2023.08.02.78
Files > Volume 8 > Vol 8 No 2 2023
Antibacterial
and anti-biofilm action of cobalt oxide nanoparticles beside persister Pseudomonas
Aeruginosa isolates
Alaa
M. Ahmed1,*, Mohammed F. Al Marjani2, and Ahmed M.
Rheimah3
1Department of Biology,
College of Science, Mustansiriyah University, Baghdad, Iraq
2Department of Biology,
College of Science, Mustansiriyah University, Baghdad, Iraq
2Department of Chemistry,
College of Science, Mustansiriyah University, Baghdad, Iraq
*
Correspondence: [email protected] , [email protected] , [email protected]
Available
from: http://dx.doi.org/10.21931/RB/2023.08.02.78
ABSTRACT
Persister
cells of Pseudomonas aeruginosa have developed a wide-reaching public
health problem. Although this is a medical concern, there is currently no
effective means to remove P. aeruginosa persister cells. Nanoparticles
containing cobalt oxide (Co3O4-NPs) were examined for their ability to impact
the formation of biofilms and inhibit the growth of bacteria. Researchers found
that Co3o4-NPs were effective against P. aeruginosa, with inhibition
zones ranging from 11 to 19 mm and MIC (Minimum Inhibition Concentration)
results of 156 to 312 g/ml. The Co3O4-NPs with a titration of 10 mg/mL (76.54
percent) had the maximum biofilm suppression activity, while a titration of
0.156 mg/ml had the lowest (11.50 percent). According to the findings, P.
aeruginosa biofilms and persister cells can benefit from applying
co3o4-NPs.
Keywords: Persister cell, SEM, co3o4-NPs, Anti-biofilm
action
INTRODUCTION
Antibiotic resistance and
biofilms have become a severe hazard to incurable infectious infections.
Combating them should be a high priority, with the primary technique being to
explore strategies to avoid their establishment 1. For further
information on biofilms, see Biofilms: A community of adherent Bacteria with
different metabolites from free-living bacteria 1. Bacterial
infections are thought to be caused by biofilms in 80% of cases 2.
These super constructs are inappropriately resistant to medications and the
immunological defense system, creating a significant health concern. A good
stress response and survival strategy for bacteria is the formation of biofilms3,4. P. aeruginosa is
a life-threatening human opportunistic pathogen that flourishes in hosts that
lack typical defenses, such as those suffering from severe illness or
immunologically impaired. In addition to highly ill patients such as those in
intensive care units (ICUs) 5, healthful persons can also take it.
The most prevalent nosocomial infections that cause death and severe illness in
hospitalized patients include 6. Bacterial persistence, known to
create chronic intractable infections, is one of the primary reasons for
antibiotic therapy failure. Sub-inhibitory levels of antibacterial medications
cause persisters, which are antimicrobial-tolerant cells that do not grow and
lack a conventional genetic resistance mechanism, to emerge in populations of bacteria.
Persister cells are hereditarily equal to antibacterial sensitive cells in a
resident that is not sure of MIC rate or persister cell. However, they take a
diverse behavior in that they can survive antibacterial at titer that may be
harmful to other cells7,8. Persister cells have been identified in
bacterial populations before the antibiotic was applied inactive slow growing
due to phenotypic variation 9. In general, nanomaterials have been
demonstrated to have stronger microbicide activity than standard antibiotics
and have even shown reactivity against numerous bacteria species 10.
The existing work aims to examine the anti-biofilm and anti-persister cell
development ability of Cobalt oxide nanoparticles against P. aeruginosa.
METHODS AND MATERIALS
Isolation, Collection,
Identification and Growth Circumstances of Bacteria
From a variety of Baghdad-Iraqi
hospitals. Fifty different clinical isolates of P. aeruginosa were studied.
