Detecting the Variation in the lasI Gene and Their Relation with Biofilm in Pseudomonas aeruginosa Isolates

One of the most causative agents for many opportunistic diseases is the Pseudomonas aeruginosa which has a high percentage of multidrug resistance disease through construction of biofilm. The current study aimed for evaluating the correlation between quorum sensing genes (which is lasI gene) and biofilm formation. The biofilm construction and antibiotics susceptibility test were achieved for all the isolates under the study. The PCR and sequencing techniques were also carried out to detect the type of variation in lasI gene for each scheme of biofilm formation (weak, strong, and moderate). High antibiotic resistance was recorded among biofilm producing isolates. The genic pattern for the weak biofilm producer, which is isolate No 19, carries lysine instead of arginine in position 92 and aspartic acid instead of asparagine in position 115. Phylogenetic analysis indicated that the horizontal genetic transfer between isolates was very high. Finally, we conclude that the lasI gene does participate in biofilm construction of P. aeruginosa


Introduction
Pseudomonas aeruginosa is one of the famous opportunistic pathogen that causes hospital mediating infections [1,2].The mortality correlates with nosocomial infections for multidrugresistant strains of P. aeruginosa has increased significantly [3,4].P. aeruginosa undergoes evolution in cystic fibrosis (CF), therefore their genomic and phenotypic is different from P. aeruginosa in other sites of human body [5].Different morphological colonies have appeared in order to adapt to selective pressure in CF airways.These are small colony variants and mucoid [6].P. aeruginosa possesses many virulence factors that contribute in the development of acute and chronic infections such as pili, alginate/biofilm, lipopolysaccharide, pyocyanin, siderophores and exotoxin [7].P. aeruginosa can persist for a long time inside the host due to their ability to adapt with stress condition such as adaptation to the inflammatory defense mechanism and antibiotic therapy [8].Overproduction of exopolysaccharide helps in the formation of biofilm in P. aeruginosa, hence causing chronic infection in CF [9].Biofilm is described as accumulation of many organisms under sheath of polysaccharide in presence of exogenous DNA (eDNA), and proteins and it contributes in the resistance to immune system and antibiotic resistance [10].Biofilm formation consists of different processes starting by reversible binding of the cells then irreversible binding and development of microcolonies, before finally establishing mature biofilm [11].Transfer the P. aeruginosa from planktonic cell to biofilm producer relies on forming extracellular matrix that forms from polysaccharides, proteins, extracellular DNA and lipids [12].Biofilm of P. aeruginosa may lead to multidrug resistance evolution due to spreading of antibiotic resistance genes through horizontal gene transfer among bacterial species [13].One study showed that the PA3 782 and PA37 1 genes upregulated with biofilm formation, therefore they may have a major role in constructing biofilm [14].Biofilms and quorum sensing manages P. aeruginosa behavior in their host through colonizing and evading different tissues [15].Quorum sensing which is intercellular communication help in formation of biofilm through autoinducers signaling controlling targets genes [16].Quorum sensing has a major role in bacterial motility in addition to expression of virulence factors, hence forming biofilm [17].In P. aeruginosa lasB plays an important role in biofilm formation by acting as polysaccharide secretion regulator [18].A study showed that about 1% from total genes in P. aeruginosa were enrolled with biofilm formation, among thesewere the las and rhl which are quorum-sensing genes that mediate biofilm formation [19].A study showed that MDR ST111 and ST235 formed biofilm in hospital waste-water pipes that leads to serious problems [11].Due to the lack of sufficient information regarding genic variation of lasI gene in isolates under study that formed strong, moderate and weak biofilm, this study aimed for comparing the variation in lasI gene among biofilm non-forming and forming isolates and for finding the type of DNA and amino acid variation in non-biofilm producer compared with biofilm producer isolates.

Collection of the Isolates
All in all, 20 P. aeruginosa isolates were detected in different samples, which were collected from sputum and urine, from different hospital in Baghdad city using morphological and biochemical tests.For morphological detection, all the samples were cultured in different media such as cetrimide agar, MacConkey agar and blood agar.Catalase and fermentative, motility, oxidase and indol biochemical tests were carried out for further identification of isolates.

Antibiotic Susceptibility Test
The susceptibility of different antibiotics against P. aeruginosa isolates was tested using disc diffusion method.Four different disc diffusion of antibiotics were used: ceftazidime (30), amikacin (10), imipenem (10) and colistin (10).Detection of the resistance, susceptible or intermediate isolates, relied on CLSI guideline [18].

