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Antibacterial Activity Pseudomonas sp. Isolated
Rhizosphere against Methicillin Resistance
Staphylococcus aereus from Clinical Samples
Asli kousha H. and Vatankhah M. R.
Department of Microbiology, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran
Email: [email protected], [email protected], [email protected], [email protected]
Abstract—These methicillin-resistant Staphylococcus aureus
(MRSA) is a substantial public health problem worldwide,
causing significant morbidity and mortality. Methicillin-
resistant S. aureus is the leading cause of skin and soft tissue
infection in patients reporting to emergency departments
for treatment.
The different types of biosurfactants with antimicrobial
activity include lipopeptides, glycolipids, phospholipids and
lipopolysaccharides. A few lipopeptides have been reported
to produce by Gram-negative bacteria like Pseudomonas.
The aim of this study was to antibacterial activity of a
compound from Pseudomonas sp. against MRSA strains
from clinical samples.
The Pseudomonas sp. was isolated from Rhizosphere in
North Iran. The antimicrobial activity of cell-free
supernatant and partially purified bacteriocin was
determined by well diffusion method. A total of 100 S.
aureus isolated from clinical specimens. The bacteria were
primarily identified by colony morphology, microscopy of
Gram’s stain and routine biochemical tests and antibiotic
disk sensitivity tests (Cefoxitin; Ceftriaxon; Amoxicillin;
Cefotaxime; Co-trimoxazole; Penicillin; Tetracyclin;
Azitromycin).
Results of this study showed the isolate Pseudomonas sp.
broadest antimicrobial spectrum against MRSA isolated
clinical samples. However, the spectrum of inhibitory
activity of these bacteria suggests a potentially useful means
for controlling the growth of food-borne pathogens bacteria
such: S. aereus and MRSA isolated food samples.
Index Terms—MRSA, rhizosphere, antiboitic activity,
clinical sample.
I. INTRODUCTION
Staphylococcus aureus is a major cause of serious
hospital, and community-acquired bacteremia worldwide
and is associated with a high morbidity and mortality [1].
The emergence of methicillin-resistant S. aureus
(MRSA) strains since 1961 has complicated the treatment
of S. aureus infections, and glycopeptides (vancomycin
or teicoplanin) are, in many cases, the only therapeutic
alternative. In recent years, new antistaphylococcal
antibiotics, such as linezolid or daptomycin, have been
developed, but their cost and the absence of large clinical
trials demonstrate clear superiority over vancomycin
Manuscript received December 26, 2014; revised March 18, 2015
maintains glycopeptides as a first-line option when
infection due to MRSA is suspected or diagnosed [2], [3].
In the 1960s and1970s, replacement of anti-biotic-
resistant bacteria with another kind but similar antibiotic
susceptible bacteria was used to inter-rupt S. aureus
outbreaks in nurseries in order to reduce recurrent
furunculosis in persistent carriers. Unfortunately, a
“nonpathogenic” strain, S. aureus 502A, was not always
nonpathogenic and its use fell out of favor [4], [5].
One non- antibiotic strategy to combat the bacterial
infections involves the selection and promotion of
endogenous barrier flora to interfere with pathogenic
bacterial adhesion [4].
Currently, MRSA strains account for many of
staphylococcal infections and reports of MRSA strains
are increasing worldwide [6]. There are also several
reports from Iran showing the prevalence of methicillin
resistance among clinical isolates of S. aureus [7], [8].
Since the first isolates of MRSA were identified in the
United Kingdom in 1961, MRSA has been a primary
cause of health care–associated infections throughout
Europe, Asia, Australia, and the United States [9], [10].
Methicillin-resistant Staphylococcus aureus (MRSA)
is a formidable bacterial pathogen responsible for a
variety of infections commonly seen in patients of all
ages [11], [12]. The MRSA strains associated with hospitals are
referred to as hospital-acquired MRSA (HA-MRSA) and
are the most common cause of hospital-acquired
infections [13], [14]. Methicillin-resistant S. aureus is the
leading cause of skin and soft tissue infection in patients
reporting to emergency departments for treatment [15],
with a rising rate in primary care clinics [16] and
intensive care units. Invasive MRSA related conditions
most commonly reported include septic shock (56%),
pneumonia (32%), endocarditis (19%), bacteremia (10%),
and cellulitis (6%) [17].
