Molecular characterization of virulence genes in quorum sensing system of different pathogenic strains of Pseudomonas aeruginosa from neonates with pulmonary infections using PCR as efficient technique

doi:10.21203/rs.3.rs-5234762/v1

Background: The expectation from the current research study was to genetically identify the virulence genes involved in quorum sensing (QS) in different strains of Pseudomonas aeruginosa, as the QS controls production of many virulence factors.

Objective: The intention of current research study was to isolate the pathogenic strains of P. aeruginosa from sputum samples of neonates and infants and their molecular characterization.

Methods: P. aeruginosawere obtained from Microbiology Laboratory, Department of Zoology, Government College University Lahore were grown on selective media. Biochemical and molecular characterization was done. Molecular characterization was done by specific primers of quorum sensing virulent genes. Amplified genes were sequenced, and accession numbers were obtained from the NCBI site. Genes lasR, lasI, rhlR, and rhlI were identified in P. aeruginosa strains (ss5, ss6, and ss11).

Results: Current study revealed that these virulence genes are the main contributors of resistance of P. aeruginosa strains against different antibacterial agents.

Conclusion: P. aeruginosa is a Gram-negative bacterium with diverse metabolic capacity to regulate survival under many different conditions and can be highly resistant to antibiotics, facilitating its spread in diverse habitats, particularly in hospitals. These virulent genes contribute to the overall pathogenic potential of P. aeruginosa that facilitates its ability to cause disease.

P. aeruginosa

Virulence genes

Pulmonary infections

neonates

Quorum sensing

lasR

lasI

rhlR

and rhlI

Bacteria are interesting microorganisms that have changed the course of the planet's history and play a significant role in many aspects of existence. They are often considered the oldest living lineage on Earth. Bacteria are famous for spreading diseases and infections, and they play a crucial role in maintaining the health of many other environmental systems [1, 2]. They participate in processes like nitrogen cycling and decomposition. Gram-negative bacteria are classified based on their staining properties after a chemical technique, commonly referred to as Gram staining [3]. Gram-negative bacteria maintain pink stains, while gram-positive bacteria maintain blue stains. Gram-negative bacilli are bacteria shaped like rods. These bacteria include Escherichia coli, Pseudomonas, Klebsiella, Salmonella, Citrobacter, and others [4–6].

Mucosal cells and hair-like cells called cilia play important roles in the immune system's respiratory system. They irritate the upper respiratory tract, including the nose and throat, and work together to capture and destroy airborne pathogens, viruses, and bacteria [7]. The lower respiratory tract produces phlegm, trapping foreign particles like pollutants and dust, which later get expelled through coughing or transferred upwards for removal via the cilia. Defensive cells in the respiratory system, such as neutrophils, lymphocytes, and macrophages, actively identify and eliminate attacks and infections. Respiratory tract is affected by Gram-negative bacteria in various ways, from mild discomfort to severe infections [8–10].

P. aeruginosa causes two main types of respiratory tract infections: acute (especially if the patient has been breathing for a long time) and chronic (if the patient has cystic fibrosis or a similar lung disease) [11, 12].The main factors involved in morbidity and mortality are the type three secretion factors (TTSS), ExoS, ExoT, and ExoU, particularly as validated in murine acute respiratory infection models. The presence or absence of TTSS components may influence the outcome of human infection. P.aeruginosa secretes pyocyanin, a blue pigment that has antibacterial properties against other microbial pathogens. Intranasal infection of adult CD-1 mice showed that pyocyanin production also caused severe lung injury. Quorum-sensing systems in P.aeruginosa involving LasI and LasR and RhlI also contribute to acute infections [13–16].

Many other acute infection models show that a deficiency in quorum-sensing capacity, notably LasR mutants, leads to reduced virulence compared to wild-type strains. Additional results showed that the loss of LasR function led to increased antibiotic resistance by mechanisms such as increased beta-lactamase activity. The loss of LasR function can also reduce the production of various virulence factors via this single mutation leading to strains better fit to cause chronic infections [17–21].

P. aeruginosa initially colonizes host tissues in the planktonic form, but the cells subsequently convert to the sessile state in biofilms. Biofilms have heterogeneous populations with phenotypic and genotypic diversification and can be formed by more than one species. P. aeruginosais one of the dominant pathogens along with other pathogens such as the Gram-negative Burkholderiacenocepacia and Gram-positive S. aureus. The heterogeneous bacterial populations in. P.aeruginosa biofilms often occupy distinct microenvironments [22, 23].

