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Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 21  |  Issue : 1  |  Page : 23-28

Molecular technology for the detection of Pyoviridine gene in Pseudomonas aeruginosa isolated from burn cases


1 Department of Babylon Health, Ministry of Health, Baghdad, Iraq
2 Department of Microbiology, College of Medicine, AL Mustansiriyah University, Baghdad, Iraq
3 Institute of Genetic Engineering and Biotechnology for Postgraduate Studies, University of Baghdad, Baghdad, Iraq

Date of Submission30-Jul-2021
Date of Acceptance06-Sep-2021
Date of Web Publication30-Jun-2022

Correspondence Address:
Mrs. Eman Nassir Hussan AL-Jesmany
Department of Babylon Health, Ministry of Health, Baghdad
Iraq
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mj.mj_14_21

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  Abstract 

Objectives: Using molecular technology for the purpose of confirmation of the diagnosis of Pseudomonas aeruginosa which depends on the detection of proteins like Pyoverdine gene of P. aeruginosa as diagnostic test. Materials and Methods: This work was done on 110 patients who had sustained burn injury from hospitals Baghdad, Al-Yarmouk and the Medical City Teaching Hospital during the period from October 2020 to the end of March 2021. The collected samples were cultured on different media (blood agar, MacConkey agar, nutrient agar, and Cetrimide agar) for isolation of P. aeruginosa bacteria as well as automated biochemical tests such as Api20E and VITEK 2 systems. The results showed that 76 (69%) samples had bacterial growth of P. aeruginosa. Antibiotic susceptibility testing was evaluated using the VITEK 2 compact. The Pseudomonas was detected using species-specific gene 16SrRNA gene using polymerase chain reaction (PCR) method and also detected by (PCR) using newly designed primers with a molecular size (389 bp) for pyoviridine gene. Results: This work showed that the predominant growth of burn wound infections was P. aeruginosa 76 samples (69%). Antibiotic susceptibility testing results showed the same sensitivity pattern of P. aeruginosa isolates to ceftriaxone and cephalothin (67.1%), gentamicin, piperacillin, ceftazidime, cefepime, and cefotaxime (65.7%). Resistance to imipenem, tobramycin, ticarcillin, and meropenem were (57.8%), (51.3%), (56.5%) and (55.2%) respectively, and had the highest sensitivity to amikacin (34.2%). Moreover, the highest resistance was to ciprofloxacin (69.7), and norfloxacin (71%). The results of using the 16SrRNA gene for the detection Pseudomonas give positive results (100%). Pseudomonas spp was detected by (PCR) for pyoviridine gene, revealed that 50% isolates give positive results. PCR product pyoviridine of the isolates (3) with the highest resistance to fluoroquinolones (ciprofloxacin and norfloxacin) was sent to nitrogen-based sequencing, and the sequencing results revealed the mutation presence. Conclusion: This study shows the current resistant pattern of P. aeruginosa against different classes of antibiotics and the involvement of several virulence genes in resistance mechanisms by using PCR which ultimately helps to select appropriate antibiotics useful for the treatment of many burned complicated by P. aeruginosa.

Keywords: Burn patients, multi-drug resistance, Pseudomonas aeruginosa, pyoviridine gene


How to cite this article:
AL-Jesmany EN, Abbas OK, Hasan AlSaadi BQ. Molecular technology for the detection of Pyoviridine gene in Pseudomonas aeruginosa isolated from burn cases. Mustansiriya Med J 2022;21:23-8

How to cite this URL:
AL-Jesmany EN, Abbas OK, Hasan AlSaadi BQ. Molecular technology for the detection of Pyoviridine gene in Pseudomonas aeruginosa isolated from burn cases. Mustansiriya Med J [serial online] 2022 [cited 2022 Nov 30];21:23-8. Available from: https://www.mmjonweb.org/text.asp?2022/21/1/23/349304


