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LIST OF CONTENTS
Subjects
Page
LIST OF ABBREVIATION
INTRODUCTION
12
LITERATURE REVIEW
15
1- Human burns
15
21
24
35
41
59
60
61
67
71
80
80
80
80
medical specimens
4- Identification for bacterial isolates
84
94
95
selected isolates
7- Bactericidal activity of different antimicrobial agent
96
97
bacterial isolates
9- Screening for the virulence factors and degrading enzymes
produced by tested S. aureus (12, 30, 50 and 95) using agar well
99
diffusion assay
10-Effect of certain bactericidal concentration on the enzyme activities 100
(virulence factors)
11-Effect of tea tree oil on the enzyme activities (virulence factors)
101
RESULTS
1- History of burned patients
107
111
118
131
136
179
SUMMARY
203
REFERENCES
210
LIST OF ABBREVIATIONS
AR
Antimicrobial resistance
ARDS
AST
BSI
Bloodstream infection
CA-MRSA
CoNS
Coagulase-negative staphylococci
DHFR
Dihydrofolate reductase
EF-G
Elongation factor G
FIC
IRS
Isoleucyl-tRNA synthetase
MRSA
NNIS
PBPs
penicillin-binding proteins
PVL
Panton-Valentine leukocidin
PCR
Resistance
SSIs
TSS
UTI
VFs
Virulence factors
VISA
vancomycin-intermediate S. aureus
VRE
VRSA
Vancomycin-resistant S. aureus
Microgram
DNA
EPS
Exopolysaccharide
Erm
Female
Gram
GISA
Hours
Kbp
Kda
Kilo Dalton
Liter
Male
MBC
MIC
Min.
Minute
MR
Methyl red
MRSA
OF
Oxidation fermentation
S. aureus
Staphylococcus aureus
Unit
Introduction
Introduction
Infection in the burned patient is a leading cause of morbidity and
mortality and remains one of the most challenging concerns for the burn
unit (Cochran et al, 2010).
Burn is one of the most common and devastating forms of trauma.
Patients with serious thermal injury require immediate specialized care in
order to minimize morbidity and mortality. Significant thermal injuries
induce a state of Immuno-suppression, which predisposes infectious
complications in burned patients (Church et al, 2011).
Burn patients are ideal hosts for opportunistic infections, the burn
site remains relatively sterile during the first 24 hour; thereafter,
colonization of the wound by Gram negative bacteria is common (Pruitt et
al, 2008)
Sources of organisms are found in the patients own endogenous
(normal) flora, from exogenous sources in the environment, and from
healthcare personnel. Exogenous organisms from the hospital environment
are generally more resistant to antimicrobial agents than endogenous
organisms. Organisms associated with infection in burn patients include
Gram-positive, Gram-negative, and yeast. The distribution of organisms
changes over time in the individual patient and such changes can be
ameliorated with appropriate management of the burn wound and patient.
The typical burn wound is initially colonized predominantly with Grampositive organisms, which are fairly quickly replaced by antibioticsusceptible Gram-negative organisms, usually within a week of the burn
injury. If wound closure is delayed and the patient becomes infected,
requiring treatment with broad-spectrum antibiotics, these flora may be
replaced by yeasts and antibiotic-resistant bacteria (Church et al, 2011).
Introduction
Introduction
Introduction
staphylococci
and
Staphylococcus aureus.
The
Introduction
and
reduce
sensory
impact.
Furthermore,
these
combinations may also control some bacteria that are known to show
consistently high resistance to antimicrobials (Aqil et al., 2005).
10
11
2.
3.
4.
5.
6.
7.
8.
9.
10. Investigation the effects of tea tree oil on the production of virulence
factors by multiresistant isolates.
11. Molecular studies on multiresistant isolates:
12
13
14
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1. Human burns
Burn infection is problematic because it delays healing, encourages
scarring and may result in bacteremia, sepsis or multiple-organ dysfunction
syndrome (a.k.a. organ failure) whereby organs from several systems are
unable to maintain homeostasis on their own, requiring immediate medical
attention. Bacteria are the most common pathogens of burn wounds. These
microbes form multi-species biofilms on burn wounds within 48 72 hours
of injury (Agnihotri et al., 2004). In patients with severe burns over more
than 40% of the total body surface area (TBSA), 75% of all deaths are
currently related to sepsis from burn wound infection or other infection
complications and/or inhalation injury (Atiyeh et al., 2005).
1.1. HUMAN skin - A major host innate defense:
Basic skin anatomy, showing the depth of injury for first, second, and third-degree burns(Roth
and Hughes, 2004).
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The skin is derived from ectoderm and mesoderm and has two
anatomic layers: the epidermis or outermost nonvascular layer consists of
several layers of epidermal cells that vary in thickness over various body
surfaces, and the dermis or corium is largely made of collagen and contains
the microcirculation, a complex vascular plexus of arterioles and venules.
The two skin layers are bound together by a complex mechanism that is
essential for normal function. Burn injury is a very painful form of trauma
because of the multitude of pain receptors and nerves that traverse the skin
layers. Beneath the skin lie the subcutaneous tissues, muscle, and bone
(Dyer, 2008).
1.2. Causes and classification of burned injury :
1.2.1-Causes of burned injury:
a. Thermal injury
Direct contact with flame, a hot surface or hot liquid (scald), or a
source of heat conduction, convection, or radiation causes a degree of
cellular damage to the skin that varies with the temperature and duration of
exposure (Baker et al., 2009). As the temperature rises further, protein
denaturation occurs, oxygen radicals are liberated, and eventually cells die
with the formation of the burn eschar (Moritz and Heuriquez, 2005).
b. Chemical injury
Chemical interaction may also damage protein structures. A
classification system that was described and remains in use groups
chemicals according to their mode of action. (Amshel et al., 2000).
c. Electrical injury
Electrical burns are caused by either an electric shock or an
uncontrolled short circuit (a burn from a hot, electrified heating element is
not considered an electrical burn). Common occurrences of electrical burns
include workplace injuries, or being defibrillated or cardioverted without a
16
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Layer
involved
Epidermis
Appearan
ce
Redness
(erythema)
Textur
e
Dry
Sensati
on
Painful
Time to
healing
1week or
less
Complication
None
Second
degree
(superficial
partial
thickness)
Extends
into
superficial
(papillary)
dermis
Red with
clear
blister.
Blanches
with
pressure
Moist
Painful
2-3wks
Local
infection/
cellulit
17
Example
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Second
degree
(deep
partial
thickness)
Extends
into deep
(reticular)
dermis
Red-andwhite with
bloody
blisters.
Less
blanching.
Moist
Painful
Weeks may
progress
to
third
degree
Scarring,
contractures
(may
require
excision
and
skin
grafting)
Third
degree
(full
thickness)
Extends
through
entire
dermis
Stiff and
white
/brown
Dry,
leather
y
Painles
Requires
excision
Scarring,
contractures
,
amputation
Fourth
degree
Extends
through
skin, and
muscle and
bone
Black;
charred
with eschar
Dry
Painles
Requires
excision
Amputation
, functional
impairment,
gangrene,
and death.
Age 10-50yrs: partial thickness burns >25% of total body surface area
Age <10 or >50: partial thickness burns >20% of total body surface area
Electrical burns
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Persons suffering these burns often need to be hospitalised for burn care.
c. Minor burns are:
Age 10-50yrs: partial thickness burns <15% of total body surface area
Age <10 or >50: partial thickness burns involving <10% of total body
surface area
Full thickness burns <2% of total body surface area, without associated
injuries.
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Body diagram for estimation of total burned surface area (%TBSA) in adults, using the rule of
nines (numbers are for anterior only and posterior only) (Roth and Hughes. 2004)
Body diagram for estimation of total burned surface area (%TBSA) in children, using the rule of
nines (numbers include anterior and posterior) (Roth, and Hughes. 2004).
20
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expected
that
burn
patients
with
other
types
of
severe
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wound bed makes an excellent medium, which can support the growth of
microorganisms as well as prevent the penetration of systemically,
administered antimicrobial drugs. Burn wound infection can be subdivided
into local or non-invasive infection and invasive infection. Local wound
infection is characterized by erythema or cellulitis, purulent, drainage, graft
loss, fever >38.5C and leukocytosis. Invasive wound infection is
characterized by conversion of partial-thickness to full-thickness injury,
necrosis of small blood vessels, edema, erythema, and tenderness at the
wound edges. Systemically, the patient may be hypothermic or
hyperthermic, hypotensive, have a decreased urine output and illeus.
Laboratory
results
will
reveal
leukocytosis
or
leukopenia,
22
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Adult
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Gram-negative
organisms
Species
Staphylococcus aureus
Vancomycin-resistant Staphylococcus aureus
Methicillin-resistant S. aureus
Coagulase-negative staphylococci
Enterococcus spp.
Vancomycin-resistant enterococci
Pseudomonas aeruginosa
Escherichia coli
Klebsiella pneumonia
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Serratia marcescens
Enterobacter spp.
Proteus spp.
Acinetobacter spp.
infections to severe illnesses such as septicaemia, endocarditis and toxic
shock syndrome. S.aureus is particularly a problem in hospitals because it
spreadseasilyintheseenvironmentsandcausespotentiallyfatalinfectionsin
immuno-compromised hospitalpatients(Liu and Chambers, 2003).
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and
life-threatening
conditions
such
as
endocarditis,
circulatory
system,
respiratory
tract,
urinary
tract,
and
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are
long-term
carriers
of
S.
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methicillin-resistant
S.aureus
(MRSA)
have
been
28
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recently
emergence
of
resistance
29
to
glycopeptides
and
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strains
of
''S.
aureus''
are
capable
of
producing
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Coagulase
Fibrinolysin
Leukocidin
Hyaluronidase
DNAse
ProteinA
EpidermolytictoxinsA
andB
Enterotoxin(s)
Toxicshocksyndrome
toxin
Proteases
Activity
Cytolytic;lyse erythrocytesofvariousanimal
species
Clotsplasma, alsousedinclinicalmicrobiology
laboratoriestodifferentiatebetweenS.aureus
and CNS
Digetsfibrin
Killsleukocytes
Breaksdownhyaluronicacid
HydrolysesDNA
Lypolytic(producesopacityinegg-yolk
medium
Epidermalsplittingandexfoliation
Foodpoisoningtoxinsthatcausevomitingand
Diarrhea
Shock,rashanddesquamation
S. aureus produces several extracellular proteases, including metalloserine and cysteine proteases (Dubin, 2002). These proteases are thought to
32
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that live in the human gut. These bacteria are known for their tendency to
cause disease in immuno-compromised patients, such as those with AIDS
or cystic fibrosis, but rarely in healthy individuals P. aeruginosa use
quorum sensing to induce the production of virulence factors such as
proteases, hemolysins, exotoxin A and pyocyanin (Lyczak et al., 2002).
3.2.1. The virulence factors of P. aeruginosa
These bacteria posses' two quorum-sensing systems: one that
regulates proteases and another that regulates hemolysins. Though separate
systems, the two interact through products of the protease system
controlling
the
hemoysin
system
at
both
transcriptional
and
posttranslational levels. While it has been widely held that quorum sensing
is required for biofilm formation, new research has been put forth that
challenges that position. A team of scientists out of Auburn University
investigated the development of P. aeruginosa biofilms using a mouse burn
model (Lyczak et al., 2002). Third-degree thermal injury was induced and
mice were then infected with two strains of P. aeruginosa, one wild type
(WT) and the other quorum-sensing deficient (QS). Eschar samples were
examined using flouresence and scanning electron microscopy at 8, 24 and
48 h after infection. Both strains developed biofilms at the same pace,
although WT biofilms were somewhat denser than the QS strain. It had
been previously demonstrated that the WT strain was more virulent than
the QS. This study suggests that the difference in virulence is not due to
impaired biofilms formation; rather it is likely due to differences in the
expression of virulence factors. It seems that quorum-sensing knockout
strains lost some other genes in the process of mutation. Proteases are
virulence factors that degrade the integrity of the hosts physical barriers by
splitting proteins and amino acids, allowing for deeper infiltration of
infection (Schaber et al., 2007). Exotoxin A halts the synthesis of proteins,
causing local tissue damage, immune-suppression and cell death.
Hemolysins act like detergents to break down lipids in epithelial cells
34
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allowing, like the proteases, the bacilli to penetrate the host resulting in the
spread of infection. In addition, genes encoding proteins for pili and
flagella are also important to P. aeruginosa since without these structures,
bacteria are unable to navigate their environment and form biofilms. P.
aeruginosa infections are difficult to treat because these microbes possess
multiple strategies for eluding predators, including multidrug efflux pumps,
antibiotic-modifying enzymes, and tough outer membranes with low
permeability (Vidaillac et al., 2009)
4. The antibiotics activity against pathogenic bacteria.
4.1. Definition of antibiotics:
Antibiotics are low-molecular weight microbial metabolites that at
low concentrations inhibit the growth of microorganisms. The term low
molecular weight substance refers to molecule with a defined chemical
structure having a relative mass of at most a few thousand Daltons
(Franklin and Snow, 1990).
Antibiotics may be informally defined as the sub-group of anti-infective
that are derived from bacterial sources and are used to treat bacterial
infections (Mackie and McCartney, 1980). Also, defined as a drug used
to treat infections caused by bacteria and other microorganisms (Davies,
1990). Originally, an antibiotic was a substance produced by one
microorganism that selectively inhibits the growth of another. Synthetic
antibiotics, usually chemically related to natural antibiotics, have since
been produced that accomplish comparable tasks (Bush et al., 1997).
4.2. Mode of action of antibiotics on bacterial cell:The development of antibiotics for the bacterial infections represents
one of the most remarkable achievements of last century. The most
contributing factors for developing resistance were the excessive use and/or
abuse of antibiotics (WHO, 1980).
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The antibacterial (antibiotics) can be divided into two categories: 1those that inhibit the growth of Gram positive bacteria and are inactive
against Gram negative bacteria, and 2- those that inhibit both Gram
positive and Gram negative bacteria. These latter are often called broadspectrum antibiotics. It is very seldom that an antibiotic is found that is
active only against Gram negative bacteria (Walker, 1998).
Antibiotics are characterized by their chemical composition and
mode of action in the target organism. The main targets for action in
bacteria include the prokaryotic cell wall, the cell membrane, protein
synthesis, enzymatic pathways, and DNA replication (Brooks et al., 2001).
The microbiological and clinical factors considered in the selection
of appropriate antimicrobial therapy of anaerobic infections include: (1) the
in-vitro activity of the drug against the pathogens; (2) the ability of the
compounds to penetrate the infected site and resist inactivation; (3) the
proven efficacy of the antimicrobial agents in well controlled, randomized
prospective studies as compared to established therapy and (4) comparison
of the side effects of the drugs. The resistance of anaerobic bacteria to a
number of antimicrobial agents has an impact on the first three factors
(Giacommetti et al., 2000).
Diagram indicated the common mechanism of killing by bactericidal antibiotics with diverse targets
(Dwyer et al., 2007).
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The activity and mode of action of some antibiotics were listed by CLS I.
(2008) as the following.
Antibiotic
Penicillin
Cephalosporin
Activity
Gram-positive bacteria
Broad spectrum
Polymyxin B
Gram-negative bacteria
Cell membrane
Erythromycin
Gram-positive bacteria
Protein synthesis
Neomycin
Broad spectrum
Protein synthesis
Streptomycin
Gram-negative bacteria
Protein synthesis
Tetracycline
Broad spectrum
Protein synthesis
Gentamicin
Broad spectrum
Protein synthesis
Rifamycin
Tuberculosis
Protein synthesis
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Erythromycin
Rifamycin
Tetracyclin
Type of
mechanism
Location of genetic
determinate
Description of
mechanism
Inactivation
Generally Extra
chromosomal
Inactivation,
Modification of
target
Modification of
target
Modification of
target
Cellular
permeability
Extrachromosomal,
Chromosomal
Acetylation by inducible
enzyme Modification of
ribosomal receptors
Alteration of protein of
ribosomal 50S subunit.
Alteration of -subunit
of RNA polymerase
Decreased effeciancy of
transport. The resistance
is partially induced
Chromosomal,
Extrachromosomal
Chromosomal
Extrachromosomal
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resistance
(Schaaff
et
al.,
2002).
Resistance
may
be
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agents
and
biocides
caused
by
the
biofilm
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Diagram illustrating drug flows over the cell envelope, drug inactivation and target binding.
Drugs enter the cell by passive transport (rate constant k passive) and exit by passive transport
(rate constant k passive), active pumping (rate constant k active), drug degradation in the
cytoplasm (rate constant k deg.) (Fange et al., 2009).