The rhizosphere, representing the thin layer of soil
surrounding plant roots and the soil occupied by the roots,
supports large active groups of bacteria [18]. Several
rhizobacterias have been used extensively as biological
agent to control many soil borne plant pathogens [19].
The different types of biosurfactants with antimicrobial
activity include lipopeptides, glycolipids, phospholipids
and lipopolysaccharides [20] A few lipopeptides have
International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 2, April 2015
©2015 Int. J. Life Sci. Biotech. Pharm. Res. 137
been reported to produce by Gram-negative bacteria like
Pseudomonas [21].
II. MATERIALS AND METHODS
A. Clinical Specimens
One hundred swab samples were collected in 2014 on
mannitol salt broth medium from different hospital
Tehran (IRAN). Samples were transported in an ice box
(at 4 ºC) Microbiological examination was performed
within 24 h.
B. Confirmation of Saureus
The samples collected on mannitol salt broth media
were incubated at 37ºC for 24 h and then inoculated onto
mannitol salt agar media and incubated at 37ºC for 24 h.
Selected isolates with colony morphology,Gram stain
reactions and biochemical characteristics (ie, Coagulase,
DNase, Manitol utilization).
C. Confirmation of MRSA and Antibiotic Resistance
Testing
All isolates of S aureus that demonstrated any level of
Oxacillin (Methicillin) and Cefoxitin (FOX) resistance.
All cultures were grown on Mueller-Hinton agar plates at
37 C for 18 hours in the presence of the following
antibiotics: ampicillin(10μg), Cefotaxime (30 μg),
Cefoxitin (30μg), Penicillin (10U), Oxacillin (1.0μg),
Trimethoprim-sulfamethoxazole (SXT; 1.25μg/23.75μg),
Tetracycline (30μg), Azitrimycin(15μg) and
Ceftriaxon(30μg) (PT Diagnostic Systems). The zone of
inhibition (in mm) around each disk was documented and
compared with a standard interpretivechart [22].
D. Isolation of Pseudomonas sp. from Rhizosphere Soil
1g of rhizosphere soil sample was suspended in 99 ml
of sterile saline solution. Samples were serially diluted
(10-5)
and 0.1 ml of sample was spreaded on King’s B
agar plates and incubated at 37°C for 24 h. The isolated
bacteria was confirmed by morphological and
biochemical tests, based on Bergeys’ Manual of
Systematic Bacteriology.
E. Purification of Protein
A 18-h-old culture of the bacteriocinogenic LAB strain
was centrifuged (×9000g, 20 min, 4ºC) and the peptidic
fraction precipitated from the cell-free supernatant with
70% saturated ammonium sulphate [23]. Some
bacteriocins can precipitate at lower ammonium sulphate
concentrations, or even in a small range of saturation,
then is important to assay which is the concentration of
salt that precipitates the peptide of interest. The
suspension was incubated overnight at 4°C and agitated
with a magnetic stirrer. Salted-out proteins were
precipitated by centrifugation (×10000g for 20 min) and
dissolved in a small volume of 10 mM phosphate buffer
(pH 7.0) or distilled water. The suspension can be
desalted by dialysis with phosphate buffer at 4°C during
12h by using benzoylated membranes (molecular weight
cut off 1200; Sigma-Aldrich) or with dialysis cassettes
with cut-off of 2000 to 3500 (Pierce Biotechnology, Inc).
Since most bacteriocins have a size smaller than 10000
Da, the use of regular dialysis bags with cut-off of 10000
-12000 Da is inappropriate for this procedure.
F. Evaluation of the Antibacterial Effect by the Well
Diffusion Technique
The antimicrobial activity of cell-free supernatant and
partially purified protein was determined by well
diffusion method [24]. Screening of Antibacterial
Activity of Pseudomonas sp. isolated from rhizosphere
soil by Agar well diffusion method. Antibacterial activity
of Pseudomonas sp. isolated from rhizosphere soil was
tested against target bacterial pathogens of health
significance like MRSA isolated by in vitro techniques
using Muller-Hinton agar plates at 37 °C for 24 h. MRSA
suspensions of 108 colony forming units (CFU)/ml were
grown to log phase, and the well diffusion were treated
with the antibacterial compounds. The plates were
incubated at 37°C for 24 h, and the size of the inhibition
halos diameter was evaluated (mm). The antibacterial
effect was determined by measuring the size of inhibited
halos formed around clinical samplese wells.