  Introduction Top


Pseudomonas aeruginosa is a Gram-negative rod bacterium, which has a remarkable ability to adapt and thrive in a variety of environments: Water, soil, occupational places, such as metalworking, fluids.[1] P. seudomonas causes the most common bacterial infection coupled with hospital infections in burns and pneumonia that are related to ventilation. It is responsible for otitis media, inflammation of the follicle, inflammation of the cornea, Burns wounds, diabetic foot infections, urinary tract infections, bacteremia, and inflammation in cystic fibrosis patients.[2] P. aeruginosa can produce multiple dyes in the agricultural medium, including the dye of green pyocyanin, pyoverdine pigment, the red poyrubin pigment, and the black pyomelanin pigment.[3] many virulence factors are produced that include secreted factors such as cytotoxic pigment pyocyanin, siderophores, alkaline protease, elastase, exotoxin A, lipopolysaccharide, pili, flagella, and biofilm formation (BF).[4] Pyoverdine, a siderophore produced by Pseudomonas spp., is also known to contribute to its virulence.[5]

Pyoverdine can also hijack iron from other host sources, including mitochondria, which apply to considerable damage on the host.[6] Iron-bound pyoverdine (known as ferripyoverdine) functions as a signaling molecule that triggers the release of the alternate sigma factor PvdS from sequestration by the intermembrane FpvA/FpvR complex.

Once released, PvdS promotes the expression of at least two secreted toxins (exotoxin A and the protease PrpL) and also its own biosynthetic machinery. The iron provided by pyoverdine is required for BF.[7]

Burn infections are caused by both Gram-positive and Gram-negative microorganisms, currently, the common pathogens isolated from burn patients are P. aeruginosa, Staphylococcus aureus, β-hemolytic streptococci, Escherichia coli, Klebsiella species, and various coliform bacilli.[8]


  Materials and Methods Top


A total of 110clinical specimens were collected at the period between the beginning of October 2020 to the end of march 2021from patients suffering from burns infections. Samples were collected from different age groups, ranging from 5 to 65 years, from different parts of the body including lower extremities, upper extremities, and trunk, in different proportions. The isolates were identified based on the colony morphology on different culture media, MacConkey agar, and blood agar and incubated at 37°C for 24 h after the incubation period, nonlactose fermented colonies were transferred on cetrimide agar medium, which is a special selective medium for P. aeruginosa and incubated at 37°C for 24 h. Bacterial isolates were diagnosed via Oxidase Test, Catalase Test. and confirmed by using IMViC biochemical tests, Api 20 E and Vitek compact 2 system. Furthermore, Pseudomonas was detected by using species-specific gene 16SrRNA gene using polymerase chain reaction (PCR) method and species-specific primers. Kirby–Bauer method was followed for antibiotics susceptibility for all isolated specimens as P. aeruginosa. The enrolled isolates were subjected to DNA extraction using Presto Mini g DNA bacteria Kits extraction genomic DNA, Purification depending on the instruction of manufacturing company (iNtron) (Kore).

Genomic DNA was prepared from overnight cultures grown, 1000 microliter of broth was centrifuged, 100 μl buffer lysis + 100 μl protease were added and incubated for 1 h at 56°C, then 220 μl ethanol was added; 600 μl were transferred to spin column, centrifuged at 8000 rpm/1 min. The rest of the sample was transferred to spin column and centrifuge. Then, 500 μl of wash 1 buffer was added centrifuged. Transfer to new wash tube and 500 μl wash 2 was added. Spin column transferred to new wash tube, centrifuged at 13,000/3 min, then transfer to elution tubes and add 50 μl preheated elution Buffer (60°C). Incubate 3 min at room temperature; centrifuge 14000 rpm/1 min.[9] Primer Preparation Lyophilized forward and reverse primers for the Pyoverdine gene and 16S rRNA [Table 1]. Were suspended in nuclease-free water to give a final concentration of (100 pmol/μl) as stock solution; to prepare 10 pmol/μl concentration as work primer re-suspended 10 μl in 90 μl of nuclease-free water.
Table 1: The sequence forward and reverse primers of pyoverdine gene and 16S ribosomal RNA

