SILVER NANOPARTICLES

International Journal of Medicine and
Pharmaceutical Sciences (IJMPS)
ISSN 2250 - 0049
Vol. 3, Issue 3, Aug 2013, 29-38
© TJPRC Pvt. Ltd.
PHENOTYPIC AND GENOTYPIC INVESTIGATION ON SILVER NANOPARTICLES OF
MORGANELLA MORGANII RECOVERED FROM (CAUTI), IRAQ
FRIAL GEMEEL ABD, ALI JABBAR ABDUL HUSSAIN AL-KAWAZ & HUSSEIN OLEIWI
M. AL-DAHMOSHI
Department of Biology, Faculty of Science, Babylon University, Babylon, Iraq
ABSTRACT
One hundred eighty eight mid-stream urine samples were collected from patients suffering from Catheter
Associated Urinary Tract Infections, (CAUTI), who visit urological consultant clinic of Hillah Teaching Hospital in Hillah,
Babylon province- Iraq during a period from November 2012 to January 2013. All urine samples were subjected for
standard bacteriological procedures to check bacteriuria. The results show that 9/159 (5.66%) give positive culture for
Morganella morganii. For detection the ability of this bacteria to produce silver nanoparticles we are used the XRD the
result of XRD for the control shows amorphous phase but after loading the silver nitrate (AgNO3) on the surface of the
isolates we get new peacks (111,200,220,311) for isolates number 2, 3, 4,5,6,7 and the isolates and the isolate number 1,8
and 9 show negative result (the amorphous phase). These peaks approved that silver ions doping on the surface and this
confirmed by using TEM. The UV-VIS used to study the behavior of silver nanoparticles this shows it is clear at zero hour
there is no reaction of her and at 24 ,72 and 120 the increased in intensity will be observed in the range of (400 & 500)nm.
KEYWORDS: CAUTI, Nanoparticles, XRD, Morganella morganii, TEM
INTRODUCTION
Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection, accounting for
more than 1 million cases each year in the US hospitals and nursing homes. The significant number of infections and
dissemination of resistant bacteria in hospitals make it important to find ways to decrease their incidence [1].
Morganella morganii is a gram-negative rod commonly found in the environment and in the intestinal tracts of
humans, mammals, and reptiles as normal flora. Despite its wide distribution, it is an uncommon cause of community-
acquired infection and most often encountered in postoperative, catheter-associated bacteriuria and other nosocomial
settings [2].In the human urinary tract, the ability of Morganella morganii to hydrolyze urea has been linked to formation
of xanthine calculi as well as the more typical struvite and carbonite apatite stones [3].
Morganella morganii possesses many virulence associated factors especially the production of urease as well as
hemolysin, protease ,and some adhesive factors which play a major role in colonization of bacteria to human tissues
[4].Risk factor for the development of Morganella morganii infection includes surgical or non-surgical trauma, prior
exposure to B-lactam antibiotics, diabetes mellitus, malnutrition, debilitation, alcoholism, and certain snake bites [5].
However, Morganella morganii infections respond well to appropriate antibiotic therapy; however, its natural resistance to
many beta-lactam antibiotics and cephalosporin may lead to delays in proper treatment [6].
As nanotechnology is emerging as an interdisciplinary field with potential to influence various aspects of human
life through a myriad of applications, biological synthesis of nanomaterials is gaining particular attention as a rapidly
growing discipline of Bionanotechnology with an enormous application potential in the coming future [7].