D- Genetic resistance:
Resistance to antimicrobial agents may be mediated by agents that
are encoded on the host cell chromosomes, plasmids or transpospons,
which are capable of integrating into the plasmid and/or into the
chromosome. Bacteria may acquire exogenous genetic material that leads to
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penicillinases
and
cephalosporinases
were
48
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penicillinase
(beta-lactamase),
similarly,
the
successive
baumannii-calcoaceticus
complex,
Pseudomonas
aeruginosa, and Klebsiella species. These latter pathogens are notable for
their increasing resistance to a broad array of different antimicrobial agents.
Emerging antimicrobial resistance trends in burn wound bacterial
pathogens represent a serious therapeutic challenge for clinicians caring for
burned patients (Gerard and Arlene, 2007).
5.7. Historical aspects of antibiotic resistant staphylococci.
The appearance of antibiotic resistant staphylococci over the past
years has been regarded as an inevitable genetic response to the selective
pressure imposed by antimicrobial therapy; it also illustrates the ability of
microbial population to readily adapt to changes in their environment
(Saunder et al., 1984).
5.7.1. Resistance to -lactam and glycopeptides antibiotics
Resistance to -lactam antibiotics in staphylococci can be mediated
by three different mechanisms; -lactamase mediated resistance, intrinsic
49
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organisms
such
as
MRSA,
(VRSA),
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choice, but has since been replaced by oxicillin due to significant kidney
toxicity. MRSA (methicillin-resistant S. aureus) was first detected in
Britain in 1961 and is now "quite common" in hospitals. MRSA was
responsible for 37% of fatal cases of sepsis in the UK in 1999, up from 4%
in 1991. Half of all S. aureus infections in the US are resistant to penicillin,
methicillin, tetracycline and erythromycin (Todar, 2008).
Community-acquired MRSA (CA-MRSA) has now emerged as an
epidemic that is responsible for rapidly progressive, fatal diseases including
necrotizing pneumonia, severe sepsis and necrotizing fasciitis. MRSA is
the most frequently identified antimicrobial drug-resistant pathogen in US
hospitals. The epidemiology of infections caused by MRSA is rapidly
changing. In the past 10 years, infections caused by this organism have
emerged in the community. The 2 MRSA clones in the United States most
closely associated with community outbreaks, USA400 (MW2 strain, ST1
lineage) and USA300, often contain Panton-Valentine leukocidin (PVL)
genes and, more frequently, have been associated with skin and soft tissue
infections. Outbreaks of CA-MRSA infections have been reported in
correctional facilities, among athletic teams, among military recruits, in
newborn nurseries, and among men who have sex with men. CA-MRSA
infections now appear to be endemic in many urban regions and cause most
CA-S. aureus infections. (Clark et al., 2005).
A-Mechanism of resistance methicillin in S. aureus
Methicillin resistance is defined as the strains of S. aureus that are
resistant to the isoxazoyl penicillins such as methicillin, oxacillin and
flucloxacillin. MRSA are cross-resistant to all currently licensed betalactam antibiotics. The expression of methicillin resistance by S. aureus
strains is by virtue of acquired penicillin binding proteinPBP2a, encoded by
mec A gene.
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vancomycin use has led to the emergence of two types of glycopeptideresistant S. aureus. The first one, designated vancomycin intermediateresistant S. aureus, is associated with a thickened and poorly cross-linked
cell wall, resulting in accumulation of acyl-D-alanyl-D-alanine (X-D-Ala-DAla) targets in the periphery that sequester glycopeptides. The second type,
vancomycin-resistant S. aureus (VRSA), is due to acquisition from
Enterococcus spp. of the vanA operon, carried by transposon Tn1546,
resulting in high-level resistance (Murphy et al., 2003).
The resistance of Staphyococi to vancomcin has been found to be
reversible under laboratory conditions. (Rybak and Akins, 2001). One
study indicated that vancomycin resistance in S. aureus may occur under
the selective pressure of prolonged vancomycin use and that the resistance
to vancomycin in S. aureus is reversible on removal of the drug .
Moreover, mechanism for vancomycin resistance in these bacteria (Walsh
and Howe, 2002).
Actually, thickening of the cell wall of vancomycin- resistant
staphylococci has been found to be associated with complex reorganization of cell wall metabolism with extra cell wall material showing
reduced peptidoglycan cross - linking of D-Ala-D-Ala termini of side
chains (Zaragoza et al., 2003).
Vancomycin act as the only effective agent available at the time.
However, strains with intermediate (4-8 g/ml) levels of resistance, termed
GISA (glycopeptide intermediate S. aureus) or VISA, began appearing in
the late 1990s. The first identified case was in Japan in 1996, and strains
have since been found in hospitals in England, France and the US. The first
documented strain with complete (>16 g/ml) resistance to vancomycin,
termed VRSA appeared in the United States in 2002 (Cui et al., 2009).
The transfer of van resistance genes from Enterococcus species to S.
aureus, which results in high levels of resistance to vancomycin, was
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obtained in vitro and in an animal model. Most important, this transfer also
occurs in vivo. Three MRSA) isolates with high or moderate levels of
resistance to vancomycin and teicoplanin have been isolated from patients
in Michigan (MI-VRSA), Pennsylvania (PA-VRSA), and New York after
acquisition of the van A gene cluster (Kacica and McDonald 2004).
Analysis of the nucleotide sequences flanking Tn1546 indicated that
the transposon was flanked by 5-bp duplications of target DNA typical of
the Tn3 family of elements to which Tn1546 belongs. This observation
confirms that Tn1546 has transposed into the plasmid of MI-MRSA.
Comparative analysis of peptidoglycan precursors and of D,D-dipeptidase
(vanX) and D,D-carboxypeptidase (vanY) activities indicated high similar
levels of expression of the vanA gene clusters in the MI-VRSA and PAVRSA strains. Thus, the difference in glycopeptide resistance between the
2 isolates is not due to a difference in van gene expression (Liu and
Chambers, 2003).
Mechanism of vancomycin resistance in VISA strains due to
vancomycin binds with the D-alanyl-D-alamine C terminus of the bacterial
cell precursors, thereby preventing cross-linking by transpeptidation
resulting in inhibition of cell wall production by attacking sites responsible
for cell wall production. VISA and hetero-VISA strains have been found to
have thickened cell wall with reduced glycoprotein. This could be due to
changes in peptidoglycan synthesis resulting in increased residues of D
alanyl-D-alanine, which bind vancomycin molecules and prevent them
from reaching the target sites(Zhu, 2008).
Mechanism of vancomycin resistance in VRSA strains also have
been found to have thicker cell walls than the sensitive strains. As with
VISA strains, there is also increased peptidoglycan synthesis. It has been
shown that vancomycin is only trapped in the outer layers and sequestered
by the bacteria and not deactivated. Exchange of genetic material is yet
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SchematicrepresentationofthemechanismsofS.aureusintermediateresistanceto vancomycin.The
vancomycin-intermediate S. aureus strains synthesise additional quantities of peptidoglycan with
increased numbers of D-Ala-D-Ala residues thatbind vancomycin, thus preventing the molecule to
bindtoitsbacterialtarget(cellwall) (Zhu, 2008).
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that have been studied, is similar to that of van A and van B. Van D-type
strains share other characteristics that distinguish them from van A- and
van B-type enterococci (Reynolds et al., 2005).
VanC. The van C phenotype is expressed constitutively or inducibly as a
result of the production of peptidoglycan precursors ending in D-Ser. Three
vanC genes encoding D-Ala:D-Ser ligases have been described: vanC-1 in
E. gallinarum, vanC-2 in E. casseliflavus, and vanC-3in E. flavescens
(Abadia et al., 2002).
VanE. The vanE phenotype corresponds to low-level resistance to
vancomycin and susceptibility to teicoplanin due to synthesis of
peptidoglycan precursors terminating in D-Ala-D-Ser as in intrinsically
resistant Enterococcus species. The vanE cluster has an organization
identical to that of the vanC operon (Abadia et al., 2002).
VanG. Acquired vanG type is characterized by resistance to low levels of
vancomycin (MIC, 16 g/mL) but susceptibility to teicoplanin (MIC, 0.5
g/mL) and by inducible synthesis of peptidoglycan precursors ending in
D-Ala-D-Ser. The chromosomal vanG cluster is composed of 7 genes
recruited from various van operons (Reynolds et al., 2005).
6. Antimicrobial resistance in P. aeruginosa.
P. aeruginosa remains a serious cause of infection and septic
mortality in burn patients, particularly when nosocomially acquired. A
prototypic burn patient who developed serious nosocomially acquired
Pseudomonas infection is described as an index case which initiated
investigations and measures taken to identify the source of the infection.
The effect of changes in wound care to avoid further nosocomial infections
was measured to provide data on outcome and cost of care. The
bacteriology of Pseudomonas is reviewed to increase the burn care
providers understanding of the behaviour of this very common and serious
pathogen in the burn care setting, before reviewing the approach to
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Following
the
over
use
of
the
expanded
spectrum
cephalosporins, sever outbreaks caused by expanded spectrum Lactamases (ESBL) producing E. coli and K. pneumoniae have been
reported (Kohler et al., 1999).
The most disturbing feature of P. aeruginosa is its broad resistance
to antibiotics. This resistance may also be plasmid mediated. There are at
least thirteen group of plasmids based on compatibility. They carry markers
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explain what is going to happen and assure them that the fizzing will not be
painful (Mayaudon & EL-Zayat, 2005).
Hydrogen peroxide solutions that contain more than 20% hydrogen
peroxide can damage the skin and mucus membranes and can even lead to
infection, rather than preventing it. Solutions this strong should be used
only occasionally and in small amounts, if at all. Care should also be taken
if hydrogen peroxide is used in the mouth, as a mouthwash or a gargle. Spit
out the hydrogen peroxide after gargling (Mertens et al., 2000).
Mode of Action.
Hydrogen peroxide works by producing destructive hydroxyl free
radicals that can attack membrane lipids, DNA, and other essential cell
components. Catalase, produced by aerobic organisms and facultative
anaerobes that possess cytochrome systems, can protect cells from
metabolically produced hydrogen peroxide by degrading hydrogen
peroxide to water and oxygen. This defense is overwhelmed by the
concentrations used for disinfection (Mertens et al., 2000).
Microbicidal activity.
Hydrogen peroxide is active against a wide range of microorganisms,
including bacteria, yeasts, fungi, viruses, and spores. A 0.5% accelerated
hydrogen peroxide demonstrated bactericidal and virucidal activity in 1
minute and mycobactericidal and fungicidal activity in 5 minutes.
Bactericidal effectiveness and stability of hydrogen peroxide in urine has
been demonstrated against a variety of health-careassociated pathogens;
organisms with high cellular catalase activity (e.g., S. aureus, S.
marcescens, and Proteus mirabilis) required 3060 minutes of exposure to
0.6% hydrogen peroxide for a 108 reduction in cell counts, whereas
organisms with lower catalase activity (e.g., E. coli, Streptococcus species,
and Pseudomonas species) required only 15 minutes exposure. In an
investigation of 3%, 10%, and 15% hydrogen peroxide for reducing
64
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65
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9.2. Alcohol
Overview.
In the healthcare setting, "alcohol" refers to two water-soluble
chemical compoundsethyl alcohol and isopropyl alcoholthat have
generally underrated germicidal characteristics. FDA has not cleared any
liquid chemical sterilant or high-level disinfectant with alcohol as the main
active ingredient. These alcohols are rapidly bactericidal rather than
bacteriostatic against vegetative forms of bacteria; they also are
tuberculocidal, fungicidal, and virucidal but do not destroy bacterial spores.
Their cidal activity drops sharply when diluted below 50% concentration,
and the optimum bactericidal concentration is 60%90% solutions in water
(volume/volume) (Rutala, 1999).
Mode of Action
The most feasible explanation for the antimicrobial action of alcohol
is denaturation of proteins. This mechanism is supported by the observation
that absolute ethyl alcohol, a dehydrating agent, is less bactericidal than
mixtures of alcohol and water because proteins are denatured more quickly
in the presence of water. Protein denaturation also is consistent with
observations that alcohol destroys the dehydrogenases of E. coli, and that
the lag phase effect could be reversed by adding certain amino acids. The
bacteriostatic action was believed caused by inhibition of the production of
metabolites essential for rapid cell division (Pitts et al., 2003).
Microbicidal activity
Methyl alcohol (methanol) has the weakest bactericidal action of the
alcohols and thus seldom is used in healthcare. The bactericidal activity of
various concentrations of ethyl alcohol (ethanol) was examined against a
variety of microorganisms in exposure periods ranging from 10 seconds to
one an s hour. P. aeruginosa was killed in 10 seconds by all concentrations
of ethanol from 30% to 100% (v/v), and Serratia marcescens, E, coliand
66
Review of Literature
antiparasitical,
insecticidal,
medicinal
and
cosmetic
67
Review of Literature
essential
oil
of
Thymus
leptophyllus
showed
higher
68
Review of Literature
Voaltile oil can affect bacteria in ways other than the expected
bactericidal or bacteriostatic action. Volatile oils cause alteration in the cell
morphology (Caelli, et al., 2000).
The thymol from thyme oil (Thymus vulgaris) and carvacrol from
oregano oil (Origanum vulgaris) both disrupt the cell membrane thereby
decreasing the intracellular ATP pool and increasing the extracellular ATP
pool in S. aureus (Dorman and Deans, 2000).
The essential oil of Melaleuca alternifolia (tea tree) has broadspectrum antimicrobial activity. The mechanisms of action of tea tree oil
and three of its components, 1,8-cineole, terpinen-4-ol, and -terpineol,
against S. aureus were investigated. Treatment with these agents at their
MICs and two times their MICs, particularly treatment with terpinen-4-ol
and -terpineol, reduced the viability of S. aureus. None of the agents
caused lysis, as determined by measurement of the optical density at 620
nm, although cells became disproportionately sensitive to subsequent
autolysis. Loss of 260-nm-absorbing material occurred after treatment with
concentrations equivalent to the MIC, particularly after treatment with 1,8cineole and -terpineol. S. aureus organisms treated with tea tree oil or its
components at the MIC or two times the MIC showed a significant loss of
tolerance to NaCl. When the agents were tested at one-half the MIC, only
1,8-cineole significantly reduced the tolerance of S. aureus to NaCl.
Electron microscopy of terpinen-4-ol-treated cells showed the formation of
mesosomes and the loss of cytoplasmic contents. The predisposition to
lysis, the loss of 260-nm-absorbing material, the loss of tolerance to NaCl,
and the altered morphology seen by electron microscopy all suggest that tea
tree oil and its components compromise the cytoplasmic membrane (Ela et
al., 1996).
Some abnormalities in E. coli and S. aureus cells treatment with
cinnamon and oregano oils (Rios et al., 1998). The mechanism of the
69
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Polymerase chain reaction based methods have been shown to have
shortened the turn-around time (2 h to 4 h) in identifying MRSA isolates
resulting inprompttreatmentforMRSA associatedinfection(Van Halet
al., 2007). These PCR-based methods are able to detect multipleother S.
aureusspecificgenesincludingthe16SrRNAandfemgenes(McClureet
al., 2006). This PCR method of detecting multiple genes in S. aureus
simultaneouslyiscalledmultiplexPCR(M-PCR).
The detection and identification of microorganisms based on their
16S rRNAs have many advantages. First, each bacterial cell contains
multiple copies of the 16S rRNA in its ribosomes. Hence, the technique is
sensitive enough to detect single bacterial cells. Second, 16S rRNA genes
are highly conserved throughout bacterial evolution. They consist of
regions which are common to all eubacteria and other regions which are
extremely species specific. By using the appropriate gene probes, it is
possible either to detect any bacterial pathogen or, when highly specific
probes are used, to identify single bacterial species. Also, this technique
allows for the identification of microorganisms independently of bacterial
growth rates and metabolic activities. This is a special advantage for the
detection of dormant and metabolically inactive bacteria, since the number
of ribosomes is not significantly affected in such organisms (Walsh and
Howe, 2002).