Biofilm Forming Assay
Weak, moderate and strong biofilms formation was checked in 20 of P. aeruginosa isolates using microtiter plate [20].0.25% glucose was mixed with tryptic soy broth for the purpose of cultivating P. aeruginosa isolates at 37 0 C for 24 h before forming biofilm.Next P. aeruginosa suspension with 125 μl was added to 96-well microtiter plates and left at 37 0 C for 24 h.The wells of microtiter plates were then washed with distilled water and the plate was inverted on filter paper at room temperature for drying.Staining the wells with 125 μl crystal violet (0.1%) was carried out and left for 5 min.The wells were then de-stained using acetic acid.The density of biofilm that was formed in each well, was measured using ELISA reader at 570nm.For each one of 20 of P. aeruginosa isolates, testing the biofilm formation was achieved in triplicate while the media without inoculum represented as a negative control while inoculum without media represented as a positive control and then added to well of microtiter plate.ODi and ODc were measured to detect strong, moderate, weak and non-biofilm producing isolates.ODi refers to average of triplicate ODs of each sample after subtracting from OD of negative control.Whereas ODc refers as three standard deviations plus mean O.D of the control.In case of (ODi < ODc), (ODc < ODi < or = 2 multiply ODc), (2 multiply ODc < ODi < or = 4 multiply ODc), and (4 multiply ODc < ODi) indicate that isolates are non-, weak, moderate or strong biofilm producer respectively.

LasI gene Detection and Sequencing
Genomic DNA mini extraction kit (Geneaid Company kit) was used to extract the DNA from 6 different P. aeruginosa isolates.Some of these isolates were multidrug resistance isolates (MDR).From these 6 different P. aeruginosa isolates, two isolates were moderate biofilm producers and the rest four were 2 weak-biofilm producers and 2 non-biofilm producers.In the PCR, the primers were taken from Uzunbayir-Akel et al. 2020s which are ATGATCGTACAAATTGGTCGGC for forward primer and GTCATGAAACCGCCAGTCG for reverse primer for amplification amplicon with size of 605 bp.PCR contents were 5 μl bacterial DNA as template, 1 μl from forward primer, 1 μl from reverse primer, 25 μl master mix and 18 μl distilled water with final volume of 50 μl.PCR was carried out with different cycles (Table 1).Visualization and staining were carried out on gel electrophoresis with 1% agarose and ethidium bromide respectively.Purified amplicons were then submitted to sequencing.The alignment of lasI gene was carried out using BioEdit program version 7.2 and BLAST tool was used to align our target gene with reference genome.

Results
Biofilm results for 20 P. aeruginosa isolates showed that isolates non-forming biofilm had OD less than 0.15 and their percentage was 20%, while isolates forming weak biofilm had OD between 0.15 and 0.3 with 45% of the total isolates and isolates forming moderate biofilm had OD between larger than 0.3 with 35% of the total isolates (Table 2).Comparing the percentage of isolates forming and non-forming biofilm showed significant value with P = 0.0002.Antibiotic susceptibility test (Figure 1) showed that not only all isolates were resistant to colistin antibiotic (100%) but also high resistance rate was detected against ceftazidime, amikacin and imipenem with (80%), (75%) and (55%) respectively (Table 3).The percentage of MDR among 20 isolates of P. aeruginosa was 5 (25%) (multidrug resistance is the resistance to 3 or more antibiotics belong to different classes for each isolate).The resistance for ceftazidime, amikacin and imipenem antibiotics was significantly higher through comparison between resistance and sensitive isolates for these antibiotics with P = 0.0002, P = 0.006 and P = 0.02 respectively.Six P. aeruginosa isolates by which 4 forming biofilm and 2 non-forming biofilms, were subjected to different concentration of antibiotics.The DNA from 6 P. aeruginosa isolates was then extracted and the PCR technique was used for detecting lasI gene presence.Results showed the lasI gene was present in all isolates except isolates 12 and 15 which were weak and moderate biofilm producer isolates (Figure 2).To confirm the deletion in lasI gene in isolates 12 and 15, the extracted DNA from 6 P. aeruginosa isolates was sent for sequencing.The sequencing results confirmed that the deletion only occurred in lasI gene in isolate belonging to 15.The genic variation among isolates under the study showed that three substitutions occurred at positions 92, 115 and 130 in isolate 19 which is a weak biofilm producer isolate (Figure 3 and Table 4).The variation at position 92 involved A converted to G by which lysine converted to arginine.At position 115, G converted to A by which aspartic acid converted to asparagine, while C converted to A at position 130 and it was missense mutation.Further analysis was carried out through comparing the DNA sequence of our isolate with reference genome in the databases and the results showed three substitutions changes that wereobserved at positions 9, 32 and 47 converting G to A, A to G and A to G respectively (Figure 4 and Table 3).Finally, phylogenetic tree was constructed between isolates under study and isolates in databases which showed high similarity index with our isolates.In general, the tree showed two clusters, the first cluster for the isolate under the study with one isolate collected from Iraq as well highlighted with red color while the second cluster showed the isolates collected from USA (Figure 5).

