Journal of King Saud University – Science 34 (2022) 101706

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Journal of King Saud University – Science

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Original article

In vitro bactericidal and imipenem synergistic effect of nano-silver

against multiple drug-resistant Pseudomonas aeruginosa
Sulaiman A. Al Yousef
Department of Clinical Laboratory Sciences, College of Applied Medical Science, University of Hafr Al Batin, P. O. Box 1803, Hafar Al Batin 31991, Saudi Arabia

a r t i c l e i n f o a b s t r a c t

Article history: Background: Pseudomonas aeruginosa (P. aeruginosa) is an aerobic gram-negative pathogen. It induces
Received 12 June 2021 decline of lung functions and increase mortality rate. It has a high antimicrobial resistance rate limiting
Revised 3 November 2021 number of antibiotics which can be used.
Accepted 7 November 2021
Objectives: Evaluation of bactericidal and antibiotic synergistic effect of Nano-silver (Ag-NP) against
Available online 12 November 2021
Multiple drug-resistant P.aeruginosa (MDR P. aeruginosa).
Material and methods: P.aeruginosa were isolated from clinical specimens. MicroScan WalkAway-96SI
Keywords:
System was used for laboratory Identification, antimicrobial susceptibility. Minimal inhibitory concentra-
Nano-silver
Pseudomonas aeruginosa
tion (MIC) was determined by a microdilution method. Time kill assay determined by incubation MRSA
Imipenem with different concentrations of Ag-NP (0, 50, 100, and 200 lg/ml) in a shaking incubator at 37 °C for
Drug resistant 24 h. Growth curves of bacterial cell cultures were attained through repeated measures of the optical den-
sity (O.D.) at 600 nm. The disc diffusion method was used to evaluate synergistic of Ag-NP with antibiotics.
Results: The MIC value of Ag-NPs against MDR P. aeruginosa was in at of 5 lg/ml. The bacterial growths of
cells treated with 1.25, 2.5, 5 and 10 mg/ml Ag-NPs were inhibited. After 4 h, almost all treated bacterial
cells were dead. All of the combinations showed significant synergistic effect (P-value 0.003), and the
result showed the highest synergism at concentrations of Imipenem at MIC (32 mg/ml) and 16 mg/ml.
Conclusion: Nano-Silver has high therapeutic activity against MDR P. aeruginosa, it can be suggested as an
alternative or adjuvant with antibiotics for MDR P. aeruginosa treatment. Further studies are required for
understand synergistic effect of Ag-NP combining, and assessment of its safety.
Ó 2021 The Author. Published by Elsevier B.V. on behalf of King Saud University. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction The rapid incidence and spread of MDR P. aeruginosa strains

leads it a high morbidity and mortality of infections caused by
Pseudomonas aeruginosa is a widespread nosocomial pathogen MDR P. aeruginosa (Fowler and Nancy, 2014). Clinically isolated
(Fowler and Nancy, 2014; Breidenstein et al., 2011), can cause to strain, specially, have witnessed a serious situation. Therefore,
cause dangerous opportunistic infections among immunocompro- there is an urgent need to develop novel antimicrobial compounds
mised patients, for example, chronic lung infections in cystic fibro- or combinations with potent antibacterial activity against clinically
sis patients (Winstanley et al., 2016). P. aeruginosa has been isolated MDR P. aeruginosa.
classified as a superbug because of its extraordinary adaptability, Among the range of materials whose antimicrobial property is
intrinsic resistance to antibiotics and ease of acquiring microbial being investigated, AgNP appear as a promising new antibacterial
resistance, which also contribute to its multidrug resistance and agent that could be helpful to confront this and other drug-
pan-drug resistance to existing antibiotics (Livermore, 2002; resistant bacteria. Different studies have established the bacterici-
Oliver et al., 2015). dal effect of Ag-NP in Gram negative and Gram-positive bacteria,
but the bactericidal mechanism of this compound has not been
clearly elucidated. Morones et al. (Yoon et al., 2008) defined the
E-mail address: drsulaiman@uhb.edu.sa antibacterial activity of silver nanoparticles in four types of
Peer review under responsibility of King Saud University.
Gram-negative bacteria: Escherichia coli, Vibrio cholera, P.aerugi-
nosa, and Salmonella tiphy and suggested that Ag-Np attach to the
surface of the cell membrane and disturb its function, penetrate
bacteria, and release silver ions (Morones et al., 2005).
Production and hosting by Elsevier