16S rRNA gene sequencing will continue to be the gold standard for
the identification of bacteria, and the automation of the technique could
enable it to be used routinely in clinical microbiology laboratories, as a
replacement of the traditional phenotypic tests. Modern technologies have
made it possible to construct a high density of oligonucleotide arrays on a
chip with oligonucleotides representing the 16S rRNA gene sequence of
various bacteria. Such a design will facilitate automation of the annealing
and detection of the PCR products of 16S rRNA gene amplification, and
hence routine identification of most clinical isolates will be possible
72
Review of Literature
(Arthur et al., 1993). The use of 16S rRNA gene sequencing has several
advantages. First, the turnaround time is short. Because amplification of the
16S rRNA gene takes only four to six hours, and the annealing and
detection of PCR products takes only another few hours, theoretically the
identification can be completed within one day. Second, it can be used for
slow growing bacteria, unlike most commercially available kits that are
based on phenotypic tests that require the detection of growth of the
organism in the presence of certain specific substrates, and hence the slow
growing bacteria are usually unidentified when the growth control shows
a negative result. Third, the problem of unidentifiable strains will be
overcome and there would be minimal misidentificationthe identification
of a clinical strain is clearly defined by the number of base differences
between it and the existing species. Fourth, oligonucleotides representing
all bacterial species, including those rarely encountered clinically, can be
included in the array, making it easy to identify the rare species. Lastly,
such a technique will be applicable not only to gynogenic bacteria, but also
to other organisms such as mycobacteria, which are not identified in most
clinical microbiology laboratories because special expertise and equipment,
such as gas liquid chromatography, are required. Therefore, eventually it
should reduce not only manpower, but also capital costs and costs of
consumables. Although the cost effectiveness of using 16S rRNA gene
sequencing in routine clinical microbiology laboratories remains to be
evaluated, our study reports the first case of tube coagulase negative S
aureus bacteremia identified by 16S rRNA gene sequencing(Sudagidan et
al., 2010).
Gene amplification techniques are increasingly used in diagnostic
microbiology and the first applications for the detection of antibiotic
resistance have been described. Studies comparing classical methods such
as disc diffusion and MIC determinations with PCR for the detection of
resistance have been performed for a number of antibiotics or specific
73
Review of Literature
of antibiotic
resistance
resistance detection,
but many
Review of Literature
A DNA probe for van A hybridized to the 120-kb plasmid. This is the
second VRSA isolate reported in the United States (Biswajit et al., 2008).
Thus, it surprised many scientists that the first S. aureus isolate
reported to manifest reduced susceptibility to vancomycin did not contain
vanA or any of the other known vancomycin resistance determinants.
Instead, the reduced susceptibility has been attributed to unusually
thickened cell walls containing
D-alanyl-D-alanine
targets capable of
Forward
ATGAATAGAATAAAAGTTGC
and
Reverse
75
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Review of Literature
PCR amplification of the vanA gene for vancomycin resistance in methicillin resistant S.
aureus. Lanes 1-6 van A positive VRSA; Lane 7-12: van A negative (Tiwari and Sen, 2006)
The results of multiplex PCR assay revealed the isolates with van Apositive genes among others (van A, van B, van C1, van C2/C3) are
predominant. van B and Van C2/C3 were not detected while van C1 and
van C1+B were each observed in only 1 (1%) of vancomycin resistant
isolates (Emaneini et al., 2008).
78
79
80
(g /L):
Peptone
5.0
Yeast extract
2.0
Lab-lemco powder
1.0
Sodium chloride
5.0
Agar
15
Distilled water
1Liter
20.0
Lactose
10.0
1.50
Sodium chloride
5.00
Neutral red
0.03
Crystal violet
0.001
Agar
15.0
Distilled water
1Liter
(g/L):
Peptone
5.0
Yeast extract
2.0
Lab-lemco powder
1.0
Sodium chloride
5.0
Agar
15
Distilled water
1Liter
Blood
100 ml
82
(g/L):
`Lab-Lemco powder
1.00
Peptone
10.0
Mannitol
10.0
Sodium chloride
7.50
Phenol red
0.025
Agar
15.0
(g/L)
Peptone
4.0
`Lab-Lemco powder
3.0
Tryptone
4.0
Lactose
10.0
L-cystine
0.128
Bromothymol blue
0.02
Agar
15.0
30.0
Casein hydrolysate
17.5
83
Starch
1.50
Agar
17.0
Distilled water
1000 ml
2g.
Ethyl alchol,95%
20 ml.
Ammonium oxalate
0.8 ml.
Distilled water
100 ml.
Iodine solution
2g.
Potassium iodide
Iodine crystals
84
Distilled water
1g.
Decolerized:
100 ml
Acetone
50 ml
20 ml
Counter stain:
Safranine
2.5 g
100 ml
Distilled water
100 ml
Procedure:
A heat-fixed smear from an 18-24 h. bacterial culture was prepared
on a clean slide. Smear was stained with crystal violet solution for 30 sec.
and then washed with distilled water. The prepared film was covered with
Grams iodine solution for 10 sec.
The iodine was poured off and the slide was washed with a
decolorized until no more purple stain run from the slide. The slide was
gently washed with distilled water, stained with the counter stain (safranin)
for 30 sec. and then washed with distilled water and dried. The dried slide
was examined under the oil immersion lens of the light microscope.
1000 ml.
85
Sugar
10.0 g.
Bromothymol blue
1.00 ml.
sorbitol, sucrose
Procedure:
Two tubes containing the OF medium were required for each sugar.
Test tubes contain inverted Durhams tube were inoculated with the tested
organisms. One tube of each pair was covered with 1 cm. of sterile mineral
oil (fermentation test), while the other not covered (oxidation test). Tubes
were incubated at 37C and examined daily for gas formation and change
in medium color (yellow and blue color represented positive and negative
oxidativefermentation tests, respectively).
b-Nitrate reduction test: Nitrate broth medium
Beef extract
Peptone
Potassium nitrate
Distilled water
pH value was adjusted at 7.1
Reagents of nitrate reduction test
(g/L):
3.0
5.0
1.0
1liter
Reagent A
A-Naphthylamine
5.0 g.
1liter
Sulfanilic acid
Acetic acid (5N),30%
8.0 g.
1 liter
Procedure:
Sterile test tubes each contained the same volume of nitrate broth
medium were inoculated with bacterial culture (24h. age) and incubated at
86
c- Motility test:
(g/L):
80
Agar-Agar
15
Peptone
10
Sodium chloride
5.0
Beef extract
3.0
pH was adjusted at
7.1
Detection of the motility was made using the stab inoculation technique
according to Tittsler and Sandholzer (1936).
d- Arginine hydrolysis test:Arginine broth medium
(g/L):
Peptone (tryptone)
5.0
Yeast extract
5.0
K2HPO4
2.0
Glucose
0.5
Arginine monohydrochloride
3.0
Distilled water
1 liter
7.0
The ingredients were dissolved by boiling; the mixer was filtered and
sterilized at 115 C for 20 minutes by autoclaving.
87
'
(g/L):
Peptone
1.0
NaCl
5.0
KH2PO4
2.0
Glucose
1.0
Urea
20
Phenol red
0.012
Agar
20
1Liter
Distilled water
pH was adjusted at 6.8
Procedure:
900 ml
Defibrinated blood
100 ml
Procedures:
Melted nutrient agar medium was cooled to 50 C and the blood was
added aseptically, and then mixed in plates. Plates were inoculated by the
tested strain by streaking, then incubated at 37 C for 24-48 h. the positive
results were indicated by formation of clear colourless (-hemolysis) or
clear greenish (-hemolysis) zones around the bacterial growth. Non
hymolytic microorganism was not reflected any change in the medium
(negative reaction).
g-Gelatin liquification test:
Nutrient gelatin medium
(g/L):
Beef extract
3.0
Peptone
5.0
Gelatin
120
Distilled water
1Liter
89
h- Oxidase test:
Reagents:
One gram of P- amino dimethyl alanilin oxalate was dissolved in 100
ml of distilled water (1% solution).The reagent was freshly prepared.
Procedure:
A filter paper strip-moistened with freshly prepared 1% aqueous
solution of P- amino dimethyl alanilin oxalate, was rubbed with colony
from a culture grown on nutrient agar. A positive reaction was indicated by
intense deep purple color appearing with 5-10 sec. A delayed positive
reaction may appeare within 60 sec. Negative organisms dont produce any
changes.
i- Citrate Utilization test: Simmon's medium:
(g /L):
Sodium chloride
5.00
MgSO4.7H2O
0.20
NH4H3PO4
1.00
KH3PO4
1.00
Sodium Citrate
2.00
Agar
20.0
40 m1
Distilled water
1 Liter
pH was adjusted at
6.8
(g /L):
Peptone
5.0
K3HPO4
5.0
Distilled water
1Liter
The solids were steamed until dissolved and filtered; pH value was
adjusted at 7.5.
Five g. of glucose were added, mixed and distributed 1.5ml volumes
into tubes, then sterilized at 121oC for 15 min.
Procedure:
The MR/VP broth was inoculated with a pure culture of the test
organism. The broth incubated at 35C for 48 to 72 h. At the end of this
time, 5 drops of the methyl red reagent added directly to the broth. Pink
color indicates positive result.
k- Vogas-Proskauer test (VP):
It consists of Glucose Phosphate Peptone water, as for the methyl red
test.
Procedure:The MR/VP broth inoculated with a pure culture of the test
organism. The broth incubated for 24 h. at 35c. At the end of this time,
aliquot 1 ml of broth was added to a clean test tube. 0.6 ml of 5% alfa
naphthol was added followed by 0.2 ml of 40% KOH. The tube was
shacked gently to expose the medium to atmospheric oxygen and the tube
remains undisturbed for 10 to15 min. A positive test is represented by the
development of a red colour in 15 min or more after addition of the reagent.
91
(g /L):
Lab-lemco powder
3.0
Yeast extract
3.0
Peptone
20
Sodium chloride
5.0
Lactose
10.0
Sucrose
10.0
Glucose
1.00
Ferric citrate
0.30
Sodium thiosulphate
0.3
Agar
12
12 ml
Distilled water
1Liter
(g / L):
Peptone
10.0
NaCl
5.00
Distilled water
1 Liter
92
The solids were dissolved by heating in the water. pH adjusted to 88.4 and boiled for 10 minutes then filtered, pH adjust to 7.2 - 7.4 and
sterilized at 121C for 20 min.
Procedure:
Tryptophan broth was inoculated with the test organism then
incubated at 35c for 18 to 24 h. After incubation, 20 drops of fresh kavac's
reagent added to the tube. The upper layer of the broth was observed for a
red ring. This ring indicates that indole was produced in the tube and that
tryptophan was digested. If the ring remains yellow or light brown, Indole
was not produced and tryptophan digestion failed to occur and this mean
Negative test.
n- Catalase activity:
Most bacteria that utilize O2 in their respiration produce hydrogen
peroxide which is toxic to their own enzyme system. Their survival in the
presence of hydrogen peroxide is possible because they produce catalase
enzyme which converted hydrogen peroxide into water and oxygen. This
test was used to differentiate between producing catalase enzyme (catalase
+ ve) and other non producing catalase (catalase -ve) organisms.
Procedure (Tube or agar plate method of catalase activity):
A few drops (about 1 ml) of 3% H2O2 reagent were added directly to
the surface of the growth on agar plate or slant. The evolution of bubbles of
gas indicating a positive test.
o- Coagulase test
Tube test (free coagulase): a small amount of the colony growth of
the organism were emulsify in a tube containing 0.5 ml of citrated rabbit
plasma diluted 1:10 in sterile saline. The tube was incubated at 35C for 4
h. and observed for clot formation by gently tilting the tube. The clot was
93
observed at that time, the tube was reinsulated at room temperature and the
result read again after 18 h. if the clot not observed.
Controls
The test was considered positive if any degree of coagulum
formation appeared.
The test was considered negative if the plasma remained wholly liquid or
showed only a flocculent precipitate.
5. Antibiotic susceptibility test.
5.1. Antibiotic disks:
Sixteen of different antibiotics were selected for carrying out the
antimicrobial susceptibility test. The antibiotic disks used in this research
were purchased from Oxoid Ltd., England. The evaluation of inhibition
zone diameter (mm) in antibiotic susceptibility test indicated in Table (1).
The evaluation of inhibition zone diameter (mm) in antibiotic susceptibility
test according to Jacques (1980) and NCCLS (1999).
Disc conc.
Susceptible Intermediate Resistant
by ug/ml
Imipenem (IPM)
10 g
16
14-15
13
Rifampicin (RF)
5 g
17
16-15
14
Tetracyline (TE)
30 g
19
15-18
14
Oxycilline (OX)
10 g
14
11-12
10
Vancomycin (VAN)
30 g
12
10-11
9
Chloromphenicol (C)
30 g
18
13-17
12
Cefotaxime (CTX)
10 g
23
15 22
14
Cefepime (FEP
30 g
18
15 17
14
Methicillin (MET)
5 g
20
17 19
16
Cephardin (CE)
30 g
18
15 17
14
Amoxycillin (AX)
20 g
14
12 13
11
Ampicillin\sulbactam
30 g
15
12 14
11
(SAM)
Ampicillin (AM)
30 g
17
14 16
13
Ciprofloxacin (CIP)
10 g
21
16 20
15
Erythromycin (E)
15 g
23
14 22
13
Tobramycin (TOP)
10 g
20
16 19
15
94
With each group of tests, tubes +of uninoculated medium with and
without antibiotic were included to act as a control to ensure sterility and
clarity of the medium. A third control tube containing inoculated medium
but without antibiotic was also included to ensure the ability of the
organism to grow in the medium. All the tubes were incubated at 37C for
24 h. and examined for turbidity as an indicator of bacterial growth.
The MICs of the resistant isolates to vancomycin were also
determined by E-test (AB Biodisk, Sweden). MICs breakpoints for
vancomycin
were
determined
according
to
the
manufacturers
recommendation.
The minimum inhibitory concentration (MIC) is defined as the
lowest concentration that inhibits a visible growth in liquid media
(Washington & Sutter, 1980). One hundred micro liters (l) were taken
from each MIC concentration as well as the lower concentrations and
introduced onto nutrient agar to determine the MBC. The plates were
incubated at 37C for 24 h. The Minimum bactericidal concentration
(MBC) is the lowest concentration at which no growth occurs on solid
media (Washington & Sutter, 1980).
7. Bactericidal activity of different antimicrobial agent.
Different concentration of the bactericidal usually used in burn units
as chemical disinfects were tested for its effect on the viability of the tested
organisms.
Ethanol produced by EL NASR Company for chemical manufacture, Cairo,
Egypt. Ethanol was diluted to different concentration (50, 60 and 70%)
using sterile distilled water.
Hydrogen peroxide (10%) manufacture by El Ameria Company for
chemical manufacture, Cairo, Egypt. Hydrogen peroxide was diluted to
different concentration (6, 7 and 8%) using sterile distilled water.
96
Procedure:
Sterile test tubes containing 9 ml required certain concentration of
disinfectants were prepared, then each tube was inoculate with 1 ml of
tested bacterial suspension for different period (5, 10, 15, 20 & 30 min.).
The most resistant bacterial isolates in this study were tested for each
disinfectant concentration and certain exposure time. Sterile swab was
dipped into tested tube and gently pressed against inside the tube to get rid
of any excess, then gently the wet swab was rubbed against the surface of
nutrient agar plate, incubate for 24hr. at37 C, then the numbers of bacterial
isolates was observe.
8.
English name
Scientific name
Family
Thyme oil
Thymus vulgaris
Labiatae
Cinnamon oil
Cinnamomum zeylanicum,
Lauraceae
Clove oil
Syzygium aromaticum
Myrtaceae
Garlic oil
Allium sativum
Alliaceae
Olive oil
Oleum olivae
Oleaceae
Melaleuca alternifolia
Myrtaceae
Peppermint oil
Mentha piperita.
Lamiaceae
Ranunculaceae
97
Poaceae
99
(g/L):
17.0
3.0
Sodium chloride
5.0
2.5
Glucose
2.5
Distilled water
1000 ml.
Agar
20
9.3. Methods:
(1) Wells of 5mm diameter were cut into the plates of specific agar media
of each test previously mentioned.
(2) Supernatants of tested isolates were prepared as follows:
Ten ml culture of tested isolates were grown for 7 to 8 hours in
nutrient broth, then centrifuged at 3500 g/15-20 min. neutralized with 5
mol/l NaOH to the final pH= 7.0 and then filter sterilized (Reinheimer et
al., 1990; Misra and Kuila, 1992). Wells previously prepared were
separately filled with 50 l of the supernatants of the tested isolates. Plates
were preincubated at 4 C for about 2h to allow diffusion of any inhibitory
metabolites into the surrounding agar, and then incubated at 37C for 24
hours. The plates were examined for a clear zone in the agar surrounding
the well. The measurements were recorded in three repetitions.
10. Effect of certain bactericidal concentrations on the enzyme
activities (virulence factors).
