Pseudomonas aeruginosa associated pulmonary infections and in vitro amplification virulent rhamnolipid ( rhlR ) gene Infecções pulmonares associadas a Pseudomonas aeruginosa e gene ramnolipídeo virulento ( rhlR ) da amplificação in vitro

This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Pseudomonas aeruginosa is a common opportunistic pathogenic bacterium with the ability to develop a strong communication pathway by quorum sensing system and different virulent factors. Among the various important secretions of P. aeruginosa rhamnolipid is important biological detergent, believed to be involved in the development of the biofilm and intercellular communication. It readily dissolves the lung surfactants that are then easily catalyzed by the phospholipases and in this way is involved in the acute pulmonary infection. Objective: research work was designed to investigate virulence and gene associated with virulence in P. aeruginosa responsible for pulmonary infections. Methods: In current study polymerase chain reaction (PCR) was used for the detection of the rhlR (rhamnolipid encoding) gene of isolated strains. A number of assays were performed that ensured its virulent behavior. Disc diffusion method was used to check its antibiotic resistance. Isolated strains were resistant to a number of antibiotics applied. Result: It was found that males are more prone to respiratory infections as compared to females. Male members with age of 44-58 and 59-73 are at a higher risk, while females with age of 44-58 are also at a risk of pulmonary infections. Antibiotic resistance was observed by measuring zone of inhibition in strains GCU-SG-M4, GCU-SG-M3, GCU-SG-M5, GCU-SG-M2, GCU-SG-M1 and GCU-SG-M6. GCU-SG-M2 was resistant to fluconazole (FLU), clarithromycin (CLR), cefixime (CFM) and Penicillin (P10). No zone of inhibition was observed. But it showed unusual diffused zone around the Ak and MEM antibiotic discs. rhl R gene and 16s rRNA gene were characterized and analyzed. Conclusion: Findings from current study would help in raising awareness about antibiotic resistance of P. aeruginosa, and also the sequence of rhl R gene can be used as the diagnostic marker sequence to identify the virulent rhl R gene sequence from the samples when isolated from sputum of Pneumonia patients.


Pseudomonas aeruginosa associated pulmonary infections and in vitro amplification virulent rhamnolipid (rhlR) gene
Infecções pulmonares associadas a Pseudomonas aeruginosa e gene ramnolipídeo virulento (rhlR) da amplificação in vitro formed it enables the pathogen to detect any change in its environment, and it can respond in a very appropriate manner after the processing of information received. Additional activities that have recently been observed these are thought to be (Gray, 1997). Aims and objectives of the current study included screening of Pseudomonas aeruginosa involved in acute pulmonary infections. Also to determine the virulence of Pseudomonas aeruginosa via detection the rhlR gene of Pseudomonas aeruginosa associated with acute pulmonary infections in humans.

Sample Collection
Blood samples, sputum samples and samples of lower respiratory tract pneumonic patients were provided from a running project in GCU Microbiology laboratory. Among these samples secretions of lower respiratory tract had been obtained by endotracheal suction catheter (nelton). Pediatric mucus extractor had been used for the samples from bronchoscopy whereas blood culture vials (TSB with SPS aerobic) were being used for blood samples (Figure 1).

Isolation and characterization of Pseudomonas aeruginosa
Bacterial isolation from sputum samples of pneumonic patients was done using citrimide agar medium ( Figure 2). Pure culture was obtained by spread plate method and streaking method. Initial confirmation of P. aeruginosa was made by morphological and biochemical test ( Figure 3) (Tables 1 and 2).

Molecular characterization of Pseudomonas aeruginosa and detection of rhlR gene
PCR based confirmation of isolated strains of P. aeruginosa was carried out using universal primers for 16srRNA. Marcogen genomics was used to manufacture universal primers and gene specific primers.

Results
Percentile of the pulmonary infections caused by Pseudomonas aeruginosa was determined. These infections include respiratory failure Type-I and Type-II, Septicemia, Asthma, pulmonary edema, pneumothorax and chest filial injury. According to the resulted percentile pneumonia can be declared as most common pulmonary infection in patients, and septicemia & Type-II respiratory failure as the 2 nd most common pulmonary infection.

