Heavy metal resistant bacteria

Heavy Metal Resistant Bacteria:
Pseudomonas aeruginosa as a model
By
Raafat M. J. Al-Enzi Alaa H. Al-Charrakh
M.Sc Microbiology Ph.D Microbial Biotechnology
Babylon University, Iraq

Dedication
To….
All martyrs and Iraqi peoples who died or vanished during
violence waves that devastated Iraq in the last few years
We dedicate this work
Authors

Acknowledgements
Praise to the Almighty Allah, the glorious creator of the universe, for
his kindness and mercy, and blessing upon Mohammad the prophet and
upon his family and followers.
The authors would like to thank Department of Microbiology, College of
Medicine, Babylon University for providing all the needed facilities, which were
essential for successful completion of the present work. Our thanks are also
extended to all members of the Department of Microbiology for their generous
help and co-operation. We would like to thank Dr. Hussein Oliewi AL-
Dahmoshi for his help and advice in this work.
Authors

a
List of Contents
No. Subject Page
List of contents a
List of tables c
List of figures d
List of Abbreviations e
Chapter One: Introduction and Literature Review 1-34
1.1. Introduction 1
1.2. Literatures review 2
1.2.1. History and description of Pseudomonas aeruginosa 2
1.2.2. Pathogenicity of Pseudomonas aeruginosa 5
1.2.3. The Antigenic Structure 8
1.2.4. Antibiotic resistance 8
1.2.5. Occurrence of heavy metals in the Environment 9
1.2.6. Bacterial tolerance against heavy metals 12
1.2.7. Beneficial heavy metals 14
1.2.8. Toxic heavy metals 15
1.2.9. Review of some important heavy metals 16
1.2.9.1. copper 16
1.2.9.2 silver 17
1.2.9.3 Mercury 19
1.2.9.4 Lead 29
1.2.9.5 Zinc 21
1.2.9.6 Cadmium 21
1.2.9.7 Nickel 22
1.2.10 Health application of heavy metals 23
1.2.11 Detection of heavy metals resistance in laboratory 24
1.2.11.1 Phenotypic characteristics 24

b
1.2.11.2 Genotypic methods 25
Chapter two : Materials and Methods 29-46
2.1. Materials 29
2.1.1. Samples 29
2.1.2. Laboratory apparatus and instruments 29
2.1.3. Chemical and biological materials 30
2.1.4. Culture media 31
2.1.5. Standard strain used in the study 32
2.1.6 Plasmid extraction kit 32
2.1.7. DNA marker ladder 33
2.2. Reagent and Solutions 33
2.3. Preparation of culture media 35
2.4 Methods 38
2.4.1 Specimen collection 38
2.5. Laboratory diagnosis 39
2.6. Detection of virulence factors 41
2.7 Detection of biofilm production 43
2.8 Genotyping assays 44
2.9 Plasmid curing 46
Chapter three: Results and Discussion 47-70
3.1. Isolation, identification and distribution of isolates 47
3.2. Heavy metals susceptibility test of Pseudomonas
aeruginosa 54
3.3 Plasmid profile of heavy metals resistant isolates 62
3.4 Plasmid curing by elevated temperature 66
3.5 Biofilm assay 68
4.1. Conclusions and Recommendations 71
References 72-93

c
List of Tables
Table
No. Title Page
1-2
Different inheritable shared mechanisms of metal and
antibiotic resistance systems in Pseudomonads and other
prokaryotes.
11
2-1-2 Laboratory apparatus and instruments 29
2-1-3 Chemical and biological materials 30
2-1-3-1 Heavy metals used in the study 31
2-1-4 Culture media 31
2-1-5 Standard strain used in the study 32
2-1-7 DNA Marker 33
3-1 Distribution of Patients according to Sex and Residence 48
3-2 Morphological properties and Biochemical testes for
identification of Pseudomonas aeruginosa isolates
50
3-3 Distribution of isolates from different Clinical samples
collected during study
50
3-4 Distribution of isolates from different hospital
environmental Samples collected during study
52
3-5 Numbers and percentage of clinical and environmental
Pseudomonas aeruginosa detected by screening test
54
3-6 Antibacterial activity of heavy metals against
Pseudomonas aeruginosa Isolated from clinical and
hospital environmental samples
55
3-7 MIC values of Pseudomonas aeruginosa isolates to
Silver sulfate, Zinc sulfate, Cadmium sulfate, Nickel
sulfate in Molar concentrations
57
3-8 MIC values of Copper sulfate, Mercury chloride, and
Lead nitrate in (g/ml) concentrations towards
Pseudomonas aeruginosa
60
3-9 MIC values of Pseudomonas aeruginosa Ps.3 after
curing exposed to following heavy metals
68
3-10
Biofilm production by P. aeruginosa isolates recovered
by clinical and hospital environment samples 69

d
List of Figures
Figure
No.
Title Page
1-1 Model showing examples of three major molecular
mechanisms responsible for dual tolerance in
Pseudomonads.
13
3-1 Number and percentages of Pseudomonas aeruginosa
isolates in clinical sample.
47
3-2 Distribution of clinical Pseudomonas aeruginosa
isolates according to age groups.
48
3-3 Number and percentage of Pseudomonas aeruginosa
isolates in hospital environment samples.
52
3-4-a Gel electrophoresis of plasmid DNA content of
Pseudomonas aeruginosa isolates after (1:30) hr. at 60
voltages.
63
3-4-b Gel electrophoresis of plasmid DNA content of
voltages.
64
3-4-c Gel electrophoresis of plasmid DNA content of
voltages
65
3-5 Gel electrophoresis of plasmid DNA content of Ps.
aeruginosa isolate before and after curing after (1:30)
hr. at 60 voltages.
67

e
List of Abbreviations
Abbreviation Key
µg Microgram
µl microliter
AgSo4 Silver Sulfate
ATPase Adenosine triphosphate synthetase
BHI Brain heart infusion
bp base pair
CDC Center of disease control and prevention
CDF Cations diffusion facilitation
CdSo4 Cadmium sulfate
CuSo4 Copper Sulfate
EDTA Ethylene di-amine tetra acetic acid
EPS Extracellular polymeric substance
H2O2 hydrogen peroxide
Hgcl2 Mercury chloride
HM Heavy metal
HMR Heavy metal resistance
hrs hours
LPS Lipopolysaccharide
LTA Lipoteichoic acid
mg Milligram
MIC Minimum Inhibitory Concentration
min minute
MR-VP
Reagent
Methyl red-vogues proskauer reagent
MTC Minimum tolerance Concentration
NiSo4 Nickel sulfate
nm Nanometer
NNIS National nosocomial infections surveillance

1.1. Introduction
Pseudomonads present a versatile group of gram negative motile, rod bacteria
of genus Pseudomonas and considered to be cosmopolitan in environment
(Johansen et al., 1996) having multiple metal and antibiotic resistance (Oyetibo et
al., 2010). The genus Pseudomonas is Gram-negative, rod-shaped bacterium,
strict aerobic with unipolar flagella motility ((Forbes et al., 2007).
Pseudomonas aeruginosa is an ubiquitous environmental species widely
spread in soil and natural water, and an opportunistic human pathogen responsible
for severe infections in immune-compromised patients. This organism infects the
pulmonary tract, urinary tract, burns, wounds, and also cause blood infections.
Due to its large occurrence in hospital water systems and its capacity to persist on
medical devices, Pseudomonas aeruginosa is a leading cause of hospital acquired
pneumonia. In addition, P. aeruginosa is the major pathogen in cystic fibrosis
(CF) lung pathology (Lipuma, 2010).
Heavy metals are ubiquitous and persistent environmental pollutants that are
introduced into the environment through anthropogenic activities, such as mining
and smelting, as well as through other sources of industrial waste. In fact, over
one-half the superfund sites in the United States are contaminated with at least
one heavy metal. Heavy metals contaminate drinking water reservoirs and
freshwater habitats and can alter macro- and microbiological communities (Ivorra
et al., 2000).
Pseudomonas aeruginosa is a ubiquitous, environmentally important microbe
that may employ many resistance mechanisms, such as the mer operon that
reduces toxic Hg2+
to volatile Hg0
, which then diffuses out of the cell (Outten,
2000). However, in bacteria, efflux systems are a more common resistance
mechanism for dealing with heavy metals.
Studies on the effects of heavy metals copper, lead, and zinc on biofilm and
planktonic P. aeruginosa determined that biofilm were 2 to 600 times more

resistance to heavy metal stress than free- swimming cells (Teitzel and Parsek,
2003).
There are many methods used for detection of heavy metals resistance in bacteria.
These methods include agar dilution methods; determination of MIC and MTC of
different heavy metals. Molecular techniques such as the polymerase chain
reaction (PCR), DNA– DNA hybridization and an analysis of restriction fragment
length polymorphism (RFLP) (Nakamura and Silver, 1994) can also be used.
The aim of this work was to study the prevalence of heavy metals resistance
(HMR) in Ps. aeruginosa recovered from clinical and environmental samples, as
well as studying production of the biofilm and its correlation with heavy metals
resistance.
This book is suited for all kinds of students (Undergraduate and postgraduate) and
doesn’t require any prerequisite knowledge of biology or chemistry. If any student
is interested in entering the health care profession in some way, this book will
give him a strong background in the biology of HMR bacteria, without
overwhelming unnecessary details.
1.2. Literatures review
1.2.1. History and description of Pseudomonas aeruginosa:
The opportunistic bacteria pathogen currently known as P. aeruginosa has
received several names throughout its history based on the characteristic blue-
green coloration produced during culture. Sédillot in 1850 was first to observe
that the discoloration of surgical wound dressings was associated with a
transferable agent. The pigment responsible for the blue coloration was extracted
by Fordos in 1860, and in 1862 Lucke was the first to associate this pigment with
rod-shaped organisms (Pitt, 1998).

Pseudomonas aeruginosa was not successfully isolated in pure culture until
1882, when Carle Gessard reported in a publication entitled “On the Blue and
Green Coloration of Bandages” the growth of the organism from cutaneous
wounds of two patients with bluish-green pus (Gessard, 1984). In several
additional reports between 1889 and 1894, P. aeruginosa (Bacillus pyocyaneus)
was described as the causative agent of blue-green purulence in the wounds of
patients (Villavicencio, 1998). A more thorough presentation on the routes of
invasion and dissemination of P. aeruginosa leading to acute or chronic infection
was provided by Freeman in a 1916 article (Philips et al., 2009).
Pseudomonas aeruginosa is a non-fermentative, aerobic Gram negative rod,
measuring 0.5 to 0.8 m by 1.5 to 3.0 m. Almost all strains are motile by means
of a single polar flagellum. It normally lives in moist environments, and uses a
wide range of organic compounds for growth, thus giving it an exceptional ability
to colonize ecological niches where nutrients are limited, from water and soil to
plant and animal tissues. Typical biochemical features of P. aeruginosa isolates
are: positive oxidase test, growth at 42°C, hydrolysis of arginine and gelatine, and
nitrate reduction. P. aeruginosa strains produce two types of soluble pigments,
pyoverdin and pyocyanin. The latter blue pigment is produced abundantly in
media of low-iron content and functions in iron metabolism in the bacterium.
Pyocyanin (from "pyocyaneus") refers to "blue pus", which is characteristic for
suppurative infections caused by P. aeruginosa (Murray et al., 2003).
Pseudomonas aeruginosa also produces pyocin, these substance resembles
antibiotics and have active effect on different strain from the same species or
other species. These substances used in typing of P. aeroginsa and diagnosis of
species (Aboud, 2001).
Pseudomonas aeruginosa is seldom a member of normal microbial flora in
humans. Representative colonization rates for specific sites in humans are 0 to 2%
for skin, 0 to 3.3% for nasal mucosa, 0 to 6.6% for throat, and 2.6 to 24% for fecal

samples (Morrison and Wenzel, 1984). However, colonization rates may exceed
50% during hospitalization
(Pollack, 1995), especially among patients who have experienced trauma to or a
breach in cutaneous or mucosal barriers by mechanical ventilation, tracheostomy,
catheters, surgery, or severe burns (Blanc et al., 1998; Erol, 2004; Ohara, 2003;
Thuong, 2003; and Valles, 2004).
Patients with impaired immunity have higher risks for colonization by this
organism (Morrison, and Wenzel, 1984; Pollack, 1995), and disruption in the
normal microbial flora as a result of antimicrobial therapy has also been shown to
increase colonization by P. aeruginosa (Blane et al., 1998; Bonten et al., 1999;
Takesue et al., 2002).
Despite the wide distribution of P. aeruginosa in nature and the potential for
community-acquired infections, serious infections with P. aeruginosa are
predominantly hospital acquired. A review of surveillance data collected by the
center of disease control and prevention (CDC), National Nosocomial Infections
surveillance system from 1986 to 1998 shows that P. aeruginosa was identified as
the fifth most frequently isolated nosocomial pathogen, accounting for 9% of all
hospital-acquired infection in united states (Emori, and Gaynes, 1993; NNIS,
1998).
Pseudomonas aeruginosa was the second leading cause of nosocomial
pneumonia (14 to 16%), the third most common cause of urinary tract infections
(7 to 11%), the fourth most frequently isolated pathogen in surgical site infections
(8%), and seventh leading contributor to bloodstream infections (2 to 6%). Data
from other studies continue to show P. aeruginosa as the second most common
cause of nosocomial pneumonia, health care-associated pneumonia, and
ventilator-associated pneumonia (Gaynes and Edwards, 2005; Kolleff et al., 2005)
and the leading cause of pneumonia among pediatric patients in the intensive care
unit (ICU) (Richards et al., 1999).