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The molecular detection of 16S rRNA gene, this step was carried out by adding 12.5 μl from OneTaq (NEB) master mix, 5 μl of DNA sample, 1.5 μl 10 pmol/μl from each primer, and 4.5 μl of free-nuclease water. The reaction was done under the optimal PCR conditions for gene. The annealing temperature in amplifying Pyoverdine gene was 58°C. PCR products were separated by gel electrophoresis on 2% agarose gel containing 0.5 μg/ml ethidium bromide. with 80 Volt/cm for 80 min [Table 2].[10]
Table 2: The polymerase chain reaction conditions for amplifying Pyoverdine gene

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  Results Top


The total 110 clinical samples gain from study patiens were submitted to microbiological culture technique, 76 (69%) were P. aeruginosa, while 34 (31%) belong to other genera of bacteria (Klebsiellapneumonia = 9 (8.2%), Escherichia coli = 10 (9.0%), Acinetobacter baumannii = 3 (2.8%), Proteus mirabilis = 8 (7.3%), and 4 (3.7%) patient have mix growth.

The results on culture media (blood agar, MacConkey agar, and cetrimide agar) reveal that the colony on the blood agar appear as white to gray color, sticky textures bacteria and examined their ability to hemolyze blood and showed type beta hemolysis,[11] while on MacConkey agar appeared as small pale colonies due to lactose non-fermenting[12] and capable of growing on cetrimide agar as blue-greenish colonies (at 37°C for 24 h). The outcome of biochemical tests gave the same results for all 76 specimen isolation as shown in [Table 3], Identification of the isolates using Api 20E system and Vitek compact 2 system.[13]
Table 3: Morphological and biochemical features for the identification of Pseudomonas aeruginosa isolates

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Antimicrobial susceptibility of the 76 selected P. aeruginosa isolates from burns wound infection to antibiotics. Antimicrobial sensitivity tests were conducted for P. aeruginosa using (14) types of antibiotics used in the hospitals According to the results obtained in [Table 4].
Table 4: Antibiotic susceptibility patterns of Pseudomonas aeruginosa by Bauer - Kirby method

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PCR has the potential to identify microbial species rapidly and precisely by amplification of gene sequences unique to a particular organism.[14] All isolates gave positive results for 16S rRNA gene that used the same primer with molecular size (956 bp) then the isolates screened for the presence of Pyoverdine gene. The result of gel electrophoresis for amplification PCR for pyoviridine gene product showed that the presence of bands of the samples. That means the primers of this genes bind specifically to complementary sequences within the DNA template. The molecular weight of the band was (389 bp) compared with DNA ladder 1500 bp as shown in [Figure 1]. The prevalence of the pyoviridine gene in the target isolates was (50%).
Figure 1: Agarose gel electrophoresis (2% agarose, 100 vol/80 min) of traditional polymerase chain reaction amplification products of Pseudomonas aeruginosa pyoviridine gene (389bp) 1–10: Show positive results: Ladder 100 bp

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PCR products for three selected isolates (1, 2, and 3) for Pyoviridine gene were choice for sequencing that depending on their resistance for fluoroquinolones (ciprofloxacin, norfloxacin). These sequences were analyzed for the detection of differences in the nucleotides (mutations) and changes in amino acids.

The results of the alignment of the Pyoverdine sequences of the local isolates (1, 2, and 3) with the reference strain P. aeruginosa as shown in [Table 5].
Table 5: Mutation in Pvc gene in isolates numbers 1, 2, and 3

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  Discussion Top


Severe burns are very devastating forms of trauma which require immediate and specialized medical care. This pathogen is responsible for morbidity and mortality in immunocompromised patients[15] Within this study These findings may be due to develops the resistance by different mechanisms, including multidrug-resistance (MDR), efflux pumps, BF.[16] The prescience of virulence factors lets the pathogen to create efficient invasion, colonization, and persistence inside the host organism.[17]