10.1. Materials:
The Liquid culture of the tested S. aureus isolates (24 h. age).
Different concentration of Ethanol (50, 60 and 70%) and Hydrogen
peroxide (6, 7 and 8%) were prepared.
100
10.2. Media:
Hydrolysis of lecithin was detected on egg yolk agar (TSA
supplemented with 10% egg yolk emulsion). Haemolytic activity was
determined on blood agar (TSA supplemented with 10% defibrinated sheep
blood).
Hydrolysis of proteinase was detected on Casein agar (TSA
supplemented with 10% casein or skim milk).The plates were incubated at
37C for 24 hours.
10.3. Methods:
Determination of the enzymatic activities of different staphyloccoci
were performed using agar diffusion assay as previously mentioned
11. Effect of tea tree oil on the enzyme activities (virulence factors).
The tested S. aureus isolates treated with different concentration of
tea tree oil were examined for their capability of producing extracellular
degrading enzymes and virulence factors. Tween 80 (v/v) was included in
all dilutions to enhance oil solubility.
Determination of the enzymatic activities of different staphyloccoci
were performed using agar diffusion assay according to Klaenhammer,
(1988) as previously mentioned.
12. Molecular identification and characterization assays of van A &
mec A gene.
The molecular ivestigations have been done in sequence unit, city of
scientific research and technological applications, Burg El Arab, Alex,
Egypt.
101
Final concentration
1x
0.2 pM
Primer a
10 pM
Primer b
10 pM
Template DNA
Ca. 0.1 g
DNA-polymerase ( Taq )
1 or 2 U
MgCl2 25 mM
2 mM
ATGAATAGAATAAAAGTTGCA
vanA R:
CCCCTTTAACGCTAATACGAT
103
After using different condition for PCR there are not give any PCR
product..so we make another design for primers.
NEW DESIGN
Detection of mec A gene
Forward primer TGGCTCAGGTACTGCTATCCACCC
Reverse primer ACCACCCAATTTGTCTGCCAGTT
This pair of primer gives product 818
Forward primer GGCTCAGGTACTGCTATCCACCCT
Reverse primer AACCACCCAATTTGTCTGCCAGT
This pair of primer gives product 818
Forwardprimer
CCAGGAATGCAGAAAGACCAAAGCA
104
Final concentration
1x
10 pM
Primer van A 1 R
10 pM
Template DNA
Ca. 0.1 g
DNA-polymerase ( Taq )
1 or 2 U
MgCl2 25 mM
2 mM
105
106
Results
Results
1- History of burned patients.
Infections are the major cause of morbidity and mortality in burned
patients. Three fourth of deaths in burned patients occur due to infections.
The present investigation covered a tRWDO RI VHYHQW\ SDWLHQWV IURP
Sidnawy Hospital, Zagazig University, Egypt and 30 patients from king
Fahd Hospital, Al Qassim, Saudi Arabia.
Type of injury, age, gender, total body surface area burned and
antibiotic treatment were included in table (1 & 2). In the present study
females (57.1%) were affected more than that of males (42.9%) (Figure1).
Most common age group affected was from 3 up to 64 years. Results in
table (3) revealed that the patients had different causes of burned injury as
exposure to flame which led to thermal injury in 33 patients out of 70 total
patients. This accounted for 47.1%. Hot water thermal injury was
implicated in 16 cases. Electricity revealed 17 patients all of them from
males. Only four patients out of 70 total patients represented in (5.7%)
suffered from chemical burns.
All the patients suffered burn from 7% up to 94% of total body
surface area burned (TBSA). More than 58 % of the patients died either
from infection, by the burn itself or by both of them as represent in table
(3). Data in tables (4 and 5) show the results of blood, swabs and other
culture. Tables (6 and 7) show the results of account of platelets, white
blood cells, the presence or absence of inhalation injury and the final
outcome for each of the seventy patients.
Prophylactic antibiotics were routinely used. Thus peflacin,
sulperazone, fortum, pencillin, flummox, cefotaxim, ciprocin, ampicillin,
cefoxitin, garamycin, amikacin and erythromycin were administrated from
the first day of admission.
107
Results
Table (1): Type of injury- age- gender - total body surface area burned
and antibiotic treatment of burned patients hospitalized at
Sidnawy Hospital, Zagazig, Egypt.
No.
Type of
Injury
Age
Gender
TBSA%
Out come
Antibiotic
intake
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
Flame
Hot water
Electricity
Flame
Flame
Flame
Hot water
Electricity
Flame
Flame
Hot water
Flame
Electricity
Flame
Electricity
Flame
Flame
Flame
Electricity
Hot water
Hot water
Hot water
Chemicals
Flame
Flame
Flame
Hot water
Hot water
Hot water
Hot water
Hot water
Flame
Flame
Electricity
Electricity
Flame
Flame
Flame
Flame
Electricity
17
22
45
54
12
50
13
57
48
42
39
10
20
3
50
44
24
64
30
4
56
11
46
43
22
20
53
39
44
58
23
21
38
15
25
50
40
36
45
62
F
M
M
F
F
F
M
M
F
F
F
F
M
F
M
F
F
F
M
F
F
F
M
F
F
F
M
F
M
M
F
M
F
M
M
F
F
F
F
M
35
48
40
71
41
60
18
90
58
53
55
18
15
20
80
40
15
94
7
18
70
16
40
50
18
20
55
40
48
32
16
15
48
20
45
90
49
40
62
90
recovered
recovered
died
died
recovered
died
recovered
died
died
died
died
recovered
recovered
recovered
died
died
recovered
died
recovered
recovered
died
recovered
died
died
recovered
recovered
died
died
died
died
recovered
recovered
died
recovered
recovered
died
died
died
died
died
Peflacin
Sulperazone
Fortum
Penicillin
Fortum
Penicillin
Flummox
Sulperazone
Sulperazone
Fortum
Penicillin
Sulperazone
Sulperazone
Penicillin
Penicillin
Fortum
Cefotaxime
Penicillin
Sulperazone
Penicillin
Penicillin
Erythromycin
Penicillin
Fortum
Penicillin
Sulperazone
Penicillin
Penicillin
Sulperazone
Penicillin
Penicillin
Penicillin
Flummox
Penicillin
Penicillin
Sulperazone
Penicillin
Penicillin
Ciprocin
Penicillin
108
Results
Table (2): Type of injury - age- gender - total body surface area burn
and antibiotic treatment of burned patients hospitalized at
king Fahd Hospital, Al Qassim, Saudi Arabia.
No.
Type of
injury
Age
Gender
TBSA%
Outcome Antibiotic
intake
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
Flame
Electricity
Hot water
Flame
Flame
Chemicals
Flame
Electricity
Electricity
Hot water
Electricity
Electricity
Hot water
Flame
Hot water
Electricity
Flame
Flame
Hot water
Flame
Electricity
Flame
Electricity
Chemicals
Flame
Chemicals
Flame
Flame
Electricity
Flame
26
40
28
18
13
50
40
60
44
37
23
43
11
54
37
53
60
18
45
48
49
5
25
55
60
38
24
15
39
62
F
M
M
F
F
M
F
M
M
F
M
M
F
M
F
M
M
F
F
F
M
F
M
M
F
M
F
F
M
F
20
87
15
22
20
50
60
70
72
50
30
75
40
60
37
50
60
22
35
55
85
20
30
80
24
recovered
died
recovered
recovered
recovered
died
died
died
died
died
recovered
died
recovered
died
recovered
died
died
recovered
died
died
died
recovered
recovered
died
died
40
15
30
7
18
died
recovered
recovered
died
died
109
Sulperazone
Sulperazone
Penicillin
Sulperazone
Penicillin
Ampicillin
Penicillin
Penicillin
Cefoxitin
Garamycin
Amikicin
Penicillin
Cefotaxim
Penicillin
Fortum
Penicillin
Penicillin
Flummox
Penicillin
Sulperazone
Erythromycin
Penicillin
Penicillin
Cefotaxim
Penicillin
Fortum
Cefotaxime
Penicillin
Sulperazone
Penicillin
Results
Table (3): Distribution of burned injury types among male and female:
Type of injury
Total no.
Gender
Outcome
Male Female recovery Death
3
30
15
18
Flame
33 patients
Hot water
16 patients
10
Electricity
17 patients
17
11
Chemicals
4 patients
Total
70 patients
30
40
29
41
&
&
,
Fig (1): Distribution of burned injury types among male and female.
5.7%
24.3%
47.1%
flame
hot water
22.9%
110
Results
Results
other 30 died. From the 27 patients who had no organisms in their blood
cultures, 24 recovered and 3 died. The above data were analyzed by Chi
square test (X2 test) and revealed that there was a significant correlation
between patient death (P < 0.01) and the presence of systemic infection.
2.5. Inhalation injury
In the present investigation 25 patients had inhalation injury, while
the other 45 patients did not show inhalation injury. The inhalation injury
had its effect on platelets count and white blood cells count as follows:
Platelets:
The results in table (6 and 7) had proved that the patients with
inhalation injury had mean platelets count 185.03 * 1000/ cmm. The others
who had no inhalation injury and give a mean platelets count of 296.6 *
1000/ cmm. This finding suggested that the platelets count significantly
decreased in patients with positive inhalation than those with no inhalation
injury.
White blood cells
The mean of white blood cells (WBCs) count in patients with
inhalation injury was 12.6* 1000 / cmm, while in patients with no
inhalation injury the mean of white blood cells count were 14.08 * 1000 /
cmm as showed in tables (6 and 7). Thus no significant relationship
between white blood cells count and the presence of inhalation injury.
2.6. Effect of total body surface area burn on the kind of organisms
The data demonstrated that there was a significant relationship
between the organisms that appeared in the swab cultures and the size of
burn. Burned patients with large TBSA burns were more liable to be
infected with mixed organisms. On the other hand, burned patients with
small TBSA burns showed absence of contaminating organisms. The mean
of TBSA of the recovered patients were 24.7%, while the mean of TBSA of
died patients were 55.7%.
112
Results
Table (4): Relation between total body surface area burned, tested
cultures and outcome of patients hospitalized at Sidnawy
Hospital, Zagazig University, Egypt.
No.
TBS
A%
35
48
40
71
41
60
18
90
58
10
53
11
55
12
18
13
14
15
20
15
80
16
40
17
18
15
94
19
20
7
18
21
70
22
16
23
40
24
50
Swabs
+ve gave1
organism
+ve gave1
organism
+ve gave 2
organisms
+ve
gave1organism
+ve gave1
organism
+ve gave 2
organisms
+ve gave 1
organism
+ve gave 3
organisms
+ve gave1
organism
+ve gave2
organisms
+ve gave1
organisms
+ve gave1
organism
-ve no growth
+ve gave1
organism
+ve gave1
organism
+ve gave1
organism
-ve no growth
+ve
gave1organism
-ve no growth
-ve no growth
Cultures
Blood
Outcome
Other
cultures
ND
ND
Recovered
-ve no growth
+ve gave1
organism(S)
ND
Recovered
+ve gave1
organism(S)
ND
Died
Recovered
ND
Died
ND
Recovered
+vegave1organis
m
+vegave1
organisms
+vegave2
organisms
+vegave1
organism
ND
+ve gave2
organisms(S)
+ve gave1
organism(U)
ND
Died
+vegave1
organism(S)
ND
Died
-ve no growth
-ve no growth
ND
-ve no
growth(U)
ND
Recovered
Recovered
+ve gave 1
organism(S)
ND
+ve gave1
organism(S)
ND
+ve gave1
organism(S)
+ve gave1
organism(U)
+ve gave1
organism(U)
ND
Died
+ve gave1
organism
+vegave2
organisms
+ve gave1
organism
+vegave1
organism
ND
+ve gave1
organism
+ve gave1
organism
-ve no growth
+vegave1
organisms
-ve no growth
-ve no growth
+ve
gave1organism
-ve no growth
+vegave2
organisms
-ve no growth
+ve gave2
organisms
+ve gave2
+vegave2
organisms
+vegave2
113
ND
Died
Died
Died
Recovered
Died
Recovered
Died
Recovered
Recovered
Died
Recovered
Died
Died
Results
organisms
-ve no growth
-ve no growth
+ve gave1
organism
+ve gave1
organism
+ve gave2
organisms
+ve gave1
organism
-ve no growth
-ve no growth
organisms
-ve no growth
-ve no growth
+ve gave2
organisms
+ve
gave1organism
-ve no growth
ND
ND
ND
Recovered
Recovered
Died
-ve no
growth(U)
ND
Died
-ve no growth
ND
Died
ND
-ve no growth
Recovered
Recovered
+vegave2
organisms
-ve no growth
ve no growth
+ve gave 1
organism
ND
-ve no growth
ND
+ve gave1
organism(S)
ND
ND
-ve no
growth(S)
+ve
gave2organisms
(U)
+ve gave2
organisms(S)
ND
Recovered
Recovered
ND
Died
ND
Died
25
26
27
18
20
55
28
40
29
48
30
32
31
32
16
15
33
48
34
35
20
45
36
90
+ve gave2
organisms
+ve gave1
organism
37
49
38
40
39
62
+ve gave1
organism
+ve gave1
organism
ND
40
90
+ve gave2
organisms
+ve gave1
organism
+ve gave 1
organism
+ve gave1
organisms
+ve gave1
organism
Died
Died
Died
Died
Died
Table (5): Relation between total body surface area burned, tested
cultures and outcome of patients hospitalized at king Fahd
Hospital, Al Qassim, Saudi Arabia.
No. TBSA
%
41
42
20
87
43
15
44
22
45
20
Swabs
-ve no growth
+ve gave2
organisms
+ve gave1
organism
+ve gave1
organism
+ve gave1
Cultures
Blood
Other
cultures
Outcom
e
-ve no growth
+vegave2
organisms
-ve no growth
ND
+ve gave 1
organism(S)
ND
Recovered
Died
-ve no growth
+ve gave1
organism(U)
ND
Recovered
-ve no growth
114
Recovered
Recovered
Results
46
47
50
60
48
70
49
72
50
50
51
30
52
75
53
54
40
60
55
56
37
50
57
60
58
59
22
35
60
55
61
85
62
63
20
30
64
80
65
organism
-ve no growth
+-ve no
growth
+ve gave2
organisms
+ve gave2
organisms
+ve gave2
organisms
ve no growth
+ve gave1
organism
-ve no growth
+ve gave2
organisms
-ve no growth
+ve gave1
organism
+ve gave1
organism
-ve no growth
+vegave1
organism
+vegave1
organism
-ve no growth
+vegave1 organism
+vegave1 organism
ND
ND
Died
Died
+vegave1 organism
ND
Died
ND
ND
Died
ND
ND
Died
-ve no growth
+ve gave1
organism(U)
ND
Recovered
ND
+ve gave1
organism(S)
ND
+ve gave1
organism(U)
+ve gave1
organism(S)
ND
+ve gave1
organism(U)
ND
Recovered
Died
Died
Recovered
Recovered
ND
-ve no growth
+vegave1 organism
-ve no growth
-ve no growth
+vegave1 organism
+vegave1 organism
+vegave1 organism
+vegave1 organism
ND
Died
Recovered
Died
Died
Recovered
Died
Died
-ve no growth
-ve no growth
ND
ND
Died
24
-ve no growth
+vegave1
organism
+ve gave1
organism
-ve no growth
+ve gave1
organism(S)
ND
ND
-ve no growth
Recovered
66
67
40
15
-ve no growth
-ve no growth
+vegave1 organism
-ve no growth
68
30
-ve no growth
-ve no growth
69
-ve no growth
70
18
+ve gave1
organism
-ve no growth
-ve no
growth(U)
ND
-ve no
growth(U)
+ve gave1
organism(S)
ND
ND
ND
Recovered
115
Died
Recovered
Recovered
Recovered
Results
Table (6): Result of platelets and white blood corpuscles from Sidnawy
Hospital, Zagazig University, Egypt.