G. Identification Pseudomonas sp. Isolated from Soil by
16S rRNA Gene Sequence
Chromosomal DNA used for polymerase chain
reaction (PCR) was prepared by using phenol-chloroform
method [25]. The DNA fragments containing 16S rDNA
were amplified from chromosomal DNA with primers
pairs 27F (5- AGAGTTTGATCMTGGCTCAG -3) and
1492R (5- GGTTACCTTGTTACGACTT - 3) [26]. PCR
reactions were performed in a DNA thermal cycler
(Biorad, USA) in a total volume of 50 µl containing
Master mix (Takara, Japan).
Amplification consisted of a 1 min denaturation step at
94°C, a minute annealing step at 58°C and a minute
extension step at 72°C. The first cycle was preceded by
incubation for 5 min at 94°C. After 35 cycles, there was a
final 10 min extension at 72°C. Negative controls
containing no DNA template were included in parallel.
PCR products were separated in a 1.5% (w/v) agarose gel
and were subsequently visualized by ultraviolet (UV)
illumination after ethidium bromide staining.
III. RESULTS
A. Antibiogram Profile MRSA Isolated Clinical Samples
The one hundred S aereus isolated from clinical
sample twenty (20%) isolate confirm MRSA. Antimicrobial sensitivity test of the MRSA isolates
recorded high resistance rate to different antibiotics
(Table I). The current data showed that only 10% of
MRSA isolates were susceptible to SXT. In the current
study, 20 out of 20 isolates (100%) of MRSA were
multidrug resistant. All were resistant to penicillins;
amoxicillin; Cefoxitin; Tetracycline; Oxacillin; Ceftriaxon;
Azitrimycin and Cefotaxime. The multi-drug resistance
in MRSA isolates might be due to the antibiotic’s
selective exposure.
International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 2, April 2015
©2015 Int. J. Life Sci. Biotech. Pharm. Res. 138
B. Antimicrobial Activity Cell Free Supernatant and
Purified Protein
The antimicrobial activity of CFCS and purified
protein (PP) from CFCS were tested against MRSA by
well diffusion assay (Fig. 1). The CFCS and PP exhibited
an antibacterial effect on a broad range of MRSA strains.
TABLE I. PREVALENCE OF ANTIBIOTIC RESISTANCE AMONG MRSA ISOLATES
Antibiotic Concentration(μg) Resistant Sensitive
Ampicillin 10 20 0
Cefoxitin 30 20 0
Oxacillin 1 20 0
SXT 1.25/23.75 18 2
Tetracycline 30 20 0
Penicillin1 10 20 0
Ceftriaxon 30 20 0
Azitrimycin 15 20 0
Cefotaxime 30 20 0
Figure 1. Antimicrobial activity of PP and CFCS Pseudomonas sp, (a) purified protein and (b) cell-free culture Supernatant.
TABLE II. ANTIMICROBIAL ACTIVITY SPECTRUM OF THE CELL-FREE CULTURE SUPERNATANT AND PARTIALLY PURIFIED PROTEIN OF
PSEUDOMONAS SP.
Code isolate Diameter of Zones Inhibition (mm)
Cell free Supernatant(100μl/well) Purified Protein(100μl/well)
Clin1 18 30 Clin2 17 31
Clin3 17 29
Clin14 17 30 Clin25 18 28
Clin36 19 28 Clin57 16 30
Clin59 17 29
Clin63 16 28 Clin67 18 25
Clin 69 19 26 Clin74 17 27
Clin75 16 30
Clin78 17 29 Clin83 18 27
Clin86 18 28 Clin89 18 29
Clin91 19 30
Clin96 18 31 Clin98 16 30
In the current study, it showed that CFCS and PP
Pseudomonas sp. isolated from rhizosphere broadest
antibacterial spectrum against MRSA isolated clinical
specimens. All isolates were sensitive to Cell free
supernatant and purified protein Pseudomonas sp. (Table
II).
(a) (b)
International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 2, April 2015
©2015 Int. J. Life Sci. Biotech. Pharm. Res. 139
IV. DISCUSSION
Over the past few years, studies concerning
bacteriocins produced by LAB have received an
increasing interest because of the potential use of
bacteriocins as food preservatives [27]. Bacteriocin-
producing isolate Pseudomonas sp. was isolated from by
rhizosphere Soil, direct plating method. The isolate
Pseudomonas sp. showed the broadest antimicrobial
spectrum against MRSA isolated clinical samples.
