Association of pvc genes expression with Biofilm formation in Clinical Isolates of Pseudomonas aeruginosa

are cluster of genes found in Pseudomonas aeruginosa. The research was designed to examine the relationship between the pvc genes expression and cupB gene, which plays a crucial role in the development of biofilm, and rhlR, which regulates the expression of biofilm-related genes, and to investigate whether the pvc genes form one or two operons. The aims were achieved by employing qRT-PCR technique to measure the gene expression of genes of interest. It was found that out of 25 clinical isolates, 21 isolates were qualified as P.aeruginosa. Amongst, 18(85.7%) were evaluated as biofilm producers, 10 (47.6%), 5 (23.8%), and 3 (14.2%) were evaluated as strong, moderate and weak producers respectively, while, 3 (14.2%) were considered as a non-biofilm forming isolate. The pvcA and pvcB were shown to be over-expressed (>2) fold in all biofilm-producer isolates, similar to that observed in cupB and rhlR, while pvcC and pvcD showed to be down-regulated (<0.5) fold in these isolates. These findings imply that the pvc genes are organized into two operons, pvcAB, and pvcCD, and genes involved in biofilm formation are regulated by pvcAB operon. This is the first study in Iraq to investigate these genes.


Introduction
Pseudomonas aeruginosa (PA) is one of the most common nosocomial pathogens in Iraqi hospitals that can cause serious infections with fatal complications 1,2 .The constant misuse and abuse of antibiotics for treating the infections resulted in the widespread development of multi-drug resistant (MDR) strains and complicated medical therapy against P. aeruginosa [3][4][5] .P. aeruginosa possesses a large genome that provides the bacteria with a wide range of virulence factors, one of them is Biofilm.
Biofilms are complex structures where communities of bacteria are embedded in the extracellular matrix.Biofilms hold a significant role in healthcareassociated infections (HAI).According to the National Institutes of Health (NIH), biofilm formation is linked to 65% and 80% of all microbial and chronic illnesses, respectively 6 .Pseudomonas aeruginosa's cup (chaperone/usher pathways) gene clusters encode fimbrial structures involved in cell-to-cell aggregation and the These adhesions may enable P. aeruginosa to adapt quickly and colonize a wide range of surfaces 7,8 .
The differential expression of cup adhesive structures is mediated by complex regulatory networks, which may include two-component regulatory systems for monitoring environmental stimuli.P. aeruginosa possesses two main Quorum Sensing systems las and rhl, the rhl system has been reported to involve in P. aeruginosa biofilm formation 8,9 .
The P.aeruginosa pvcABCD gene cluster is associated with biofilm formation through positively regulating the expression of different cup genes by the production of Paerucumarin a novel isonitrile-functionalized cumarin.(Derived from P. aeruginosa cumarin) paerucumarin enhances the expression of the chaperon/usher pathway (cup) proteins in a pathway that includes ptxR 7,10,11 .
This study sought to understand the relationship between pvc genes and cupB and rhlR genes in different biofilm-forming phenotypes clinical isolates of P.aeruginosa.This investigation was carried out using RT-qPCR to measure the expression levels of mentioned genes.Simultaneously determining whether the pvc consists of one operon pvcABCD 12 or two operons pvcAB and pvcCD.

Bacterial Isolates and Molecular identification
This study was carried out on 25 isolates of P. aeruginosa which were obtained from various wards in Baghdad's hospitals including, Burn wards, ICUs, Dialysis centers, and urology departments from October 2021 until February 2022.The identification of isolates was done by molecular analysis; after the DNA was extracted using Wizard® Genomic DNA Purification Kit (Promega, USA), 16S rRNA gene sequence analysis was performed with the following conditions: initial denaturation at 95 °C for 15 minutes as the first stage of amplification, followed by the 2 nd stage of amplification with denaturation at 95.0°C for 15 seconds, annealing at 60.0°C for 30 seconds repeated for 45 cycles 13 .This program was performed using Mastercycler® nexus-PCR Thermal Cycler (Eppendorf, Germany).
To accurately confirm the identification of all isolates, qRT-PCR technique was employed to detect gyrB gene following the program that consists of two stages: the first stage of initial denaturation for 15 minutes at 95°C, and the second stage of denaturation for 15 seconds at 95 °C, and annealing for 1 minute at 60 °C14 the second stage was repeated for 50 cycles.The program was performed using Rotor-gene Q (Qiagen, Germany).
The primers used in this study are listed in Table .1

