Research Article | Open Access
Shawgi A. Elissidig1,2, and Samy A. Selim1,3
1Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Jouf University, Sakaka, P.O. 2014, Saudi Arabia.
2Faculty of Medical Laboratory Sciences, Omdurman Islamic University, Omdurman, Sudan.
3Department of Botany, Microbiology Section, Faculty of Science, Suez Canal University, Ismailia, Egypt.
J Pure Appl Microbiol, 2019, 13 (3): 1769-1774 | Article Number: 5817
https://doi.org/10.22207/JPAM.13.3.52 | © The Author(s). 2019
Received: 10/08/2019 | Accepted: 09/09/2019 | Published: 25/09/2019
Abstract

This study analysed Pseudomonas aeruginosa and Staphylococcus aureus burn wound infections, in order to evaluation their incidence, histological change and antimicrobial susceptibilities. Out of 39 burn wound cases admitted to surgery department, 20 and 12 strains of P. aeruginosa and S. aureus respectively were isolated from pyogenic burned skin lesions. Antibiotic resistance sketches of these strains to antibiotics were strategized. All the tested strains were multiple antibiotic resistance. Developed rates of susceptibility were confirmed for P. aeruginosa isolates against cefotoxim, gentamicin and nitrofurantoin. Also this study was to examine relation between the demonstrated antibiotic resistance and the occurrence of plasmids. Molecular sizes of the noticed plasmids were 24,321 kbp in P. aeruginosa and 23,25 kbp in S. aureus. Plasmid curing in grouping with MIC purpose revealed that resistance of P. aeruginosa and S. aureus isolates was plasmid allied. Histological analysis of burn wound infection is created on the thought of microorganisms attacking viable tissue below the eschar surface. The great MAR recognised marks it required for antibiotic resistance testing to be piloted former to antibiotics remedy for burn wound infection.

Keywords

P. aeruginosa, S. aureus; Burns, Antimicrobial Agents, Plasmid; Histology.

Introduction

Burns hurt is a chief community health delinquent in many nations in the world. In case of a burn injury, the organism experiences complex changes in homeostasis; these changes are rarely comparable to changes in case of any other trauma or disorder. For this reason, mortality in the early phase of burns in such traumas is common1,2. Contamination in the burn injury stretches the therapeutic of the wound in all phases of healing; for this reason, it is important, that the treatment of the burn infection injury includes antibiotic therapy, deletion of necrotic tissues in time, ensuring the blood and oxygen source to the wound, the augmentation of the resistance of the organism, and the adequate diet3.

Bacterial contamination is one of the greatest severe difficulties in burn basis serious complications and death following thermal injury4. P. aeruginosa and S. aureus are the furthermost chief contagious and risky bacteria in injury patient5. P. aeruginosa is one of the maximum significant and greatest reasons of grave contamination in injury patients6-8. Treatment of these infections is problematical by antibiotic resistance9-12.

In this study examined the histologic examination of biopsy and antimicrobial resistance of bacterial isolates from Burns. Likewise, the persistence of this study was to study any relative between antibiotic-resistance of bacteria and the company of plasmids.

Materials and Methods

Sample Collection and Identification of Bacteria
Thirty-nine cases were selected from patients carried out at the Surgery Department, General Hospital, Port Said City, Egypt (Bioethics agreement according to the Ethical Committee, Suez Canal University, Egypt). The average age was 25 years. A swab of each pus sample was suspended in 3 mL of water peptone. Drops of the prepared postponements were banquet on surface of plates holding Pseudomonas selective medium and Staphylococcus 110 medium13. All plates were incubated face down and the bacteria were allowed to grow at 37°C for 24-48 hours prior to enumeration and further identification. Pure well-isolated colonies were preceded for their biochemical tests: Glucose oxidation/fermentation, lactose fermentation, oxidase test, nitrate reduction, indole reaction, urease production, coagulase test, Voges-Proskaure, arginine and ornithine utilization tests14.

Antibiotic Susceptibility Studies
All trials were piloted using the inventive typical cultures to escape the unstructured hurt of antibiotic resistance. Antibiotic resistance was tested using a modified Kirby-Bauer disc diffusion method15-17.

