Research Article | Open Access
Muazzam Sheriff Maqbul1, Ali Mohamed Alshabi2, Aejaz A. Khan3, S.M. Shakeel Iqubal3 , Tasneem Mohammed3, Ibrahim Ahmed Shaikh4, Areej Dawoud3, Uday M. Muddapur5, Mohammed Shahid Hussain6 and S.K. Singh7
1Faculty of Microbiology and Immunology, Ibn Sina National College of Medical Sciences, Al Mahjar Street: 31906, Jeddah 21418, Kingdom of Saudi Arabia.
2Department of Clinical Pharmacy, College of Pharmacy, Najran University, Najran, Saudi Arabia.
3Department of General Science, Ibn Sina National College of Medical Sciences, Al Mahajar Street: 31906, Jeddah 21418, Kingdom of Saudi Arabia.
4Department of Pharmacology, College of Pharmacy, Najran University, Najran, Saudi Arabia.
5Department of Biotechnology, KLE Technological University, BVB Campus, Hubballi, 580031, India.
6Department of Orthodontics and Dentofacial Orthopedics, M. A. Rangoonwala Dental College, Pune, Maharashtra, India.
7Department of Chemistry, GGV (Central University), Bilaspur (C.G) – 495009, India.
J. Pure Appl. Microbiol., 2020, 14 (1): 255-260 | Article Number: 5994
https://doi.org/10.22207/JPAM.14.1.26 | © The Author(s). 2020
Received: 13/12/2019 | Accepted: 18/03/2020 | Published: 28/03/2020
Abstract

The present study aimed to determine the susceptibility of Pseudomonas aeruginosa strains isolated from patients with nosocomial infections to standard synthetic chemical antibiotics and organic ethanoic acids derived from local produce. The minimum inhibitory concentrations (MIC) of the  standard synthetic antibiotics determined from standard e-test results and antibiotic sensitivity tests showed many multidrug-resistant strains among the isolates. We compared the susceptibility of  these strains  to organic ethanoic acids derived from different sources using standard microbiological assays. All strains of P. aeruginosa isolated from the patients were susceptible to the organic ethanoic acids with a satisfactory MIC and minimum bactericidal concentrations. Therefore, organic ethanoic acids were more effective against P. aeruginosa than standard synthetic antibiotics.

Keywords

Antimicrobial activity, Pseudomonas aeroginosa, ethanoic acid, Nosocomial infection, Immuno suppressed, Vinegar.

Introduction

Pseudomonas aeruginosa is a Gram-negative motile bacterium that is a major cause of community-acquired, and hospital-borne nosocomial infections1,2. The major concern about nosocomial infections with P. aeruginosa is that > 10% of infections are caused by multidrug-resistant species1,3,4 that arise through antibiotic abuse. Pseudomonas is only one among many bacterial species that have become resistant to multiple antibiotics, and the most resistant strain is P. aeruginosa5. Gessard isolated P. aeruginosa from green pus in 1882 and since then it has been studied in detail, especially with regard to nosocomial infections1. The characteristic virulence factors of this bacterium are the exopigments pyoverdin and pyocyanin, which are potentially involved in the occurrence of these infections1. Pseudomonas aeruginosa causes versatile infections in humans, especially when immunosuppressed, and it is categorized as the most important bacterial cause of infections acquired during prolonged hospitalization. This bacterium causes frequent infections due to its natural adaptability and abundance, and it has emerged as the most important species with which to analyze multidrug resistance1,6. Here, we isolated multidrug-resistant strains from clinical specimens derived from patients with nosocomial infections, then compared the susceptibility of these strains to commercial synthetic antibiotics and to ethanoic acids prepared from organic apple cider, sugar molasses, dates, grapes, and grains using conventional standard microbiological techniques4,7,8,9. Pseudomonas aeruginosa ATCC27853 was the standard control strain. Because  ethanoic acids contains carboxylic acids that control the formation of P. aeruginosa biofilm, they should help to suppress infection with this bacterium6,8,9.

Materials and Methods

All chemicals, reagents, and media components for this study were purchased from Sd Fine Chem Ltd, (Kolkata, India) Loba Chemie PvT., Ltd. (Mumbai, India), HiMedia (Mumbai, India), bioMérieux SA., (Marcy l’Etoile, France or Sigma Aldrich Corp. (St. Louis MO, USA).