https://doi.org/10.1016/j.jksus.2021.101706
1018-3647/Ó 2021 The Author. Published by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
S.A. Al Yousef Journal of King Saud University – Science 34 (2022) 101706

Silver nanoparticles showed antibacterial activity against some 2.5. Synergistic effect of nano-silver with antibiotics
drug-resistant bacteria (Inoue et al., 2010; Birla et al., 2009). In
addition, several studies regarding the synergistic activity of Ag- The disc diffusion method was used to evaluate the synergistic
Np in combination with other compounds have been reported: a of silver nanoparticles with antibiotics. Based on the CLSI standards
combination of amoxicillin and Ag-NP showed greater bactericidal (Wayne, 2011). By using the spread plate method, the Mueller-
efficiency towards E.coli than when they were applied separately Hinton agar plates were inoculated with the turbidity adjusted
(Li et al., 2005), and interactions between Ag-NP and polymyxin bacterial suspension, and antibiotic discs (Hi Media Chemicals
B showed synergistic effects for Gram negative bacteria (Ruden Pvt. Ltd., Mumbai, India), were placed on plates containing MDR
et al., 2009). P. aeruginosa and was incubated at 37 °C for 24 h after which the
Carbapenem such as imipenem and meropenem play key role inhibition zone diameter of plates containing colloidal silver,
treatment of P. aeruginosa infections. Lately, a problem of car- MDR P. aeruginosa, and antibiotic disc and those containing only
bapenem resistance has loomed with emergence of Carbapenem the antibiotic disc and P. aeruginosa organisms were measured,
resistant P. aeruginosa (Maltezou, 2009; Lister et al., 2009). Treat- and zone of inhibition (ZOI) was measured by subtracting the disc
ment of imipenem-resistant infection is very difficult, due to imi- diameter from the total inhibition zone diameter. The synergistic
penem resistance genes are located on transferable genetic effect was quantified by the equation (B A)/A  100, where A
elements such as plasmid. These infections are associated with and B are the ZOI for antibiotic and antibiotic + silver nanoparticles,
high mortality and morbidity rates (Bebrone, 2007). In the present respectively.
study, we investigated the antimicrobial activities of Ag-NP alone
and in combination with Imipenem against MDR P. aeruginosa. 2.6. Statistical analysis

2. Material methods All experiments were conducted in triplicate to validate the

producibility of the experiments. Statistical analysis was carried
2.1. Bacterial isolate out by one-way ANOVA at a P-value of 0.05 by Microsoft Excel soft-
ware (Microsoft, USA).
Ninety P. aeruginosa isolate from clinical specimens at Microbi-
ology laboratory, Department of clinical analysis at Gynecology
3. Results
and Children hospital, Hafr Elbatin, KSA. Mid-stream urine, suction
tip, pus and blood specimens were collected aseptically for bacte-
3.1. Bacterial strains
riological examination. Handling, transporting, and storing of col-
lected samples were made at refrigeration temperature. All
Of ninety P. aeruginosa isolates, 15 were MDR P. aeruginosa.
samples were inoculated on Blood agar, incubated at 37 °C for
Multiple drug-resistant P. aeruginosa was defined as isolate showed
overnight, and colonies were processed.
resistance to three or more of the following eight sentinel
antimicrobial agents: amikacin; aztreonam; cefepime; ceftazidime;
2.2. Antimicrobial testing ciprofloxacin; colistin; imipenem; and piperacillin/tazobactam.