Plasmid Analysis
The plasmid isolated by mini-prep alkaline extraction method [18]. Concentrations and purity of DNA were assessed spectrophotometrically using Spectro 22, Labo Med, Inc., USA. Gels were prepared by adding 1% agarose and 5 µL ethidium bromide (10 mg/mL) to the TBE buffer. Pure DNA sample (3µL) was added to 12µL deionized water and 1µL endonuclease (EcoR I, Hind III (Sigma Production), BamH I (Roche Diagnostics GmbH). Sequential dilutions of acridine orange were used for curing of isolated plasmids18.

Histopathological Study
Twenty-five tissue biopsies were collected, all biopsies were practise and stains according to basic histopathological technique. Specimen were taken in 10% Buffered Neutral Formalin 2h and fixed immediately at 37°C. All specimens were taken and processed manually in which all specimens were dehydrate in 70%, 80% for 2hours, and then 90% alcohol for overnight and absolute alcohol for 2 hours. Then cleared in xylene for 3 hours for two coplin jar. Then tissues were saturate with molten paraffin wax 2 changes for 2hours each. Then fixed with paraffin wax in mould and left to harden at room temperature and then chilled in refrigerator. Twenty-five blocks were prepared, albumenized slides were set and with rotary microtome. Five-micrometre paraffin tissue sections of skin were examined to evaluate the Burn infection. haematoxylin and eosin staining method was used to evaluate the changes in skin 19.

RESULTS

Thirty-two isolates were recovered from pyogenic lesions burned skin over the course of this period. P. aeruginosa (20 strains) accounted for 51% of total isolates (Fig. 1). This trailed by S. aureus (12 strains, 31 %) and other organisms (18 %). As can be seen in table I, the rate of cephradine and kanamycin were similar among both P. aeruginosa and S. aureus isolates, at 100%. Advanced degrees of susceptibility were confirmed for P. aeruginosa isolates against cefotoxim, gentamicin and nitrofurantoin. All S. aureus strians were sensitive to vancomycin.

Fig. 1. Frequency of P. aeruginosa and S. aureus bacterial strains form studied wound burn infection
Fig. 2. Skin tissue contain bacterial contamination fixed in Buffered Neutral Formalin [hematoxyline and eosin Stain (x40)]

Plasmid profiles of the two bacterial isolates under study were detected. Only six out of 10 isolates ( 5 P. aeruginosa and 5 S. aureus) were found to contain plasmids. No plasmids could be detected for other 4 isolates. Two isolates (one P. aeruginosa and one S. aureus) were selected for further study as representative of plasmid-bearing isolates. Each of them was found contain only one plasmid (Table II). Molecular sizes of the detected plasmids were 24,321 kbp in P. aeruginosa and 23,25 kbp in S. aureus. Concentration and degree of purity of the plasmid DNA(S) were as in table II. Plasmid curing in combination with MIC determination revealed that resistance to ampicillin was plasmid linked. Digestion of the two isolated plasmids, singly with Hind III, EcoR I and Bam H1 restriction enzymes showed in table III. Number of recognition sites, number of fragments and the approximate of molecular size of restricted fragments as shown in table III. The histologic analysis of the paraffin sections with the haematoxylin and eosin staining method showed significant increase  in bacterial counts per counted high power field (40׳).

Table (1):
Antimicrobial resistance profile of P. aeruginosa and S. aureus isolates from studied wound burn infection

P. aeruginosa S. aureus
Isolates (n) 20 12
Ampicillin (10 μg) 100 33
Amoxycillin (10 μg) 60 50
Cephradine (30 μg) 100 100
Cefotoxim (30 μg) 0 ND
Cephalexin (30 μg) 59 Nd
Streptomycin (10 μg) ND 10
Colistin sulfate (10 μg) 100 25
Chloramphenicol(30 μg) ND 0
Velosef (30 μg) 75 25
Tetracycline (10 μg) 100 ND
Erythromycin (30 μg) ND 100
Kanamycin (30 μg) 100 100
Rifampicin (5 μg) 25 0
Gentamicin (30 μg) 0 43
Nitrofurantoin (300 μg) 0 ND
Vancomycin (30 μg) ND 0