Bacterial isolates were detected by their characteristic on selective and
differential culture media, biochemical assays, and the Vitek-2 system. All
bacterial isolates were cultivated on Luria Bertani (LB, Difco Laboratories)
agar or in Luria Bertani broth for 18-24 hr. at 37 °C.
Synthesis and Characterization of
Cobalt Oxide Nanoparticles
The Sigma-Aldrich Company
provided all of the components and the highest quality. UV irradiation was used
to create cobalt oxide nanoparticles11,12,13,14,15. A 125-watt UV
source (with a maximum wavelength of 365 nm) and an ice-bath-cooled Pyrex tube
make up the photocell, eliminating an increase in temperature due to UV
radiation. Our results were as follows: we added 0.06-mole Ethylene glycol
(CH2OH)2 to 75 ml of 0.03-mole cobalt (II) acetate for 20 minutes with a
magnetic stirring rod. For 30 minutes, the mixture was exposed to light from a
photocell. This morning's precipitation was a dark brown. The material was
repeatedly separated and washed using ion-free water in a centrifuge. The
material has been dehydrated for 24 hours. After calcining it for three hours
at 450 °C, a black powder of co3o4-NPs was produced.
Persister Cells Detection
The test for detecting persister
cells was produced by applying rabidly killing techniques and freshly cultured
cells with lytic solutions 16. For the McFarland standard of 0.5
McFarland, P. aeruginosa isolates were cultured for 18-24 hours at 37 °C in
Luria Bertani broth. Then, 200μL of buffer lysis solution was poured into a
sterile 10 mL sterile test tube, vortex spun for 10 seconds, and then for 10
minutes were incubated at room temperature. After that, 2 μL 00 of the
enzymatic lysis solution was gently poured over the mixture. Finally, incubate
for 15 minutes at 200 rpm at 37 °C in a shaking incubator. A 10 μL smear on
Luria Bertani agar was used to determine the frequency of persister cells, and
the plates were incubated for 18-24 hours at 37 °C.
Biofilm Assay
According to 17, the
following was done to evaluate the biofilm of P. aeruginosa: First, in brain
heart infusion (BHI) broth, they were cultivated for 18-24 hours. The growth
was then diluted to 1:100 and pipetted onto the wells of a 96-well polystyrene
flat bottom plate containing BHI broth medium with 2 percent (w/w) sucrose,
where it was cultivated for 24 hours at 37 °C, after being rinsed 3 times in
phosphate-buffered saline and leaving it to dry at room temperature earlier,
being colored for fifteen minutes with 200μL of 1% crystal violet. The stain
was eluted using 200μL of absolute ethanol after aeration at room temperature,
and OD was measured using an ELISA reader at 630 nm. The experiment was done
three times for each isolate. Separate groups of test isolates were tested for
adherence, each with a different mean optical density value.
No biofilms if the OD ≤ ODc, weak
biofilms, If ODc < OD ≤ 2 × ODc
Moderate biofilms, if 2 × ODc
< OD ≤ 4 × ODc and if 4 × ODc < OD, strong biofilms
Antimicrobial Effect and MIC of
Co3O4-Nps
To study the inhibitory impact of
Co3O4-Nanoparticales against persistent P. aeruginosa Strains, according to 30,
agar well diffusion methods were utilized. DMSO in the other served as a
negative control, and an antibiotic disc in the other well was a positive
control, the co3o4-Nanoparticales in the latter well. In addition, the MIC was
determined using a 96-well polystyrene microtiter plate. 100μL of (MHB) Muller
Hinton broth was put on a microtiter plate for each well, and then 100μL
Co3O4-NPs (10 mg/mL) was added to the first vertical row from A1 to A10, followed
by 1:2 serial dilutions. The remaining 100 mL of the last well had been
abandoned. After 24 hours of incubation, 5 μL of bacterial
suspension (108 CFU/mL or 105 CFU/well) was added to all wells except the
negative control row from A12 to H12. After dyeing with 10μL of resazurin dye
were put to each well and then incubated for around four hours at 37 °C to get
prepared for reading, the effects of diverse titer of Co3O4-NPs for the
microbiological growth were determined by utilizing UV visible spectrophotometer
18.