30 Frial Gemeel Abd, Ali Jabbar Abdul Hussain Al-Kawaz & Hussein Oleiwi M. Al-Dahmoshi
There has been a strong interest in developing environmentally benign protocols for biological synthesis of
nanomaterials that do not involve toxic chemicals in synthesi process. Biological synthesis of various metal nanoparticles
by using prokaryotic as well as eukaryotic organisms including bacteria, fungi, plants [8]. However, among various
organisms studied until to date, prokaryotes remain the choice of organism for biological synthesis of nanomaterials . This
is predominantly because prokaryotes offer well-defined advantages over eukaryotic organisms such as easy handling, ease
of downstream processing and ease of genetic manipulation [9].
Bacterial synthesis of silver nanoparticles (AgNPs) is particularly attractive from microbiology perspective due to
existence of well-known silver resistance machinery in few silver resistant bacterial species, thus making their study
significantly important for biomedical applications [10]. Moreover, silver nanoparticles have remained an attractive choice
of nanomaterial because of their ability of encompassing broad application area from electronics to medicine to food
technology [11].
Most of the natural processes also take place in the nanometer scale regime. Therefore, a confluence of
nanotechnology and biology can address several biomedical problems, and can revolutionize the field of health and
medicine nanotechnology is currently employed as a tool to explore the darkest avenues of medical sciences in several
ways like imaging sensing targeted drug delivery and gene delivery systems and artificial implants . The new age drugs are
nanoparticles of polymers, metals or ceramics, which can combat conditions like cancer and fight human pathogens like
bacteria [12]
The development of new resistant strains of bacteria to current antibiotics has become a serious problem in public
health; therefore, there is a strong incentive to develop new bactericides [13]. Bacteria have different membrane structures
which allow a general classification of them as Gram-negative or Gram positive. The structural differences lie in the
organization of a key component of the membrane, peptidoglycan. Gram negative bacteria exhibit only a thin
peptidoglycan layer (~2–3 nm) between the cytoplasmic membrane and the outer membrane [14]. In contrast, Gram-
positive bacteria lack the outer membrane but have a peptidoglycan layer of about 30 nm thick [15].
Silver has long been known to exhibit a strong toxicity to a wide range of micro-organisms for this reason silver-
based compounds have been used extensively in many bactericidal applications , Silver compounds have also been used in
the medical field to treat burns and a variety of infections[16]. Commendable efforts have been made to explore this
property using electron microscopy, which has revealed size dependent interaction of silver nanoparticles with bacteria
[17]. Nanoparticles of silver have thus been studied as a medium for antibiotic delivery [18]. The synthesize composites for
use as disinfecting filters [19] and coating materials [20] However, the bactericidal property of these nanoparticles depends
on their stability in the growth medium, since this imparts greater retention time for bacterium– nanoparticle interaction.
There lies a strong challenge in preparing nanoparticles of silver stable enough to significantly restrict bacterial growth.
Studies were carried out on both antibiotic resistant (ampicillin- resistant) and nonresistant strains of gram-negative
(Escherichia coli) and a non-resistant strain of gram-positive bacteria (Staphylococcus aureus). A multi-drug resistant
strain of gram-negative (Salmonella typhus, resistant to chloramphenicol, amoxycilin and trimethoprim) bacteria was also
subjected to analysis to examine the antibacterial effect of the nanoparticles [21]. Efforts have been made to understand the
underlying molecular mechanism of such antimicrobial actions. The effect of the nanoparticles was found to be
significantly more pronounced on the gram-negative strains, irrespective of whether the strains were resistant or not, than
on the gram-positive organisms, antibacterial effect of the nanoparticles depend on their stability in the medium as a
colloid, which modulates the phosphotyrosine profile of the bacterial proteins and arrests bacterial growth. The bactericidal
effect of silver ions on micro-organisms is very well known; however, the bactericidal mechanism is only partially

Phenotypic and Genotypic Investigation on Silver Nanoparticles of 31
Morganella morganii Recovered from (CAUTI), Iraq
understood. It has been proposed that ionic silver strongly interacts with thiol groups of vital enzymes and inactivates them
[22]. Experimental evidence suggests that DNA loses its replication ability once the bacteria have been treated with silver
ions . Other studies have shown evidence of structural changes in the cell membrane [23]. Silver ions have been
demonstrated to be useful and effective in bactericidal applications, but due to the unique properties of nanoparticles
nanotechnology presents a reasonable alternative for development of new bactericides. Metal particles in the nanometer
size range exhibit physical properties that are different from both the ion and the bulk material. This makes them exhibit
remarkable properties such as increased catalytic activity due to morphologies with highly active facets [24].
The current study aimed to phenotypic and genotypic investigation of silver nanoparticles among Morganella
morganii isolates recovered from catheter-associated urinary tract infection (CAUTI).
MATERIALS AND METHODS
Patient and Samples
One hundred eighty eight mid-stream urine sample were collected from patients suffering from (CAUTI) who
visit urological consultant clinic of Hillah Teaching Hospital in Hillah, Babylon province- Iraq during a period from
November 2012 to January 2013. all samples were subjected to standard bacteriological procedure including culturing on
blood and MacConkey's agar plates for isolation and incubated for 24-48 hours at 37C0
[25].
All suspected Gram negative isolates were screening by traditionally tests and then confirmed By Viteck 2
compact system (Biomérieux).
DNA Extraction, Primer Designing and PCR Conditions
All Morganella morganii isolates were subjected for DNA extraction according to the protocol provided by
manufacturer (Geneaid/Taiwan). The primer pair used to investigate silE gene was designed in this study using Workbench
- Primer 3 software. The primer sequence was DAKW F:5- GTGAATATCCATGAGCGGGT-3 ; DAKW R: 5-
CAACTGCAGCTCTTTCATGC-3. The PCR product size was 280 bp.
The PCR conditions indicated in this study was Simple 3-step PCR protocol as shown in table (1).
Table 1: The Cycling Conditions of Phylogeny Groups
Steps Temperature Time No. of Cycles
Initial denaturation 95 C° 2 min 1
Denaturation 95 C° 30 sec
30Annealing 59 C° 30 sec
Extension 72 C° 30 sec
Final extension 72 C° 5 min 1
Hold 4C° 10
Table 2: The 30 µl PCR Mix
Item Volume
Master mix 15 µl
Target DNA 5 µl
Forward Primer (10pm/ µl) 2.5 µl
Reverse Primer (10pm/ µl) 2.5 µl
Nuclease free water 5 µl
Total Volume 30 µl
Method of Biosynthesis and Phenotypic Investigation of Silver Nanoparticles
The method of silver nanoparticles production by Morganella morganii was achieved as indicated by Song et