Pseudomonas aeruginosa is especially problematic for seriously ill patients in
ICUs. From 1992 to 1997, data from the National Nosocomial Infection
Surveillance System showed that P. aeruginosa was responsible for 21% of
pneumonias, 10% of urinary tract infection, 3% of bloodstream infections, and
13% of eye, ear, nose, and throat infections within ICUs in the United State
(Richards et al., 1999). A similar study conducted in Europe identified P.
aeruginosa as the second most frequently isolated organism in reported cases of
ICU-acquired infections (Spencer, 1996).
1.2.2. Pathogenicity of Pseudomonas aeruginosa:
Pseudomonas aeruginosa can infect almost any external site or organ. Most
community infections are mild and superficial, but in hospitalized patients,
infections are more common, more severe and more varied.
P. aeruginosa is pathogenic only when introduced into areas devoid of normal
defenses, e.g. when mucous membranes and skin are disrupted by direct tissue
damage; when intravenous or urinary catheters are used; or when neutropenia is
present, as in cancer chemotherapy. The bacterium attaches to, and colonizes the
mucous membranes or skin, invades locally and produces systemic disease
(Sambrook and Rusell, 2001).
These processes are promoted by the Pili, enzymes and toxins.
Lipopolysaccharide plays a direct role in causing fever, shock, oliguria,
leukocytosis and leucopenia, disseminated intravascular coagulation and adult
respiratory distress syndrome (Pollack, 2000).
Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen
implicated in sight-threatening ocular infectious diseases such as keratitis
(Sharma et al., 2006; Willcox, 2007; Green et al., 2008). Pseudomonas keratitis is
a serious ocular infection that can lead to corneal scarring and severe visual
disability if aggressive and appropriate therapy is not promptly initiated (Keay et
al., 2006; Stapleton et al., 2007).

Pseudomonas aeruginosa is a cause of community-acquired pneumonia (CAP)
and most of nosocomial infections are ascribed to this organisms, as most of the
hospital environments are colonized by it (Hatchette et al., 2000).
Bacteremia and septic shock due to P. aeruginosa continue to be the major
problems in hospitalized patients with underlying malignancies, cardiopulmonary
disease, renal failure or diabetes. Bacteremia due to Pseudomonas aeruginosa in
intravenous drug users is usually associated with bacterial endocarditis and may
also develop osteomyelitis of a variety bones (Boffi et al., 2001).
For an opportunistic pathogen such as P. aeruginosa, the disease process
begins with some alteration or circumvention of normal host defenses. The
pathogenesis of Pseudomonas infections is multifactorial, as suggested by the
number and wide array of virulence determinants possessed by the bacterium.
Most Pseudomonas infections are both invasive and toxinogenic. The ultimate
Pseudomonas infection may be seen as composed of three distinct stages: (1)
bacterial attachment and colonization; (2) local invasion; (3) disseminated
systemic disease (Brooks et al., 2010).
1- Colonization:
Although colonization usually precedes infections by P. aeruginosa, the exact
source and mode of transmission of the pathogen are often unclear because of its
ubiquitous presence in the environment. It is sometimes present as part of the
normal flora of humans, although the prevalence of colonization of healthy
individuals outside the hospital is relatively. The pili of P. aeruginosa will adhere
to the epithelial cells of the upper respiratory tract and, by inference, to other
epithelial cells as well. These adhesins appear to bind to specific galactose or
mannose or sialic acid receptors on epithelial cells. Colonization of the respiratory
tract by Pseudomonas requires pili adherence and may be aided by production of
a protease enzyme that degrades fibronectin in order to expose the underlying
pilus receptors on the epithelial cell surface. Mucoid strains, which produce an

exopolysaccharide (alginate), have an additional or alternative adhesin which
attaches to the tracheobronchial mucin. Also, it is possible that surface-bound
exoenzyme S could serve as an adhesin for glycolipids on respiratory cells.
Alginate slime forms the matrix of the Pseudomonas biofilm which anchors the
cells to their environment and in medical situations; it protects the bacteria from
the host defenses such as lymphocytes, phagocytes, the ciliary action of the
respiratory tract, antibodies and complement. Mucoid strains of P. aeruginosa are
most often isolated from patients with cystic fibrosis and they are usually found in
lung tissues from such individuals (Brooks et al., 2010).
2-Invasion:
The ability of P. aeruginosa to invade tissues depends upon production of
extracellular enzymes and toxins that break down physical barriers and damage
host cells, as well as resistance to phagocytosis and the host immune defenses. P.
aeruginosa produces three other soluble proteins involved in invasion: a cytotoxin
and two hemolysins. The cytotoxin is a pore-forming protein. It was originally
named leukocidin because of its effect on neutrophils, but it appears to be
cytotoxic for most eukaryotic cells. Of the two hemolysins, one is a phospholipase
and the other is a lecithinase. They appear to act synergistically to break down
lipids and lecithin. The cytotoxin and hemolysins contribute to invasion through
their cytotoxic effects on neutrophils, lymphocytes and other eukaryotic cells
(Brooks et al., 2010).
3-Dissemination:
Blood stream invasion and dissemination of Pseudomonas from local sites of
infection are probably mediated by the same cell-associated and extracellular
products responsible for the localized disease, although it is not entirely clear how
the bacterium produces systemic illness. P aeruginosa is resistant to phagocytosis
and the serum bactericidal response due to its mucoid capsule and possibly LPS.

The Lipid A moiety of Pseudomonas LPS (endotoxin) mediates the usual
pathologic aspects of Gram-negative septicemia, e.g. fever, hypotension,
intravascular coagulation, etc. It is also assumed that Pseudomonas exotoxin A
exerts some pathologic activity during the dissemination stage (Brooks et al.,
2010).
1.2.3. The Antigenic Structures:
Somatic antigen: There are seventeen somatic antigens designated as O1 to O17
that divide the strains of P. aeruginosa into 17 serogroups.
Flagellar antigen: Two flagellar antigens have been isolated from strains of P.
aeruginosa. One of these is uniform and the other is complex and may consist of
5- 7 factors (Brooks et al., 2010). Flagellum yields heat–labile antigens (H–
antigen).Consequently it is not specific (Liu, 1974).
1.2.4. Antibiotic resistance:
Pseudomonas aeruginosa is pathogen that causes a substantial portion of
hospital infections. It is frequently multi resistant, which contributes to the high
morbidity and mortality of patients in intensive care units (ICUs), burn units and
surgery wards. A major reason for its prominence as a pathogen is its high
intrinsic resistance to antibiotics, such that even for the most recent antibiotics
(Fluit et al., 2000).
Its high intrinsic resistance to antibiotics and ability to develop multidrug
resistance pose serious therapeutic problems. Broad-spectrum B-lactams, such as
carbapenems, are potential drugs for the therapy of infections caused by P.
aeruginosa (Aloush, 2006; Rossolini, 2005), However, the increasing use of these
compounds has resulted in the emergence of carbapenem- resistant P. aeruginosa
isolates, limiting treatment options (Falagas et al., 2006; Zavascki et al., 2005).
Most carbapenem resistance is due to impermeability, which arises via loss of
the OprD (D2) porin, but carbapenem hydrolysing metallo-B-lactamases (MBLs)
are increasingly reported (Livermore et al., 2000).

Genes encoding MBLs are located as cassettes in integrons that provide them
with the potential for expression and dissemination (Walsh et al., 2005; Queenan,
2007).
Nosocomial isolates of P. aeruginosa are found in high proportion of resistance
to a new advanced–generation Fluorquinolone with a high potency and a broad
spectrum of antimicrobial activity, in European hospitals (Brisse et al., 2000).
A study at the Centers for Disease Control and prevention revealed that
resistance to Imipenem for P. aeruginosa isolated from the respiratory tract of
patients in (ICUs) and in teaching hospitals has increased (Pierson and Friedman,
1992; Cavallo et al., 2000).
Pseudomonas aeruginosa showed higher levels of resistance to gentamicin and
imipenem. Rossolini reported that P. aeruginosa isolates were highly resistant to
carbapenems (imipenem and meropenem) and to carbenicillin, ticarcillin,
clavulanate, piperacillin, tazobactam, mezlocillin, ciprofloxacin, gentamicin,
tobramycin, netilmicin, ceftazidime and cefeprime (Rossolini et al., 2005).
Anti Pseudomonal antibiotics include the third-generation cephalosporins
(cefoperazone, cefsulodin, ceftazidime), fourth-generation cephalosporins
(cefepime, cefpirome, cefclidin), extended–spectrum penicillins (ticarcillin,
piperacillin, azlocillin), monobactams (aztreonam), carbapenems (imipenem,
meropenem) and Quiolones (ciprofloxacin, enoxacin, ofloxacin). The addition of
beta–lactamase inhibitors to extended–spectrum penicillins and cephalosporins
has expanded the antibacterial spectra of these agents against most of gram–
positive and gram–negative organisms (Wateret and Wunderink, 2001).
1.2.5. Occurrence of Heavy metals in the Environment:
Metal pollutants pose a severe threat to ecological system due to their negative
impact on most life forms. Though some heavy metals are essential trace
elements, most can be, at high concentrations, toxic to all forms of life, including
microbes, by forming complex compounds within the cell. The heavy metal

protagonists have been divided into three main groups: (i) micronutrient cations
(copper, cobalt, nickel, zins), (ii) toxic cations (cadmium, lead, mercury), and (iii)
toxic oxyanions (arsenate/arsenite, chromate and tellurite) (Aguilar et al., 2010).
Although, higher organisms readily succumb to the toxic influence of metals,
microbes are known to possess a wide array of genetic composition that allows
them to circumvent metallic stress (Valdman et al., 2001).
Since heavy metals are increasingly found in microbial habitats due to natural
and environmental processes, microbes have evolved several mechanisms to
tolerate the presence of heavy metals (Adrash et al., 2007).
This increasing heavy metal tolerance has another implication in the
environment as it may contribute to the maintenance of antibiotic resistance gene
by increasing the selective pressure of the environment. The occurrence of
multiple metals and antibiotic resistance property in microbial community poses a
potential threat towards human and environmental health. There is concern that
metal contamination functions as a selective agent in the proliferation of antibiotic
resistance. There is substantial overlap between known mechanisms for metals
and antibiotic resistance, such as those for copper and tetracyclines, copper and
ciprofloxacin, and arsenic and -lactamse (Baker-Austin et al., 2006).
Many researchers have speculated and have even shown that a correlation exists
between metal tolerance and antibiotic resistance in bacteria (Table 1-2) (Oyetibo
et al., 2010; Abd et al., 2011; Al- Imran, 2006).

Table 1-2: Different inheritable shared mechanisms of metal and antibiotic resistance systems
in Pseudomonads and other prokaryotes (Sarma et al., 2010).
Microbial species, such as Pseudomonas, have been shown to be relatively
efficient in bioaccumulation of uranium, copper, lead, and other metal ions from
polluted effluents (Mullen et al., 1989; Gupta et al., 2001).
Environmental pollution with toxic heavy metals is spreading throughout the
world along with industrial progress. The important toxic metals cadmium, nickel
and lead find its way to the water bodies through wastewater (Ajmal et al., 1998).
Hazardous characteristics of the pollutants cause renal dysfunction, bone
degeneration, liver, lungs and blood damage (Ebdon et al., 2001).
Cadmium is the most dangerous metal ion for human health due to its hazardous
characteristics and non biodegradability. It has been reported that cadmium
damages the cells by a broad spectrum of effects metabolism. It is known to bind
with essential respiratory enzymes (Nies, 2003), cause oxidative stress
(Banjerdkji et al., 2005), and inhibits DNA repair (Jin et al., 2003).

Nickel is the most abundant heavy metal contaminants of the environment due
to its release during mining and smelting practices (Prasad and Strazalka, 2000).
Nickel is required as an essential co- factor in several bacterial enzymes, which
carry out metabolic functions (Mulrooney and Hausinger, 2003). But it disrupts
processes when it is present in excess (Babich and Stotzky, 1983).
Lead is hazardous waste and is highly toxic to human, plants, animals (Low et
al., 2000). In human chronic lead exposure produces neurotoxicity, anaemia, and
kidney damage and acute lead toxicity can be fatal (Rensing et al., 1998).
The known mechanisms of heavy metal toxicity include inducing oxidative
stress and interfering with protein folding and function (Nies, 1999). Chemical
and physical weather of igneous and metamorphic rocks and soils often release
heavy metals into the sediment and into the air (Moore and Ramamoorthy, 1984).
Other contributions include the decomposition of plant and animal detritus,
precipitation or atmospheric deposition of airborne particles from volcanic
activity, wind erosion, forest fire smoke, plant exudates, and oceanic spray
(Kennish, 1992).
1.2.6. Bacterial tolerance against heavy metals:
Bacteria, being one of the most primitive life forms on earth, naturally
developed tolerance to wide range of toxic heavy metals including As, Cd, Co,
Cu, Hg, Ni ,Sb , Te, Zn (Silver and Walerhaug 1992; Silver and Ji 1994) in its
genome.
Copper, nickel, cobalt and iron are essential to bacteria because of their use as
catalytic and structural elements in enzymes and other molecule (Cobine et al.,
1999). In many cases, the first response toxic metal contamination is a large
reduction in microbial activity (Pennanen et al., 1996).
Microbes may play a large role in biogeochemical cycling of toxic heavy metals
also in cleaning up or remediating metal-contamination environments.

Bacteria have developed a variety of resistance mechanisms to counteract
heavy metal stress. These mechanisms include the formation and sequestration of
heavy metals in complexes, reduction of a metal to a less toxic species, and direct
efflux of a metal out of the cell (Nies, 1999; Outten et al., 2000).
Pseudomonas aeruginosa is a ubiquitous, environmentally important microbe
that may employ many resistance mechanisms, such as the mer operon that
reduces toxic Hg2+
to volatile Hg0
, which then diffuses out of the cell (Outten et
al., 2000). However, in bacteria, efflux systems are a more common resistance
mechanism for dealing with heavy metals (Figure 1-1).
Figure 1-1. Model showing examples of three major molecular mechanisms responsible for
dual tolerance in pseudomonads.
(i) Co-resistance towards Ni and ampicilin due to physical linkage of resistance determinants for the two
antimicrobial factors on pBC15 plasmid (Raja and Selvam, 2009). (ii) Cross resistance due to presence
of a membrane-bound DsbA–DsbB disulfide bond formation system that confers resistance to five
antibiotics accompanied by two metals (Hayashi et al., 2000), (iii) Co-regulatory resistance due to the
linkage of mex and czc operons leads to expression of metal efflux and imipenem resistance (Perron et
al., 2004).