Biofilm production assay
To evaluate the quantity of biofilm formation in isolates, a modified microtiter plate approach was employed following the method reported by Lima et al 15 .All isolates were cultured on nutrient agar for 24 hours at 37˚C.Then a Single-colony was inoculated in Brian heart infusion broth (BHI) supplemented with 50 g/L sucrose; the broth was then incubated at 37˚C for 24 hours.
-A 200 μl of the bacterial suspension was transferred in triplicates on 96-well polystyrene plates (Nunc, Denmark).The negative control was BHI broth without bacterial inoculum, while the positive control was the PA01 strain since this strain is recommended as the positive control for biofilm assays.
-After transferring the suspensions to the plates, the plates were then incubated at 37 C for an additional 24 hours, and wells were washed three times with (PBS) Phosphate-buffered saline.
-Methanol was used to fix the bacterial biofilm that had attached to the wells for 15 minutes.And crystal violet solution was used to stain the biofilm for 5 minutes.
-ELISA reader (Biorad, UK) was used to measure the absorbance of the stained biofilm at a wavelength of 570 nm.The average of the optical density (OD) readings for the three wells was used to determine the optical densities (ODi) for each isolate.This value was then contrasted with the optical density value of the negative control (ODc).
According to Stepanović et al 16 , the isolates were divided into the following 4 groups based on the findings of this comparison.

RNA extraction and cDNA synthesis
The bacterial colonies were suspended in TRIzol reagent and the total RNA was extracted using TransZol up plus RNA kit (TransGen/ Biotech/ China) as directed by the manufacturer.To measure the purity and concentration of isolated RNA, NanoDrop2000c (Thermo Fisher Scientific, USA) was used.As for the complementary DNA (cDNA) synthesis it was performed in a thermocycler (Applied Biosystem, USA) where 4 μl of template RNA was reversely transcribed using EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, Biotech.China) the prepared cDNA was used to perform the RT-PCR amplification.

Primers Design
The oligonucleotides used in this study were designed by Asmaa M. Salih Almohaidi using the Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/)where cupB, rhlR primers were designed especially for this study, while pvcB, pvcC and oprD were modified to better serve the purpose of this study from the reference sequence 7,18 .All the primers used in this study were synthesized by Alpha DNA Ltd (Canada) and stored at -23°C (Table1).

Real-Time Quantitative PCR (RT-qPCR)
The identified P.aeruginosa isolates were subjected to real-time quantitative PCR (RT-qPCR), to evaluate the expression levels of pvc genes (pvcA, pvcB, pvcC, pvcD) and cupB, rhlR genes.The expression was investigated using TransStart® Top Green (TransGen, Biotech.China) as per the manufacturer's instructions.Each PCR tube had a reaction mixture of the following components: 10 µL of the master mix; 3 µL of cDNA; 1 µL of each primer; and 5 µL of nuclease-free water in a final volume of 20 µL.The qRT-PCR was carried out using Rotor-gene Q Real-time PCR System (Qiagene,Germany) and the program for the prepared reactions started with an initial denaturation step at 94°C for 30sec; denaturation at 94°C for 5 sec.; annealing (refer to Table 1) for 15 sec; and extension at 72°C for 20 sec these steps were repeated for 40 cycles.PAO1 was used as a reference strain since many studies considered it as a standard strain 15 , while oprD and oprM were used as housekeeping genes.The reactions were carried out in triplicate, and the expression levels for the genes were examined using the mean ct value

Statistical analysis
Statistical analyses were performed using the SPSS Statistics software V 17.0 (SPSS Inc., Chicago, IL, USA).The expression values of all the data are displayed as the average ± standard error (SD).Student's t-test was used for the statistical analysis, and significance levels were chosen at *p 0.05 and **p 0.01.