Isolation and purification of P. aeruginosa
Green pus, green catheters, urine, greenish ventilators, abscesses, throat swabs, nasal swabs, aspirates and collected from patients with nosocomial infections, and samples collected at surgery were processed using standard aseptic microbiological techniques. The samples were streaked onto plates containing cetrimide agar, a selective and differential medium, and incubated overnight at 37°C to allow P. aeruginosa to secrete exopigments. The isolates were identified and purified based on positive oxidase tests. The authenticity of the standard P. aeruginosa ATCC 27853 strain was confirmed in the same manner5,10,11.

Antimicrobial susceptibility tests
The antimicrobial susceptibility of the clinical isolates and the standard strain to standard synthetic chemical antibiotics was assessed using rapid e-tests4,5,7, Briefly, the isolates were inoculated onto separate plates containing Mueller-Hinton agar and standard e-test plastic strips were infused with each antibiotic and incubated with the isolates at 37°C overnight to develop zones and ellipses. The interaction of the ellipse was taken as the minimum inhibitory concentration (MIC), whereas the zone indicated the susceptibility of the antibiotic to the bacterium. Conventional standard was employed to observe The susceptibility of the P. aeruginosa isolates and the standard strain to five organic ethanoic acids was assessed by the standard diffusion method using Kirby-Bauer discs. Briefly, the isolates were inoculated separately on Mueller-Hinton agar plates with the infused discs for 24 h at 37°C to form zones, indicating the sensitivity of the bacterium toward the organic ethanoic acids. The MIC and minimum bactericidal concentration (MBC) reflecting the ability of ethanoic acid to kill the bacterium were estimated using the standard tube dilution method. Briefly, the isolates were separately inoculated into ethanoic acids diluted in peptone water, and incubated for 24 h at 37°C.  The absence of turbidity indicated the sensitivity of the bacterium to the test agent. The last dilution with turbidity determined the MIC value of the test agent against the bacterium.

The MBC was determined by inoculating each dilution of MIC onto separate agar plates for each isolate5,10,11,12, then incubating them for 24 h at 37°C. The first dilution with no growth defined the MBC of the acid toward the bacterium Pseudomonas aeruginosa ATCC278.

RESULTS AND DISCUSSION

The results obtained from the e-tests of the susceptibility of P. aeruginosa to standard synthetic chemical antibiotics compared with organic ethanoic acid showed that none of the clinical P. aeruginosa isolates were resistant to organic ethanoic acid, whereas all the clinical isolates were resistant all the standard synthetic antibiotics tested. The best and worst antibiotic zone diameters for the standard synthetic chemical antibiotics against the clinical isolates of P. aeruginosa were 23 and 2 mm, respectively, compared with 29 and 20 mm, respectively, for organic ethanoic acids.  Among the 10 clinical isolates, the strain from the wound sample was resistant to 8 of the 12 standard synthetic chemical antibiotics, and sensitive to piperacillin/tazobactam (PTC) and tetracycline (TC) with a zone diameter of 20 mm. This strain also had intermediate sensitivity toward gentamicin (GM) and meropenem (MP) with zone diameters of 9 and 11 mm, respectively. The clinical P. aeruginosa isolates from the wound sample were the most multidrug-resistant among the strains isolated herein,  with different degrees of sensitivity to all tested antimicrobial agents. These results showed that organic ethanoic acids were more effective than standard synthetic antibiotics even against the most multidrug-resistant of the clinical P. aeruginosa isolates obtained herein.

Fig. 1. Comparison of mean e-test susceptibility of 10 clinical Pseudomonas aeruginosa isolates to standard synthetic chemical antibiotics

Fig. 2. Comparison of mean conventional susceptibility of 10 clinical Pseudomonas aeruginosa isolates to organic ethanoic acids derived from different sources.

Table (1):
Comparison of Pseudomonas aeruginosa susceptibility to standard antibiotics according to e-test zone diameters (mm).

