fewer
antimicrobial drugs. They carry a number of different virulence genes, for
example, pvl gene that encodes panton-valentine leukocidin (pvl) toxin,
vancomycin resistance gene (van-A), also a different type of the gene complex
system called ‘Staphylococcal cassette chromosome mecA (SCCmec) encodes for
methicillin resistance (Naimi et al.,
2003). This cassette contains mobile genetic elements (e.g. mecA genes) which
have been categorized into five distinct types I to V and it’s identify based
on location (Martins and Cunha, 2007).
The genetic variety observed among strains of Staphylococcus aureus has helped in the formation of various grades
of virulence patterns and antibiotic resistance (De et al., 2007). Primarily seen as a challenge in hospitals is MRSA (Ahmed et
al., 2010).
In the 1950’s, many strains of S. aureus produced penicillinase to
overcome penicillin; a β-lactam drug methicillin was introduced which was
available in 1950s and become a drug preferred among others in the management
of penicillin-resistant staphylococcal infections. One year after the launch of
this drug, S. aureus resistant
strains of Staphylococcus aureus
appeared and it developed as a hospital acquired (nosocomial) pathogen early in
the 60’s (Jorgensen, 1986). MRSA
emerged as a very severe challenge problem in some US hospitals during 1970s
and by the 1990s; MRSA became an
international problem (Klevens et al.,
2007).
1.2.1 Mechanism of
methicillin resistance
Resistance to most β-lactam drugs including
methicillin in Staphylococci is mediated by an altered penicillin-binding
protein (PBP) this protein is programmed (encoded) by a gene known as the mec
gene (Brown et al., 2005). If the
bacterium possesses the mecA gene, it renders β-lactam including penicillin,
methicillin, and even cephalosporin ineffective.
1.2.2 Phenotypes
of MRSA
Globally,
majority of the MRSA resistant
phenotypes with multi resistance characteristic have been reported e.g. MRSA-MLSB (macrolides, streptogramines B and
lincosamides,) phenotypes. The MLSB resistance phenotypes (MLSBC and MLSBi
phenotypes) transfer multiple-resistance to most classes of antibiotics
including, macrolides, lincosamides, and streptogramines B (Lewis and
Jorgensen, 2005). Most of these phenotypes have been reported worldwide
including emerging countries (Siberry et
al., 2003; Ahmed et al., 2010).
1.2.3 S. aureus resistance to other
antibiotics
Clindamycin
is useful drug for treating MRSA infections; however, treatment failures has
been reported in patients with MRSA infections caused by inducible clindamycin
resistant using these drugs (MRSA-MLSBi) strains has been reported by several
workers (Siberry et al., 2003, Lewis
& Jorgensen, 2005). MRSA-MLSBi strains cannot be detected by standard
susceptibility tests. However, a test known as D test (Clinical Laboratory
Standard, 2006) is being used to detect such strains (Ahmed et al., 2010).
Another
antibiotic vancomycin was seen as the most preferred drug in the management
of S.
aureus infections but Vancomycin resistant S. aureus (VRSA) phenotype has evolved which confers further
resistance to vancomycin (Martins and Cunha, 2007). Some challenging factors
related as with one contracting MRSA
infection have been seen e.g. prolonged hospitalization and antimicrobial
therapy. Nasal colonization has also been known as one of the risk factors for
infection and carriage of MRSA in
various healthcare settings (Von et al.,
2001). People who have no link with hospital sites or any other risk factors
have been seen to have MRSA. Such
strains have been recognized as community acquired (CA-MRSA),
(Martins and Cunha, 2007). Several reports of clinical CA-MRSA have appeared since its first report in 1980s
(Fey et al., 2003).
1.3 JUSTIFICATION OF THE STUDY
Globally, Staphylococcus
aureus (S. aureus), a recalcitrant bacteria has been seen as a major source of
infection in hospitals or within communities. The organism has established
resistance to commonly prescribed antimicrobial agents. Its striking ability to
acquire resistance to new antimicrobial agents is the most important factor
contributing to the successful extensive distribution of this nosocomial
pathogen (Diekema, 2001).
The information on multidrug resistance of S. aureus will guide the clinician in
prescription and will reduce mortality rate hence help in more efficient health
care delivery in Umuahia.
1.3.1 Aims
The aim of this study is to determine the
prevalence of multidrug resistant S.
aureus isolates from wound infection patients obtained at Federal Medical
Centre Umuahia and also to characterize S.
aureus.
1.3.2 Specific Objectives.
i.
To isolate and identify S. aureus
isolates from wound infections.
ii.
To determine the antibiotic resistance profile and multiple antibiotic
resistance index of the isolates.
iii.
To determine the biofilm forming potential, beta lactamase production
and methicillin resistance of the isolates.
iv.
To determine the plasmid profile of the organism.
v.
To determine the resistance pattern before and after plasmid curing.
vi.
To characterize the S.aurues
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