No
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
1 day
4 day
PLTs
WBCs
PLTs
WBCs
150
319
ND
115
158
ND
ND
216
189
425
235
ND
189
342
254
ND
202
281
204
225
ND
ND
135
95
170
326
152
460
ND
138
ND
481
272
326
143
450
200
ND
310
120
20
12.7
ND
22
4.2
ND
ND
23.8
9
21.7
15
ND
20.3
14
28.9
ND
14.3
41.7
18.1
14
ND
ND
16.2
3.3
23
18.7
5.3
24.5
ND
7.8
ND
9.8
21.8
20.4
22.7
8.7
10
ND
6.5
7.7
200
300
150
ND
ND
131
230
ND
139
265
246
570
152
ND
ND
236
ND
ND
ND
ND
151
450
110
ND
ND
300
ND
ND
344
ND
153
446
ND
ND
107
ND
198
90
310
ND
12
8.9
12
ND
ND
3
4.7
ND
5.9
3
24.9
16.7
8.9
ND
ND
5.3
ND
ND
ND
ND
4.1
7.2
8.2
ND
ND
18
ND
ND
12
ND
14.7
7.8
ND
ND
5.6
ND
3.8
10
2.4
ND
WBCs
PLTs
ND
10 day
PLTs
250
ND
ND
ND
ND
ND
808
ND
ND
344
183
500
291
ND
ND
ND
ND
ND
ND
ND
ND
864
306
ND
ND
250
ND
ND
ND
ND
ND
405
ND
ND
ND
502
ND
63
ND
ND
116
Inhalation
Outcome
(-)
(-)
(-)
(+)
(+)
(+)
(-)
(+)
(-)
(-)
(+)
(-)
(-)
(-)
(+)
(-)
(-)
(+)
(+)
(-)
(+)
(-)
(+)
(+)
(-)
(-)
(-)
(-)
(-)
(+)
(-)
(-)
(+)
(-)
(+)
(-)
(-)
(-)
(+)
(-)
Recovered
Recovered
Died
Died
Recovered
Died
Recovered
Died
Died
died
died
recovered
recovered
recovered
died
died
recovered
died
recovered
recovered
died
recovered
died
died
recovered
recovered
died
died
died
died
recovered
recovered
died
recovered
recovered
died
died
died
died
died
WBCs
10
ND
ND
ND
ND
ND
14.2
ND
ND
6.8
14.5
4
3.9
ND
ND
ND
ND
ND
ND
ND
ND
24.3
11.4
ND
ND
10
ND
ND
ND
ND
ND
8.6
ND
ND
ND
11.4
ND
4
ND
ND
(+)
(-)
Not detected
Positive inhalation
No inhalation
Results
Table (7): Result of platelets and white blood corpuscles from Fahd
king Hospital, Al Qassim, Saudi Arabia.
No.
1 day
4 day
10 day
inhalation outcome
PLTs WBCs PLTs WBCs PLTs WBCs
41
42
261
100
8.5
5.5
ND
80
ND
3
ND
ND
ND
ND
(-)
(+)
recovered
died
43
414
18.5
ND
ND
ND
ND
(+)
recovered
44
45
372
ND
13.9
ND
230
164
10.3
12.6
ND
ND
ND
ND
(-)
(-)
recovered
recovered
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
167
139
405
ND
236
321
269
ND
157
594
156
345
319
ND
125
ND
189
342
130
425
235
170
340
167
200
8.4
13.8
35.7
ND
15.7
37.1
39.4
ND
16.7
16.3
14.5
20
12.7
ND
7.5
ND
20.3
14
16.0
21.7
15
23
18.5
9
10
93
ND
165
172
100
45
99
ND
51
ND
ND
58
300
150
ND
236
152
ND
110
265
246
ND
300
93
195
5.5
ND
7.5
7.3
10.5
12
3.6
ND
9
ND
ND
5
8.9
12
ND
5.3
8.9
ND
8.0
3
24.9
ND
18
6.2
3.8
ND
ND
ND
200
ND
172
ND
499
ND
ND
ND
ND
ND
ND
ND
ND
291
ND
306
344
183
ND
250
ND
ND
ND
ND
ND
9
ND
19.2
ND
16.4
ND
ND
ND
ND
ND
ND
ND
ND
3.9
ND
11.5
6.8
14.5
ND
10
ND
ND
(+)
(+)
(-)
(-)
(-)
(-)
(+)
(-)
(+)
(-)
(+)
(+)
(-)
(-)
(+)
(-)
(-)
(-)
(+)
(-)
(+)
(-)
(-)
(+)
(+)
died
died
died
died
died
recovered
died
recovered
died
recovered
died
died
recovered
died
died
died
recovered
recovered
died
died
died
recovered
recovered
died
died
WBCs
PLTs
ND
(+)
(-)
Not detected
117
Positive inhalation
No inhalation
Results
118
Results
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
WOUND CULTURE
+ ve
+ ve
- ve
+ ve
- ve
- ve
- ve
+ ve
- ve
- ve
+ ve
+ ve
-ve
+ ve
- ve
+ ve
+ ve
+ ve
+ ve
- ve
+ ve
- ve
- ve
- ve
+ ve
+ ve
- ve
+ ve
+ ve
+ ve
+ ve
- ve
Results
Bacilli short rods
Cocci in cluster
Bacilli short rods
Cocci in cluster
Cocci in pairs
Bacilli straight thick rods
Bacilli straight thick rods
Cocci in cluster
- ve
+ ve
- ve
+ ve
+ve
- ve
- ve
+ ve
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
- ve
+ ve
+ ve
- ve
+ ve
- ve
+ve
- ve
- ve
+ ve
- ve
+ ve
+ ve
+ ve
+ ve
- ve
- ve
+ ve
+ ve
+ ve
- ve
+ve
+ ve
+ ve
+ ve
+ ve
+ ve
-ve
-ve
+ ve
- ve
+ ve
- ve
+ ve
+ ve
+ ve
- ve
+ ve
+ ve
- ve
BLOOD CULTURE
33
34
35
36
37
38
39
40
120
Results
116
117
118
119
120
121
BLOOD CULTURE
URINE AND SPUTUM
CULTURE
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
WOUND CULTURE
+ ve
- ve
+ ve
-ve
-ve
+ ve
+ ve
- ve
- ve
- ve
+ ve
+ ve
-ve
+ ve
+ ve
+ ve
+ ve
+ ve
+ ve
- ve
+ ve
+ ve
- ve
- ve
+ ve
+ ve
- ve
+ ve
+ ve
- ve
- ve
- ve
+ ve
+ ve
+ ve
Bacilli in chain
Cocci in cluster
- ve
+ ve
Cocci in cluster
Bacilli short rods
Cocci in cluster
+ ve
- ve
+ ve
- ve
121
Results
Wound culture
blood culture
Urinary and sputum
culture
Total
Gram positive
isolates
No.
%
Gram negative
isolates
No.
%
Total
No.
22
17
8
46.8
36.2
17
18
9
6
54.5
27.3
18.2
40
26
14
50
32.5
17.5
47
58.75
33
41.29
80
100
'
'
:RXQGFXOWXUH
EORRGFXOWXUH
8ULQDU\DQG
VSXWXPFXOWXUH
122
Results
Wound culture
blood culture
Urinary and sputum
culture
Total
Gram positive
isolates
No.
%
Gram negative
isolates
No.
%
Total
No.
13
6
6
52
24
24
8
5
3
50
31.25
18.75
21
11
9
51.22
26.83
21.95
25
60.98
16
39.02
41
100
'
'
:RXQGFXOWXUH
EORRGFXOWXUH
8ULQDU\DQG
VSXWXPFXOWXUH
123
Results
Gram positive
isolates
No.
%
Gram negative
isolates
No.
%
Total
No.
Wound culture
blood culture
35
23
57.4
62.2
26
14
42.6
37.8
61
37
50.4
30.6
14
60.9
39.1
23
19.0
72
59. 50
49
40.50
121
100
'
'
:RXQGFXOWXUH
EORRGFXOWXUH
8ULQDU\DQGVSXWXP
FXOWXUH
124
Results
125
Results
126
Results
127
Results
128
Results
+
+
+
+
-
Species 2
+
+
+
+
+
+
Species 3
+
+
+
+
+
-
+
+
+
+
-
+
+
+
-
Species 4 Species 5
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
129
Results
S. aureus on
mannitol salt agar
Photo no.(3):Staphylococcusaureusonabloodagarplate.
S.aureuscoloniesappearsmooth,convex,creamwhite Photo no. (4): K. pneumoniae
on MacConkey agar
Figure(6):Indole reaction,
Figure(7):Voges-Proskauer,
130
Figure(8):Methyl red
Results
Results
Number of isolates
28
6
19
7
1
61
Percentages
45.9%
9.9%
31%
11.5%
1.7%
100%
S. aureus
S. epidermidis
P. aeruginosa
K. pneumoniae
Micrococcus
132
Results
Number of isolates
18
3
10
5
1
37
Percentages
48.7%
8.1%
27.0%
13.5%
2.7%
100%
Prevalence
percentage%
S. aureus
S. epidermidis
P. aeruginosa
K. pneumoniae
Micrococcus
133
Results
S. aureus
S. epidermidis
P. aeruginosa
K. pneumoniae
Micrococcus
Total
11
3
7
2
0
23
47.8%
13.0%
30.5%
8.7%
0%
100%
W
S. aureus
S. epidermidis
P. aeruginosa
K. pneumoniae
Micrococcus
134
Results
Number of isolates
Percentages
57
12
36
14
2
121
47%
9.9%
29.8%
11.6%
1.7%
100%
S. aureus
S. epidermidis
P. aeruginosa
K. pneumoniae
Micrococcus
Total
S. aureus
S. epidermidis
P. aeruginosa
K. pneumoniae
Micrococcus
135
Results
chloromphenicol,
rifampicin,
erythromycin,
136
Results
amoxycillin,
ampicillin\sulbactam,
chloromphenicol,
137
Results
C
R
R
S
I
S
S
S
S
S
S
I
R
R
S
R
S
R
S
S
S
I
R
R
S
I
R
S
S
S
S
S
S
S
S
S
S
R
I
R
I
R
S
RF
S
R
S
R
R
R
S
S
S
S
S
R
R
S
R
S
S
R
S
S
S
S
S
S
S
I
R
S
S
R
S
S
S
S
R
S
S
R
S
R
R
S
138
E
I
R
S
R
S
R
R
S
S
S
S
R
R
S
R
S
S
R
S
R
S
R
S
S
S
R
S
S
S
S
S
R
S
S
R
S
S
S
S
R
R
S
TE OX CE VA
R R R S
R S R S
R S R S
R R R R
R R R S
S R R S
S S R S
S R S S
S R R S
R S S S
S S R S
S S R R
R R I R
R R S S
S S R S
I R S S
R S I S
S R R S
S S R S
S S S S
S I I S
S R R R
R R S S
S S R S
R R S I
S S R S
S R R S
I S I S
S R R S
S S I S
R R I S
I S S S
R S I S
R R R S
R R R S
R R S S
S R R S
S S R S
R R S S
S S R S
S S R S
S S I S
Results
95S
97S
98S
99S
101S
102S
106S
108S
109S
113S
114S
115S
117S
118S
120S
R
R
S
S
S
S
R
S
S
R
R
S
S
S
R
R
S
S
R
R
R
R
I
R
I
I
S
R
S
S
R
S
R
R
S
R
R
S
R
I
I
S
S
S
R
Table continue
R S R S R
S R S R S
S S S S I
S R S S R
R S R R S
I S S S S
R S S R S
R I S S S
S S R S S
S R S S R
S R S S R
R I R R S
S R R R S
S S S S S
R S S S R
R
S
R
S
R
S
S
I
R
R
R
S
S
S
R
R R
R S
S S
I S
S S
R S
S S
I S
R S
S S
S S
R S
R S
I S
S S
R
S
R
S
R
I
R
R
I
R
R
R
S
R
R
S
S
S
S
S
S
S
S
S
S
S
S
S
S
I
R
R
R
R
R
R
I
S
S
R
R
R
R
R
S
R
S
I
S
S
S
I
R
R
S
S
S
R
R
S
CIP
CTX
AX
SAM
C
RF
E
TE
OX
CE
VA
Am
IPM
MET
FEP
S
No of
suscepetibile
isolates
37
20
17
23
32
37
36
31
27
17
51
14
49
12
28
%
64.9
35.08
29.8
40.35
56.14
64.91
63.15
54.38
47.36
29.82
89.47
24.56
85.96
21.05
49.12
I
No of
intermediate
isolates
1
8
10
8
8
1
1
4
2
11
1
7
4
4
9
139
%
1.7
14.03
17.5
14.03
14.03
1.75
1.75
7.01
3.50
19.29
1.75
12.28
7.01
7.01
15.78
R
No of
resistant
isloates
19
29
30
26
17
19
20
22
28
29
5
36
4
41
20
%
33.3
50.87
52.6
45.61
29.82
33.33
35.08
38.59
49.12
50.87
8.77
63.15
7.01
71.92
35.08
Results
^
/
/W
y
Ky
s
/WD
&W
R I S R R
65
S R S S
CIP
CTX
AX
SAM
C
RF
E
TE
OX
CE
VA
Am
IPM
MET
FEP
S
No of
suscepetibilty
isolates
2
0
0
1
1
0
2
1
0
1
2
0
2
0
1
%
100
0
0
50
50
0
100
50
0
50
100
0
100
0
50
I
No of
intermediate
isolates
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
140
%
0
0
0
0
0
50
0
0
0
0
0
0
0
0
0
R
No of
resistant
isolates
0
2
2
1
1
1
0
1
2
1
0
0
0
2
1
%
0
100
100
50
50
50
0
50
100
50
0
0
0
100
50
Results
^
/
/W dy
y ^D
Z&
d
Ky
s
10
13
s20
23
24
27
32
33
35
44
49
51
56
57
68
69
71
73
141
Results
77
80
82
84
85
88
89
90
93
100
103
104
107
111
112
119
CIP
CTX
AX
SAM
C
RF
E
TE
TBR
CE
IPM
FEP
S
No of
suscepetibile
isolates
%
16
44.44
14
38.88
12
33.33
17
47.22
12
33.33
4
11.11
15
41.66
22
61.11
36
100
9
25
25
69.44
17
47.22
P.aeruginosa
I
No of
intermediate
isolates
0
5
0
0
6
2
1
5
0
8
5
4
142
%
0
13.88
0
0
16.66
5.55
2.77
13.88
0
22.22
13.88
11.11
R
No of
resistant
isloates
20
17
24
19
18
30
20
9
0
19
6
15
%
55.55
47.22
66.66
52.77
50
83.33
55.55
25
0
52.77
16.66
41.66
Results
^
/
/W
y
dZ
&W
RF
isolate
15
22
38
39
41
46
48
61
110
116
121
143
Results
S
No of
suscepetibile
isolates
12
9
4
3
8
4
4
8
4
8
12
10
CIP
CTX
AX
SAM
C
RF
E
TE
TBR
CE
IPM
FEP
I
No of
intermediate
isolates
0
0
1
0
3
0
0
0
0
2
1
0
%
85.71
64.28
28.57
21.42
57.14
28.57
28.57
57.14
28.57
57.14
85.71
71.42
%
0
0
7.14
0
21.43
0
0
0
0
14.28
7.14
0
R
No of
resistant
isloates
2
5
9
11
3
10
10
6
10
4
1
4
%
14.28
35.71
64.28
78.57
21.43
71.42
71.42
42.86
71.42
28.57
7.14
28.57
^
/
/W
dy
y
^D
Z&
d
dZ
/WD
&W
144
Results
145
Results
146
Results
MBC(g/ml)
S. aureus (12)
63
63
S. aureus (30)
31.25
63
S. aureus (50)
63
125
S. aureus (95)
31.25
63
Tested bacteria
D/
D
147
Results
Tested bacteria
MIC(g/ml)
MBC(g/ml)
62.5
S. aureus (12)
62.5
S. aureus (30)
30
62.5
S. aureus (50)
15.625
15.625
S. aureus (95)
62.5
62.5
D/
D
148
Results
MIC(g/ml)
MBC(g/ml)
S. aureus (12)
63
125
S. aureus (30)
63
125
S. aureus (50)
125
125
S. aureus (95)
31.25
63
Tested bacteria
D/
D
149
Results
Tested bacteria
MIC(g/ml)
MBC(g/ml)
S. aureus (12)
125
250
S. aureus (30)
63
125
S. aureus (50)
31.25
63
S. aureus (95)
15.625
15.625
D/
D
150
Results
Results
minutes was satisfied to inhibit all tested bacteria. On the other hand at the
highest concentration 8%, the lowest exposure time was at 10 minutes, so
we could concluded that the best concentration of hydrogen peroxide to be
used as disinfectant was 8 % for 10 minutes it was enough to inhibit all
tested bacterial isolates.
Table (32): Exposure time inhibition of different concentration of
ethanol for multiresistant S. aureus no. (12, 30, 50 and 95).
Concentration of
Exposure time inhibition (minutes)
ethanol % S. aureus12 S. aureus
S. aureus
S. aureus
30
50
95
70
15
13
11
10
60
20
18
13
11
50
28
>30
20
25
S. aureus12
S. aureus30
S. aureus50
S. aureus95
Ethanol concentration %
Figure (21): Exposure time inhibition of different concentration of
ethanol for multiresistant S. aureus no. (12, 30, 50 and
95).