One such system is the cop system of Pseudomonas syringae, which contains
the structural genes copABCD and is homologous to the pco system in
Escherichia coli. The copB and copD genes are involved in the transport of
copper across the membrane, while the products of the copA and copC genes are
outer membrane proteins that bind Cu2+
in the periplasm, protecting the cell from
copper (Cooksey, 1994; Silver, 1996). Other types of efflux systems simply
pump toxic metal ions out of the cell; these systems include the P-type ATPase
cadA, which was first identified in Staphylococcus aureus and is found in other
gram-positive bacteria, that pumps out Cd2+
and Zn2+
by using a phospho-
aspartate intermediate (Nies, 1999; Nusifora, 1989; Silver, 1996).
The genetics and biochemistry of heavy metal resistance mechanisms have been
carefully studied in free-swimming organisms; however, many bacteria in the
environment exist in surface-attached communities called biofilms. Biofilm
bacteria are usually embedded in an extracellular polymeric substance (EPS)
matrix composed of polysaccharides, proteins, and nucleic acids (Flemming et al.,
2001; Platt, 1985; Sutherland, 2001; Whitchurch et al., 2002, Whitfield, 1988).
Exposures have been shown to increase the incidence of bacterial antibiotic co-
resistance through the transfer of genetic elements containing both metal and
antibiotic resistance genes (Ugur et al., 2003). also through the selection of
organism that contain elements, such as non specific efflux pumps, which can
convey cross- resistance to both metals and antibiotic (Summers, 2002).
Active efflux from either the cytoplasm or the periplasm is well-known as a
major mechanism of bacterial heavy metal resistance (Nies, 2007; Summers,
2009).
1.2.7. Beneficial heavy metals:
Metals play a vital role in biological systems as a living cell cannot exist
without metals ions. It's estimated that over 50% of all protein are metalloprotiens
and that about a third of all structurally characterized proteins contain metals
(Degtyarenko, 2000). Copper, nickel, cobalt and iron are essential to all

organisms because of their use as catalytic and structural elements in enzymes and
other molecules (Cobine et al., 1999). Trace amounts of some heavy metals are
also required by living organisms, including, manganese, molybdenum, nickel,
vanadium, strontium and zinc (Gort et al., 1999).
Excessive levels of essential metals, however, can be high toxic to the organism
(Gadd, 1992; Franke et al., 2003). Essential heavy metal ions present a dual
challenge to both eukaryotic and prokaryotic cells in that they are useful but can
also be lethal, Therefore, a cell must meet its physiological requirement for
essential metal ions while prevention their deleterious effect (Adalkkalam and
swarup, 2002).
1.2.8. Toxic heavy metals:
Some of the heavy metals are purely toxic with unknown cellular role (Shi et al.,
2002). Other metals essential for life at low concentration but become toxic at
high concentration, (Badar et al., 2000). High concentration of all the heavy
metals Fe, Mo, Mn are classified to have low toxicity, Zn, Ni, Cu, V, Co, W, Cr,
are categorized to have average toxicity, while As, Ag, Sb, Cd, Hg, Pb, U, are
grouped in high toxic heavy metals (Nies, 1999).
Wide range of essential cell components is potential targets for metal induced
damage such as DNA for replication as a result of which cell death can occur
(Malakul et al., 1998).
Slightly elevated metal levels in natural waters may cause the following sub lethal
effects in aquatic organism:
1- Histological or morphological change in tissue.
2- Change in physiological, such as suppression of growth and development, poor
swimming performance, change in circulation.
3- Change in biochemistry, such as enzyme activity and blood chemistry.
4- Change in behavior.
5- Change in reproduction (Connell and Miller, 1984).

1.2.9. Review of some important heavy metals:
1.2.9.1. Copper:
Copper is an essential metal, mainly required by aerobic cells as a factor for
electron transport and redox enzyme system (Rensing, C. and Grass, 2003). It is
used as a biocide for centuries (Dollwet et al., 2001).
In ancient Egypt (2000 BC), copper was used to sterilize water and wounds.
The ancient Greeks in the time of Hippocrates (400 BC) prescribed copper for
pulmonary diseases and for purifying drinking water. Gangajal, “Holy water”,
given to Hindu devotees to drink as a blessed offering, is stored in copper utensils
as it keeps the water sparkling clean.
During the Roman Empire, copper cooking utensils were used to prevent the
spread of disease. The early Phoenicians nailed copper strips to ships’ hulls to
inhibit fouling and thus increase speed and maneuverability. The Aztecs used
copper oxide and malachite for treating skin conditions. Early American pioneers
moving west across the American continent put copper coins in large wooden
water casks to provide safe drinking water for their long journey.
By the 18th century, copper had come into wide clinical use in the Western
world in the treatment of lung and mental disorders. In the Second World War,
Japanese soldiers put pieces of copper in their water bottles to help prevent
dysentery. Copper sulphate was (and is still) highly prized by some inhabitants of
Africa and Asia for healing sores and skin diseases.
All these civilizations right in using copper for the above mentioned purposes.
The following sections review scientific studies demonstrating the potent biocidal
properties of copper and copper compound and their current uses in health related
application. Interestingly, bacteria exposed to metallic copper surfaces do not
enter a viable but on culturable physiological state, in which they are viable but
do not multiply, but are completely inactivated (Wilks et al., 2006).
Importantly, based on the vast amount of antimicrobial efficacy testing (180 tests,
utilizing 3235 control and test samples, conducted in independent microbiology

laboratories) sponsored by the Copper Development Association (CDA), the U.S.
Environmental Protection Agency (EPA) has recently (March 2008) approved the
registration of copper alloys as materials with antimicrobial properties, thus
allowing the CDA to make public health claims (Copper Development
Association. 2008).
The following statement is included in the registration: "When cleaned
regularly, antimicrobial copper alloys surfaces kill greater than 99.9% of bacteria
within two hours, and continue to kill more than 99% of bacteria even after
repeated contamination". These public health claims acknowledge that copper,
brass and bronze are capable of killing harmful, potentially deadly bacteria, such
as Methicillin-resistant S. aureus (MRSA). MRSA is one of the most virulent
strains of antibiotic-resistant bacteria and a common cause of hospital and
community-acquired infections. Copper is the first solid surface material to
receive this type of EPA registration ( Borkow and Gabbay, 2008).
1.2.9.2. Silver:
Ionic silver (Ag ions) is considered to be effective against a broad range of
micro-organisms, with low concentrations documented to have therapeutic
activity (Russell et al., 1994; Lansdown et al., 1997).
Silver has been described as being ‘oligodynamic’ because of its ability to exert
abactericidal effect at minute concentrations (Clement et al., 1994).
Consequently, a large number of healthcare products now contain silver,
principally due to its antimicrobial activities and low toxicity to human cells.
Such products include silver-coated catheters
(Sampath et al., 1994; Dasgupta, 1994), municipal water systems (Chambers et
al., 1962; Kool et al., 1999) and wound dressings (Adams et al., 1999).
Wounds often provide a favorable environment for the colonization of
microorganisms (Chambers et al., 1962; Adams, 1999; Bowler and Davies 1999).

In order to improve the opportunity for wound healing, it is important to create
conditions that are unfavorable to micro-organisms and favorable for the host
repair mechanisms, and topical antimicrobial agents are believed to facilitate this
process. Antiseptic agents are now considered for the treatment of localized skin
and wound infections because they have a lower propensity to induce bacterial
resistance than antibiotics. One example of the early use of silver in wound care is
silver sulphadiazine (AgSD) cream, developed in the 1960s, for the treatment of
burns.
Recently, a trend towards the use of wound cover dressings that contain
silver has been evident, and today, a selection of foam, film, hydrocolloid, gauze
and dressings with Hydrofiber technology impregnated with silver are
commercially available. However, concerns are being expressed regarding the
overuse of silver and the possible emergence of bacterial resistance to silver,
particularly within the clinical environment (Pirnay et al, 2003; Gupta et al.,
2001).
Silver-resistant bacteria have been reported since 1975 (McHugh et al., 1975;
Deshpande and Chopade, 1994) and research within this area is clearly increasing
(Silver et al., 2003).
A preliminary understanding of the genetics underlying silver resistance has
been known since 1998 (Gupta and Silver, 1998, Silver et al., 1999) with a greater
understanding of the biochemistry documented a year later (Gupta A., et al.,
1999).
Clinical evidence of silver-resistant bacteria has been principally in hospitals,
specifically in burns wards, where silver salts (in the form of silver nitrate) are
used as antiseptic agents (Mchugh et al., 1975; Klasen, 2000).
Many clinicians and researchers have questioned whether the widespread usage
of silver could lead to cross-resistance to antibiotics, as has been suggested with a
number of biocides, specifically triclosan, chlorhexidine and quaternary
ammonium compounds (QACs) (Aiello and Larson 2003; Russell, 2003)

However, in reference to the available evidence to date, this appears to represent
an unjustifiable concern.
Silver Nitrate has antiseptic properties. A very dilute solution is sometimes
dropped into newborn babies' eyes at birth to prevent contraction of Neisseria
gonorrhoea or Chlamydia from the mother.
1.2.9.3. Mercury:
Mercury (Hg), the heavy metal cation with the strongest toxicity, it is affinity to
thiole groups is even stronger than the affinity of cadmium for sulfide; the
solubility product of Hgs is 6.38 x 10.53 (Weast, 1984).
Consequently, it is the most toxic of all tested elements for Escherichia coli, the
MIC was 10 mM (Mergeay et al, 1985).
Because of high toxicity mercury has no beneficial function. However, since
bacteria may be confronted with toxic Hg2+
with high probability, mercury
resistance determinants, Mer, are very wide spreading (Hobman and Brown 1997;
Reniero et al., 1998; Smith et al., 1998).
Resistance to mercury is based on unique peculiarities of mercury; it is redox
potential and the vapour pressure/ melting/ boiling point of metallic mercury
which extraordinary low for a metal, melting point
-38. 87o
C, boiling point 356.56o
C (Weast, 1984).
Thus, living cells are able to reduce Hg to the metal, and this metal does not
remain inside the cell with the potential to become oxidized again, but it leaves
the cell by passive diffusion (Silver and Phung 1996; Silver 1996), metallic
mercury may also oxidizing again by bacteria (Smith, et al., 1998).
In Gram negative bacteria, the periplasmic Hg2+
binding protein Mer p binds the
cation as the first step of detoxification (Qian et al., 1998).
Mer P probably delivers the toxic cation to the mercury transporter Mer T which
transports the cation into the cytoplasm (Hobman and Brown, 1996).

Alternatively or in addition to Mer PT, an alternative up take route exists which
involves the Mer C protein ( Hamlett et al., 1992; Sahlman et al., 1997).
Once inside the cell, Hg is reduced by NADPH- depended to Hg(0) by Mer A
protein which is related to glutathione reductase and other proteins (Schiering et
al., 1991).
Mercury resistance is tightly regulated, mainly by the Mer R protein (Brunker et
al., 1996; Hobman and Brown 1997; Yurieva et al., 1997).
This protein binds as a dimer to the Mer promoter and inhibits transcription
initiation without Hg2+
.
1.2.9.4. Leads:
Lead (Pb) has been used in high amount since 2500 years (Hong et al., 1994),
e.g. as fuel ad dictum, although lead toxicity is well known for a long time
(Johnson, 1998). In sea water, it is even rare than mercury (Weast, 1984). Due to
it is low solubility, especially lead phosphate is insoluble with a solubility product
of 10-54; it is biologically available concentration is low. Therefore; it is MIC for
Escherichia coli is only 5 mM (Mergeay et al., 1985). Thus, lead is not
extraordinary toxic for microorganism, only it is excessive use by man makes lead
problem. There is no molecular information about uptake of lead. Lead tolerant
bacteria have been isolated (Trajanovska et al., 1997), and precipitation of lead
phosphate within the cells of these bacteria has been reported (Levinson et al.,
1996; levinson and Mahler 1998). The only information about molecular biology
of any lead resistance comes from Ralstonia eutropha CH34. It has been shown
that resistance to lead in this bacterium is mediated by a P-type ATPase
(Borremans et al., 2001). Thus, lead resistance may also be based predominantly
on metal ion efflux.

1.2.9.5. Zinc:
Zinc (Zn) is another essential trace element. It is not biologically redox reactive
and is thus not used in respiration. It is, however, important in forming complexes
(such as zinc fingers in DNA) and as a component in cellular enzymes (Nies,
1999). Bacterial cells accumulate zinc by a fast, unspecific uptake mechanism and
it is normally found in higher concentrations (but is less toxic) than other heavy
metals (Nies, 1999). Uptake of zinc ions is generally coupled to that of
magnesium, and the two ions may be transported by similar mechanisms in
bacteria (Nies and Silver, 1995). Industrial plants producing metallic zinc by
roasting or heating are always potential sources of environmental zinc (Diels and
Mergeay, 1990).
Zinc is component in such a variety of enzyme and DNA – binding protein in
like zinc finger proteins, which also exist in bacteria (Chou et al., 1998), that life
seems to be not possible without zinc. The toxicity of zinc to Escherichia coli is
similar to the toxicity of copper, nickel and cobalt, the MIC was 1Mm (Mergeay
et al., 1985). The P- type ATPase transport zinc only across the cytoplasmic
membrane, the RND-systems are hypothized to efflux a cross the complete cell
wall of Gram-negative bacteria the first of these systems was cloned as the
Cobalt-zinc-cadmium resistance CZC from Ralstonia eutrophus CH34 (Mergeay,
et al.1985; Nies et al., 1987). Resistance is based on energy-depended metal ion
efflux (Nies et al., 1989b).
1.2.9.6. Cadmium:
Cadmium is extremely toxic for all living organisms even when present at a low
concentration. It has been reported that Cd2+
damages the cells by a broad
spectrum of effects on cell metabolism for instances by binding to essential
respiratory enzymes (Nies, 2003), inducing oxidative stress (Banjerdkij et al.,
2005) or inhibiting DNA repair (Jin et al., 2003).

Cadmium ions can easily enter bacterial cells by the transport systems for
essential divalent cations such as Mn+2
(Tynecka et al., 1981) or Zn ions (Laddaga
and Silver, 1985), so almost all prokaryotes and eukaryotes have developed
mechanisms to prevent excessive accumulation of Cd2+
in the cells. The relatively
low intracellular Cd2+
concentration is maintained through the regulation of
sequestration or efflux. Microbial resistance to Cd2+
is usually based on energy
dependent efflux mechanisms (Silver, 1996; Nies, 2003).
A distinct Cadmium resistance mechanism reported in Pseudomonas involves
Cadmium – binding proteins called Pseudothioneins (PT) (Higham et al., 1986).
Three major groups are responsible for this cadmium efflux: CBA-type
chemiosmotic antiporters, P-type ATPases and cation diffusion facilitators (CDF).
The CBA-type anti porters are protein complexes that are able to span the
complete cell wall of a gram-negative bacterium, while CDF proteins and P-type
ATPases are located in the cytoplasmic membrane and are single-subunit systems
(Aguilar et al., 2010).
1.2.9.7. Nickel:
Nickel (Ni) is widely distributed in nature and 85% of the earth crust is nickel
(Carson et al., 1986). Nickel is used in production of stainless steel, as a chemical
catalyst in electroplating, ceramics, pigments, batteries and coins (IARC, 1990).
Nickel is classified as a borderline metal ion because it has both soft and hard
metal properties and can bind to sulfur, Nitrogen, and oxygen groups (Moore and
Ramamoothy, 1984b). In many bacteria nickel is required for enzyme such as
urease, dehydrogenase, hydrogenase (Van valiet et al., 2001). However excess
nickel is toxic, nickel binds to protein and nucleic acid and frequently inhibits
enzymatic activity, DNA replication transcription and translation (Grosse et al.,
1999).