al.,(2009)26
as follow: the Morganella morganii isolates were initially grown at 37 C for 24 h in a 500-mL Erlenmeyer flask
that contained LB broth (100 mL) in a shaker incubator set at 200 rpm and then The bacterial growth were incubated with
aqueous 5 mM solutions of AgNO3 at 37C in a shaker incubator at 200 rpm in the dark, and the reactions were carried out
for up to 120 h (5 days).
The extracellular synthesis of AgNPs was initially detected by visual inspection of the culture flask for a change
in color of culture medium from clear light-yellow to brown/green. The separations of AgNPs from bacterial cells were
performed by centrifugation of aliquots of culture supernatants (1.5 mL) at 3000 rpm for 6 min at 25C.
The UV-vis analysis was done as follow, the AgNPs suspensions were diluted 10 times using MilliQ deionized
water at every time point and UV-vis spectra were obtained. For X-Ray Diffraction (XRD) analysis, the samples were
prepared by precipitating AgNPs obtained after 20 h of biosynthesis at 13,000 rpm for 20 min, followed by four washings
with MilliQ deionized water, and drop casting the samples onto a glass substrate.
For TEM analysis, AgNPs samples obtained after 20 h of reaction were prepared by drop casting the colloidal
suspensions of AgNPs onto carbon-coated Cu grids followed by drying under air for 24 hours [26].
RESULTS AND DISCUSSIONS
One hundred eight eighty samples were collected during the period from November 2012 to January 2013 patients
who were suffering from (CAUTI) patients in Hilla Teaching Hospital in Hilla Province, a mid-stream urine the sample
were primarily grown on to blood and MacConkey's agar plates for isolation and incubated for 24-48 hours at
37C0[27].All samples culturing on traditional and conventional media.
As showed in figure (1), a total of 159 positive cultures, just nine showed positive for Morganella morganii, all
from male. The identification of these isolates depends on the main characteristics of these bacteria according to [28].
Figure 1: Percentage of M. morganii in CAUTI Patients
API 20E was used to confirm the results of identification and the results of API 20E come with those obtained in
traditional tests. Also the system two Vitek was used to confirm the results of identification and the results of API 20E
there was different between species but the result probability between (95-99%) are show in table (3).
And the result As follows, catalase positive, oxidase negative, urease positive and negative to gelatinase and
phenylalanine deaminase positive. Also, the isolates were found to be unable to ferment the sugars: Lactose, Mannitol,
Sorbitol, Sucrose, Arabinose, Inositol, Rhamnose, Melibiose, but able to ferment Glucose only with gas.

Table 3: Viteck 2 Compact System Results
This study was performed to determine whether extracellular silver nanoparticles (AgNPs) production by
Morganella morganii. Morganella morganii isolate were able to synthesize extracellular Ag nanoparticles. To undertake
this study Morganella isolates were exposed to 5 mM colorless AgNO3 solutions Morganella morganii formed dark brown
coloured solutions within 20 h of reaction figure (2) , and the color of the solutions did not significantly change from that
point onward , even after continuing the reaction for up to 5 days. To understand the nature of nanoparticles, detailed
physico-chemical characterization of extracellular AgNPs formed by all Morganella morganii was carried out using UV-
Vis absorbance spectroscopy, and X-ray diffraction (XRD).
Figure 2: Medium with AgNO3 (1mM) and Controlled Sets (Negative and Positive) at Room Temperature
Figure (3) shows the UV-vis absorbance spectra of colloidal solutions obtained after reaction of all Morganella
isolates with 5 mM AgNO3 for zero, 24,72 and 120 –h . The presence of a characteristic Ag surface Plasmon resonance
(SPR) between 400 and 500 nm is clearly evident in all the samples, thus confirming the formation of extracellular AgNPs
by all Morganella morganii[29].
The differences in the position of absorbance of SPR features of AgNPs synthesized by different isolates of
Morganella morganii was notable, which was most likely due to the difference in the size and/or shape of Ag nanocrystals
synthesized by these bacteria [ 30]. It is also interesting to note as the result showed that the Morganella morganii started
synthesizing AgNPs as early as within 1 h of reaction and the yield of AgNPs increased as the reaction progresses over a
period of time.