152
Results
10
11
15
10
15
18
10
11
20
8
11
20
S. aureus12
S. aureus30
S. aureus50
s
S. aureus95
H2O2 concentration %
Figure (22): Exposure time inhibition of different concentration of
H2O2 for multiresistant S. aureus no. (12, 30, 50 and 95).
Results
clove, lemon grass. Cinnamon oil had the lowest antibacterial activity
against the four S. aureus (12, 30, 50 and 95) with inhibition zones (0, 1.2,
1.3 and 3.5 mm), respectively.
Table (34): Effect of essential oils against the four multiresistant S.
aureus.
Clear zone (mm).
Essential oil
S. aureus12 S.aureus30
Thyme
Cinnamon
Lemmon grass
Clove
Garlic
Olive
Tea tree
Peppermint
Nigella sativa
8.2
-ve
4.5
6.5
-ve
-ve
8.0
2.0
4.3
7.5
1.2
3.0
4.0
1.6
3.5
8.3
2.0
-ve
S. aureus
50
8.0
1.3
2.0
5.5
-ve
1.5
7.5
-ve
5.0
S.aureus
95
9.0
3.5
-ve
4.5
1.2
3.0
9.0
1.6
2.2
^
^
^
^
>
'
d
E
154
Results
155
Results
with 30 l tea tree oil were more than that of either VAN disc or thyme oil
or tea tree oil each separately.
Table (35): Determination of the MIC and MBC of thyme and tea tree
oil for multiresistant S. aureus no. (12, 30, 50 and 95).
S. aureus12
Essential
oils
Thyme
oil
Tea tree
oil
MIC
MBC
62.5
7.8
S. aureus 50
S. aureus30
MIC
S. aureus 95
MBC
MIC
MBC
MIC
62.5 15.62
31.25
7.8
15.62
15.62 15.62
7.8
15.62
15.62
31.25
15.62
MBC
7.8
15.62
D/
D
^
^
^
^
Fig (24): Determination of the MIC and MBC of thyme oil for
multiresistant S. aureus (12, 30, 50 and 95).
D/
D
^
^
^
Fig (25): Determination of the MIC and MBC of tea tree oil for
multiresistant S. aureus (12, 30, 50 and 95).
156
Results
S. aureus 12
10.0
THYME OIL
(30g)
8.2
S. aureus 30
11.0
7.5
18.5
S. aureus 50
9.0
8.0
17.0
S. aureus 95
10.0
9.0
19.0
^
^
^
^
sE
d,zDK/>
sEd,zD
S. aureus 12
10.0
S. aureus 30
11.0
8.5
19.5
S. aureus 50
9.0
7.5
16.5
S. aureus 95
10.0
9.0
19.0
157
Results
^
^
^
^
sE
d/>
sE
Vancomycin
VAN+
Thyme oil
Thyme oil
158
Results
159
Results
Lecithinase
Haemolysin
+VE
+VE
+VE
+VE
+VE
+VE
+VE
+VE
160
Proteinase
+VE
+VE
+VE
+VE
Results
Table (39): The virulence factors (VFs) of selected S. aureus (12, 30, 50
and 95) using different volumes 10, 20 & 30 l.
Bacterial
isolates
S. aureus 12
S. aureus 30
20
10
24
12
26
15
24
20
28
19
30
13
15
25
18
30
20
32
S. aureus 50
22
24
28
14
12
20
24
22
S. aureus
95
18
21
27
18
15
12
18
22
24
Table (40): The effect of different conc. of H2O2 (volume 10l) on VFs
of tested isolates.
Diameter of inhibition zone (mm)
Lecithinase
H 2O 2
Conc.
0%
6%
7%
8%
Haemolysin
Proteinase
S12
S30
S50
S95
S12
S30
S50
S95
S12
S30
S50
S95
20
10
0
0
10
7
0
0
22
12
0
0
18
0
0
0
24
16
10
0
20
13
8
0
14
8
4
0
18
11
0
0
15
6
0
0
25
18
5
0
20
15
0
0
18
10
0
0
Lecithinase
Haemolysin
Proteinase
Conc.
0%
50%
60%
70%
80%
S12
S30
S50
S95
S12
S30
S50
S95
S12
S30
S50
S95
20
10
8
0
0
10
7
5
0
0
22
12
9
0
0
18
9
5
0
0
24
16
8
0
0
20
13
9
0
0
14
8
5
0
0
18
11
6
0
0
15
12
9
0
0
25
18
10
0
0
20
15
11
0
0
18
10
7
0
0
10.3. Effect of tea tree oil on the enzyme activities (virulence factors).
The effects of tea tree oil on the production of virulence factors
(VFs) by S. aureus (12, 30, 50 and 95) had been investigated.
161
Results
The production of the lecithinase by S. aureus (12, 30, 50 and 95) showed
lower levels when cultured with 0.3% tea tree oil. Four S. aureus (12, 30,
50 and 95) isolates, significantly lower levels of hemolysin and protease
were produced in the presence of 0.6 % tea tree oil when compared to
control cells. In general, these experiments demonstrate significant
reduction in levels of protease, hemolysin and lethinase after growth for 24
h. in the presence of 0.3 and 0.6% tea tree oil.
The above data in table (42) were analyzed by Chi square test and
revealed that there was a significant correlation between oil concentration
and the reduction in virulence factors of tested isolates (P < 0.01).
Table (42): The effect of tea tree oil on virulence factors of
multiresistant S. aureus.
Lecithinase
Hemolysin
Protease
Tea
tree oil
Conc.
S12
S30
S50
S95
S12
S30
S50
S95
S12
S30
S50
S95
0%
0.1%
0.3%
0.6%
20
18
0
0
18
7
0
0
25
12
0
0
20
18
0
0
24
15
5
0
20
11
3
0
12
11
8
0
18
15
7
0
15
7
0
0
25
20
5
0
20
18
8
0
24
22
10
0
162
Results
DNA
of
the
selected
bacteria
was
extracted
using
12 30 50 95
163
Results
Figure (29): The 16S rDNA gene amplified by polymerase chain reaction
(PCR) of the selected bacterial isolates no. (12, 30, 50 and 95).
Automated DNA sequencing
The 16S rDNA genes were sequenced using 3130 genetic analyzer at
sequencer unit of city for scientific research and technology application.
Isolate no. 12
CTCCTTCGTTCATATCATGCTAGTCGAGCGACGGACGAGAAGCTTGCTTCTC
TGATGTTAGCGGCGGACGGGTGAGTAACACGTGGATAACCTACCTATAA
ACTGGGATAACTTCGGGAAACCGGAGCTAATACCGGATAATATTTTGAACC
GCATGGTTCAAAAGTGAAAGACGGTCTTGCTGTCACTTATAGATGGATCCG
CGCTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCAACGATGCAT
AGCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGG
TCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAA
GCCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTCTTCGGATCGTAAAAC
TCTGTTATTAGGGAAGAACATATGTGTAAGTAACTGTGCACATCTTGACGGT
ACCTAATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACG
TAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGTAGGCGG
TTTTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGG
Analysis after using ncbi
Description
164
Max Total
Query
E
Max
score score coverage value ident
1074 1074 97%
0.0
99%
1070
1070
98%
0.0
99%
1068
1068
97%
0.0
99%
Results
DQ170822.1 Uncultured Staphylococcus sp. clone
WS03A_F12 16S ribosomal RNA
gene, partial sequence
HM330396.1 Uncultured bacterium clone
ncd948h07c1 16S ribosomal RNA
gene, partial sequence
HM329075.1 Uncultured bacterium clone
ncd948g03c1 16S ribosomal RNA
gene, partial sequence
HM329057.1 Uncultured bacterium clone
ncd947g03c1 16S ribosomal RNA
gene, partial sequence
AM945662.1 Staphylococcus equorum partial 16S
rRNA gene, isolate equorum 1
FR821779.1 Staphylococcus aureus subsp. aureus
LGA251 complete genome sequence
JF322979.1 Staphylococcus sp. 1036(2011) 16S
ribosomal RNA gene, partial sequence
JN084556.1 Staphylococcus aureus strain
Pn1016/Pant-Uttar/2007 16S
ribosomal RNA gene, partial sequence
JN084552.1 Staphylococcus aureus strain
366/Chen 16S ribosomal RNA gene,
partial sequence
JN084551.1 Staphylococcus aureus strain
254/Chen 16S ribosomal RNA gene,
partial sequence
1068
1068
98%
0.0
99%
1066
1066
99%
0.0
98%
1066
1066
99%
0.0
98%
1066
1066
99%
0.0
98%
1066
1066
97%
0.0
99%
1064
6372
98%
0.0
99%
1064
1064
97%
0.0
99%
1064
1064
98%
0.0
99%
1064
1064
98%
0.0
99%
1064
1064
98%
0.0
99%
Phylogenetic analysis
BLAST program www.ncbi.nlm.gov/blast phylogenetic analysis was
used to assess the DNA similarities of obtained 16S rDNA gene sequence.
Multiple sequence alignment and molecular phylogeny were performed
using BioEdit software. The phylogenetic tree was displayed using
TREEVIEW program.
165
Results
JN084556.1| Staphylococcus aureus strain Pn1016/Pant-Uttar/2007
JN084552.1| Staphylococcus aureus strain 366/Chen
JF322979.1| Staphylococcus sp. 1036(2011)
JN652894.1| Uncultured Staphylococcus sp. clone C19
JN652882.1| Uncultured Staphylococcus sp. clone C7
JN652891.1| Uncultured Staphylococcus sp. clone C13
JN652890.1| Uncultured Staphylococcus sp. clone C12
JN652892.1| Uncultured Staphylococcus sp. clone C14
JN652881.1| Uncultured Staphylococcus sp. clone C2
DQ170822.1| Uncultured Staphylococcus sp. clone WS03A_F12
GU459256.1| Staphylococcus aureus isolate K54-3
Isolate no 6
GU459255.1| Staphylococcus aureus isolate K54-2
AM945662.1| Staphylococcus equorum partial
0.0025
0.0020
0.0015
0.0010
0.0005
0.0000
Isolate no. 30
CCCGGGTCGGTTATGCTCTGCTAGTCGAGCGACGGACGAGAAGCTTGCTTCT
CTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGATAACCTACCTATAAG
ACTGGGATAACTTCGGGAAACCGGAGCTAATACCGGATAATATTTTGAACC
GCATGGTTCAAAAGTGAAAGACGGTCTTGCTGTCACTTATAGATGGATCCG
CGCTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCAACGATGCAT
AGCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTCCAGA
CTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGA
CGGAGCAACGCCGCGTGAGTGATGAAGGTCTTCGGATCGTAAAACTCTGTT
ATTAGGGAAGAACATATGTGTAAGTAACTGTGCACATCTTGACGGTACCTA
ATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGT
GGCAAGCGTTAT
Description
166
928
96%
0.0
99%
928
96%
0.0
99%
Results
GU459255.1 Staphylococcus aureus isolate
K54-2 16S ribosomal RNA gene,
partial sequence
GQ467748.1 Uncultured bacterium clone
R4J3C4_B10 16S ribosomal
RNA gene, partial sequence
GQ001329.1 Uncultured bacterium clone
nbu171g03c1 16S ribosomal
RNA gene, partial sequence
JF411480.1 Bacterium 1H302 16S ribosomal
RNA gene, partial sequence
EU373089.1 Bacterium ACCC1 16S
ribosomal RNA gene, partial
sequence
FR690784.1 Uncultured Staphylococcus sp.
partial 16S rRNA gene, clone
9.32
FR690779.1 Uncultured Staphylococcus sp.
partial 16S rRNA gene, clone
9.26
FR690582.1 Uncultured Staphylococcus sp.
partial 16S rRNA gene, clone
3.20
FR690555.1 Uncultured Staphylococcus sp.
partial 16S rRNA gene, clone
2.28
FR821779.1 Staphylococcus aureus subsp.
aureus LGA251 complete
genome sequence
JF322979.1 Staphylococcus sp. 1036(2011)
16S ribosomal RNA gene, partial
sequence
JN084559.1 Staphylococcus aureus strain
Pn3576/Pant-Orissa/2008 16S
ribosomal RNA gene, partial
sequence
JN084558.1 Staphylococcus aureus strain
Pn2087/Pant-Jhark/2008 16S
ribosomal RNA gene, partial
sequence
JN084557.1 Staphylococcus aureus strain
Pn1035/Pant-Uttar/2007 16S
ribosomal RNA gene, partial
sequence
JN084556.1 Staphylococcus aureus strain
Pn1016/Pant-Uttar/2007 16S
ribosomal RNA gene, partial
sequence
JN084555.1 Staphylococcus aureus strain
Pn833/Pant-Jhar/2007 16S
ribosomal RNA gene, partial
sequence
JN084554.1 Staphylococcus aureus strain
557/Chen 16S ribosomal RNA
gene, partial sequence
JN084553.1 Staphylococcus aureus strain
928
928
96%
0.0
99%
928
928
96%
0.0
99%
928
928
96%
0.0
99%
924
924
96%
0.0
99%
924
924
96%
0.0
99%
922
922
96%
0.0
99%
922
922
96%
0.0
99%
922
922
96%
0.0
99%
922
922
96%
0.0
99%
922
5518
96%
0.0
99%
922
922
96%
0.0
99%
922
922
96%
0.0
99%
922
922
96%
0.0
99%
922
922
96%
0.0
99%
922
922
96%
0.0
99%
922
922
96%
0.0
99%
922
922
96%
0.0
99%
922
922
96%
0.0
99%
167
Results
383/Chen 16S ribosomal RNA
gene, partial sequence
JN084552.1 Staphylococcus aureus strain
366/Chen 16S ribosomal RNA
gene, partial sequence
JN084551.1 Staphylococcus aureus strain
254/Chen 16S ribosomal RNA
gene, partial sequence
922
922
96%
0.0
99%
922
922
96%
0.0
99%
Phylogenetic analysis
FR690555.1| Uncultured Staphylococcus sp. partial
JF322979.1| Staphylococcus sp. 1036
FR690582.1| Uncultured Staphylococcus sp. partial
FR690779.1| Uncultured Staphylococcus sp. partial
FR690784.1| Uncultured Staphylococcus sp. partial
JN652894.1| Uncultured Staphylococcus sp. clone C19
JN652882.1| Uncultured Staphylococcus sp. clone C7
JN652891.1| Uncultured Staphylococcus sp. clone C13
JN652890.1| Uncultured Staphylococcus sp. clone C12
JN652892.1| Uncultured Staphylococcus sp. clone C14
JN652881.1| Uncultured Staphylococcus sp. clone C2
JF411480.1| Bacterium 1H302
AM945662.1| Staphylococcus equorum partial
AB674510.1| Staphylococcus aureus
Isolate no 19
HM311188.1| Uncultured bacterium clone ncd1164c05c1
GU459256.1| Staphylococcus aureus isolate K54-3
GQ467748.1| Uncultured bacterium clone R4J3C4_B10
GQ001329.1| Uncultured bacterium clone nbu171g03c1
AB674511.1| Staphylococcus aureus gene for
GU459255.1| Staphylococcus aureus isolate K54-2
0.0030
0.0025
0.0020
0.0015
0.0010
0.0005
0.0000
Isolate no. 50
TTGCTCGGTTATATCATGCTGTCGAGCGACGGACGAGAAGCTTGCTTCTCTG
ATGTTAGCGGCGGACGGGTGAGTAACACGTGGATAACCTACCTATAAGACT
GGGATAACTTCGGGAAACCGGAGCTAATACCGGATAATATTTTGAACCGCA
TGGTTCAAAAGTGAAAGACGGTCTTGCTGTCACTTATAGATGGATCCGCGCT
GCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCAACGATGCATAGCC
GACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTC
CAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGC
CTGACGGAGCAACGCCGCGTGAGTGATGAAGGTCTTCGGATCGTAAAACTC
TGTTATTAGGGAAGAACATATGTGTAAGTAACTGTGCACATCTTGACGGTAC
CTAATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTA
GGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGTAGGCGGTT
TTTTAAGTCTGATGTGAAAGCCCACGGC
168
Results
Description
169
1046
1046
98%
0.0
99%
1046
1046
98%
0.0
99%
1046
1046
98%
0.0
99%
1046
1046
98%
0.0
99%
1046
1046
98%
0.0
99%
1046
1046
98%
0.0
99%
1044
1044
98%
0.0
99%
1044
1044
98%
0.0
99%
1044
1044
98%
0.0
99%
1044
1044
97%
0.0
99%
1044
1044
98%
0.0
99%
1042
1042
98%
0.0
99%
1042
1042
97%
0.0
99%
1042
1042
98%
0.0
99%
1040
1040
98%
0.0
99%
1040
1040
97%
0.0
99%
1040
1040
98%
0.0
99%
Results
HM074037.1
HM317980.1
GQ001329.1
FR821779.1
JN084559.1
JN084556.1
JN084552.1
JN084551.1
JN092619.1
JF784041.1
JF513981.1
CP002643.1
JF015377.1
Phylogenetic analysis.