Metabolically active cells are mostly found in the periphery and consume most of the oxygen, creating an oxygen gradient in the biofilm. The upper layers of the biofilm contain less metabolically active organisms and are anoxic. Therefore, peripheral bacterial cells actively growing in a biofilm are more susceptible to antibiotics, which have difficulty penetrating the mucus layer of the biofilm to reach deeper cell [24]. These help maintain channels and void spaces in mature biofilms so some nutrients can flow thought the matrix and are also involved in dispersion of cells from biofilms. Thus, various factors that modulate the properties of biofilms need to be produced by isolates of P. aeruginosa in order to maximize its ability to chronically colonize and infect human lungs [25–28].

Sample Collection:

Sputum samples from neonates and infants suffering from RDS associated pneumonia were collected from Fatima Memorial Hospital Lahore by qualified and experienced pulmonologists with the consent of parents and guardians of the infants. Samples were transported to microbiology lab in Government College University Lahore in insulated ice bags. Study was approved by Board of studies (BOS), Department of Zoology and Advance Studies and Research Board, Government College University, Lahore (REG-ACAD-ASRB/57/24/021) with the approval of Office of Research Innovation and Commercialization, Government College University, Lahore (ORIC, GCUL) vide number 9390/ORIC/24.

Isolation of Bacteria:

MacConkey agar was prepared and samples were streaked on agar plates and incubated for overnight at 37^oC. Plates were checked after incubation period to confirm whether the bacteria are gram negative or gram positive[25].

Eosine Methylene blue agar (EMB Agar):

EMB was used as a differential as well as selective media for isolation of negative gram bacteria. EMB inhibits the growth of gram positive bacteria and gram negative bacteria show pinkish colonies. For confirmation of P.aeruginosa, EMB agar test was also performed, to confirm whether gram negative bacteria present or not, for this isolated colonies were streaked on EMB agr plates and incubated for overnight at 37^oC[26] (Fig 1).

Gram’s and Endospore staining

To differentiate Gram negative and gram positive bacteria, Gran staining was done. Shape of bacterial colonies were identified under microscope (Fig 2).

Qualitative Assay:

For confirmation of P. aeruginosa in samples, preparation of King B agar media was done, bacterial colonies were streaked on the media and incubated for 24 hours at 37^oC. After incubation period, colonies were checked under trans-illuminator to check the production of fluorescein and pyocyanin[27, 29] (Fig 3).

Isolation of Pure culture:

For isolation of pure colonies of P.aeruginosa cetrimide agar media was prepared and sterilized. After sterilization, media was poured into plates and let the media to get settle down. After solidification of media, bacterial colonies were picked up with sterilized inoculating loop and streaked on the cetrimide agar pates. After streaking, plates were inverted and incubated for 48 hours at 37^oC. After incubation period, Plates were checked to confirm whether the bacterial colonies grow on cetrimide agar or not[30] (Fig 4).

Preparation of inoculums and glycerol stock of bacterial strains:

Nutrient broth media was prepared and sterilized. After sterilization, brith was poure into falcon tubes and bacterial colony was inoculated into nutrient broth. Nutrient broth culture was incubated for overnight at 37℃.

For glycerol stock preparation, 200µl glycerol was added into Eppendorf and sterilized, after sterilization 800µl of bacterial strain was inoculated into sterilized glycerol and glycerol stock of bacterial strains were prepared and stored at 20^oC[26].

Pathogenicity Test:

For isolation of pathogenic strains of P.aeruginosa, Blood agar test which is also known as pathogenicity test was performed. For this test, Nutrient agar media was prepared and sterilized in autoclave. After sterilization, as the media become normal cool at room temperature non-coagulated blood was added into nutrient agar media to make blood agar media. After mixing of blood into nutrient agar, media was poured into plates and let the plates to get solid. After solidification, pure colonies of P.aeruginosa were picked up with inoculating loop and streaked on blood agar plates. After streaking, Plates were incubated and type of hemolysis were checked after 24 hours[31] (Fig 5)

Molecular characterization of virulence genes P.aeruginosa:

Molecular characterization of virulence genes was done by isolationg genomic DNA from bacterial strains.