ND: Not Determined

Table (2):
Characterisation of isolated plasmids including numbers, concentration, purity, curing and size

Plasamid Characterization P. aeruginosa S. aureus
No. of Plasmids 1 1
Conc.(μg/mL)/ Purity 320/2.1 320/2.0
% Curing 100% 100%
Total plasmid Size (Kbp) 24.321 23.25

Table (3):
The restriction patterns of plasmids from P. aeruginosa and S. aureus isolates from studied burned skin

Plasmids of: Restriction Enzymes No. of Recognition Sites No. of Fragments Size of Fragments (Kbp)
P. aeruginosa

Hind III

EcoR I

BamH I

3

3

0

2

2

0

13.3 and11.021

17.3 and 7.02

0

S. aureus

Hind III

EcoR I

BamH I

4

0

0

3

0

0

9.2, 7.75 and 6.3

0

0

DISCUSSION

Contagion is one of the supreme thoughtful worries in burn patients, and Pseudomonas, particularly P. aeruginosa, is the record vital, hardy, and hazardous organism10-12. P. aeruginosa blossoms on the humid burn wound external8,19 and is extremely pathogenic in thermally wounded immunosuppressed patients. Notwithstanding developments in therapeutic and surgical overhaul, the scenario residues deprived, with a death rate of about 80% in such patients8. In several cautiously unindustrialized nations such as Iran20-21, Zimbabwe22, South Korea4, Jordan23, Libya24, Nigeria25-26, India1,27-29, Turkey6,30 and Syria31. P. aeruginosa was described to be public bacteria between burn patients. Though P. aeruginosa is not a typical microbe of injury wound contagions in the industrialised countries, a few burn centres in Canada and the USA32, France33, and Italy34 have informed P. aeruginosa as significant bacterium in burn divisions.

The second best shared separate in this study was S. aureus, in studies from cautiously unindustrialized nations4,6,8,10-11,24-28. In the Surgery Department, S. aureus was initiate in 31% of sections occupied from the burn damages. In case of a severe burn, the chance of S. aureus taint rises, subsequently the patients expend more time in the Sector, and suffer more regular dressings. Thus, surgery should be achieved as primary as conceivable, hence reducing the period of hospital stay, infection-related treatment costs, and taming the value of cure2.

Antibiotics is single of the highest hazard influences for antibiotic resistance. Extreme custom of antibiotics excites the progress of antibiotics- resistant microbes, developments treatment expenses, and causes side possessions2. Antibiotics are only approved if the causal agent is found to be sensitive to antibiotics, preliminary with minor group antibiotics, and in the occurrence of the signs of wound infection. Throughout surgery, in case of average to severe burns, a sole amount of cefuroxime and vancomycin are suggested in case of P. aeruginosa and S. aureus infection correspondingly.

In many studies plasmid related resistance, particularly for pathogenic bacterial strains, are still of dangerous position35-38. Amount of gratitude locations, quantity of wreckages and the estimated of molecular size of restricted fragments few. As of the partial total of endonuclease used in this study. Digestion with EcoR I provided two fragments with P. aeruginosa while no fragments were observed for S. aureus isolate. Comprehensive description of these plasmids is required in future for more indulgent about gene appearance and constancy. Histological investigation of a burn wound biopsy documents the needed variation and is the greatest technique for finding of burn wound infection. We achieve that to make meticulous staining of skin tissue with haematoxylin and eosin staining.

Finally, it is recommending that officials in our burn units take into account recover workers’ sanitation; reflect suitable cleansing for all tools; found a novel and actual antibiotic strategy and avoid unnecessary use of antibiotics.

Declarations

Acknowledgements
The author would like to thank the Surgery Department Staff General Hospital, Egypt, for providing the urine samples which was the source of bacterial strains.

Conflict of Interest
The authors declares that there is no conflict of interest.

Authors’ Contribution
Author listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

Funding
None

Data Availability
All datasets generated and analyzed during this study are available in the NCBI database repository, Accession No: MH161378 and is included in the manuscript.

Ethics Statement
The study was approved from the Universities Ethical Committee (ENREC).

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