Nickel occurs mostly in Ni (II) cation form, Ni (III) is unstable (Weast, 1984).
The MIC of Ni2+
to Escherichia coli is 1mM (Mergeay et al., 1985).
Accumulation of Ni2+
as a resistance strategy has been reported in strains of P.
aeruginosa (Gupta. et al., 2004), but the mechanisms involved have not yet been
studied (Aguilar et al., 2010).
1.2.10. Health application of heavy metals:
Heavy metals, particularly silver and mercury, have a variety of applications in
controlling microbial population (Kenneth and Jeffery 2006). Silver salts alone or
in combination with other drugs appear to have a significant potential as topical
antimicrobial agent (Fox, 1977; de gracia, 2001). Mercury in the form of less
toxic organic compounds is being used as skin disinfectant (Gerald, 2007).
Copper is considered safe to humans, as demonstrated by the widespread and
prolonged use by women of copper intrauterine devices (Anonymous, 2002;
O'Brien, 2008). The risk of adverse reactions due to dermal contact with copper is
considered extremely low (Hostynek, 2003; Gorter, 2004).
Copper is an essential trace element involved in numerous human physiological
and metabolic processes (Olivares and Uauy 1996, Uauy and Olivares, 1998),
including in wound repair (Borkow and Gabbay 2008), and many over-the-
counter treatments for wound healing contain copper (Pereira and Felcman,
1998). The National Academy of Sciences Committee established the U.S.
Recommended Daily Allowance of 0.9 mg of copper for normal adults (Chen et
al., 2008).
Copper exerts its toxicity to microorganisms through several parallel
mechanisms, which eventually may lead to the microorganisms’ death even
within minutes of their exposure to copper (Ohsumi et al., 1988; Espirito et al.,
2008; Borkow et al., 2007). Harrison et al., (2008) identified that Cu2+
works
synergistically with quaternary ammonium compounds (QACs); specifically
benzalkonium chloride, cetalkonium chloride, cetylpyridinium chloride,

myristalkonium chloride, and Polycide) to kill P. aeruginosa biofilms. In some
cases, adding Cu2+
to QACs resulted in a 128-fold decrease in the biofilm
minimum bactericidal concentration.
1.2.11. Detection of heavy metals resistance in laboratory
1.2.11.1. Phenotypic characteristics:
There are many methods used for detection of heavy metals resistance in
bacteria. These methods include agar dilution methods and pouring methods,
determination of MIC and maximal tolerant concentrations MTC of different
heavy metals.
Regarding of resistance to cadmium, Hu and Zhao, (2007) studied
determination of cadmium resistance in P. Putida CD2 using maximal tolerant
concentrations (MTC) method, and they found that the MTC was the highest
metal concentration at which the growth of bacteria was still observed.
Raja et al., (2009) studied resistance of P. aeruginosa to Nickel, Chromium,
and lead, and they found that Pseudomonas BC15 was capable of absorbing 93%
Ni, 65% Pb, 50% Cd and 30% Cr within 48 h from the medium containing 100
mg of each heavy metal per liter. The multiple metal tolerance of this strain was
also associated with resistance to antibiotics such as ampicillin, tetracycline,
chloramphenicol, erythromycin, kanamycin and streptomycin.
Sevgi et al., (2010) studied resistance and determined the (MTCs) of six heavy
metals, Cr, Cu, Ni, Co, Cd, and Zn, and they found that Pseudomonas spp.
Revealed that resistance to Cr was found in 73.9% of the bacterial strains isolated
from soil samples, whereas 26% of the isolates exhibited resistance to Ni, 18.4%
to Zn, 11.5% to Cd, 9.2% to Co, and 7.3% to Cu. Observed maximum MTCs
were 5 mM for Zn and Cu, 3 mM for Ni, and 2 mM for Cr.

In a study undertaken to examine the effects of heavy metals arsenic, silver,
nickel, cobalt, cadmium, lead and mercury on P. aeruginosa isolated from
infected wounds using an agar dilution method, Prasad et al., (2009) found that P.
aeruginosa strains were highly susceptible to heavy metals ions and two strains
were found to be resistant against silver ions isolated from burns infections.
Regarding resistance of Pseudomonas spp. to chromium, copper, nickel, and
cadmium, Singh et al., (2010), studied the tolerance of Pseudomonas spp. isolated
from sewage of industrial effluents from waste water treatment plant, and they
found that all the isolates exhibited high resistance to heavy metals with minimum
inhibitory concentration (MIC) for heavy metals ranging from 50g/ml to
350g/ml. They also revealed that the isolates showed multiple tolerances to
heavy metals and the heavy metal tolerance test indicated maximum microbial
tolerance of Pseudomonas spp. to copper (300 g/ml) and lo west to Chromium
(60 g/ml) .
Abdelatey et al., (2011) studied resistance and screening for heavy metal
tolerance, they isolated and characterized some bacteria from an Egyptian soil.
Three Gram positive isolates were identified as Staphylococcus aureus, Bacillus
subtilis, Bacillus cereus and two Gram negative isolates were identified as
Pseudomonas spp. and Bordetella spp. Both Gram positive and Gram negative
bacteria. Showed the metal tolerance against Cd2+
and Co2+
at different
concentrations ranging from 0.0- 1.0 mg L-1
and determined that metals tolerance
in all bacteria showed the higher sensitivity to Cd2+
more than Co2+
.
1.2.11.2. Genotypic characteristics:
Investigations of adaptive responses commonly involve studying phenotypic
changes. However, a more basic understanding of adaptation is possible if the
molecular mechanisms of resistance are also characterized. Approaches that can
be used include the use of molecular techniques such as the polymerase chain
reaction (PCR), DNA– DNA hybridization and an analysis of restriction fragment

length polymorphism (RFLP) (Barkay et al., 1985; Diels and Mergeay, 1990;
Rochelle et al., 1991; Nakamura and Silver, 1994).
These techniques are, in general, more sensitive and rapid than some of the
traditional methods. A significant advantage is that these approaches can be aimed
precisely at a particular genetic determinant and thereby may provide a useful
means of investigating bacterial responses to environmental stress and reveal the
molecular mechanisms of adaptation (Abou-Shanab et al., 2007).
Exogenous plasmid isolation has been used to detect heavy metals resistance
genes to mercury in soil bacteria. This method allows capture of plasmids from
the total bacterial fraction of an environmental sample without the necessity to
culture the host organism (Smit et al., 1998). They also identified plasmids of
10−50 kb carrying resistance to copper, streptomycin and chloramphenicol by
amended soil with mercuric chloride and found this to subsequently increase the
recovery of resistance plasmids. Plasmids carrying resistance to mercury have
also been captured from polluted soils and slurries (Top et al., 1994; Smalla et al.,
2000).
In addition to multi-resistance plasmids, resistance genes are situated on
transposable elements that can associate with other elements such as
chromosomes and plasmids. These transposable elements include transposons and
integrons, which can be transferred horizontally. Integrons are thought to play an
important role in the evolution of resistance as they contain mobile gene cassettes
which bear a recombination site known as a 59-base element (59-be) that is
recognized by the integron encoded integrase IntI most cassette genes described
are antibiotic or quaternary ammonium compound resistance genes (Hall et al.,
1991).
Jaysankar et al., (2008) used the biochemical and 16S rRNA gene sequence
analyses and they revealed the role of Pseudomonas aeruginosa and other
bacteria like Bacillus spp. to detoxification efficiency for Hg, Cd, and Pb
indicates good potential for application in bioremediation of toxic heavy metals.

In a study for determination of heavy metals resistance (Cd), (Mn), (Zn), (Cu),
(Pb), (Co) using Pseudomonas aeruginosa strain E1, Xiao-xi et al., (2009) used
16S rDNA sequence analysis to identified strain E1, and they found that strain
E1was resistant to heavy metals and the order is found to be
Cd Mn Zn Cu Pb Co in solid media. Strain E1 also exhibits the
resistance to 12 antibiotics (Xiao-xi et al., (2009).
Sobral et al., (2012) used multiple locus variable number of tandem repeats
(VNTR) analysis (MLVA) has been shown to provide a high level of information
for epidemiological investigations of Pseudomonas aeruginosa chronic infection.
In this study, an automatized MLVA assay has been developed for the analysis of
16 VNTRs in two multiplex polymerase chain reactions (PCRs), they used
MLVA-16Orsay scheme to identify the genotyping of 83 isolates from eight
cystic fibrosis patients, demonstrating that the same genotype persisted during
eight years of chronic infection in the majority of cases.
De la Iglesia et al., (2010) used PCR primer pair to specifically detect copper
P-type ATPases gene sequences. And new copper P-type ATPases gene
sequences were found, and a high degree of change in the genetic composition
because of copper exposure was also determined.
Abdelatey et al., (2011) studied a number of bacteria isolated and
characterized from an Egyptian soil. The semi-quantitative reverse transcription-
PCR (RT-PCR) was used to investigate the gene expression mechanism
responsible for the metal resistance in some of these gram positive and gram-
negative bacteria that were, highly resistant to Co2+
and Cd2+
. The mer, chr, czc,
and ncc genes that are responsible for resistance to heavy metals, were shown to
be present in these bacteria by using RT-PCR. The results of the gene expression
analysis revealed that merA and chrB genes were down-regulated in the all strains
of bacteria (Staphylococcus. aureus, Bacillus subtilis, Bacillus cereus,
Pseudomonas spp. and Bordetella spp.) treated with Co2+
and Cd2+
. However,
cobalt-zinc-cadmium (czcD) and nickel-cobalt-cadmium (nccA) gene were up-

regulated in the all strains of bacteria treated with Co2+
and Cd2+.
, and they found
Co2+
and Cd2+
resistance genes are widely distributed in both gram-positive and
gram-negative isolates obtained from different samples of Egyptian soils.

29
2.1 Materials:
2.1.1 Samples:
1- Clinical sample including (Burn swaps, Ears swaps, and Wound swaps) were
collected from (150) patients suffering from burns, Otitis media and wound
infections, The age range of patients was (1-60) years of both sexes.
2- Environmental sample, 150 samples were collected from Hilla Teaching
hospital environment which included disinfectants, fomites, beds, wall, bath
rooms and catheters.
3- The period of collection was extended from November 2011 to March 2012.
2.1.2 Laboratory apparatus and Instruments:
The apparatus and instruments used throughout this study are listed in
Table (2- 1).
Table (2-1) Lab apparatus and instruments
No. Instruments Company/Country
1. Centrifuge Hettich/Germany
2. Benson burner Germany
3. Water bath Memmert/Germany
4. Inoculating loop Memmert/Germany
5. Distillator GFL/Germany
6. Autoclave Stermite/Japan
7. ELISA Mixer Denley/UK
8. ELISA Reader Beckman/Germany
9. ELISA Shaker Jean Robin/France
10. ELISA Washer Beckman/Germany
11. Eppendrof Centrifuge Hermle/Germany
12. Incubator Memmert/Germany
13. Micropipettes USA
14. Light Microscope Olympus/ Japan
15. Refrigerator Concord/Italy
16. Sensitive electronic balance A&D/Japan
17. Shaker water bath Memmert/Germany
18. Ultra Violet transilluminator San. Gabriel/ USA
19. Vortex Yamato/Japan
20. Digital camera Sony/ Japan
21. Electrophoresis Albent/ Taiwan
22. Water bath Memmert/Germany

30
23. Screw tube China
24, E. Graph Japan
25. Wooden sticks China
26. Safety cabinet Labogene /Denmark
27. polystyrene Tarson, Kolkata/India
2.1.3 Chemical and biological materials:
The chemical materials used in the study are shown in Table (2-2)
Table (2-2) Chemical and biological materials
No. Material Company/country
1. H2O2, 99% Ethanol alcohol, Glycerol,
Kovac's reagent, urea solution
Fluka chemika/
Switzerland
2. KH2PO4, Na2HPO4, NaCl, NH4Cl,
MgSo4, CaCl2, -naphthol, Methyl red,
Glycerol, Agarose,
Phosphate buffer saline (pH=7.2),
Tetramethyl-p-paraphylen diamine
dihydrochloride
B.D.H/ England
3. Ethidium Bromide Promega/USA
4. NaCl, H2SO4 Merk Darmstadt,
Germany
5. Crystal Violet Sigma/USA
6. Gram stain set KSA
7. TBE buffer Promega, USA
8. Agarose Promega, USA
9. Loading dye (bromophenole blue) Promega, USA
10. 100 bp DNA ladder Geneaid, Korea

31
2.1.3.1 Heavy metals:
Table (2-2) Heavy metals used in the study.
Heavy metals Assembly Content Origin
Mercury chloride Hgcl2 30gm Analytical reagent/ India
Nickel sulfate NiSo4 25gm Analar /England
Lead nitrate PbNo3 25gm Himedia laboratory pvt. Ltd/
India
Cadmium sulfate CdSo4 25gm lab reagent/ India
Zinc sulfate ZnSo4 25gm DIDACTIC, Barcelona/Spain
Silver Sulfate AgSo4 25gm BDH/ England
Copper Sulfate CuSo4 25gm BDH/d England
2.1.4 Culture media:
The culture media used in the study are shown in Table (2-3)
Table (2-4) Culture media
No. Media Company/country
1. Blood agar base, brain heart infusion agar, Brain
heart infusion broth, Müller-Hinton agar,
MacConkey agar, Peptone broth, Yeast extract,
Tryptic soy agar, Agar-agar.
Mast diagnostic/U.K
2. Nutrient agar, Nutrient broth, Peptone water
broth.
Oxiod
3. Simon citrate agar, Triple sugar iron agar, MR-
VP broth, Tryptic soy agar.
Diffco/U.k
4. Cetrimide agar Biolife/Italy

32
2.1.5 Cetrimide agar (Forbes et al., 2007).
This medium composed of:-
dissolved in 850 ml of distilled water, adjusted pH to 7, sterilized by autoclaving
then the volume was completed to 1000 ml. cetrimide agar was used as a selective
media for Pseudomonas aeruginosa.
2.1.6 Standard strain used in the study:
Bacterial isolate Genetic properties Source
Escherichia coli MM294 hsdR-
, hsdM+
, ednAI,
pro-
, thi-
, Rif+
Genetic engineering
Institute/Baghdad
2.1.7 Plasmid extraction kit:
Table (2-3) plasmid extraction kit (Geneaid kit, USA):
Diagnostic kits Company/origin
Geneaid kit USA
Peptone 20g
MgCl2 1.5g
K2SO4 10g
Cetrimide 0.3g
Agar 15g

33
2.1.8 DNA Marker (Ladder):
DNA
Marker
Description Origin
100 bp
Ladder
100-300 base pairs (bp). The ladder consists
of 12 double strand DNA fragment ladder
with size of (100, 200, 300, 400, 500, 600,
700, 800, 900, 1000, 1500, 3000 bp). The
500bp present at triple the intensity of other
fragments and serve as a reference. All other
fragments appear with equal intensity on gel.
Geneaid/Korea
2.2 Reagents and Solutions:
2.2.1. Reagents:
2-2-1-1 Oxidase test reagent:
This reagent was prepared by dissolving 0.1 gram of tetra methyl-P-
phenylene diamine dihydrochloride in 10 ml of distilled water. It was used
immediately after preparation (Collee et al., 1996).
2-2-1-2 Catalase test reagent:
This reagent was prepared in 3% concentration of H2O2 and it was used to
identify bacterial ability to produce catalase enzyme (McFaddin, 2000).
2.2.1.3 Methyl red reagents:
A weight of 0.1gm of methyl red was dissolved in 300ml of 99% ethanol and
then the volume was completed to 500ml by distilled water. It was used to detect
the acidity of the medium, which was produced by complete fermentation of
carbohydrates (McFaddin, 2000).