ETT v/s Blood Culture and Bronchoscopy
Percentage of gram negative bacterial content (P. aeruginosa) in three different cultures was compared

Introduction
Pseudomonas aeruginosa bacterium is living in versatile environments. It is gram negative bacterium, metabolically able to regulate its systems and highly resistance to antibiotics causing it to spread in diverse habitats mainly in hospitals. P. aeruginosa is a human adaptable pathogen causing acute infections (bacteremia, pneumonia and urinary tract infections) in individuals with HIV infections, surgical wounds, cancer, carrying catheters or burns, or an organ transplantations (Gellatly and Hancock, 2013). The heterogeneous bacterial population of P. aeruginosa showed distinct microenvironments for biofilms (Ren et al., 2016). Metabolically active cell are at periphery and consume most of the oxygen, causing oxygen gradients in the biofilm (Su et al., 2016).
P. aeruginosa has vast ecological distribution with ability to fit in changed circumstances making it a successful opportunistic pathogen. According to American surveillance data, it is the third most common opportunistic pathogen. Pseudomonas aeruginosa releases hemolysins i.e. rhamnolipid as well as phospholipase-C that have very toxic consequences to the host cells and is responsible for the spread of pathogen throughout the target tissue. Both toxins act together for the catalysis to lipids and as well as lecithin. Structure of the rhamnolipid to some extent is similar to that of detergents. These rhamnolipids readily dissolve the phospholipids which are then easily cleaved by the action of phospholipase-C. Lung surfactants as a result are lost which initiate atelectasis that ultimately then leads towards chronic infections of lungs (Liu, 1966;Wardlaw et al., 2006). P. aeruginosa's exposure to the respiratory epithelial cells of human can result into the inhibition of mucociliary transportation and several ciliary actions (Read et al., 1992;Bhutta, 2007). Two Quorum sensing systems i.e. las QS and rhlR are present in this bacterium. rhlR QS releases many toxins including rhamnosyl transferase and rhamnolipid Both toxins ultimately cause lung infection. A number of scientific projects had worked on the determination of rhamnolipids functioning which is still not clear. Firstly, they were known as surfactants because they have ability to reduce the surface tension (Smyth and Openshaw, 2006). Their study became very important because the hydrocarbons e.g. alkanes were firstly solubilized by them and then their uptake is promoted (Beal and Betts, 2000). They also have the property to enhance the hydrophobic ends of a cell surface that promotes the decomposition of the molecules that previously were slightly soluble. However, the purpose of the rhamnolipid is not just to assimilate the substrates which are not soluble that is evident by their competent growth on the substrates that were soluble (Al-Tahhan et al., 2000). A number of virulent agents are produced by the P. aeruginosa and for the transformation of these factors it has a highly developed mechanism through which bacterial density is checked and these cells communicate with each other through signals. Once the bacterial biofilm has been aeruginosa e, também, a sequência do gene rhl R pode ser usada como sequência de diagnóstico para identificar a sequência virulenta do gene rhl R das amostras quando isoladas do escarro de pacientes com pneumonia.

Prevalence and Isolation of P. aeruginosa
Sample collection in the present study has shown that in males (60%) the rate of pulmonary infection is comparatively higher than females (40%). Male members within the age of 44-58 and 59-73 are at a higher risk. In addition to that some females within the age ranging from 44-58 are also at a risk of getting pulmonary infections. Citrimide agar medium is a selective bacterial medium that only allows the growth of P. aeruginosa. Using this media culture of strains i.e. GCU-SG-M I , GCU-SG-M 2 , GCU-SG-M 3 , GCU-SG-M 4 , GCU-SG-M 5 , GCU-SG-M 6 were obtained.

Virulence Behavior assays
i. Blood Agar Test: Strains showed beta hemolysis ii. Protease Test: The result of this activity was clearly observed by the presence of a hollow zone. Different strains provided different values and it is obvious that the sensitivity of each strain varies. These values were recorded as 9.5mm, 2.5mm, 9mm, 4mm, 7mm, and 8mm for GCU-SG-M 4 , GCU-SG-M 3 , GCU-SG-M 5 , GCU-SG-M 2 , GCU-SG-M 1 and GCU-SG-M 6 respectively. iii. Lipase and Lecithinase Test: Results are shown in Figure 4. iv. Biofilm-Formation Assay: All strains produced positive results for this assay indicating the how virulent behavior these pathogenic bacterial strains have. v. Pigment Production a. Qualitative Assay: Different culture media were used to determine the quality of pigment produced by the pathogenic strains. Among all strains the two i.e. GCU-SG-M 6 and GCU-SG-M 4 were observed with a slight yellowish-green colored colonies that this is due to the production of pyoverdin pigment production ( Figure 5). Remaining strains of P. aeruginosa probably produced another pigment i.e. Pyocyanin that's why these colonies appeared with bluegreen colored colonies on different agar media e.g. king A, king B, citrimide, LB and MH agar media (Table 3).
b. Quantitative Assay: Quantitative of the produced pigment was measured by quantitative assay using spectrophotometer. Optical density was measured at OD 520 nm. One by the pyocyanin producing strains i.e. GCU-SG-M 5 produced the highest value of optical density (23µg/ml). The lowest pigment yielding strain that showed about 1.7µg/ml.