The current study showed a high prevalence of P. aeruginosa infections among burned patients which were in agreement with the result of other investigators in Iraq/Mosul,[18],[19] and in many economically developing countries such as Zimbabwe,[20] South Korea[21] and India.[22]

Although P. aeruginosa is not a classic pathogen of burn wound infections in economically developed countries, a few burn centers in Canada and the USA, France, and Italy have reported P. aeruginosa as an important microorganism in the burn unit.[23] while the results are slightly more than.[24],[25] and it was very far from the percentage reported by.[26]

Incidence of “P. aeruginosa” according to the gender of the patient showed a higher rate of “P. aeruginosa” among females compared to males which is compatible with study in Iraq/Sulaimaniyah[27] Otherwise, these results differ with the study in Nigeria[28] and Egypt[29] where males were the most affected. Isolation of Pseudomonas according to the site of infection was a result similar to a study in Sulaimaniyah, Iraq Which found that the majority of the specimens were taken from the lower limbs (43%) followed by the trunk (23%) and the upper limbs (17%).[27]

The growth of Pseudomonas on different media, blood agar, MacConkey agar, and cetrimide agar showed that only 76 isolates were capable of growing on cetrimide agar, which is a special selective medium for Pseudomonas. Depending on the shape of the bacterial isolates on a selective medium and the results of biochemical tests, as well as the results of Api 20E which showed that only seventy-six out of one hundred ten bacterial isolates were P. aeruginosa, while 31% belonged to other bacteria.[18]

Othman used API 20 E as final confirmation was made using the analytical profile index (API 20E system).[27] Zbinden reported that the accurate identification of Pseudomonus aeruginosa by VITEK 2system.[30]

The results obtained in this study revealed that P. aeruginosa isolates have shown high resistanc against Ciprofloxacin and norfloxacin. These results are close to the result belonging to AL-Buaiji[19] and higher than resistance against ciprofloxacin, 20.6% belonging to Al-Doory[31] and higher than the result of norfloxacin, 38.8% belonging to.[32] while the resistance of Meropenem was 55.2%, imipenem was 57.8% which is similar to Al-Doory.[31] Pseudomonads may develop resistance to carbapenems through combined mechanisms such as target in accessibility and overexpression of efflux systems.[33] Gentamicin group has shown high resistance to this group of antibiotics 65.7%. This result is compatible with[19],[31] results and disagreement with the results obtained by[34] who showed extremely low resistance rate to imipenem and meropenem, 8% both and 4% to imipenem in Pakistan[35] Gentamicin has shown high resistance antibiotic 65.7% This result compatible with[19],[31] results that were 60% and 65.7% respectively. and incompatible to the results of 47.6% resistance to P. aeruginosa.[36] Tobramycin resistanc rate was 51.3% which is similar to,[32] while less than the results of resistance to Tobramycin is 88.2%.[37] resistance to Amikacin was (34.2%), this result coincides with the finding of amikacin resistance which reported 34.9%,[36] and incompatible with and funding of Amikacin resistance were seen in (67%).[38]

The aminoglycosides inhibit protein synthesis in the bacterial cell by binding to 30S subunit of the ribosome and the Aminoglycoside-resistance in Pseudomonas sp. is primarily due to changes in the target enzymes and inactivation of the antibiotics[39] have mentioned.

Piperacillin resistance rate is 65.7% which is close to findings that reported 57.9%.[19] The result of this study crossed with the results finding, 35.8% resistance[31] while Ticarcillin resistance rate is 56.5% which is similar to the study which found that resistance to Ticarcillin was 61%.[40] While the difference in result is apparent compared with results found that all P. aeruginosa isolated from burn were resistant to ticarcillin 100%.[32]