170
1040
1040
98%
0.0
99%
1040
1040
97%
0.0
99%
1040
1040
97%
0.0
99%
1038
6217
98%
0.0
99%
1038
1038
97%
0.0
99%
1038
1038
98%
0.0
99%
1038
1038
98%
0.0
99%
1038
1038
98%
0.0
99%
1038
1038
98%
0.0
99%
1038
1038
98%
0.0
99%
1038
1038
98%
0.0
99%
1038
5183
98%
0.0
99%
1038
1038
98%
0.0
99%
Results
FM163053.1| Staphylococcaceae bacterium ACEMC 3-3 partial
DQ170796.1| Uncultured Staphylococcus sp. clone WS03A_B09
JN831370.1| Staphylococcus aureus strain SU-BIO3
GU459256.1| Staphylococcus aureus isolate K54-3
HM311188.1| Uncultured bacterium clone ncd1164c05c1
JN084555.1| Staphylococcus aureus strain Pn833
FJ899095.1| Staphylococcus aureus strain SC01
JN084553.1| Staphylococcus aureus strain 383/Chen
JN084554.1| Staphylococcus aureus strain 557/Chen
JN084557.1| Staphylococcus aureus strain
JN084558.1| Staphylococcus aureus strain Pn2087
JN102552.1| Staphylococcus aureus subsp. aureus strain FUA2076
JN102568.1| Staphylococcus aureus subsp. aureus strain FUA2081
JN831368.1| Staphylococcus aureus strain SU-BIO1
JN831371.1| Staphylococcus aureus strain SU-BIO4
GU459255.1| Staphylococcus aureus isolate K54-2
AM945662.1| Staphylococcus equorum partial
AB674510.1| Staphylococcus aureus
Isolate 30
HM269253.1| Uncultured bacterium clone ncd245c06c1
GQ001181.1| Uncultured bacterium clone nbu170a08c1
0.0008
0.0006
0.0004
0.0002
0.0000
Isolate no. 95
CCCGTCGGCTCATGATCATGCTAGTCGAGCGACGGACGAGAAGCTTGCTTCT
CTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGATAACCTACCTATAAG
ACTGGGATAACTTCGGGAAACCGGAGCTAATACCGGATAATATTTTGAACC
GCATGGTTCAAAAGTGAAAGACGGTCTTGCTGTCACTTATAGATGGATCCG
CGCTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCAACGATGCAT
AGCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTCCAGA
CTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGA
CGGAGCAACGCCGCGTGAGTGATGAAGGTCTTCGGATCGTAAAACTCTGTT
ATTAGGGAAGAACATATGTGTAAGTAACTGTGCACATCTTGACGGTACCTA
ATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGT
GGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGTAGGGCGGTTTT
TTTAAGTCTGATGTGAAAGCCCCACGGCT
Description
171
Max Total
Query
E
Max
score score coverage value ident
1046 1046 97%
0.0
99%
Results
gene, partial sequence
HM309869.1 Uncultured bacterium clone
ncd920d10c1 16S ribosomal RNA
gene, partial sequence
HM280136.1 Uncultured bacterium clone
ncd580a07c1 16S ribosomal RNA
gene, partial sequence
GQ033257.1 Uncultured bacterium clone
nbw1028e12c1 16S ribosomal RNA
gene, partial sequence
JF171124.1 Uncultured bacterium clone
ncd1960c11c1 16S ribosomal RNA
gene, partial sequence
GU459255.1 Staphylococcus aureus isolate K54-2
16S ribosomal RNA gene, partial
sequence
HM260683.1 Uncultured bacterium clone
ncd158b09c1 16S ribosomal RNA
gene, partial sequence
DQ170822.1 Uncultured Staphylococcus sp. clone
WS03A_F12 16S ribosomal RNA
gene, partial sequence
FR821779.1 Staphylococcus aureus subsp. aureus
LGA251 complete genome sequence
JF322979.1 Staphylococcus sp. 1036(2011) 16S
ribosomal RNA gene, partial sequence
JN084559.1 Staphylococcus aureus strain
Pn3576/Pant-Orissa/2008 16S
ribosomal RNA gene, partial sequence
JN084556.1 Staphylococcus aureus strain
Pn1016/Pant-Uttar/2007 16S
ribosomal RNA gene, partial sequence
JN084552.1 Staphylococcus aureus strain
366/Chen 16S ribosomal RNA gene,
partial sequence
JN084551.1 Staphylococcus aureus strain
254/Chen 16S ribosomal RNA gene,
partial sequence
JN092619.1 Staphylococcus aureus strain FFL34
16S ribosomal RNA gene, partial
sequence
JF784041.1 Staphylococcus aureus strain CIFRI
CH-TSB27 16S ribosomal RNA gene,
partial sequence
JF513981.1 Staphylococcus aureus strain sc-sa 16S
ribosomal RNA gene, partial sequence
CP002643.1 Staphylococcus aureus subsp. aureus
T0131, complete genome
JF015377.1 Uncultured bacterium clone
ncd197g02c1 16S ribosomal RNA
gene, partial sequence
Phylogenetic analysis.
172
1046
1046
97%
0.0
99%
1046
1046
97%
0.0
99%
1046
1046
97%
0.0
99%
1042
1042
97%
0.0
99%
1042
1042
97%
0.0
99%
1035
1035
97%
0.0
98%
1035
1035
98%
0.0
98%
1033
6183
97%
0.0
99%
1033
1033
97%
0.0
99%
1033
1033
97%
0.0
99%
1033
1033
97%
0.0
99%
1033
1033
97%
0.0
99%
1033
1033
98%
0.0
98%
1033
1033
97%
0.0
99%
1033
1033
97%
0.0
99%
1033
1033
97%
0.0
99%
1033
5155
97%
0.0
99%
1033
1033
97%
0.0
99%
Results
0.0006
0.0004
0.0002
0.0000
173
Results
Different primers were used for detection of van A gene & mec A
genes and different conditions were used for the same pair of primers.
11.2.1. Detection of mec A gene
mecA F AAAATCGATGGTAAAGGTTGGC
mecA R AGTTCTGCAGTACCGGATTTTGC
11.2.2. Detection of van A gene
VANA: ATGAATAGAATAAAAGTTGCAATAC
VANA1: CCCCTTTAACGCTAATACGAT
After using different condition for PCR there are not give any PCR
product..so we make another design for primers
NEW DESIGN
Detection of mec A gene
Forward primer TGGCTCAGGTACTGCTATCCACCC
Reverse primer ACCACCCAATTTGTCTGCCAGTT
This pair of primer gave product 818
Forward primer GGCTCAGGTACTGCTATCCACCCT
Reverse primer AACCACCCAATTTGTCTGCCAGT
This pair of primer gave product 818
Forward primer CCAGGAATGCAGAAAGACCAAAGCA
Reverse primer
GGGTGGATAGCAGTACCTGAGCCA
Results
van A 2
Forward primer: GCTCAGCAATTTGTATGGAC
Reverse primer: TGTCATATTGTCTTGCCGAT
After using different condition for PCR. Only primer of van A give
positive for S. aureus 12 and S. aureus 50 only and did not present in
another two isolates. May be another gene for vancomycin resistant may
van C or van R or other.
175
176
Discussion
Discussion
Burn patients are ideal hosts for opportunistic infections. Infections
remain the leading cause of death among patients who are hospitalized for
burns (Tancheva and Hadjiiski, 2005).
In spite of considerable advances in the treatment of burns; infection
continues to pose the greatest danger to burned patients. Approximately 73
per cent of all death within the first five days post-burn have been shown to
be directly or indirectly caused by septic processes.
Different patient
177
Discussion
Results also revealed that most common age group affected was 20
to 40. This came in concordance with study conducted by Mertens et al.
(2000) who documented that the age group of most burned patients ranged
from 20 50 years.
Data analysis revealed that exposure to flame led to thermal injury in
33 patients out of 70 total patients. This accounted for 47.1%. Hot water
thermal injury was implicated in 16 cases. Electricity revealed 17 patients
all of them from males. Only four patients out of 70 total patients
represented in (5.7%) suffered from chemicals burns. In agreement with
this found, Hill and Bowe (2002) stated that, thermal injury caused by
exposure to flame has the highest percentage among burned patients, then
hot water and the injury caused by electricity.
Moreover, there was a highly significant correlation between age and
death. The 29 patients who were able to recover were significantly younger
than those who died. Thus the recovered group had a mean age of 18 years,
while the group that suffered death had a mean age of 48.3 years. This
matched with (Zaid, 2001) who observed that the twenty three out of 50
patients were able to recover because they were younger than those who
died.
In our study, itisestimatedthat all the patients suffered burn of
7% up to 94% of TBSA burns. More than 58 % of the patients died either
from infection, by the burn itself or by both of them. Also 29 patients who
were able to recover with mean TBSA burns of 22% while, the other 41
patients mean TBSA burns of 51% died. This indicated that the death is
directly proportional to the total body surface area. This clearly matched
with Nichols (2008) who reported that incidence of infection is also
affected by the size of the patients burn injury. At BH, Burn hospital the
incidence of infection was determined for patients with <30% TBSA burn
injury compared to patients with 30% TBSA. The overall incidence of
178
Discussion
infection was low for patients with <30% TBSA burn injuries and generally
associated with the need for invasive devices. Bloodstream infection (BSI)
increases dramatically as burn wound size increases, related to increased
exposure to intravascular catheters and to burn wound manipulationinduced bacteremia. At (BH), Boston from 1996-2000 there were 3 cases of
BSI in 585 patients with <30% TBSA burn injury compared to 55 cases of
BSI in 60 patients with 30% TBSA. In this series, pneumonia occurred
only in ventilated patient, accounting for all 15 cases seen. Similarly,
urinary tract infection (UTI) occurred principally in patients with
indwelling urinary catheters, accounting for 33 of the 36 cases seen.
The present study showed that from the twenty nine patients (41.4%)
were recovered, while the other 58.7% were died. Comparing the number
of positive and negative cultures for each patient, it was found that there is
no relation between patient death and number of positive and negative
cultures. This agrees with Nour El-Dien. (1999) who reported that from
the 30 patients who recovered, five patients had negative swab cultures the
other twenty five had positive culture. On the other hand, among the other
50 patients who died, only three had negative swab cultures, while the
others showed positive results in their swab cultures. So the data revealed
that there was no relation between patient death and the presence of local
infection.
Santucci et al. (2003) reported that infections remain the leading
cause of death among patients who are hospitalized for burns. The risk of
burn wound infection is directly correlated to the extent of the burn and is
related to impaired resistance resulting from disruption of the skins
mechanical integrity and generalized immune suppression
It is quite clear in the present study that the incidence of total
microorganisms isolated from swab, blood, urine and sputum culture from
burned patients indicate that Gram-positive bacteria were the most
179
Discussion
Discussion
spread; however this view has been increasingly challenged in the era of
vancomycin-resistant Enterococcus (VRE). Also (Tredget, 2004) reported
that the most common organisms isolated from burn patients were S.
aureus and P. aeruginosa.
This on line with Tiwari and Sen. (2006) who found that S. aureus
was the most common isolate from blood followed by P.aeruginosa, and
Coagulase Negative Staphylococci (CONS). In most of the cases the
organisms isolated from blood were the same as isolated from pus.
On the other hand Ahmad and Aqil. (2006) found that P.
aeruginosa (19%) was the commonest isolate from burn wounds followed
by S. aureus (15%), S. epidermidis (11%) and E.coli (10.5%). It is evident
that P.aeruginosa has emerged as a great threat in burn wound infection
and its very important that antibiotic policy is formulated to keep a check
on it. P.aeruginosa was the most common isolate from blood also followed
by S.epidermidis, Klebsiella, S.aureus and E.coli. This in contrary with
Biswajit et al. (2008). They reported that Gram-negative organisms were
found to be more prevalent. P. aeruginosa was found to be the most
common isolate followed by S. aureus, S. epidermidis, Klebsiella and
Salmonella. In most of the cases, same organisms were found in blood and
pus sample.
Antibiotic sensitivity testing (AST) aims to determine the
susceptibility of an isolate to a range of potential therapeutic agents. This
can be with a view to individualizing the antibiotic to be administered or to
monitor resistance patterns developing in that environment, gathering this
information is important for revising and updating the standard antibiotic
prescribing policy for a particular population or institution. The antibiotic
sensitivity against bacteria is assayed by three different methods, liquid
dilution, agar dilution and disc diffusion method by following the reference
standards established by various authorities such as the Clinical and
181
Discussion
chloromphenicol,
rifampicin,
erythromycin,
S. aureus is particularly
Discussion
rapidly develop resistance to these drugs. Perhaps the most commonly
known resistance of S. aureus, is methicillin resistance, which has caused
alarming reports with regard to the spread of S. aureus in hospitals and the
community (Gtz, 2010).
Moreover, Rybak and Akins. (2001) reported that the emergence of
high levels of penicillin resistance followed by the development and spread
of isolates resistant to the semi synthetic penicillins (methicillin, oxacillin,
and nafcillin), macrolides, tetracycline, and aminoglycosides has made the
therapy of staphylococcal disease a global challenge.
Furthermore, Matsukawa et al. (2001) found that penicillin and
first to second generation cephalosporin in vitro sensitivity tests must be
undertaken as a guide for intelligent antimicrobial therapy.
The emergence of glycopeptide- resistance of S.aureus was
significantly marked in hospital that had been endemic with isolates of
methicilliin resistant staphylococci and followed the policy of empirical
use of glycopeptides. Extensive use of glycopeptides (vancomycin or
teicoplanin) allowed selection of resistant isolates of S. aureus.
Glycopeptide- resistant mutants of S. aureus have been experimentally
selected by gradually increasing the levels of vancomycin present during
in-vitro growth (Rybak and Akins, 2001).
`Glycopeptides such as vancomycin are frequently the antibiotics of
choice for the treatment of infections caused by methicillin resistant
Staphylococcus aureus (MRSA) (Arthur et al., 1993). For the last 7 years
incidence of vancomycin intermediate S. aureus and vancomycin resistant
S. aureus (VISA and VRSA, respectively) has been increasing in various
parts of the world (Gtz, 2010).
Moreover, Cui et al. (2009) study indicated that vancomycin
resistance in S. aureus may occur under the selective pressure of prolonged
183
Discussion
184
Discussion
cefotaxime,
amoxycillin,
ampicillin\sulbactam,
Discussion
186
Discussion
MIC is 8 to 32 g/ml are intermediate and those for which the MIC is 32
g/ml are resistant.
The present investigation recorded that the four VRSA isolates no.
12, 30, 50 and 95 had a resistant to vancomycin (MIC range, 16-70 mg/l)
This on line with Chang et al. (2003) who reported that the MIC for 335 of
358 isolates (93.57%) for vancomycin was 2 mg/l indicating that all were
sensitive to vancomycin. Sixteen isolates showed an MIC range between 48 mg/l, indicating vancomycin intermediate resistance. For the remaining
seven isolates, the MIC was in the range of 16-70 mg/l indicating that these
seven isolates were vancomycin resistant (VRSA). These were all MRSA
and resistant to majority of the other antibiotics tested. All these seven
isolates showed resistance to a minimum of six other antibiotics including
vancomycin and methicillin.
Furthermore, some studies have been reported the emergence of
VRSA in clinical isolates that. By definition, show in-vitro MICs of > 4 mg
/l. (Rybak and Akins 2001).
The results also indicated that MICs and MBCs of imipenem
antibiotic for S. aureus no. 12 and 95 were 62. 5 g/ml, also S. aureus no.
50 had the same MIC and MBC 15.625 g/ml, while MIC and MBC of S.
aureus no. 30 were 31.25 and 62.5g/ml, respectively. The results
demonstrated that MIC and MBC of ciprofloxacin for S. aureus no. 12 and
30 were 62. 5 and 125 g/ml, respectively. Also S. aureus no. 50 had the
same MIC and MBC 125 g/ml, while MIC and MBC of S. aureus no. 95
were 31.25 and 62.5g/ml, respectively.