34
2.2.1.4 Voges-Proskauer reagent (Barritt's reagent):
This reagent was prepared according to Collee et al (1996). It consisted of two
solutions:
A- - Naphthol- reagent 5%: It was prepared by dissolving 5gm of -
Naphthol in 100 ml of 99% ethanol.
B- Potassium hydroxide KOH 40% solution: 40 grams of KOH was
dissolved in 100 ml of distilled water. This reagent was used to detect
the partial glucose hydrolysis.
2.2.1.5 Kovac's reagents:
It was prepared by dissolving 10gm of p-dimethylamine benzaldehyde
(DAMB) in 150ml amyle-alcohol, and then 50ml concentrated HCL was
gradually added to this mixture. This solution was stored in a dark bottle, and
gently shaken before use. It was used in the demonstration of indole production
(McFadden, 2000).
2.2.2 Solutions:
2.2.2.1 Normal saline solution:
This solution was prepared by dissolving 0.85 gm NaCl in 90 ml distilled water
(D.W), and further completed to 100 ml with D.W (MacFaddin, 2000).
[
2.2.2.2 Gram Stains Solutions:
The solutions were prepared according to the required microbiological
methods. These solutions included: four solutions crystal violate, iodine, absolute
alcohol, and safranine (Collee et al., 1996).
[
2.2.2.3 Phosphate buffer saline:
Phosphate buffer solution was prepared according to the manufacturer
instructions, by dissolving one tablet of PBS (pH 7.3 checked pre-and post autoclave) in
100 ml of distilled water and then sterilized by autoclave.

35
2.2.2.4 Urea Solution (20%)
It was prepared by dissolving 20 gm of urea in small volume of distilled
water, and completed up to 100 ml D.W. and then sterilized by Millipore filter
paper. It was used in urease test for the detection of urease positive bacteria
(McFaddin, 2000).
2.2.2.5 Stock solution:
It was prepared by a content of metal dissolving in sterile normal saline, and
then shaken at 75 rpm in water bath for 15 minute at 37C (Sergio Vaca Pacheco et
al., 1995).
2.2.2.6 DNA loading buffer:
This buffer was prepared by dissolving 25mg bromophenol blue and 4mg
sucrose in 10ml distilled water, and stored at 4o
C until used (Sambrook and
Rusell, 2001).
2.2.2.4 Ethidium Bromide Solution:
It was prepared by dissolving 0.05gm of Ethidium Bromide in 10ml distilled
water and stored in dark reagent bottle (Sambrook and Rusell, 2001).
2.2.2.5 Tris-Borate-EDTA Buffer (TBE):
Tris-OH, 0.08M, Boric acid, 0.08M, EDTA and 0.02M ,were added to 800ml
distilled water, the pH was adjusted to 8 and completed to one litter by distilled
water, then autoclave at 121o
C for 15 minutes, and stored at 4o
C until used
(Sambrook and Rusell, 2001).
2.3 Preparation of culture media:
Culture media were prepared according to the instructions of the manufacturer
company and serialized by autoclaving in 1 bar at 121o
C for 15 minutes.
2.3.1 MacConkey agar medium:
MacConkey agar medium was prepared according to the method
recommended by the manufacturing company. It was used for the primary

36
isolation of most Gram-negative bacteria and to differentiate lactose fermenters
from non-lactose fermenters (Benson, 1998).
2.3.2 Nutrient agar medium:
Nutrient agar medium was prepared according to the manufacturing
company. It was used for cultivation of the bacterial isolates when it was
necessary (McFadden, 2000).
2.3.3 Nutrient broth:
This medium was used to grow and preserve the bacterial isolates
supplemented with 15% glycerol (McFadden, 2000).
2.3.4 Blood agar medium:
Blood agar medium was prepared according to manufacturer by
dissolving 40 gm blood agar base in 1000 ml D.W. The medium was autoclaved at
121ºC for 15 min, cooled to 50 ºC and 5% of fresh human blood was added. This
medium was used as enrichment medium for the cultivation of the bacterial isolates
and to determine their ability of blood hemolysis (Forbes et al., 2007).
2.3.5 Muller-Hinton agar:
Muller-Hinton agar medium was prepared according to the manufacturing
company (Himedia, India) and it was used in antimicrobial susceptibility testing.
2.3.6 Brain heart infusion broth with 5% glycerol:
This medium was prepared by adding 5ml of glycerol to 95ml of BHI agar before
autoclaving (McFadden, 2000). This medium was used for preservation of
bacterial isolation for long period of time.
2.3.7 Peptone water medium:
This medium was prepared by dissolving 8gm peptone in 1000ml of distilled
water, then distributed into test tubes, and autoclaved. It was used for the
demonstration of the bacterial ability to decompose the amino acid tryptophan to
Indole (McFadden, 2000).

37
2.3.8 Urea agar medium:
This medium was prepared according to manufacturing company, by suspended
2.4 grams urea agar base in 95 ml distilled water and the medium was boiled to
dissolve the medium completely. The pH was adjusted at 6.8 then this medium
was autoclaved and cooled to 50ºC then 5ml of 20% filtration sterilized urea
solution was added. The medium was distributed into sterile tube and allowed to
set in a slope position. This medium was used for detecting bacterial ability to
produce urease enzyme (MacFaddin, 2000).
2.3.9 MR-VP medium:
MR-VP medium has been prepared and used to detect the partial and complete
hydrolysis of glucose according to McFadden, (2000).
2.3.10 Tryptic Soy agar medium:
This medium was prepared according to manufacturer by dissolving 40gm of
tryptic soy agar in 1000ml of distilled water. To enhance growth of bacterial isolates,
this medium was modified by adding 5% fresh blood after autoclaving the medium at
121ºC for 15 minutes, and cooling it to 50ºC. This medium was used as enrichment
medium for cultivation of the bacterial isolates (McFadden, 2000). This medium was
used in Biofilm assay procedure.
2.3.11 M9 Media:
6 gm of Na2HPO4, 3 gm of KH2PO4, 0.5 gm of NaCl, and 1gm of NH4Cl
were dissolved in 950 ml of distilled water with 2% agar , then they were sterilized
into autoclave , after cooling the mixture to 50o
C , 2 ml of 1M of MgSO4 , 10 ml of
20% glucose and 0.1 ml of 1 M of CaCl2 (all of them were sterilized separately by
filtration ) were added to it ,then the volume was completed to 1000 ml (Sambrook
and Rusell, 2001).

38
2.3.13 Motility medium:
It was prepared by dissolving 0.5gm agar with 100ml of nutrient broth, and
then dispensed into sterile test tubes (5ml in each).This medium has been used to
detect the motility of bacteria (Collee et al., 1996).
2.3.12 Simmons' citrate medium:
Simmon's citrate medium was used for determining the ability of bacteria to
utilize citrate as the sole carbon source (McFadden, 2000).
2.3.13 Agarose gel medium:
The agarose gel was prepared according to the method of (Sambrook and
Rusell, 2001) by adding 1gm agarose to 100ml of 1x TBE buffer. The solution
was heated to boiling (using water bath) until all the gel particles dissolved, the
solution was allowed to cool down within 50-60o
C, and mixed with 0.5µg/ml
ethidium bromide.
2.4 Methods:
2.4.1 Specimens collection:
A Total of (300) swab specimens were collected, of which, 150 Specimens
were collected from different sites of infections (burns, wounds and otitis media.
Information about (age, antibiotic usage, residence and hospitalization) of patients
were taken into consideration.
Each swab taken carefully from the site of infection by using transport media
to maintain the swab wet until taken to laboratory. Whereas 150 samples were
collected from AL- Hilla Teaching hospital environment (beds, bathroom,
fomites, wall, catheters and disinfectants) by using wet swabs. Each specimen was
inoculated on (MacConkey agar, Nutrient agar) and then incubation at 37ºC for 24
hrs. For confirmation of bacterial isolation, isolates were then inoculated on
cetrimide medium.

39
2.5 Laboratory diagnosis:
2.5.1 Morphological tests:
2.5.1.1Colonial morphology and microscopic examination:
The grown colonies on the Muller-Hinton agar characterized by diffusible
pigments and sweet-grape odor were selected for further diagnostic tests. The
results of the following tests regarding diagnosis at P. aeruginosa were according
to (MacFadden, 2000). Cultural characteristics include; colonial morphology
(smooth mucoid), grape odor, its color and diffusible pigments on Muller-Hinton
agar (bluish green or yellowish green) and inability to ferment lactose on
MacConkey agar (Collee et al, 1996). Microscopic examination includes the
examination of shape (rods), gram–stain reaction, arrangement of cells with each
other.
2.5.2 Biochemical tests:
2.5.2.1 Oxidase test:
A piece of clean filter paper was saturated with oxidase reagent. If the color
turned rose to purple, the oxidase test would be positive (Forbes et al., 2007).
2.5.2.2 Catalase test:
A colony of organisms is transferred by sterile wooden stick to the surface of
a clean, dry glass slide, and one drop of 3% H2O2 is added to it. The formation of
gas bubbles indicated the positive result (Forbes et al., 2007).
2.5.2.3 Indole test:
It was used for determination of organism's ability to produce indole from
deamination of tryptophan by tryptophanase. The formation of red color ring at
top of broth indicates for positive reaction while a yellow color ring indicated a
negative reaction (MacFaddin, 2000).

40
2.5.2.4 Methyl Red test:
It is employed to detect the production of sufficient acid during the
fermentation of glucose. The change of color to orange was positive reaction
(Murray et al., 2003).
2.5.2.5 Voges-Proskauer (acetoin production) test:
The VP test is used to detect acetone (acetyl-methyl-carbinol), which is
produced by certain bacteria during growth in peptone glucose broth (MR-VP
broth) the positive result was changing of the color of the medium to red
(McFadden, 2000).
2.5.2.6 Simmon's Citrate test:
The citrate test was used to determine the ability of bacterium to utilize citrate
as its only source of carbon. The positive result was changing of color of media
from green to blue (Forbes et al., 2007).
2.5.2.7 Urease test:
The urea base agar was sterilized by autoclave, after that it was cooled to 50°C
and urea substrate was added to it and it was poured in sterile tubes; then it was
inoculated by bacterial culture, which was incubated for (24-48) hours at 37°C.
When urea was broken down, ammonia was released and the pH of medium
increased. This pH change was detected by a pH indicator that turned pink in a
basic environment. A pink medium indicated a positive test for urease. Failure of
deep pink color to develop indicates a negative reaction (Collee et al., 1996).
2.5.2.8 Motility:
Tubes containing motility medium were stabbed once at the center with on
inoculating needle then, incubated at 35°C for 24–48 hours. The motile bacteria
spread out from the line of inoculating (Forbes et al., 2007).
2.5.2.9 Growth at 42o
C:
P. aeruginosa has a characteristic ability to grow at 42°C in successive
subculture. Tubes containing nutrient broth were inoculated with selected

41
colonies and incubated at 42°C for 24 hours. The ability to grow was indicated as
a positive result (Collee et al., 1996).
2.7 Detection of virulence factors:
2.7.1 Hemolysin production:
Hemolysin production was carried out by inoculating of blood agar medium
with bacterial isolate at 3°C for 24-48 hrs. An appearance of clear zone around the
colonies referred to complete hemolysis ( -hemolysis) or greenish zone around
the colonies referred to partial hemolysis ( -hemolysis), while no changing
referred to non-hemolytic ( -hemolytic) (McFadden, 2000).
2.7.2 Siderophores production:
M9 medium agar was prepared and 200µl of Dipyridyl was added and mixed the
media with inoculums, incubated at 37o
C for24 hrs. The positive result is growing
of bacteria in the media (Nassif and Sansonetti, 1986).
2.7.3 Detection of HMR in bacterial isolates:
We were used (3) method to detect the MIC of HMR, included:
2.7.3.1 Screening test for one of heavy metals was leads.
2.7.3.2 Agar dilution method, for four of heavy metals included (Cadmium,
Nickel, Zinc, and Silver).
2.7.3.3 Pouring method, for three of heavy metals included (Copper, Mercury,
and Leads). All method mentions previous described in details following:
2.7.3.1 Determination of MIC using lead as a screening test:
All isolated were tested (screening test) with leads at concentration 400 µg/ml,
were prepared as follows:
a- Prepared stock solution by dissolve 500000 µg of Lead in 20ml normal
saline.
b- Stock solution of leads was prepared by dissolve 250000 µg of leads in
20ml normal saline.
c- Mueller-Hinton agar was used as inoculums medium.