Rhamnolipid Assay
Strains which showed the maximum Rhamnolipid production resulted 2.4µg/ml whereas, the minimum value obtained was 0.2µg/ml. Maximum Rhamnolipid yielding strains were those isolated from bronchoscopy samples as well (Table 4).
But it showed unusual diffused zone around the Ak and MEM antibiotic discs (Tables 5 and 6).

Fluorescence of P. aeruginosa Isolated under UV rays
Fluorescence of the isolates was clearly observed which appeared as illuminating greenish light. This green light was considered as a confirmation for the present in fluorescent pigment present in the P. aeruginosa strains.

Molecular Characterization
Molecular identification with universal primers was done (Table 7) (Figures 8 and 9 (Figures 10 and 11) Aligned sequence of P. aeruginosa rhlR gene showed 99% homology with previously reported rhlR gene sequences.
AGGAATGACGGAGGCTTTTTGCTGTGGTGGGACGGTTT GCGTAGCGAGATGCAGCCGATCCACGACAGCCAGGGCGT GTTCGCCGTCCTGGAAAAGGAAGTGCGGCGCCTGGGCTT CGATTACTACGCCTATGGCGTGCGCCACACGATTCCCTTCA CCCGGCCGAAGACCGAGGTCATGGCACCTATCCCAAGGCC TGGCTGGAGCGATACCAGATGCAGAACTACGGGGCCGTG GATCCGGCGATCCTCAACGGCCTGCGCTCCTCGGAAATGG       work of Kumar et al. (2005) according to which the most common pathogenic bacteria is P. aeruginosa (63.5%) which is followed by Staphylococcus aureus (33.3%), Acinobacter lwoffi (22.6%) in the spread of VAP infections. Similar results had been reported in different studies from other

Discussion
Among all pathogenic bacteria the most common is P. aeruginosa that is responsible for 57.7% of VAP infections and 53.3% of cystic fibrosis is also caused by this pathogen. Our results are supported by the research regions (Arora et al., 2011). Some of the previous studies have shown that the infection of P. aeruginosa are more common in females than males. For example 48% of male individuals and 52% of the female individuals and effected according to Read et al. (1992). Similarly the research work of Khattak et al., 2013 have shown that females are at higher risk of infection (54%) than in males (45%). But in present study higher rate of infection is scene in the males (i.e. 60%) but in females it is 40%. These results are supported by the previous work of Rashid and Kornberg (2000) and Khattak et al. (2013) that show higher rate of susceptibility in male (56%, 60%) respectively than in females (44%, 40%). This variation can be explained by the difference in climatic condition of various geographic regions of the world. Virulence behavior assays and antibiotic resistance to a wide range of antibiotics have shown that P. aeruginosa involved in hospital acquired pneumonia, ventilator acquired pneumonia is highly efficient multi drug resistant pathogenic bacteria. There are a number of genes that play important role in formation of P. aeruginosa biofilm (that contributes in its resistant nature), in the production of rhamnolipid as well as in synthesis of pyocyanin. Which are the virulent expressions of these genes in other words (Ochsner et al., 1994). There are undoubtedly number of these type of genes either involved in the reaction of autoinducers or in the secretion of virulent products. The results of all virulent assays of present study supports the results of previous studies of Lee et al. (2006) as P. aeruginosa have showed positive results of all virulence assessing assay (Ali et al., 2015). Our findings would help in raising awareness about antibiotic resistance of P. aeruginosa associated with pulmonary infections in patients. Arasu et al. (2016) reported that there was a 70.7% susceptibility of P. aeruginosa isolates to Meropenem but also concluded Ciprofloxacin was the most active agent (85.4% susceptibility) against P. aeruginosa strains along with Amikacin (95.1% susceptibility).