The results demonstrated that MICs of oxycilline antibiotic were
125, 62.5, 31.25 and 15.625 g/ml, for S. aureus no. 12, 30, 50 and 95,
respectively. Also MBCs of oxycilline antibiotic were 250, 125, 62.5 and
15.625 g/ml, for S. aureus no. 12, 30, 50 and 95, respectively.
187
Discussion
of
which
are
commercially
important
especially
for
the
Discussion
The results in our investigation revealed that, thyme oil and tea tree
oil had the highest antibacterial activity against the four S. aureus (12, 30,
50 and 95) with inhibition zones (8.2, 7.5, 8.0 and 9 mm), respectively,
while tea tree oil with inhibition zones (8.0, 8.3, 7.5 and 9 mm),
respectively followed by clove, lemon grass. Cinnamon oil had the lowest
antibacterial activity against the four S. aureus (12, 30, 50 and 95) with
inhibition zones (0, 1.2, 1.3 and 3.5 mm), respectively.
Our results are in agreement with Deans and Ritchie (1987) where
they found that lemon grass and cinnamon oil had no inhibitory effect
against tested isolates. These results were agreement with Zafra-Polo et al
(1989). They reported that the essential oil of Thymus leptophyllus showed
higher antimicrobial activity against S. aureus and other tested bacteria.
Also the essential oil of Thymus vulgaris (thyme oil) was very effective
against S. aureus, E. coli and Salmonella typhimurium.
The antimicrobial activity of different species of thymus was tested
against bacteria. The essential oil of Thymus broussonettii was most
efficient for killing the S. aureus, E. coli and inhibiting their growth
(Tantaoui et al., 1993).
In this relation, Ela et al. (1996) screened sixteen essential oils for
their antimicrobial activity against S. aureus, by using the agar diffusion
method and measuring their inhibition zone. They classified the tested oils
DFFRUGLQJ WR WKHLU DFWLYLW\ VWURQJO\ DFWLYH LQKLELWLRQ ]RQH ! PP
moderately active (inhibition zone > 6 to < 8 mm), and inactive (no
inhibition zone < 6 mm). Basil and parsley were inhibitory to S. aureus.
The antimicrobial activity of different species of thymus was tested
against bacteria the essential oil of Thymus vulgaris was most efficient for
killing E .coli, other bacteria & fungi and inhibiting their growth (ElGayyare et al., 2003).
189
Discussion
Also El-Aidy. (2008) reported that thyme and tea tree oil were the
most effective oil against all tested isolates E .coli, S. aureus, P.
aerugenosa and K. pneumonieae.
The volatile oils of plants exhibited various reduction in the growth
according to its chemical composition. Many research groups screened
various plant extracts as secondary metabolites to detect biological
activities (Salama & Zaid, 2000 and Abo-Ghalia et al., 2004). The results
in accordance with Rota et al. (2004) who reported that, the bactericidal
properties of thyme essential oil are supposed to be associated with high
levels of carvacrol and linalool. Kalemba and Kunicka (2003) reported
that, because of the great number of cell constituents, volatile oils seem to
have no specific cellular targets. As typical lipophiles, they pass through
the cell wall and cytoplasmic membrane, disrupt the structure of their
different layers of polysaccharides, fatty acids, phospholipids and
permeabilize them. The mode of action of antimicrobial agents also
depends on the type of microorganisms and is mainly related to their cell
wall structure and the outer membrane arrangement cytotoxicity appears to
include such membrane damage. In bacteria, the permeabilization of the
membranes is associated with loss of ions and reduction of membrane
potential, collapse of the proton pump and depletion of the ATP pool
(Turina et al., 2006).
Moreover the volatile oils can act as prooxidants affecting inner cell
membranes and organelles such as mitochondria. Depending on type and
concentration, they exhibit cytotoxic effects on living cells but are usually
non genotoxic (Bakkali et al. (2008).
It is worthy to mention here that the minimum inhibitory
concentration and minimum bactericidal concentration were determined for
thyme oil and tea tree oil. The results showed that the MIC and MBC value
for thyme oil against S. aureus12 were 62.5 ug/ml. Also the MIC value of
190
Discussion
thyme oil for S. aureus 30 was 15.62 ug/ml and MBC recorded 31.25
ug/ml, while MIC and MBC value for thyme oil against S. aureus 50 and S.
aureus 95 were 7.8 and 15.62 ug/ml, respectively. The MIC and MBC
value for tea tree oil against S. aureus 30 were 31.25ug/ml. Also the same
MIC and MBC for S. aureus (50) 125 ug/ml. The MIC of tea tree oil for S.
aureus (95) was 125 ug/ml and MBC recorded 250 ug/ml.
The present investigation was extended to include the study of
combination between (vancomycin + thyme oil) & (vancomycin + tea tree
oil) which gave a synergetic effect against each strain of S. aureus (12, 30,
50 and 95), respectively, where the inhibition zone of VAN disc saturated
with 30 l thyme oil or saturated with 30 l tea tree oil were more than
that of either VAN disc or thyme oil or tea tree oil each separately. This
experiment was carried out for the medical applications in the future
research. Rational for the use of antimicrobial combination was to decrease
emergence of resistance isolates; decreased dose-related toxicity of
antimicrobial using the other agent(s) (Mandell and Sande. 1980) and to
polymicrobial infection (it is one of the reasons for broad-spectrum
coverage).
Cappelletty and Rybak. (1996) reported that combination of
antimicrobial agents are considered to be synergetic if the effect of
combination is greater than the effect of either agent alone or greater than
the sum of the effect of individual agents. Antagonism result occurred if the
combination provides an effect worse than the effect of either agent alone
or worse than the sum the effect of individual agents.
This is in agreement with Mostafa. (2005) who reported that the
combination between oil and antibiotic was more active than each
individual alone. Also the synergistic effect from the association of
antibiotic with oil against resistant bacteria leads to new choices for the
treatment of infectious diseases. This effect enables the use of the
191
Discussion
Discussion
Discussion
Discussion
tree oil. For four S. aureus (12, 30, 50 and 95) isolates, significantly lower
levels of hemolysin and Proteinase were produced in the presence of 0.6 %
tea tree oil when compared to control cells. In general, these experiments
demonstrate significant reduction in levels of protease, hemolysin and
lethinase after growth for 24 h. in the presence of 0.3 and 0.6% tea tree oil.
The suppression of toxins
part in the
interactionof
these
S.aureusproductsand componentsisnecessarytoapplythecorrecttreatm
ent andtopreventinfections (Edwards-Jones and Foster, 2002).
These results are in accordance with data reported in study of
Iwalokun et al. (2003) who stated that the exposure to tea tree oil may alter
cell permeability leading to the leakage of extracellular proteins. It is also
hypothetically possible that the presence of tea tree oil affects the
outermost part of the cell wall in such as way as to cause the extracellular
proteins to become less highly associated with the cell wall and therefore
more extracellular proteins is found free in the supernatant. Increased
expression of VFs after treatment with sub-inhibitory levels of
antimicrobial agents has been reported previously, although infrequently. S.
aureus isolates resistant to fluoroquinolones have increased expression of
fibronectin-binding
proteins
after
treatment
with
sub-inhibitory
195
Discussion
Another study, using four S. aureus isolates, found that the presence
of tea tree oil had significantly altered lethinase production after 24 h,
compared to the controls (Edwards-Jones and Foster, 2002).
It has been observed previously that the suppression of extracellular
protein synthesis occurs only with antibiotics that inhibit protein synthesis
and that antibiotics with other modes of action may in fact have a
stimulatory effect. Although tea tree oil is not an antibiotic, tea tree oil acts
by inhibiting the synthesis of the extracellular enzyme protease (Herbert et
al., 2001).
Our results are not matched with Kernodle et al. (1995) who had
several possible explanations for the apparent increases in protein synthesis
levels after growth in the presence of sub-inhibitory tea tree oil. Another
example is increased -toxin production by S. aureus after treatment with
subinhibitory levels of the -lactam antibiotics nafcillin and methicillin
(Doss et al.2KOVHQet al., 1998).
Moreover, Herbert et al. (2001) reported that the mechanisms
behind the apparent decrease in hemolysin seen in the present study may be
elucidated by the gene encoding hemolysin is lower expressed or
transcribed in the presence of tea tree oil.
Two studies have investigated this very possibility, both assessing
the effectiveness of tea tree oil products in the eradication of MRSA
carriage (Caelli et al.'U\GHQet al., 2004). Data from the first study
showed that tea tree oil formulated into nasal ointment and skin wash
compared favourably to mupirocin nasal ointment and Triclosan skin wash
for the decolonisation of MRSA carriage (Caelli et al., 2000). Similarly,
the second study indicated similar clearance rates for both the tea tree oil
regimen and standard treatment. These clinical outcomes may have
occurred by any number of mechanisms. Firstly, the topically applied tea
tree oil may have had a direct killing effect against the target organisms.
196
Discussion
However, it has been postulated that antimicrobial agents are often present
at only sub-inhibitory levels, and cannot therefore produce killing effects.
This means that sub-inhibitory levels of tea tree oil may somehow
responsible for eradicating S. aureus carriage or infection. Sub-inhibitory
tea tree oil may have any number of effects against the infecting organism,
host cells, and any interactions between the two. Since data from this study
suggest that extracellular VFs such as coagulase, protease and toxins are
not reduced in the presence of tea tree oil, a reduction in the levels of these
proteins is not likely to be the mechanisms by which tea tree oil is acting.
One possibility is that the tea tree oil alters cells of S. aureus in such a way
as to render them susceptible to the effects of the host immune system. This
mechanism and others require investigation (Dryden et al., 2004).
Several genomic targets have been effectively used for the
identification of Staphylococcus species, including the 16S rRNA gene, the
tRNA gene intergenic spacer, the internal transcribed spacer, the heat-shock
protein 60 (HSP60) gen, the chaperonin 60 gene, the femA gene, the sodA
gene, the gap gene and the nuc gene. Recently, an enterobacterial repetitive
intergenic consensus PCR and BOX-PCR were also used in the
identification of S. epidermidis isolates (Walker, 1998). Some antibiotic
resistance genes were found on non conjugative plasmids, these genes may
also move from one plasmid to another. The unstable nature of plasmids
that can spread even to multiple species of bacteria and may be lost or
acquired spontaneously, make plasmid fingerprinting, which was the first
molecular typing method to be used for epidemiological purposes often
poorly reproducible (Stefani & Agodi, 2008).
It is worthy to mention here that our study showed that the four
isolates no. (12, 30, 50& 95) which were isolated from wounds &blood and
selected as multi-resistant to most tested antibiotics were preliminary
identified as S. aureus. PCR amplification of 16s rRNA gene of the four
selected isolates using specific primers produced amplicons of size 1500bp
197
Discussion
for all isolates. PCR products confirmed the identity of the four isolates as
S. aureus. The partial nucleotide sequences of 16S rRNA gene of the four
tested isolates named S. aureus 12 (ZWFR 12) S. aureus 30 (ZWFR 30), S.
aureus 50 (ZWFR 50) and S. aureus 95 (ZWFR 95) were then submitted to
the DDBJ/EMBL/Gen Bank with the accession number(s) 12 [AB674512],
30 [AB674513], 50 [AB674514] and 95 [AB674515], respectively.
Also molecular studies represented in detection of mec A and van A
gene
by
using
different
TheuseofPCRforthedetectionofthemec
typinghasbeendescribedpreviouslyby
Agene
Oliveira
condition.
and
van
andcolleague
MRSA,PCR
Reverse
primer
Discussion
that the four isolates were VRSA May be another gene for vancomycin
resistant may van C or van R or other.
Actually, thickening of the cell wall of vancomcin- resistant
staphylococci has been found to be associated with complex reorganization of cell wall metabolism with extra cell wall material showing
reduced peptidoglycan cross-linking of D-Ala-D-Ala termini of side chains
(Walsh and Howe, 2002).
These studies on the role of cell wall thickening may provided us
with another explantation for the absence of van a gene in two out of four
vancomycin resistant isolates in the current study. This may mean that cell
wall thickness may be the effective mechanism for vancomcin- resistant
isolates lacking van A gene. Biswajit et al. (2008) reported that in view of
low level vancomycin resistance and the absence of van genes or any
altration in D-Ala-D-Ala residues of peptidoglycan , it would be reasonable
to consider the cell wall thickning as the major contributor to vancomycin
resistance staphylococci clinical isolates. However, in the current study the
level of vancomycin resistance was not low and the presence of other van
gene was not excluded.
This came in concordance with study of DeNiederhusern et al.
(2011) who reported that from 12 VRSA isolate contained vanA gene, six
isolates were negative for vanA by PCR whereas all the isolates had not
expressed mecA.
Our results on line with Assadullah et al. (2003) who reported that
sixty five percent of the VRSA isolates were Van-negative with multiplex
PCR assay.
Moreover, Reynolds. (1990) stated that multistep genetic alteration
is required for MRSA to achieve the level of vancomycin resistance of
VISA. In the progression of vancomycin resistance, isolates with
199
Discussion
200
Discussion
The study depended on the same criteria for detection of the ten
vancomycin resistant isolates. However, other workers reported that the
most resistant isolates bear the vanA gene cluster and these are usually
found to be in the range 32-64 mg/l (Heggers et al., 2009). This may
explain why not all vancomycin resistant isolates by conventional methods
were PCR positive for vanA gene in the present study. All our VRSA
isolates showed MIC for vancomycin of 32 mg/l., which vanA gene or not
(Heggers et al., 2009). So this PCR method is confirmatory for the VRSA
isolate if give positive result (confirm nuc gene for S. aureus and vanA
gene for vancomycin resistance), but if positive for nuc gene and negative
for vanA gene, it cannot exclude the possibility to be VRSA. It should be
regarded that there are van resistance genes other than van A (which was
concerned in the current study), these genes include vanB, vanC, vanD and
vanE and vanG (Depardieu et al., 2006).
However, more studies are still needed to explore the definite
mechanisms by which these isolates acquire resistance to vancomycin
which may open the door to overcome this problem.
201
202
Summary
Summary
Multidrug-resistant microbial infections are becoming increasingly
common and difficult to treat. In the current study we aimed to study the
prevalence of antibiotic resistant bacteria specially MRSA and VRSA
strains in burn units at different Hospitals, study their antibiotic
susceptibility profile and suggest antibiotic oil combination therapy to
control and prevent the transmission of MRSA and VRSA strains to aid
burned patients in recovering from their injuries. In the future, these
alternatives may prove useful in treating not only burn infections but other
antibiotic-resistant infections as well.
Results obtained in this study can be summarized as follows:1.
2.
3.
One hundred and twenty one isolates classified into five bacterial
groups and were further on identified by studying morphological and
biochemical characteristics using conventional methods according to
the key described in Bergey's manual. Fifty seven isolates were
included in group 1, while 12 isolates in group 2, On the other hand
there were 14 isolates in group 3, while 36 Gram-negative isolates in
group 4, two isolates no. (8 and 65) were group 5. Isolated bacteria
related to groups I, 2, 3, 4 & 5 were preliminary identified as S.
203
Summary
5.
6.
b-
Summary
also S. aureus no. 50 had the same MICs and MBCs 15.625
g/ml, while MICs and MBCs of S. aureus no. 30 were 31.25 and
62.5g/ml, respectively.
c-
d-
On the other hand MICs of oxycilline were 125, 62.5, 31.25 and
15.625 g/ml, for S.aureus no. 12, 30, 50 and 95, respectively.
Also MBCs of oxycilline antibiotic were 250, 125, 62.5 and
15.625 g/ml, for S.aureus no. 12, 30, 50 and 95, respectively.
7-
b-
Summary
9-
The MIC and MBC of thyme and tea tree oil were determined to
obtain the lowest concentration that inhibits a visible growth in liquid
media and the lowest concentration at which no growth occurs on
solid media. The MIC and MBC value for thyme oil against S.
aureus12 were 62.5 ug/ml. Also the MIC value of thyme oil for S.
aureus 30 was 15.62 ug/ml and MBC recorded 31.25 ug/ml, while
MIC and MBC value for thyme oil against S. aureus 50 and S. aureus
95 were 7.8 and 15.62 ug/ml, respectively. The results reveal that the
MIC and MBC value for tea tree oil against S. aureus 30 were
31.25ug/ml. Also the same MIC and MBC for S. aureus (50) 125
ug/ml. The MIC of tea tree oil for S. aureus (95) was 125 ug/ml and
MBC recorded 250 ug/ml.
206
Summary
Summary
208
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