42
d- Muller-Hinton medium was supplement with different cationic
concentration of leads included (100,200.400, 800, 1600, 2400, 3200
µg/ml).
e- A loopfull of refresh bacterial growth was transferred and streaked on plate
supplemented with metals and incubated at 37C for 24 hrs.
f- After incubation presence of bacterial growth indicates ability of bacterial
isolate to resistance the metals ions.
2.7.3.2 Determination of MIC using Agar dilution method:
All isolates were also tested to determine the minimal inhibitory
concentrations (MICs). MICs of four metal including (Cadmium, Nickel, Zinc,
and Silver) was carried out by using supplement method was determined by an
agar dilution method with modification as described by Riley, were prepared as
follows:
a- Mueller-Hinton agar was prepared in 50 ml distilled water, and
approximate concentration 0.0001M, 0,001M, 0.01M for Cadmium and
0.0001M, 0.001M, 0.01M, 0.1M for Nickel, Zinc, and Silver, added to
flask contained 50 ml Mueller-Hinton, mixed well ,and autoclaved.
b- After autoclaving, inoculated medium was allowed to cool (45C) and then
poured into a plate.
c- Inoculating by refreshed growth and incubated at 37C for 24 hrs.
d- After incubation if there is growth, meaning resistance and if there is no
growth meaning sensitive.
2.7.3.3 Determination of MIC using Pouring method:
All isolates were also tested to determine the minimal inhibitory
concentrations (MICs). MICs of three metals (Copper, Mercury, and Lead)
carried out by used pouring method was determined by stock method with

43
modification as described by (Forbes et al., 1998), were prepared as
follows:-
a- Prepared stock solution by dissolve 500000 µg of Copper and Lead in
20ml normal saline.
b- Stock solution of Mercury was prepared by dissolving 250000 µg of
Mercury in 20ml normal saline.
c- Mueller-Hinton agar was used as medium.
d- Muller-Hinton medium was supplement with different cationic
concentration of leads included (100,200.400, 800, 1600, 2400, 3200
µg/ml).
e- A loopfull of refresh bacterial growth was transferred and streaked on plate
supplemented with metals and incubated at 37C for 24 hrs.
f- After incubation presence of bacterial growth indicates ability of bacterial
isolate to resistance the metals ions.
2.8 Biofilm assays:
Semi-quantitative measurements of biofilm formation were determined
using tissue culture-treated, 96-well polystyrene plates, based on the methods of
Christensen et al. (1985) and Ziebuhr et al. (1997). Bacteria were grown in
individual wells of 96-well plates at 37 ºC in BHI medium supplemented with 1%
glucose. After 24 h growth, the plates were washed vigorously. This involved
three rounds of plunging the plates into a large volume of distilled water and
decanting to remove unattached bacteria. The plates were subsequently dried for 1
h at 60 ºC prior to staining with a 0.4% crystal violet solution. The A492 of the
adhered, stained biofilms was measured using a microtitre plate reader. Biofilm
formation by each strain was measured. A biofilm-positive phenotype was
defined as having a value of 0.17 at absorbance of 492 nm.

44
2.9 Genotyping assays:
Plasmid profile of HMR isolates:
2.9.1 Plasmid extraction:
Plasmid extraction of gram negative bacteria was extracted by using Geneaid kit
the step of the method was according to the manufacturing company information
as following:
Step one (Harvesting):-
• 1.5 ml of bacterial culture was transferred to a micro centrifuge tube.
• Bacterial culture was centrifuged at 14 - 16,000xg for 1 minute and the
supernatant discard.
Step two (Re-suspension):
• An aliquot of 200µl of PD1Buffer (RNase A added) was add to the tube and
the cell pellet resuspend by vortex or pipetting.
Step Three (Lysis):-
• An aliquot of 200 µl of PD2 Buffer was added and mix gently by inverting
the tube 10 times. Do not vortex to avoid shearing the genomic DNA.
• The suspension was allowed to stand at room temperature for 2 minutes or
until the lysate is homologous.
Step four (Neutralization):-
• An aliquot of 300 µl of PD3 Buffer was added and mix immediately by
inverting the tube 10 times.
• Bacterial culture was Centrifuge at 14 – 16,000 x g for 3 minutes.
Step five (DNA-Binding):-
• Place a PD column in a 2 ml collection tube.
• Add the supernatant from step 4 to the PD column and Centrifuge at 14 –
16,000 x g for 30 seconds.
• The – flow through was discard and the PD column was placed back in the
2ml collection.

45
Step six (Washing):-
• An aliquot of 400 µl of W1 Buffer was in to the PD column.
• Bacterial culture was Centrifuge at 14 – 16,000 x g for 30 seconds.
2ml collection.
• An aliquot of 600 µl of Wash Buffer (ethanol added), was added into the PD
column.
• Bacterial culture was Centrifuge at 14 – 16,000 x g for 30 seconds.
2ml collection.
• Bacterial culture was Centrifuge at 14 – 16,000 x g again for 3 minute to dry
the column matrix.
Step seven (DNA Elution):-
• The PD column was transferred to anew microcentrifuge tube.
• Add 50 µl of Elution Buffer into the center of column matrix.
• The suspension was allowed to stand for 2 minutes or until the Elution
Buffer is absorbed by matrix.
• Bacterial culture was Centrifuge at 14 – 16,000 x g for 2 minute to elute the
DNA.
2.8.2 Agarose gel electrophoresis:
The method of Sambrook& Russel, (2001), was used follows:
1. One gm of the agarose was dissolved in 100 ml of TBE buffer by using water
bath at 100o
C after that it was cooled to 50o
C and 10 µl of ethidium bromide
solution was added to it.
2. Suitable amount of the agarose was poured in the tray and left to solidify.
3. The comb was raised from the agarose and the tray was transferred to the
electrophoresis chamber and covered with TBE buffer.

46
4. Ten microliter of the DNA was mixed with 3microliter of the loading buffer
and placed in the agarose wells.
5. The electrophoresis process was carried using low voltage (about 6 vol /cm
and by passing 20 milliamper) for 1-2 hours.
6. The bands of the plasmid DNA were detected by UV-trans illuminator in a
wave length of (260)Nanometesr.
2.9.2 Plasmid curing:
There are many method used for plasmid curing as (Elevated Temperature,
SDS, ethidium bromide, Novobiocin…..etc). Elevated Temperature method was
used for plasmid curing according to Kheder (2002), as follows:
Plasmid curing by physical agents (elevated temperature)
A single colony of P. aeruginosa isolate was inoculated into 10ml of
nutrient broth, after incubation at 37°C for 24 hrs, then 0.2 ml.of bacterial culture
was inoculated to 10ml of fresh nutrient broth, and incubated at elevated
temperature from 20 to 46°C for 24hr with shaking water bath 100rpm, after
incubation time several dilutions were performed up to 10-7
dilutions, then 0.1ml
of last three dilutions were spread on plates of nutrient agar which contain heavy
metals and incubated at 37° C for 24hr.
After that the isolates are cultured on Mueller Hinton agar was supplement with
heavy metals at different concentration, after incubation that the results were
recorded by the loss of ability of the tested bacteria to survive on the medium
which contains the heavy metal concentration; However if there was growth
detected the responsible gene for resistance present on chromosome.

3.1 Isolation, identificatio
Out of 150 (50%) clin
culture, 13(32.5%) were fr
Such variations in Pseudo
other studies (Anonymous
the percentage of rural (75
related to infected with P.
between residence and sex
Figure (3-1) Num
The high percentage (75%
due to the fact that people
information about health c
with patients lived in urban
on, and distribution of isolates:
nical samples (patient), only 40(26.6
rom male and 27 (67.5%) were from fe
omonas infections between both sexes
s, 1997; Suman, et al., 2005). Also re
5%) more than urban (25%) which is
. aeruginosa, (p 0.01); while there wa
x (p>0.05).
mber and percentages of P. aeruginosa isolate
%) of rural patients infected with P. a
e in rural regions are almost uneducate
care and the pathogenicity of microorg
n regions (Table 3-1).
6%) gave positive
male (Figure 3-1).
were reported by
esults showed that
highly significant
s no relationship
es in clinical sample.
eruginosa may be
ed, they have little
ganisms compared

Table (3-1) Distri
Reside
Urban
4
6
10(25%)
Regarding the age
infection of P. aeruginos
groups (16-30) years was
However this results were
and Gourley (2000) wh
susceptible to Pseudomon
some disorders such as
hygienic conditions may i
and Sriram, 2005).
Figure (3-2): Distributio
۰
۲
٤
٦
۸
۱۰
۱۲
۱٤
۱٦
ibution of Patients according to Sex and R
ence
Rural
No. of Patients
(%)
S
9 13(32.5) M
21 27(67.5) Fem
30(75%) 40(100) To
groups, the results showed difference w
sa among different age groups (Figu
s more susceptible to infections, acco
e in disagreement with the results obta
o found that children and older pa
nal infections compared with other ag
immunodeficiency, malnutrition as
increase the chance of Pseudomonal in
on of clinical P. aeruginosa isolates according
Residence:
ex
Male
male
otal
with respect to the
ure 3-2). The age
ounted for (40%).
ained by Herfindal
atients were more
ge groups because
well as the poor
nfections (Hauser
g to age groups.

For isolation and identification of P. aeruginosa, all clinical samples were
subjected to different selective media. (Cetrimide agar) medium was used for
primary isolation of P. aeruginosa, on which appeared circular, mucoid, and
smooth. On blood agar, most isolates produced -hemolysis but others did not
produce hemolysis. All isolates grew well on MacConkey agar, (but did not
ferment lactose sugar, and on Muller Hinton agar they produced the distinguished
exo- pigments that varied from yellowish-green to bluish-green as well as they
emitted a sweet grape-like odor.
In addition to the cultural characteristics of isolates on the different culture
media mentioned above, most biochemical tests were carried out (Table 3-2) and
the results compared with standard results documented by (MacFaddin, 2000). All
isolates were Gram negative rods and positive for oxidase, catalase, able to grow
at 42o
C and did not sugar fermentation.
Out of 40 positive clinical samples, 21(52.5%) were from burn, 7(17.5%)
wound, and 12(30%) ear infections (otitis media) isolates (Table 3-3). In the
present study, the distribution of P. aeruginosa isolates according to the site of
infection was studied; it was found that the most infections of this bacterium
occurred in the burn (52.5%). This may be due to the selected samples of this
study. This study was designed to isolate P. aeruginosa from selected clinical
samples (burn, wound, and ear infections). Other sites of infection like blood,
urine, stool, and sputum were excluded and not included during period of
sampling. According to this interpretation, the recovery rate of P. aeruginosa
isolates from selected clinical was more than reported by many researcher
worldwide. The result obtained by Nadeem et al., (2008) who found that among
that (2800) isolates of P. aeruginosa 420(15%) isolates were from burn
infections; Vives-Flórez and Garnica, (2006) found that among the 65 isolates of
this bacterium only 5 (7.7%) isolates obtained from burn.

Table (3-2) Morphological properties and Biochemical tests for identification of
Pseudomonas aeruginosa isolates:
ResultTest
Gram negative, Rods shapeGram's
Stain
+Capsule
+Oxidase
+Catalase
_Indole
_Methyl Red
_Voges Proskaur
+Citrate utilization
±Urease
K/K no changeTriple Sugar Iron
_H2S production
+Motility
±Swarming
Hemolysis on blood agar
_Lactose fermentation
+Pigment production
+ Positive test, Negative test, K: Alkaline
Table (3-3) Distribution of isolates from different clinical samples collected during study:
%
of isolates
No. of P.
aeruginosa
% of samplesNo.Samples
Source
52.5%
17.5%
30%
21
7
12
33.3%
33.3%
33.3%
50
50
50
Burn
Wound
Ear Swab
100%40100%150Total
Ear Swabs were obtained from patients with otitis media.

Pseudomonas aeruginosa in burn units are still associated with high (60%)
death rates, bacteremia is associated with 50% increase in mortality, and patients
are characteristically susceptible to chronic infection by P. aeruginosa
(Mendelson et al., 1994).
In the present study P. aeruginosa in wound infections were studied. It was
found that infection with this bacterium in wounds was less than the infection in
burns. The interpretation may be due to wound wards of female were excluded
from the present study. The percentage of (17.5%) of burn infection in present
study may be due to persistence of patient in hospital for long period time after
operation which made them more vulnerable lead to infection with opportunistic
bacteria like Pseudomonas aeruginosa; (Kluytmans et al., 1997) found that P.
aeruginosa were rare in wound infection (5.76%), while Lari et al., (2000) found
that P. aeruginosa predominant in wound infection (92%).
The result obtained in this study showed that twelve isolates (30%) of
Pseudomonas aeruginosa were obtained from patients with otitis media (Table 3-
3). Al-Amir (1998) showed that the isolation rate of Pseudomonas aeruginosa
was reached to (11%) among other causative agents (aerobic and anaerobic
bacteria) isolates from otitis media.
Pseudomonas aeruginosa was the most common organism isolated from mild to
severe form of otitis external and chronic suppartive otitis media, different types
of bacteria can reach to the middle ear because they can persistent or recurrent
discharge through a chronic perforation of the tympanic membrane due to
perforated tympanic membrane, bacteria can gain entry into the middle ear via the
external ear canal. Infection of middle ear mucosa subsequently results in ear
discharge (Hauser and Sriram, 2005).
From the 150(50%) clinical samples only 40(26.6%) isolates were belonged to P.
aeruginosa and other 110(73.3%) belonged to the other genera that were not
determined during this study (Figure 3-1, Table 3-3).