However, the spectrum of inhibitory activity of these
bacteria suggests a potentially useful means for
controlling the growth of food-borne pathogens bacteria
such: S. aereus and MRSA isolated food samples.
According to the study Laine et al (1996) have found
that Pseudomonas sp. Antimicrobial activity against food
poisoning bacteria and moulds [28]. We suggestion that
purified protein of Pseudomonas sp. could be used in the
food preservation.
REFERENCES
[1] A. Soriano, J. A. Martinez, J. Mensa, et al., “Pathogenic
significance of methicillin resistance for patients with Staphylococcus aureus bacteremia,” Clin. Infect. Dis., vol. 30, pp.
368-373, 2000. [2] V. G. Jr. Fowler, H. W. Boucher, G. R. Corey, et al., “Daptomycin
versus standard therapy for bacteremia and endocarditis caused by
Staphylococcus aureus,” N. Engl. J. Med., vol. 355, pp. 653-665, 2006.
[3] J. A. Cepeda, T. Whitehouse, B. Cooper, et al., “Linezolid versus
teicoplanin in the treatment of gram-positive infections in the
critically ill: A randomized, double-blind, multicentre study,” J.
Antimicrob. Chemother., vol. 53, pp.345-355, 2004. [4] J. R. Tagg and K. P. Dierksen, “Bacterial replacement therapy:
Adapting ‘germ warfare’ to infection prevention,” Trends in Biotechnology, vol. 21, pp. 217-223, 2003.
[5] M. C. Roghmann and L. McGrail, “Novel ways of preventing
antibiotic-resistant infections: What might the future hold?” American Journal of Infection Control, vol. 34, pp. 469-475, 2006.
[6] S. Stefani, D. R. Chung, J. A. Lindsay, A. W. Friedrich, A. M. Kearns, H. Westh, et al., “Methicillin-resistant Staphylococcus
aureus (MRSA): Global epidemiology and harmonisation of
typing methods,” Int. J. Antimicrob. Agents, vol. 39, pp. 273-282, 2012.
[7] J. Sadeghi and S. Mansouri, “Molecular characterization and antibiotic resistance of clinical isolates of methicillin-resistant
Staphylococcus aureus obtained from Southeast of Iran
(Kerman),” APMIS, vol. 122, no. 5, 2014.
[8] E. Abbasi-Montazeri, A. D. Khosravi, M. M. Feizabadi, H.
Goodarzi, S. S. Khoramrooz, M. Mirzaii, et al., “The prevalence of methicillin resistant Staphylococcus aureus (MRSA) isolates
with high-level mupirocin resistance from patients and personnel
in a burn center,” Burns, vol. 39, pp. 650-654, 2013. [9] J. M. Boyce, B. Cookson, and K. Christiansen, “Methicillin-
resistant Staphylococcus aureus,” Lancet Infect. Dis., vol. 5, pp. 653-663, 2005.
[10] W. R. Jarvis, J. Schlosser, R. Y. Chinn, S. Tweeten, and M.
Jackson, “National prevalence of methicillin-resistant Staphylococcus aureus in inpatients at US health care facilities,”
Am. J. Infect. Control, vol. 35, no. 10, pp. 631-637, 2007. [11] H. F. Chambers, “The changing epidemiology of Staphylococcus
aureus?” Emerg. Infect. Dis., vol. 7, pp. 178-182. 2001.
[12] A. L. Frank, J. F. Marcinak, P. D. Mangat, and P. C. Schreckenberger, “Increase in community-acquired methicillin-
resistant Staphylococcus aureus in children,” Clin. Infect. Dis., vol. 29, pp. 935-936, 1999.
[13] R. Durai, P. C. Ng, and H. Hoque, “Methicillin-resistant
Staphylococcus aureus,” Journal of Chiropractic Medicine, vol. 11, pp. 64-76, 2012.
[14] K. C. Carroll, “Rapid diagnostics for methicillin-resistant Staphylococcus aureus: Current status,” Mol. Diagn. Ther., vol. 12,
no. 1, pp. 15-24, 2008.
[15] G. J. Mor, A. Krishnadasan, R. J. Gorwitz, G. E. Fosheim, L. K. McDougal, R. B. Carey, et al., “Methicillin-resistant S. aureus
infections among patients in the emergency department,” N. Engl. J. Med., vol. 355, no. 7, pp. 666-674, 2006.