The levels of resistance to cephalosporins including ceftazidime, cefotaxime, cefepime, cephalothin, and ceftriaxone. This study, P. aeruginosa isolates has revealed high resistance (65.7%) for both ceftazidime and cefepime. This result coincides with the finding of (65.7, 68.4) for ceftazidime and cefepime, respectively.[19] Results obtained in this study revealed that the levels of resistance to cefotaxime and ceftriaxone are (65.7, 67.1). This result coincides with the finding[41] which found 50.75 resistance to cefotaxime and74.95 to ceftriaxone, while the result incompatible with high resistance 100% to both of them.[38] The MDR, defined as resistance to three classes of antimicrobials, increased amongst these organisms making it difficult to choose appropriate suitable antimicrobial therapy.[42]

Recently various methods have been developed to rapidly and accurately identify Pseudomonas species as a medically important bacterium. According to these, PCR has the potential to identify microbial species rapidly and precisely by amplification of gene sequences unique to a particular organism.[14] The results of this study showed that the number of the P. aeruginosa isolated from the burn by using PCR detection, all the P. aeruginosa isolates (100%) were positive which based on the 16S rRNA gene. The results of 16S rRNA gene matched with the study in Kurdistan.[37]

A total of 15 pyoverdine genes have now been identified that are essential for pyoverdine synthesis in P. aeruginosa PAO1 and it is likely that most, if not all, of the genes that are essential for pyoverdine synthesis in this strain are now known. The result of gel electrophoresis for amplification PCR product found that the presence of bands, the results indicated 50% of the isolates were carrying the pyoviridine genes and confirmed as P. aeruginosa.

In this study, it will be addressed the pvc genes lie at about 66-70 min on the genetic map,[43] about 240 kb away from pvdS. A separate cluster of four genes (pvcABCD) has been reported to be required for synthesis of the pyoverdine chromophore.

pvc mutants can make pyoverdine in some growth media so that these genes are not essential for pyoverdine synthesis.[44] The existence of pyoverdine-negative isolates of P. aeruginosahas prompted the need for accurate and enhanced genotyping procedures based on the determination of the gene sequence of ferripyoverdine receptor fpvA of P. aeruginosa.[45]

No matching found with pvc gene detection and sequencing study except[46] found that the proximity of the ptxR gene to the pvcABCD operon it seemed possible that PtxR plays a role in the expression of pvcABCD and, thus, pyoverdine[46] reported that the ptxR deletion in strain PAO1 completely abrogated pvc expression, with no pvc mRNA detected under iron-limited or iron-replete conditions.

The isolates that had double mutation in Pyoverdine isolation number (2) had higher ciprofloxacin and norfloxacin resistance than those with a single mutation in Pyoverdine isolation number (3).

Resistance to fluoroquinolone antibiotics is multifactorial and can be via one or a combination of target-site gene mutations, increased production of MDR efflux pumps, modifying enzymes, and/or target-protection proteins.[47]

Characterized mechanisms of fluoroquinolone resistance among P. aeruginosa isolates have been restricted to chromosomal genes, including target mutations and active efflux Similar to the case for other gram-negative bacteria, DNA gyrase is the primary target for the fluoroquinolones in P. aeruginosa.[48]

In Gram-negative bacteria, GyrA is the preferred target of fluoroquinolone, and resistance mutations thus tend to occur in this enzyme first with additional mutations in topoisomerase IV seen in some highly resistant isolates.[49]

The current study concluded that P. aeruginosa isolate is high despite modern techniques and sterilization and disinfection solution and has abundant ability to form biofilms, produce virulence enzymes, and antibiotic resistance. For these isolates that were identified by phenotypic testing used detection of PCR and DNA sequencing analyzes designed to make sure identification Burn wound infection isolates, as well as identification of mutations, if any.


  Conclusion Top


The highest prevalence of P. aeruginosa was located in burn, the highest frequency resistance was against fluoroquinolones (Ciprofloxacin and Norfloxacin) and the lowest resistance was to Amikacin. Half selected local isolates of P. aeruginosa carried Pyoverdine genes can uptake iron from host sources, which apply to considerable damage on the host. Using PCR is highly sensitive, specific and rapid method which vastly improved the detection of P. aeruginosa.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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