DNA Extraction (Phenol: Choloroform extraction)

Bacterial genomic DNA was extracted by the phenol-chloroform method. Lysogeny broth medium was inoculated with separate areas of bacteria grown on agar and cultured with shaking overnight at 37 °C. Centrifuge 10 ml of culture medium at 10,000 rpm (12,000 × g) for 20 min to pellet the brain. Remove the supernatant and resuspend the pellet in 400 μL of TEN buffer. Centrifuge the suspension at 12,000 × g for 10 min. Remove the supernatant, suspend the pellet in SET buffer (200 μL) and add 120 μL of lysozyme (20 mg/ml). Incubate the reaction mixture at 37°C for half an hour. Then add 200 μL of TEN buffer and 10 μL of 25% SDS solution. As, the mixture became normally cool 5M solution of NaCl (20 ul) was added in the mixture and then equal quantity 1:1 of phenol: chloroform was added into the mixture and centrifuge at 12,000 x g for 20 min to form a separate aqueous layer on top of the matte degraded protein. Aqueous layer was then transferred into the new pre- labeled Eppendorf, and then chloroform of equal volume was added into the Eppendorf. Centrifuge at 12,000 × g for 15 min to separate the aqueous phase from the chloroform. Place the top layer back into a new tube and soak overnight with two volumes of chilled ethanol. After that, Eppendorf was centrifuged at the speed of 12000rpm for 10 minutes, after centrifugation, supernatant was thrown away and with 70% ethanol was used to wash the pellet. After washing with ethanol, Eppendorf was air dried to avoid any contamination. DNA was adhere along the walls of Eppendorf.Store the DNA pellet in 50 µl of deionized water or TE buffer at ≤20°C until further use. [29, 32, 33].

Amplification of specific genes in P. aeruginosa:

Amplification was performed in a 25-μl volume containing DNA sample (50 ng), Taq buffer (1X), DMSO (dimethyl sulfoxide), magnesium chloride (2 mM), each primer (10 pM/μl) (Table 1), nucleotides (dATP, dCTP, dGTP, dTTP) (200 μM, Thermo Scientific), and Taq polymerase (1 U/μl, FIR. Amplification was performed in a thermal cycler for 30 cycles, including predenaturation, denaturation, and annealing represented in fig 1. Primers listed in table 1 were used to amplify the QS genes lasI, lasR, rhlI and rhlR. A DNA Ladder of 1 kb plus 100 bp was used for comparison of DNA bands. For gel electrophoresis 1 gram agarose (1% agarose gel electrophoresis) was dissolved in the 2 % 50X TAE buffer and mixed well followed by heating in a microwave oven for 1 mint to boil the solution which was then poured into the casting tray. After solidifying, a fixed comb was used to make wells into which the amplified PCR product was loaded along with dye and the DNA ladder used to determine fragment sizes. The agarose gel was run at 90 volts for 30-40 minutes. After electrophoresis, the DNA in the gel was visualized under a UV trans-illuminator for observation of the amplified product. The bands of DNA were cut and gene clean was used to extract the DNA[34, 35].

Sequencing of PCR Products

The PCR products from the clinical bacterial strains were sent for sequencing to the first BASE Laboratories, Malaysia for detailed analysis.

On Maconkey agar media, pinkish colonies showed the presence of gram negative bacteria and showed that the bacteria in samples were lactose fermented bacteria.

Pinkish colonies on Eosin Methylene Blue Agar (EMB) showed positive result of presence of P.aeruginosa (Fig 1).

Gram staining showed that bacterial strains retain oink colour which is the indication of gram negative bacteria (Fig 2).

Under transilluminator, colonies of Pseudomonas aeruginosa showed yellow-green and bright green color due to fluorescein and pyocyanin production (Fig 3)

On cetrimide agar media, isolated colonies of Pseudomonas aeruginosa were grown (Fig 4).

All strains of Pseudomonas aeruginosa showed β-hemolysis (Fig 5).

Extracted DNA samples were run on agarose gel and the bands observed in a trans-illuminator under ultraviolet radiations (figure 6) .The bacterial genes amplified were the identified by sequencing of the amplified products Using specific primer BLAST analyses of the nucleotide sequences there was 97-100% homology of the amplified sequences with previously published sequences. All PCR strains were identified and assigned an NCBI accession number (Table 2-4). Phylogenetic relationships of identified organisms were plotted in a dendrogram (Fig 7-11).

All strains had a lasB gene homologous to the LasB gene sequence in strain PA01 at the same chromosomal location and with sequence identities of 95–99%. Quorum sensing is regulated by LasB, and a lack of quorum sensing genes affects LasB expression. Quorum sensing genes LasR, LasI, rhlR, and rhlI all were identified at the same chromosomal locations in genomes of all the clinical strains of P.aeruginosa [36]. The sequences displayed identities of 98–100%. A mutation in the mvfR gene, which encodes an important regulator of quorum sensing, was found in the genome of strain PA7. The same mutation is also present in the mvfR gene but is absent in many strains and therefore lacks LasB activity. The LasB sequences and quorum sensing reports cannot be explained without LasB activity [37].