Discussion
In this study, 80% of P. aeruginosa isolates formed biofilm and this percentage was approximately close to the study conducted by O'Toole [20], and showed that 93.4% of the isolates form biofilm. Percentage of MDR isolates in our study was 25% which is higher than the percentage reported in another study with 6.52 % [21].However, it was significantly lower than that reported by Banar et al. [22] with 87%.In addition, there was no correlation with MDR and biofilm formation in our isolates and this results were compatible with a previous study outcome [23].However, most of the isolates were resistant to different antibiotics with high percentages and this agreed with Saffari et al. [21] but disagreed with O'Toole's study [20].For the isolates under study, the high percentage of the resistance for the antimicrobial agents and high rate of forming biofilm indicated that these isolates have high pathogenicity.In addition, 66.6% from isolates forming and non-forming biofilm carried lasI gene.This result indicated that there was no correlation between construction of biofilm and the lasI gene prevalence.In addition, presence of gene in non-biofilm producer could be due to affecting the formation of biofilm.However, Lahij et al. [24] showed that there is a strong correlation between construction of biofilm and the presence of lasI gene.Moreover, Perez et al. [25] showed that most of isolates carried lasI gene with 90%.Our result agreed with Sabharwal et al. who showed that only 75% of isolates carried lasI gene.However, Aboushleib et al. [26] found that only 48% of formed isolates carried lasI gene.The lasI gene was not detected in isolate 15 which is a moderate biofilm producer.We, however, think that the isolate 15 was able to form moderate biofilm in the presence other quorum sensing genes such as rhII.This result was compatible with Senturk et al. [27] who showed some missing quorum sensing genes were replaced by the presence of other in P. aeruginosa therefore this bacterium still has the ability to produce biofilm.The mutation pattern in lasI gene was detected in isolate 19 which is weak biofilm producer isolate, therefore we think that P. aeruginosa to produce weak biofilm, in position 92 lysine should be replaced by arginine and at position 115 aspartic acid should be replaced by asparagine.Another study showed that the mutation in quorum sensing genes converted P. aeruginosa from biofilm producer isolate to non-biofilm producer isolate [28].Many other studies showed that the mutation in quorum sensing genes can have a direct effect on construction of biofilm in P. aeruginosa [22].We detected genomic variation in DNA sequence through comparing our isolates with the database and this variation may reflect that P. aeruginosa may change in their pathogenicity to adapt with their host defense strategies.Finally, phylogenetic analysis showed that the isolates clustered relying on geographic distribution by which all Iraq isolates aggregate in one cluster, this gives an indication that lasI gene highly transferred among isolates in specific regions through horizontal gene transfer.

Conclusion
Studying the relation between lasI gene and biofilm formation showed that there is a powerful correlation between antibiotic resistance and construction of biofilm and between lasI gene and biofilm formation for the isolates under study.In addition, lysine replaced arginine in position 92 and aspartic acid replaced asparagine in position 115 for weak biofilm producer isolate.

Figure 1 :
Figure 1: Susceptibility pattern for different antibiotics.Inhibition zones were seen for ceftazidime, amikacin and imipenem antibiotics.

Figure 2 :
Figure 2: Gel documentation for lasI gene.lane 1: the ladder, lanes with numbers 6, 12, 14, 15, 16 and 19 are the isolates while the last lane: negative control.The concentration of agarose in gel was 1% and the gel was run on 80 V for 1 hour.

Figure 4 :
Figure 4: Alignment pattern between reference genome and one of our isolate.The genic variation showed in three different positions.

Figure 5 :
Figure 5: Construction phylogenetic tree among isolates under the study and the isolates collected from database.Isolates that highlighted with red color represented the isolates under the study.

Table 1 :
PCR cycles to amplify lasI gene Statistical testComparing the proportions of the data under the study was achieved using Chi-squared test (Campbell, 2007).

Table 2 :
Percentage and OD of isolates forming and non-forming biofilm.

Table 4 :
Analysis of the lasI gene and its protein sequence through comparison with reference genome.

Position of DNA Change DNA Change Position of Amino Acid Change
Figure 3: Alignment of the DNA sequence for lasI gene to the isolates under the study.The patterns of genic variation showed in isolate 19 compared with other isolates.

Table 3 :
Analysis of the lasI gene and its protein sequence through comparison with reference genome.