From the 150(50%) hospi
to the P. aeruginosa whi
(Figure 3-3). The hospita
their numbers and percent
Figure (3-3) Number and perc
Table (3-4) Distribution of
during study:
Samp
Cath
B
Bath
Fom
Wall o
Disinf
T
ital environment samples only 3 (2%)
ile other 147 (98%) belonged to othe
al environment samples were distribu
tages (Table 3-4).
centage of P. aeruginosa isolates in hospital e
f isolate from different hospital environmen
ples type
No. of
samples % of isolates
heters
Beds
h rooms
mites
of wards
fectants
26
25
24
25
30
20
1 (0.7%)
0
0
1 (0.7)%
0
1 (0.7)%
otal 150 3 (2)%
isolates belonged
er bacterial genera
uted according to
environment samples.
nt samples collected

In present work, only three P. aeruginosa isolates were detected in samples of
(catheters, fomites, and disinfectant) at the same percentage (0.7%). This
percentage may be that due to P. aeruginosa has resistance to antibiotic,
disinfectant, and heavy metals, and it has virulence factors enough to prevent
action of these agents. However, in other hospital environment sites like wards,
beds and bath room the percentage of Pseudomonas aeruginosa was not detected
(Table 3-4). This result may be due to good antiseptic agent used in hospitals,
good management though designed scientific schedule for using antiseptic agent
to cleaning wards and tools of hospitals, using educated poster, and increase
awareness of health workers about the pathogenicity of nosocomial infection and
method of prevention. P. aeruginosa were isolated in different percentages by
several authors worldwide.
Vives-Flórez and Garnica, (2006) found that 30% of isolates of P. aeruginosa
were obtained from hospital environment; Alia et al., (2000) had succeeded to
isolate P. aeruginosa from catheters at rate reaching to 8% while Yagoub and El-
Agbash, (2010) isolated this bacteria from hospital disinfectant at rate reached to
5%.
Pseudomonas aeruginosa was very common in hospital conditions due to its
resistance to most antibiotics and disinfectants, and heavy metals. The production
of virulence factors is affected by the differences in the site of antibiotic
resistance genes which may be chromosomal or plasmid – borne and with the
presence of transposable elements which facilitate the transmission of these genes
from the plasmid to the chromosome and vice versa (Nordmann et al., 1993).
3.2. Heavy metals susceptibility test of Pseudomonas aeruginosa:
3.2.1. Results Agar screening test:
In present study, all isolates were subjected to susceptibility testing by screening
test by using agar medium supplemented with (PbNO3 400g/ml).

Results revealed that 37 isolates 85% were resistant to lead nitrate, these isolates
were distributed into 34 clinical and 3 hospital environment samples (Table 3-5).
Whereas, Vaca Pacheco et al., (1995) used lead nitrate as a screening test for
detection of heavy metals resistant P. aeruginosa isolates and they found all their
isolated were resistance to lead nitrate (PbNO3) at concentration of (400g/ml).
Table (3-5) Numbers and percentage of clinical and hospital environment Pseudomonas
aeruginosa detected by screening test:
(%)TotalNo. of isolates (%)
Clinical
Environmental
Susceptibility to
PbNO3
( 400g/ml)
85%3734(85%) 3(100%)Resistant
15%6(15%) 06Sensitive
100%4340(92.8%) 3(6.9%)Total
3.2.2 Susceptibility to heavy metals using Agar dilution method:
In present study, the effects of four heavy metals on P. aeruginosa isolates were
investigated by using MIC of heavy metals in different molar concentration, the
heavy metals were used silver sulfate, zinc sulfate, cadmium sulfate, and nickel
sulfate. Results revealed that 34 isolates were resistant to silver sulfate at
concentration 0.0001M; 32 isolates were resistant to zinc sulfate at concentration
0.0001M, 33 isolates were resistant to cadmium sulfate, and 37 isolates were
resistance to nickel sulfate at concentration 0.0001M; However all isolates of P.
aeruginosa were sensitive 100% to all heavy metals mentioned above at
concentration (0.1M) (Table 3-6).

The interpretation of these results may be due to the fact that P. aeruginosa has
many mechanisms for heavy metals resistance; Firstly, the accumulation of
specific ions can be diminished, not by interference with uptake but by active
extrusion of the heavy metals ion from the cells this mechanism is specific only
for Pseudomonas spp., Secondly, cations can be segregated in to complex
compound by thiol- containing molecules and then ejected from cell, Thirdly,
some metal ions may be reduced to a less toxic oxidative state by the complex
enzymes and special oxidation mechanisms in the cells, Finally, for many metals
resistance and homoeostasis is a combination of two or three of the mentioned
basic mechanisms that is the case which P. aeruginosa success (Abdul-Sada,
2008). Prasad et al., (2009) found that all isolates were sensitive to heavy metals
(Cd2+
, Ag+
, Ar2+
, Co2+
, Ni2+
, Hg2+
, and Pb2+
) at concentration 0.1M, and most of
them were resistant to heavy metals at concentration (0.0001M). Singh et al,
(2010) found the MIC values of Ni2+
were ranged from (80-250 g/ml), Cd 2+
was
(80-210 g /ml).
Table (3-6): Antibacterial activity of heavy metals against Pseudomonas aeruginosa
isolated from clinical and hospital environment samples.
No. of resistant isolates at different molar conc. of heavy
metals
Metal ion
0.10.010.0010.0001
001634Silver
0153333Zinc
001332Cadmium
003737Nickel

In the present study, regarding to silver sulfate, out of 37 isolates, 16 isolates
were able to grow in concentration 0.0001M and 0.001M respectively. However,
all isolates inhibited in concentration 0.1M (Table 3-6, 3-7). According to
hospital environment isolates (P.39, P.40, and P.41) revealed resistant to silver
sulfate and the MIC ranged from (0.001-0.01g/ml ).
These results obtained by Prasad et al., (2009) who found that all isolates of
P. aeruginosa were resistant to silver nitrate at concentration 0.0001M and all
isolates were sensitive to silver nitrate at concentration 0.01M and 0.1M; Dong et
al., (2001) obtained 100 g/ml of sliver effective to prevent P. aeruginosa ATCC
27853 production biofilm and inhibition of bacterial attachment. Several authors
found that resistance to Ag+
was plasmid mediated gene in P. aeruginosa were
resistant to silver, (Tennent et al., 1985; Kucken et al., 2000) found plasmid
mediated gene in Pseudomonas spp. the reason to silver salts were resistance.
Regarding to zinc sulfate, out of 37 isolates, 33 isolates were able to grow in
both concentration 0.0001M and 0.001M (Table 3-6, 3-7); and 22 isolates were
inhibited in concentration 0.01M (Table 3-7). However, all isolates inhibited in
concentration 0.1M (Tables 3-6, 3-7).
According to hospital environment isolates (P.39, P.40, and P.41) were resistant
to zinc sulfate and the MIC was (0.01g/ml ).
In a local study, Abdul-Sada, (2008) found that P. aeruginosa isolated from
wastewater in Basrah, Iraq, were resistant to Zn2O3 at concentration 0.4M. Xiao-
xi et al., (2009) found that P. aeruginosa isolate E1 were resistant to Zn2+
in
concentrations of (16.5 mmol/L), 0,0165M. Nies (2003) interpreted that P.
aeruginosa respond to excess Zn2+
by metal-inducible resistance mechanisms,
Zn2+
were resistant in bacteria is mainly based on active efflux of metal ions to
prevent toxic effects in the cell. In many cases, Zn2+
resistance mechanisms are
indistinguishable; the efflux of Zn2+
is facilitated by P-type ATPases, CBA
transporters and CDF chemiosmotic transporters.

Table (3-7): MIC values of Pseudomonas aeruginosa isolates to Silver sulfate, Zinc
sulfate, Cadmium sulfate, Nickel sulfate in Molar concentrations.
Isolates
MIC of
Silver sulfate
MIC of
Zinc sulfate
MIC of
Cadmium
sulfate
MIC of
Nickel
sulfate
P.1 0.01 0.1 0.001 0.01
P.2 0.01 0.1 0.001 0.01
P.3 0.01 0.1 0.01 0.01
P.4 0.01 0.1 0.001 0.01
P.5 0.01 0.1 0.001 0.01
P.6 0.01 0.1 0.01 0.01
P.8 0.01 0.01 0.01 0.01
P.9 0.01 0.1 0.01 0.01
P.12 0.01 0.1 0.001 0.01
P.13 0.01 0.1 0.001 0.01
P.14 0.01 0.1 0.001 0.01
P.15 0.01 0.1 0.01 0.01
P.16 0.001 0.01 0.001 0.01
P.17 0.001 0.1 0.0001 0.01
P.19 0.0001 0.0001 0.001 0.01
P.20 0.001 0.01 0.001 0.01
P.21 0.001 0.01 0.001 0.01
P.22 0.01 0.01 0.001 0.01
P.24 0.001 0.01 0.001 0.01
P.25 0.01 0.01 0.001 0.01
P.26 0.001 0.01 0.001 0.01
P.27 0.01 0.01 0.01 0.01
P.28 0.001 0.1 0.01 0.01
P.29 0.001 0.01 0.001 0.01
P.30 0.001 0.01 0.001 0.01
P.31 0.0001 0.0001 0.01 0.01
P.32 0.001 0.1 0.01 0.01
P.33 0.001 0.01 0.01 0.01
P.34 0.001 0.01 0.001 0.01
P.36 0.0001 0.0001 0.01 0.01
P.37 0.001 0.01 0.0001 0.01
P.38 0.001 0.01 0.01 0.01
P.39 0.001 0.01 0.001 0.01
P.40 0.01 0.01 0.0001 0.01
P.41 0.001 0.01 0.01 0.01
P.42 0.001 0.1 0.0001 0.01
P.43 0.001 0.0001 0.0001 0.01

In present study, P. aeruginosa resistance to cadmium sulfate revealed that out
of 37 isolates, 32 isolates were able to grow in concentration 0.0001M (Table 3-6,
3-7). However all isolates were inhibited in concentration 0.01M and 0.1M (Table
3-7).
According to hospital environment isolates (P.39, P.40, and P.41) revealed
were resistant to cadmium sulfate and the MIC ranged from (0.0001-0.01 g/ml ).
wastewater in Basrah were resistant to cadmium chloride at concentration 0.1M.
Prasad et al., (2009) found that all isolates of P. aeruginosa were resistant to
cadmium sulfate in concentration 0.0001M but they were sensitive to this metal in
concentration 0.01M and 0.1M respectively. Xiao-xi et al., (2009) found that P.
aeruginosa isolate E1 was resistant to Cd2+
at concentrations (18.5 mmol/L)
(0.018M) respectively.
Nies, (2003) interpreted that P. aeruginosa respond to excess cd2+
by metal-
inducible resistance mechanisms. Cd2+
resistance in bacteria is mainly based on
active efflux of metal ions to prevent toxic effects in the cell. In many cases, the
efflux of Cd2+
is facilitated by P-type ATPases, CBA transporters and CDF
chemiosmotic transporters.
Several authors found that Microbial resistance to Cd2+
is usually based on energy
dependent efflux Three major groups are responsible for this cadmium efflux:
CBA-type chemiosmotic antiporters, P-type ATPases and cation diffusion
facilitators (CDF) (Silver, 1996; Nies, 2003). The CBA-type antiporters are
protein complexes that are able to span the complete cell wall of a gram-negative
bacterium, while CDF proteins and P-type ATPases are located in the cytoplasmic
membrane and are single-subunit systems. Pseudomonas species resistant to
cadmium up to 1.2 mM maximum tolerable concentration was isolated from
heavy metal contaminated soil (Roane and Kellogg, 1996).

In the present work, the resistance of P. aeruginosa to nickel sulfate revealed
that 37 isolates were able to grow at concentrations 0.0001M, and 0.001M.
However, all isolates inhibited in concentration 0.01M and 0.1M (Table 3-6, 3-7).
According to hospital environment isolates (P.39, P.40, and P.41), results
revealed were that they resistant to nickel sulfate and the MIC was (0.01g/ml ).
wastewater in Basrah, Iraq were resistant to nickel sulfate at concentration 0.4M.
Prasad et al., (2009) found that 40% P. aeruginosa isolates were resistant to Ni+2
in concentration 0.001M and all isolates were sensitive to silver nitrate in
concentration 0.01M and 0.1M respectively Singh et al; (2010) found the MIC of
Ni2+
was ranged from (80-250 g/ml). Silver, (1996) found that these mechanisms
are sometime encoded in plasmid genes facilitating the transfer of toxic metal
resistance from one cell to another; (Aguilar- Barajas, et al., 2010) revealed that
P. aeruginosa were resistant to nickel sulfate by accumulation of Ni2+
ions, and
the identification of homologous gene for metal cations resistance in the genome
of species Pseudomonas indicates that these bacteria have the potential to display
tolerance mechanism against Ni2+
.
3.2.3 Susceptibility to heavy metals using Pouring plate method:
In the present study, the effects of three heavy metals (copper sulfate, mercury
chloride, and lead nitrate) on P. aeruginosa isolates were investigated using MIC
of heavy metals in different concentrations.
Our results showed that all isolates were resistant to copper sulfate and the MIC
values ranged from (400-3200 g/ml) (Table 3-8). Results also showed that most
of isolates 33:37 were tolerant to copper sulfate at concentration 800 g/ml ;
however, two of the isolates were tolerant to copper sulfate at concentration of
1750 g/ml . According to hospital environment isolates (P.39, P.40, P.41) they
were resistant to copper sulfate and the MIC was 1600g/ml .