Figure 3: UV-VIS Absorbance Spectroscopy for AgNPs from Morganella morganii
However, the amount of AgNPs produced by these bacteria reached to a saturation state somewhere between 20 h
and 120 h of reaction. This suggests that although all Morganella isolates have the capability to reduce (Ag+) ions to form
AgNPs (Ag0), the rate of AgNPs formation may vary among them.
The maximum absorbance intensity of the Ag (SPR) feature of different isolates of Morganella morganii differ
with the time of reaction. It is also interesting from the result note that AgNPs production by Morganella reached to a
saturation state within 20 h of reaction, after which no further increase in AgNPs synthesis was observed and this noted
from the result of x-ray diffraction (XRD).
During this study In my opinion, 20 h time point provides a better representation than 120 h time point for
comparison between AgNPs synthesized by Morganella isolates, because at 20 h time point AgNPs biosynthesis is in its
log (growth) phase, which enables to capture the state of as formed particles, rather than a possibility of their further
modification while AgNPs stay in the bacterial growth media up to 120 h. And during this study I noted the rate of AgNPs
formation was found to be maximum at 5 mM AgNO3 concentration, and was reduced by increasing the precursor
concentration and over the 5 mM AgNO3 the bacteria was die and this result agree with [29].
The crystallography of AgNPs formed by different isolates of Morganella after 20 and 72 and 120 h of reaction
was investigated by XRD. As is evident from XRD patterns in Figure(4) , extracellular AgNPs synthesized by Morganella
morganii are highly crystalline in nature, that could be perfectly indexed to the {111}, {200}, {220} and {311} Bragg
reflections of the face centered cubic (fcc) form of crystalline silver so this result agree with this [30].
XRD analysis thus provided a clear indication of formation of high quality crystalline AgNPs using a Morganella
morganii mediated biosynthesis process..The UV-vis, XRD results presented in this study clearly demonstrate that
formation of AgNPs is a genus-wide characteristic phenotype of Morganella morganii.
Further experiments were performed to explore whether AgNPs formation is a characteristic phenotype restricted
to genus Morganella, or whether other taxonomically related genera of Enterobacteriaceae family also show this feature.to
obtain this I do comparative analysis of AgNPs synthesis using laboratory strains of Escherichia coli, Serratia marcescens,
Kelebisella pneumonia and Aeromonas hydrophila was performed in the presence of 5 mM AgNO3, no AgNPs formation
was observed in any of these closely related organisms.
This strongly suggests that AgNPs synthesis in the presence of Ag+ ions is a phenotypic character that is uniquely
associated with Morganella morganii.

Figure 3-4: X-Ray Diffraction Results for Morganella morganii Different Time of Reaction
Compared with Control, XRD Patterns Recorded Showing 4 Sharp Peaks Corresponding to the
Diffraction from 111, 200, 220 and 311 Planes of Silver
Figure (5) show the results of TEM analysis, AgNPs samples obtained after 20 h of reaction were prepared by
drop casting the colloidal suspensions of AgNPs onto carbon-coated Cu grids followed by drying under air for 24 hours
this test was performed at university of Technology in Baghdad.
Figure 5: Show Transmission Electron Microscopy (TEM) Images of Extracellular
AgNPs formed by Morganella morganii (36.9nm)
Concerning the investigation of the silver nanoparticles gene (silE) the results reveald that 6/9 (66.7%) of isolates
have silE as shown in figure (6).
Figure 6: 2% Agarose Gel Electrophoresis of 280bp PCR Products of silE Gene. The Lane M Represent
100bp Molecular Marker, Lane 2,3,4,5,6,7 Represent Sample No. of Positive Results while Lane 1, 8 and
9 Represent Sample No. of Negative Results. Lane Control Represents Negative Control for silE Gene

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