Anti-Biofilm Activity of
Co3o4-Nps
Co3O4-NPs' anti-biofilm impact on
persistent P. aeruginosa strains was studied following 19. The
isolates of P. aeruginosa were injected in BHI broth at 37 degrees Celsius.
After that, 20 μL of bacterial suspension was put into each microtiter plate
well, containing 80 μL of BHI broth with 2 % sucrose.
100 μL of co3o4-NPs was added and mixed well before incubating at 37°C for 24
hours. Then, the plate was allowed to dry at room temperature for two further washes
in PBS. Cells of Bacteria that cannot be linked to one another were removed.
Biofilms were stained with 200 μL of 1% crystal violet
solution. The stain was eluted with 200μL of absolute ethanol, and the OD was
calculated using an ELISA reader at 630nm. Only B.H.I broth was used as a
negative control. The experiment was done three times for each bacterial
isolate. The following equation was used to calculate the rate of inhibition
rate of co3o4-NPs:
Rate of inhibition (%) =( OD of
control – Od of treated ) / (OD of control) × 100
RESULTS
Persister Cells Detection
Rapidly killing approach, used to
examine the formation of persister cells in P. aeruginosa isolates. According
to the findings, only 5 out of 50 P. aeruginosa isolates (PA4, PA5, PA21, PA27,
and PA47) had persister cells 20
seen in Figure 1.
Figure 1. Formation of Persister cell in P. aeruginosa By Rapidly Killing Method.
As a result, it is an optimal
procedure that operates without influencing the culture's bacterial population
size, strain, or physiological state.
Cobalt Oxide Nanoparticles,
Synthesis and Characterization
A distinctive XRD design of the
precursor synthesized at 450 °C for three hours is shown in Figure 2. All of
the reflection peaks in this pattern (JCPDS Card File No. 76–1802) might be
easily attributed to crystalline cobalt with oxygen. In this pattern, there
were no noticeable impurity peaks. The diffraction peaks of 2θ = 19.50°,
31.37°, 37.02°, 39.10°, 44.97°, 55.84°, 59.58°, 65.46°, and 77.62°
appeared. The mean crystallite sizes
were 25 nm.
Figure 2. XRD Pattern Of Co3O4 -Nanoparticles
The ingredient of the created elements at 450°C was
fixed by EDS analysis. Figure 3 shows that the result contained just cobalt
(Co) and oxygen (O) elements, with a cobalt/oxygen atomic ratio of roughly 3:4,
which is compatible with the predicted value of Co3O4. No
additional elements are found, indicating that the Co3O4
NPs are highly pure.
Figure 3. EDS Measurement of S the Co3O4 Nps
SEM was used to evaluate the
morphology of the created units. The SEM image of produced Co3O4
nanoparticles is shown in Figure 4. The nanoparticles have a sphere-like
morphology in the SEM picture, and they are created of agglomerated collected
particles representing connectivity well among particles. The size of
nanoparticles, as well as their homogeneity and size distribution, can be
detected by using SEM. The SEM image shows that the particles are evenly
scattered. The mean particle size was randomly estimated from the figures to be
37nm.
Figure 4. SEM Image Of Co3O4 Nps
Co3O4-Nps, Antibacterial Activity
And MIC
On persistence,
Co3O4-Nanoparticales were proven to have antibacterial efficacies against P.
aeruginosa. Co3O4-NPs displayed antibacterial solid activity in the agar-well
diffusion technique, with inhibition zones (11-19 mm) at 10.000g /ml. According
to the MIC results, CO3O4-NPs had high antibacterial activity (156.25 -312.5
g/ml), as demonstrated in Table 1 and Figure 5 A, B. The ability of persistent
P. aeruginosa to proliferate was often reduced during 16 hours of CO3O4-NPs
exposure, as was also observed
Table.