Antibiotics
Sample AK PM TZ TX GM MP NI PTC TC TLC TM TS S I R
Green pus 2R 3R 2R 4R 2R 20S 18S 2R 19S 21S 12I 20S 5 1 6
Green cath 131 2R 3R 2R 4R 12I 20S 22S 20S 23S 21S 20S 6 2 4
Urine 4R 3R 2R 4R 2R 20S 20S 20S 20S 20S 10I 10I 5 2 5
GV 5R 2R 2R 4R 2R 10I 20S 21S 21S 20S 10I 11I 5 3 4
Abscess 4R 3R 2R 4R 2R 20S 20S 10I 10I 21S 20S 20S 5 2 5
Throat swabs 2R 4R 2R 21S 20S 2R 4R 22S 20S 20S 19S 18S 7 0 5
Aspirates 2R 4R 2R 4R 2R 9I 11I 21S 20S 20S 4R 2R 3 2 7
Wound 4R 2R 4R 2R 9I 11I 6R 20S 20S 2R 4R 2R 2 2 8
Nasal swabs 2R 22S 20S 10I 20S 20S 21S 20S 23S 20S 4R 3R 8 1 3
 Surgical sources 4R 3R 2R 4R 2R 4R 2R 9I 11I 21S 20S 20S 3 2 7
ATCC 27853 Control 11I 20S 9I 11I 22S 20S 10I 20S 20S 21S 20S 23S 8 4 0
Mean Zone value for all the specimens 7.5 12 11 11.5 11 11 11.5 12 16.5 12.5 12.5 12.5 5 2 4

I, intermediate susceptibility; R, resistant; S, susceptible.

Table (2):
Comparison of Pseudomonas aeruginosa susceptibility to standard antibiotics determined as minimum inhibitory concentrations in e-tests.

Antibiotic MIC (µg/mL)
Sample AK PM TZ TX GM MP NI PTC TC TLC TM TS
Green pus 1 1 1 0.25 0.25 0.5 0.5 0.5 0.25 0.25 0.5 0.5
Green cath 1.25 0.5 1 1 0.25 0.5 0.5 0.5 0.25 0.25 0.25 0.5
Urine 1 1 1 0.25 0.25 0.5 1 1 1 0.5 0.25 0.5
GV 1 1 1 0.25 0.25 0.5 0.5 0.25 0.25 0.25 0.25 0.5
Abscess 1 1 1 0.25 0.25 0.5 1 1 1 0.25 0.25 0.25
Throat swab 0.25 0.25 0.5 1 1 0.25 1 1 1 0.5 0.5 0.25
Aspirates 0.25 0.5 0.5 0.5 0.25 0.5 0.5 0.5 0.25 0.25 0.5 0.5
Wound 1 1 1 0.25 0.25 0.5 0.25 0.5 0.5 0.5 0.25 0.5
Nasal swab 0.25 0.25 0.5 1 1 1 1 1 0.25 0.25 0.25 0.25
Surgical 0.25 0.5 0.5 0.5 0.25 0.25 0.5 0.5 0.5 0.25 0.5 0.25
ATCC 27853 0.25 0.5 0.5 0.5 0.25 0.25 0.5 0.5 0.25 0.25 0.25 0.25
Mean MIC* 0.75 0.625 0.75 0.625 0.625 0.625 0.625 0.625 0.625 0.375 0.375 0.375

*For all samples. Cath, catheter; GV, greenish ventilator; MIC, minimum inhibitory concentrations (µg/mL); Surgical, sourced during surgery.

Table (3):
Comparison of diffusion zone diameters (mm) among Pseudomonas aeruginosa isolates that were susceptible to organic ethanoic acids in conventional assays.

  Organic ethanoic acid sources  
Sample Apple cider Sugar molasses Dates Grapes Grains
Green pus 22 22 22 22 22
Green catheter 20 22 19 22 22
Urine 21 21 20 22 24
Greenish ventilator 22 22 23 22 23
Abscess 27 22 24 22 24
Throat swab 24 22 23 26 26
Aspirates 23 26 24 22 22
Wound 24 22 22 22 22
Nasal swab 22 22 22 22 22
Surgical samples 25 22 22 22 22
ATCC 27853 (Control)  29 26 26 21 20
Mean Zone diameter (mm) 24.5 24 21.5 23.5 23

Tables 1 and 3 show detailed comparison of the antimicrobial susceptibilities of all P. aeruginosa isolates from patients with nosocomial infections to standard synthetic chemical antibiotics and organic ethanoic acids. The mean MIC of standard antibiotic dilutions in e-tests and of organic ethanoic acids ranged from 0.375 – 0.750 µL/mL and 0.25 – 0.875 µL/mL, respectively for all P. aeruginosa isolates tested. Tables 2 and 4 respectively show details of the MIC of the standard synthetic chemical antibiotics and organic ethanoic acids for all Pseudomonas aeruginosa isolates from patients with nosocomial infections. The mean values of the MBC of organic ethanoic acid ranged from 0.5 – 1.25 µL/mL toward all P. aeruginosa isolates. Table 5 shows a detailed comparison of the MBC of organic ethanoic acids for all the P. aeruginosa isolates.  