The MicroScan WalkAway-96SI System was used in laboratory

Identification, antimicrobial susceptibility testing and P. aeruginosa 3.2. Antimicrobial activity of nano-silver
detection was performed with Neg Combo Panel Type 32 (Dade
BehringÒ, USA). All procedures were performed according to the Cell viability assay was used to assess the bactericidal effect of
manufacturer’s instructions. Ag-NP, multiple-drug resistant P. aeruginosa was subjected to two-
fold Ag-NP serial dilutions for 24 h. Nano-Silver affected bacterial
cellular viability in a dose-dependent manner. Bacterial cell viabil-
2.3. Antimicrobial activity of NANO-silver
ity was measured by using a colony-forming capacity assay. MDR
P. aeruginosa was inhibited at concentrations over 5 mg/ml at 105
Nano-silver were obtained from SIGMA ALDRICH, SAINT LOUIS,
CFU where no visible bacterial growth in agar plate.
USA. MIC defined as the lowest concentration of AgNP preparation
that prevented bacterial growth. MIC The minimal inhibitory con-
centration (MIC) was determined by a microdilution method, using 3.3. Time-kill assay
LB broth (Sigma–Aldrich) and final inoculum of 105 CFU/ml. Bacte-
ria were incubated with serial twofold dilutions of Ag-NP, and the Growth curves of P. aeruginosa treated with Ag-NPs showed that
effect on cell viability was measured after a 24 h period of incuba- Ag-NPs can inhibit the growth and reproduction of bacterial cells,
tion. Bacterial cell viability was measured by using a colony- Fig. 1. The bacterial growths of P. aeruginosa treated with 0, 1.25
forming capacity assay in nutrient agar (Kohanski et al., 2007). 2.5, 5 and 10 mg/ml Ag-NPs were inhibited. After 4 h, almost all
All the assays were run in parallel with negative and positive treated bacterial cells were dead. The bacterial growth of the cells
control. treated with 1.25 and 2.5 mg/ml Ag-NPs were also lower than that
of cells in the control group.
2.4. Time-kill assay
3.4. Synergistic effect of nano-silver with imipenem
To examine the growth curves of bacterial cells exposed to Ag-
NPs, Mueller-Hinton broth with different concentrations of Ag-NPs Nano-Silver showed a significant synergistic effect when it
powder (0, 1.25, 2.5, 5, and 10 lg/ml) was used, and the bacterial combined with imipenem P-vale 0.003, Table 1. Also, the synergis-
cell concentration was adjusted to 105 CFU/ml (Salomoni et al., tic effects of Ag-NP were investigated with Imipenem against MDR
2017). Each culture was incubated in a shaking incubator at P. aeruginosa using the disc diffusion method, and the effects eval-
37 °C for 24 h. Growth curves of bacterial cell cultures were uated by determination of the synergism percentage. The result
attained through repeated measures of the optical density (O.D.) showed the highest synergism at concentrations of Imipenem at
at 600 nm. (32 mg/ml) and 16 mg/ml, Fig. 2.
2
S.A. Al Yousef Journal of King Saud University – Science 34 (2022) 101706

Fig. 1. Growth curve of MDR P. aeruginosa with different concentrations of Nano-silver.

Table 1
Statistical analysis of inhibition zones of different imipenem concentration combined with Ag-NP at different combinations.

Source of Variation SS df MS F P-value F crit

Between Groups 1066.8 2 533.4 9.212435233 0.003764567 3.885294
Within Groups 694.8 12 57.9
Total 1761.6 14

Fig. 2. Synergistic effect of combination of Ag-NP with Imipenem at different concentrations for them.

4. Discussion The surveillance results showed a high incidence of MDR P. aerug-

inosa in our hospitals.
The increasing trend of antimicrobial resistance alarms about Many studies evaluated the antibacterial activity of Ag-NP
the dilemma that could be faced when treating these infections. (Chaloupka et al., 2010; Gade et al., 2010). Nano-Silver reported

3
S.A. Al Yousef Journal of King Saud University – Science 34 (2022) 101706

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