1
Antibacterial Effect of Co3o4-NPs Against Persister Cell of P. aeruginosa Isolates.
Figure
5
Antibacterial Activity Of Co3o4-Nps Against Persister Cell Of P. aeruginosa Isolates By A-Agar-Well
Diffusion Method B-Micro Titer Plate Method.
Biofilm Inhibition Activity
The ability of co3o4-NPs to
suppress biofilm formation by persister cells of P.aeruginosa isolates was
examined in this work. Co3o4-NPs were examined for their average inhibition
rate against P. aeruginosa. Co3o4 nanoparticles greatly assisted biofilm
reduction. Biofilm was inhibited to a percentage of 76.54 percent at 10 mg/ml
and 0.156 mg/ml. Because of this, co3o4-NPs had a considerable effect on P.
aeruginosa persister cell biofilms.
Table 2. AntiBiofilm Ability Of Co3o4-Nps Against
Persister P. aeruginosa Isolates.
If we can diminish persister, we
can reduce bacterial resistance to medicines and therapies, which will help
treat chronic infections. We assessed the existence of the creature utilizing a
quick-killing strategy. Following testing with lysis buffer solution and
enzymatic lysis solutions, persister cells in P. aeruginosa isolate. Current
work indicates the inhibitory zones of co3o4-NPs ranged from 11- 19 mm in a
concentration of 10.000μg/ml to remove P. aeruginosa persister cells. For P.
aeruginosa isolates, the MIC of Co3O4-Nanoparticales ranged from 156.25 to
312.5 μg/ml. Results showed that Co3O4-NPs demonstrated a significant
anti-biofilm activity at 76.54 percent present in co3o4-Nanoparticales at a
concentration of 10000 μg/mL correspondingly.
DISCUSSION
Methods that depend on
antibacterial titer are substantially slower than the rapid killing method of
persister cell isolation. Further studies can be conducted on the isolated
persister cells because the fast killing approach is stable after isolation 16
The reflection peaks could be
easily attributed to crystalline cubic phase Co3O4. The
crystallite sizes of the as-produced were calculated by using the Scherer
formula21,22,23.
P. aeruginosa is a hazardous
microorganism that is a source of various infections in the community and
hospitals. In recent years, the incidence of multidrug-resistant P. aeruginosa
isolates has become a global public clinical issue 24. Bacterial
persistence refers to the survival of a minor proportion of germs after being
eliminated by bactericidal drugs at lethal levels. Co3o4-NPs were also tested
against P. aeruginosa persister cells. It was evaluated as an antibacterial and
suppression of biofilm formation agent concern to diverse reports 27.
The antibacterial activity of Co3O4-NPs was improved against a wide range of
microbial diseases 28. In general, the effects of Nanoparticles
ca3o4 against bacteria were summarized as follows: electrostatic interaction
causes mechanical harm to the cell wall, oxidative stress from the formation of
reactive oxygen species (ROS), and damage to protein activities and cell
structures from metal cation release 29.
CONCLUSIONS
To boost their
bactericidal potency, co3o4-nps were synthesized utilizing a photolysis method.
The current work was the first to indicate that co3o4-NPS may effectively
suppress biofilm and persister cell production of P. aeruginosa.
Acknowledgement
To each person
who aided us in accomplishing this project at Mustansiriyah University,
Department of Biology and Department of Chemistry
Conflict of
interest authors states that they do not have a
conflict of interest.
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Received: May 15, 2023/ Accepted: June 10, 2023 / Published:
June 15, 2023
Citation: Ahmed, A.M.; Al Marjani, MF;
Rheimah, A.M. Antibacterial And Anti-Biofilm Action Of Cobalt Oxide
Nanoparticles Beside Persister Pseudomonas Aeruginosa Isolates. Revis Bionatura 2023;8 (2) 78. http://dx.doi.org/10.21931/RB/2023.08.02.78