Table (3-8): MIC values of Copper sulfate, Mercury chloride, and Lead nitrate in
( g/ml) concentrations towards Pseudomonas aeruginosa.
Isolate
MIC of
Copper
sulfate
MIC of
Mercury
chloride
MIC of
Lead
nitrate
P.1 1600 2.7 3200
P.2 1750 54.3 3200
P.3 1750 86.4 3200
P.4 1600 86.4 3200
P.5 1750 43.2 3200
P.6 1750 54.3 3200
P.8 1750 86.4 3200
P.9 1750 86.4 3200
P.12 1600 54.3 3200
P.13 1750 43.2 3200
P.14 1750 86.4 800
P.15 1600 86.4 3200
P.16 1600 86.4 3200
P.17 1600 54.3 3200
P.19 1600 86.4 3200
P.20 3200 86.4 3200
P.21 1600 86.4 800
P.22 1600 2.7 2400
P.24 400 54.3 3200
P.25 1600 43.2 3200
P.26 1600 86.4 3200
P.27 1600 21.6 2400
P.28 800 43.2 2400
P.29 400 21.6 3200
P.30 1600 21.6 3200
P.31 1600 21.6 1600
P.32 1600 21.6 3200
P.33 1600 21.6 3200
P.34 1600 21.6 3200
P.36 1600 86.4 3200
P.37 800 21.6 3200
P.38 3200 21.6 3200
P.39 1600 43.2 1600
P.40 1600 86.4 3200
P.41 1600 54.3 3200
P.42 1600 54.3 3200
P.43 1600 43.2 3200

In a local study, Abdul-Sada, (2008) found that P. aeruginosa isolate from
wastewater in Basrah were resistant to copper chloride at concentration 0.3M.
Also, results were similar to that obtained by Karbasizaed et al., (2003) who
revealed that coliforms were tolerant to copper sulfate in 1750 g/ml.
Singh et al., (2010) found the MIC of Pseudomonas spp. to Cu2+
ranged (120-300)
g/ml. Xiao -xi et al., (2009) found P. aeruginosa isolate E1 were resistant to Cu2+
in concentration (12.0 mmol/L) (0.01M).
Pseudomonas aeruginosa were resistant to Cu2+
in which contains the structural
genes copABCD and is homologous to the pco system in Escherichia coli. The
copB and copD genes are involved in the transport of copper across the
membrane, while the products of the copA and copC genes are outer membrane
proteins that bind Cu2+
in the periplasm, protecting the cell from copper (Silver,
1996). Cooksey, (1994) found the plasmid pPT23D
was one of the first
Pseudomonas spp. copper resistance system.
Regarding to mercury chloride, results reported that all isolates were resistant to
mercury chloride and the MIC value ranged from (2.7-86.4 g/ml) (Table 3 -8).
Results also showed that most of isolates 20:37 were tolerant to mercury chloride
at concentration from (10.8- 43.2 g/ml ), However most of isolate 35:37 were
tolerant to Mercury chloride at concentration of 2.7 g/ml (Table 3 -8). According
to hospital environment isolates (P.39, P.40, P.41) they were resistant to mercury
chloride and the MIC values ranged from (43.2-86.4g/ml ).
The result obtained by karbasizaed et al., (2003) revealed that coliforms were
tolerant to mercury chloride was in 54.3 g/ml. Prasad et al., (2009) found that all
isolates of P. aeruginosa were sensitive to mercury chloride in concentration
0.0001M, 0.001M , 0.01M and 0.1M.
Pseudomonas aeruginosa were able to resist to mercury because it has mer
operon that reduced toxic Hg2+
to volatile Hg0
, which then diffuses out of the cell.
The result obtained by karbasizaed et al., (2003) revealed that coliforms were

tolerant to mercury chloride was in 54.3 g/ml. Prasad et al., (2009) found that all
isolates of P. aeruginosa were sensitive to mercury chloride at concentrations
0.0001M, 0.001M, 0.01M and 0.1M respectively.
According to lead nitrate, the results showed that all isolates were resistant to
lead nitrate and the MIC values ranged from (800-3200 g/ml) (Table 3-8). The
Results also showed that most of isolates 30:37 were tolerant to lead nitrate at
concentration 2400 g/ml ; however six of isolates were sensitive to lead nitrate at
concentration of 400 g/ml . According to hospital environment isolates (P.39,
P.40, P.41) they were resistant to lead nitrate and the MIC values ranged from
(1600-3200 g/ml ).
The present study showed, the results were similar to that obtained by
Karbasized et al., (2003) who revealed the coliforms were tolerant to lead nitrate
was in 3200 g/ml. Xiao -xi et al., (2009) found P. aeruginosa isolate E1 were
resistant to pb+2
in concentration (10.0 mmol/L)(0.01M). Prasad et al., (2009)
found that all isolates of P. aeruginosa were sensitive to lead nitrate at
concentrations 0.001M, 0.01M and 0.1M.
Many authors found that P. aeruginosa were resistant pb2+
by the system
localized in cad AC operon, cad A catalyzed the active efflux of Cd2+
, Zn2+
, and
Pb2+
, also they found Pseudomonads have P-type ATPase that can resist Pb2+
(Nucifora et al., 1989).
3.3. Plasmid profile of heavy metals resistant isolates:
The plasmid content was investigated for all 37 isolates of P. aeruginosa (34
clinical and 3 hospital environments). The results revealed that most isolates
32:37 harbored large (mega) plasmid with huge molecular weight cannot be
detected using 3000 bp size marker (ladder) used in the present study (Figure 3-4-
a).
Out of 32 plasmid-carrying isolates, 31(97%) were from clinical samples and
only one isolate was from hospital environment samples.

Out of 34 clinical isolate
when they detected by ele
from clinical samples and
(Figure 3-4-a, b).
From these results, this st
HMR isolates may be corr
Many researchers world
large (mega) plasmids. Ra
aeruginosa that were hea
revealed the P. aeruginosa
molecular weight 100 kbp
was not available at the p
all of the plasmid bands w
Figure (3-4a): Gel electrop
Lane (M): DNA molecular si
Lane (E): Show negative con
Lanes: show clinical isolates
Lane (P39): Show hospital en
e, 32 isolates carried only one plasmid
ectrophoresis. The results also showed
d two isolate from hospital environmen
tudy can conclude that the presence of
related with their ability to resist differ
dwide reported that HMR in P. aerugi
aja and Selvam, (2009) found that the
avy metal resistant was 23 kbp. Nikbin
a isolated from hospital in Tahran, Iran
p. However in this study a large size m
eriod of the study, so only 3000bp ma
were in the gel out of ladder.
phoresis of plasmid DNA content of P. aerug
(1:30) hr. at (60) voltage.
ize marker (3000-bp ladder).
ntrol (E. coli standard strain MM294).
(P1), (P5), (P8), (P9), (P12), (P22), (P24), a
nvironment isolate.
d (only one band)
d that three isolate
nt have no plasmid
f plasmids in these
rent HM.
inosa is carried on
plasmid size of P.
n et al., 2007 that
n has plasmid with
marker (40000bp)
arker was used and
ginosa isolates after
and (P43).

Figure (3-4b): Gel electrophoresis of plasmid DNA content of P. aeruginosa isolates after
(1:30) hr. at 60 voltages.
Lane (M): DNA molecular size marker (3000-bp ladder).
Lane (E): Show negative control (E. coli standard strain MM294).
Lanes: (P13), (P14), (P15), (P16), (P17), (P19), (P20), (P21), (P25), (P27), (P28), (P29),
(P30), (P31), (P32), (P33), (P34), (P36), (P37), and (P38) shows clinical isolates.
Lanes :( P40), (P41) Show hospital environment isolates.

Figure (3-4-c): Gel electropho
(1:30) hr. in (60) voltage.
Lane (M): DNA molecular si
Lane (E): Show negative con
Lanes: (P2), (P3), (P4), (P6), (P
The presence of plasm
authors. (Chakrabarty, 19
also known to code for
compounds. (Weiss et al
resistance to HgCl2, but
phenylmercuric acetate.
Collard et al., (1994) fou
determinats move from ch
(Raja and Selvam 2009)
heavy metals such as c
plasmid DNA was designa
oresis of plasmid DNA content of P. aerugino
ize marker (3000-bp ladder).
ntrol (E. coli standard strain MM294).
P26), (P42) show clinical isolates.
mids in HMR isolates was also rep
976) showed that plasmids in Pseudom
r heavy metal resistance and degrad
l., 1978) reported that many plasmids
t also resistance to several organom
und that in P. aeruginosa some heavy
hromosome to plasmid (or in the revers
) revealed isolate P. aeruginosa exhib
cadmium, chromium, nickel and lead
ated as pBC15, The size of the plasmid
osa isolates after
ported by several
monas species are
dation of organic
s carried not only
mercurials such as
y metal resistance
se direction).
bited resistance to
d, due to present
d DNA was

approximately 23 kb, and suggest that nickel and ampicillin resistance gene was
conferred by plasmid DNA. Cadmium resistant gene was present on chromosomal
DNA along with the gene for chromium resistance.
Lead resistance gene was shown to be present on the chromosomal DNA
rather than the plasmid DNA.
(Silver, 1996) obtained that plasmid genes facilitating the transfer of toxic metal
resistance from one cell to another in P. aeruginosa.
The reason of presence plasmid that confers resistance to HM in P. aeruginosa
may be due to the fact that most of this large plasmid carrying in addition to HM,
resistance other biocides likes antibiotics (Lawrence, 2000).
Many isolates of P. aeruginosa have no plasmid content and still show heavy
metals resistance that lead to think that gene responsible for these resistances
found on the chromosome ( Raja and Selvam, 2009).
On the other hand (Silver, 1992) revealed the mercuric ions chemically reduced,
and detoxified by plasmid- encoded enzyme.
3.4 Plasmid curing by elevated temperature:
Plasmids are small autonomously replicating circular pieces of double-stranded
circular DNA, The bacterial cells may loss their plasmids during cell division;
these types of cell were said to be cured. Curing may occur naturally through cell
division or by treating the cells with chemical and physical agents (Snyder and
Champness, 1997).
Plasmid curing was achieved in the present study using elevated temperature for
detection whether HMR trait was plasmid or chromosome – mediated.
Only one P. aeruginosa isolate (P.3) was chosen and treated with physical agent
(elevated temperature 46o
C) to detect the plasmid curing for isolate used in this
study.

After curing, isolates were retested for its ability to resist HM using Mueller
Hinton agar supplement with heavy metal at different concentrations.
Our Results showed that the tested isolate exposed to curing has lost it is plasmid
(Figure 3-5).
Figure (3-5): Gel electrophoresis of plasmid DNA content of Ps. aeruginosa isolate before and
after curing after (1:30) hr. at(60) voltage.
Lane (M): DNA molecular size marker (3000-bp ladder).
Lane (P3/1): shows clinical isolate (first dilution).
Lanes (P3/2): shows clinical isolate (second dilution).
Lanes (P3/3): shows clinical isolate (third and last dilution).
P.3 isolate was missing their plasmid as revealed that DNA content of their cured
cells on gel electrophoresis. Also this isolate still survived and grow in the present
of HM, but resistance to some HM decreased compared to wild type isolates
(Table 3-9).

This result indicates that the HM resistance trait was carried on chromosome
rather than plasmid. This also can be interpreted that in Pseudomonas aeruginosa
some heavy metal resistance determinants move from plasmid to chromosome
(Collard et al., 1994).
Many isolates of P. aeruginosa have no plasmid content and still show heavy
metals resistance that lead to think that gene responsible for these resistances
found on the chromosome ( Raja and Selvam, 2009).
Table (3-9): MIC values of Pseudomonas aeruginosa P.3 before and after curing
exposed to different heavy metals:
Heavy metals MIC before
curing
MIC after
curing
Silver sulfate (Ag2So4) 0.01M 0.01M
Zinc sulfate (ZnSo4) 0.1M 0.01M
Cadmium sulfate
(3CdSo4)
0.01M 0.01M
Nickel sulfate (NiSo4) 0.01M 0.01M
Copper sulfate (CuSo4) 1750 g/ml 1600 g/ml
Mercury chloride
(HgCl2)
10.8 g/ml 86.4 g/ml
Lead nitrate (PbN2O6) 3200 g/ml 3200 g/ml
Perron et al., (2004) revealed that P. aeruginosa resistant to metals ions by
metals efflux express czc operons. Raja and Selvam, (2009) found that P.
aeruginosa resistant to cadmium; chromium and lead could be chromosomal
encoded.
3.5. Biofilm assay:
In the present study, the results showed that 20 isolates of P. aeruginosa had
biofilm. All hospital environment isolates were biofilm producers (3 isolates),
whereas only 17:40 (42.5%) of clinical isolates were biofilm producers. The
relationship between biofilm production and HMR was studied. It was found that

the HMR of P. aeruginosa isolates was not correlated with production of the
biofilm.
The results shown in (Tables 3-10, 3-8) reveal that the isolate P. 22 (planktonic)
and the isolate 43 (biofilm) were similar in their resistance to copper sulfate (1600
g/ml ), which indicate that the biofilm production had no role in increase of
resistance to HM compared to the resistance of free-swimming (planktonic)
organisms.
This result was also detected for other HM studied (silver, zinc, cadmium,
nickel, copper, and lead) in which there was no relationship between biofilm
formation and HM resistance (Table 3-10, 3-8, 3-7). However there was a clear
correlation between biofilm production and mercury resistance (Table 3-10, 3-8).
It was found that the resistance to mercury was increased in biofilm isolates P.43
(43.2 g/ml ) in comparsion with planktonic isolate P. 22(2.7 g/ml ) (Table 3-8).
Table (3-10): Biofilm production by P. aeruginosa isolates

From these results, this study concluded that the increase of HM resistance was
correlated with biofilm production for some (but not all) HM used. A hallmark
trait of biofilms is increased resistance to antimicrobial agent compared to the
resistance of free-swimming organism (Costerton et al., 1999; Hentzer et al.,
2001; Teizel and Parsek, 2003).
A proposed mechanism that contributes to this increased resistance is binding
and sequestration of antimicrobial agents by EPS component, such as negatively
charged phosphate, sulfate, and carboxylic acid groups (Hunt, 1986).
Another factor that may contribute to the resistance of biofilms is that many
antimicrobial agents target metabolically active cells. Biofilms are subjected to a
wide range of chemical gradients that result in decreased metabolic activity within
the depths of a biofilm.
Studies of biofilm and heavy metal interactions have mainly focused on the
sorption of heavy metals. Several researchers have reported that biofilms are
capable of removing HM ions from bulk liquid (Ferris et al., 1989, Huang et al.,
2000, Labrenz et al., 2000, Liehr et al., 1994).
Teizel and Parsek (2003) reported that biofilms were from 2 to 600 times more
resistant to HM stress than free-swimming cells. They also showed that biofilms
are more resistant to HM than either stationary-phase or logarithmically growing
plankotonic cells. The exterior of the biofilm was preferentially killed after
exposure to elevated concentrations of copper. A potential explanation for this is
that EPS that encase a biofilm may be responsible for protecting cells from heavy
metals stress by binding the heavy metals and retarding their diffusion within the
biofilm (Teizel and Parsek, 2003).
In another study, Dong Gu et al., (2001) found that100 g/ml of sliver was
effective to prevent P. aeruginosa ATCC 27853 production biofilm and inhibition
of bacterial attachment.

Conclusions and Recommendation_______________________________________
71
Conclusions:
1- Burns were more accessible for being contaminated with Ps.
aeruginosa.
2- Most of isolates were resistant to most of heavy metals studied.
3- Most of the isolates were harbored plasmid, which could have a
role in resistance to HM. However curing experiment of HM
resistant isolate showed the HM resistance was harbored on
chromosome, which may be transmitted from plasmid to
chromosome.
4- About half of HM resistant isolates had biofilm and the increase of
to HM resistance was correlated with biofilm production for some
(but not all) HM used.
Recommendations:
1- Studying HMR of Ps. aeruginosa using advanced molecular
techniques (PCR).
2- Heavy metals should be used for treatment of Ps. aeruginosa, like
silver and mercury salts which were found to be very effective.
These heavy metals can be used for topical treatment instead of
antibiotics cream which may induce resistance in Ps. aeruginosa
when applied in sub-inhibitory concentrations.
3- Heavy metals should be used in disinfectants and antiseptics
production.
4- Studying the HMR in organism other than Pseudomonas
aeruginosa especially that associated with nosocomial infections.

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