It is known that virulence factors such as extracellular proteases are produced by P. aeruginosa during late development and that their production is controlled by quorum sensing. P. aeruginosa causes severe diseases. LasR mutants have been studied for various properties. LasR mutants can grow in selective carbon and nitrogen sites compared to the wild type. The results showed that the loss of LasR function led to an increase in antibiotic resistance. In beta-lactamase activity, increased resistance was noted. This showed the loss of function of LasR. LasB, is a pseudolysin, which is an extracellular protease damages the tissue in P. aeruginosa infections [38]. Virulence factors LasB and AprA are regulated by the Las quorum system. Rhl quorum system also regulate LasB. AprA and LasB proteases require calcium for functioning and stability. LasB is more sensitive to calcium depletion. Protein structure of these two proteases are different; AprA contains eight calcium ion binding sites, and LasB has one. LasB produced a precursor protein involved autocatalytic processing and exported across the outer membrane. During infection, the high amount of extracellular DNA present in the extracellular matrix of P. aeruginosa biofilms has been shown to effectively chelate cations such as calcium, thus creating a cation-limited environment. Loss of LasB is observed when LasR and RhlR inactivate LasB [39].

Isolates of P. aeruginosa from the Lahore region showed presence of virulence genes involved in resistance to many of the commercially available antibiotics. Mutation of these genes may develop more resistant strains. To control the infections caused by P.aeruginosa, a detailed study of the expression of genes is required.

Consent for publication

There is no conflict of interest among authors

Availability of data and material

Available

Conflict of Interest

No competing interests among authors

Funding

None

Ethical Approval

The study was approved by Board of studies (BOS), department of Zoology and Advance Studies and Research Board, Government College University Lahore (REG-ACAD-ASRB/57/24/021). Certificate of from ethical committee is available.

Patients’ approval

There was no research work on humans. Only sputum samples were collected from expert doctors who took samples with consent of patients.

Authors’ contributions: N.M.A. prepared first and final draft of manuscript. M.C. and S.R. worked on figures. N.R. and I.L. did methodology and references section. S.Z. contributed in final draft of manuscript in results section.

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Table1. Primers used for detection of virulence genes involved in quorum sensing of P. aeruginosa

Sr. no.	Gene	Primer	Nucleotide sequence
1	Universal	forward	16S-27F 5´-AGAGTTTGATCMTGGCTCAG-3´
		reverse	16S-1492R 5´-TACGGYTACCTTGTTACGACTT-3´
2	rhlR	forward	5’ TGCATTTTATCGATCAGGGC 3’
		reverse	5’ CACTTCCTTTTCCAGGACG 3’
3	lasI	forward	5’ CGTGCTCAAGTGTTCAAGG 3’
		reverse	5’ TACAGTCGGAAAAGCCCAG 3’
4	rhlI	forward	5’ TTCATCCTCCTTTAGTCTTCCC 3’
		reverse	5’ TTCCAGCGATTCAGAGAGC 3’
5	rhlB	forward	5’ GCCCACGACCAGTTCGAC 3’
		reverse	5’ CATCCCCCTCCCTATGAC 3’.
6	lasR	forward	5’ AAGTGGAAAATTGGAGTGGAG 3’
		reverse	5’ GTAGTTGCCGACGACGATGAAG 3’
7	rhlA	forward	5’GATCGAGCTGGACGACAAGTC3’
		reverse	5’GCTGATGGTTGCTGGCTTTC3’

Table 2. Virulence genes of P. aeruginosa (ss5)

Sr. no.	Gene	Accession no
	rhlR	MH373641
	lasI	MH373643
	rhlI	MH373645
	rhlB	MH373647
	lasR	MH373649
	rhlA	MH333800

Table 3. Virulence genes of P. aeruginosa (ss6)

Sr. no.	Gene	Accession no
	rhlR	MH373642
	lasI	MH373644
	rhlI	MH373646
	rhlB	MH373648
	lasR	MH373650
	rhlA	MH349090

Table 4. Virulence genes of P. aeruginosa (ss11)

Sr. no.	Gene	Accession no
	rlhR	MH388293
	lasI	MH373651
	rlhI	MH388292
	rhlB	MH388291
	lasR	MH373652
	rhlA	MH373653

No competing interests reported.

Molecular characterization of virulence genes in quorum sensing system of different pathogenic strains of Pseudomonas aeruginosa from neonates with pulmonary infections using PCR as efficient technique

Status:

Version 1

Abstract

Figures

INTRODUCTION

METHODOLOGY

RESULTS

DISCUSSION

CONCLUSION

Declarations

References

Tables

Additional Declarations

Status:

Version 1