[16] M. L. Parchman and A. Munoz, “Risk factors for methicillin-
resistant Staphylococcus aureus skin and soft tissue infections presenting in primary care: A South Texas Ambulatory Research
Network (STARNet) study,” J. Am. Board Fam. Med., vol. 22, no. 4, pp. 375-379, 2009.
[17] R. M. Klevens, M. A. Morrison, J. Nadle, S. Petit, K. Gershman, S.
Ray, et al., “Invasive methicillin-resistant Staphylococcus aureus infections in the United States,” Journal of the American Medical
Association, vol. 298, no. 15, pp. 1763-1771, 2007. [18] M. Villacieros, B. Power, M. Sanchez-Contreras, J. Loret, R. I.
Oruzebal, M. Martin, et al., “Colonization behaviour of
Pseudomonas fluorescens and Sinorhizobium meloti in the alfalfa (medicago sativa) rhizosphere,” Plant Soil, vol. 251, pp. 47-54,
2003. [19] E. D. Amico, L. Cavalca, and V. Andreoni, “Analysis of
rhizobacterial communities in perennial Graminaceae from
polluted wate meadow soil, and screening of metal resistant potentiality plant growth promoting bacteria,” FEMS Microbial.
Ecology, vol. 52, pp. 153-162, 2005. [20] H. Hashizume, M. Igarashi, R. Sawa, H. Adachi, Y. Nishimura,
and Y. Akamatsu, “A new type of tripropeptin with anteiso-
branched chain fatty acid from Lysobacter sp. BMK333-48F3,” J. Antibiot., vol. 61, pp. 577-582, 2008.
[21] U. Schillinger and F. Lucke, “Antibacterial activity of Lactobacillus sake isolated from meat,” Appl. Environ. Microbiol.,
vol. 55, pp. 1901-1906, 1989.
[22] Clinical and Laboratory Standards Institute, “Performance standards for antimicrobial susceptibility testing,” Nineteenth
informational supplement, CLSI document M100-S19, Clinical and Laboratory Standards Institute, Wayne, PA, 2009.
[23] E. V. Pingitore, E. Salvucci, F. Sesma, and M. E. Nader-Macías,
“Different strategies for purification of antimicrobial peptides from Lactic Acid Bacteria (LAB),” in Communicating Current
Research and Educational Topics and Trends in Applied Microbiology, A. Mendezvilas, Ed. Spain: formatex, 2007.
[24] U. Schillinger and F. Lucke, “Antibacterial activity of
Lactobacillus sake isolated from meat,” Appl. Environ. Microbiol., vol. 55, pp. 1901-1906, 1989.
[25] J. Sambrook and D. W. Russell, “Purification of nucleic acids by extraction with phenol: Chloroform,” in Commonly Used
Techniques in Molecular Cloning, 3rd ed. J. Sambrook and D. W.
Russell, Eds. USA, NY: Cold Spring Harbor Laboratory Press, 2001.
[26] S. Turner, K. M. Pryer, V. P. W. Miao, and J. D. Palmer, “Investigating deep phylogenetic relationships among
cyanobacteria and plastids by small subunit rRNA sequence
analysis,” Journal of Eukaryotic Microbiology, vol. 46, pp. 327-338, 1999.
[27] J. Cleveland, T. J. Montville, I. F. Nes, and M. L. Chikindas, “Bacteriocins safe, natural antimicrobials for food preservation,”
Int. J. Food Microbiol., vol. 71, pp. 1-20, 2001.
[28] M. H. Laine, M. T. Karwoski, L. B. Raaska, and T. M. Mattila Sandholm, “Antimicrobial activity of Pseudomonas sp. against
Food poisoning bacteria and moulds,” Letters in Applied Microbiology, vol. 22, no. 3, pp. 214-218, 1996.
Hanieh Asli Kousha received the B.S in Biology from Azad University
of central Tehran (2012) and the M.S in Microbiology from the Islamic Azad University of Tonekabon Branch, 2014. Both activities and
interest of Hanieh Asli Kousha are in enviromental microbiology and
food microbiology, especially in probiotics. Hanieh Asli Kousha has 5
ISI articles in international journals and has participated in an
international congress.
International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 2, April 2015
©2015 Int. J. Life Sci. Biotech. Pharm. Res. 140