Table (4):
Comparison of minimum inhibitory concentrations of organic ethanoic acids among Pseudomonas aeruginosa isolates.

Organic ethanoic acids (µg/mL)
Sample Apple cider Sugar molasses Dates Grapes Grains
Green pus 0.25 0.5 1 1 1
Green catheter 0.25 0.25 1 1 0.75
Urine 0.25 0.5 1 0.75 1
Greenish ventilator 0.25 0.25 1 0.5 1
Abscess 0.25 0.5 0.75 0.5 0.75
Throat swabs 0.25 0.25 0.5 0.5 1
Aspirates 0.25 0.75 0.75 1 0.5
Wounds 0.25 0.75 0.75 0.75 0.5
Nasal swab 0.25 0.25 1 0.5 1
Surgical sources 0.25 0.75 1.25 1 0.5
ATCC 27853 0.25 0.25 0.5 0.5 0.5
Mean MIC (µg/mL)* 0.25 0.5 0.875 0.75 0.75

MIC, minimum inhibitory concentrations. *Derived from all samples.

Table (5):
Comparison of minimum inhibitory concentrations of organic ethanoic acids among Pseudomonas aeruginosa isolates determined by conventional assays.

Organic ethanoic acids (µg/mL)
Sample Apple cider Sugar molasses Dates Grapes Grains
Green pus 0.5 0.75 1.25 1.25 1.25
Green catheter 0.5 0.5 1.25 1.25 1
Urine 0.5 0.75 1.25 1 1.25
Greenish ventilator 0.5 0.5 1.25 0.75 1.25
Abscess 0.5 0.75 1 0.75 1
Throat swabs 0.5 0.5 0.75 0.75 1.25
Aspirates 0.5 0.5 1 1.25 0.75
Wound 0.5 0.5 1 1 0.75
Nasal swabs 0.5 0.5 1.25 0.75 1.25
Surgical 0.5 0.1 1.5 1.25 0.75
ATCC 27853 0.5 0.5 1 0.75 0.75
Mean MIC (µg/mL) 0.5 0.75 1.25 1 1

*For all samples. MIC, minimum inhibitory concentrations.

The comparative findings of the antimicrobial susceptibility test values for the 10 clinical isolates of P. aeruginosa derived from patients with nosocomial infections suggests that organic ethanoic acid is significantly more effective than standard synthetic antibiotics.

CONCLUSION

The most important finding of the study was that the ATCC 27853 standard strain of P. aeruginosa was more susceptible to organic ethanoic acids than to standard synthetic antibiotics.

The susceptibility of the 10 clinical isolates of P. aeruginosa derived from the patients was variable compared with that to organic ethanoic acids. This finding indicates that the multidrug-resistant strains in the clinical isolates might be the result of excessive antibiotic administration, especially during prolonged hospitalization. Multidrug-resistant strains emerge due to constant disregard for warnings about antibiotic use published by the World Health Organization (WHO). The present findings showed that natural remedies such as organic ethanoic acids can serve as an alternative to synthetic antibiotics not only for eradicating multidrug-resistant organisms but also for other infections, which together with zero side effects could help save many lives.

Declarations

ACKNOWLEDGMENTS
We acknowledge Ibn Sina National College, Jeddah (Kingdom of Saudi Arabia)  Administration for their constant support and encouragement due to which this project was come to existence.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.

AUTHORS’ CONTRIBUTION
All authors reviewed the data and the manuscript.

FUNDING
None.

ETHICS STATEMENT
This article does not contain any studies with human participants or animals performed by any of the authors.

AVAILABILITY OF DATA
All datasets generated or analyzed during this study are included in the manuscript and/or the Supplementary Files.

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