Isolates molecular identification
In this study 16S rRNA and gyrB genes were used to identify P. aeruginosa clinical isolates.A conventional PCR technique was used to detect the 16S rRNA gene in 25 clinical isolates.All the tested isolates were positive for 16S rRNA 25/25 (100%) as seen in Fig. 1, while real-time quantitative PCR (RT-qPCR) was used to detect gyrB gene in 25 clinical isolates (that tested positive for 16S rRNA).The results for this test showed that 21 out of the tested 25 isolates were positive for gyrB gene 21/25 (84%) (Values ranged from16 to ~22cycles), these 21 isolates were the ones selected to carry on with the study.The ability of 21 P. aeruginosa isolates to generate biofilms was examined.Of the biofilmproducing phenotypes, 18 (85.7%) of the P. aeruginosa isolates were considered biofilm producers: 10 (47.6%) were qualified as strong, 5 (23.8%) were considered intermediate, and 3 (14.3%)were qualified as weak biofilm producers, while 3 (14.3%)were unable to produce biofilm (Table .2).

Antimicrobial Susceptibility:
Figure 2 provides a summary of the susceptibility profile of P.aeruginsa isolates toward 12 antimicrobial agents.Following the resistance interpretative criteria used for antimicrobial drugs as it has been previously described, the highest levels of susceptibility were to Gentamicin (80.9%),Tobramycin (78.19%),Levofloxacin(76.1%),Cefepime (71.4%),Amikacin and Ceftazidime (66.6%),) while the Meropenem (52.3%) and Imipenem (47.6%).According to Table .3, there was a relationship between the patterns of biofilm production and antibiotic susceptibility profile.Among 21 isolates, analysis of the different resistance phenotypes showed that 14 (66.7%) were MDR isolates and 7(33.3%) were sensitive to the antibiotics used.Out of 14 MDR isolates, 13/ 14(92.9%)were biofilm-producer isolates, including 8 belonged to the strong biofilm producers group, 3 belonged to the moderate biofilm producers group and 2 belonged to the weak biofilm producers group, only 1/14(7.1%)MDR isolates was non-biofilm producers.The ability to form biofilms was substantially more likely to have the resistance phenotype MDR.

Gene expression analysis:
In this study, RT-qPCR was performed for 8 genes with a significant expression between the clinical isolates and the reference strain, this includes two housekeeping genes.The RT-qPCR results showed that pvcA, pvcB, cupB genes were over-expressed >2.0 fold in 85.7% of isolates (strong, moderate and weak biofilm-forming isolates) (Fig. 3).
While pvcC and pvcD showed down-regulation <0.5 fold in all isolates 100%.Fig. 3 and rhlR only showed over-expression in two groups (strong and moderate) over 71% of the isolates.
The difference in the expression levels is visualized as an array in the amplification curve, where the expression level of pvcAB operon was continued and passed the threshold line in the early cycle, whereas a delay in the expression of pvcCD operon was observed.The different letters in the same column means there are significant differences.
As noticed in Table 5, the statistical comparison between current genes expression and the biofilm formation ability.The results showed a significant increase in gene folding for pvcA, pvcB, and cupB, rhlR genes that were synergistic in a compatible way with increasing the strength of biofilm for present isolates.pvcAB may up-regulat cupB while pvcCD may not.

Discussion
The results for 16S rRNA sequence identified all samples as P.aeruginosa.The sensitivity of 16S rRNA gene is 100% which agrees with the results submitted by Al-Taai et al and Al-Tememe 19,20 , while other study 21 recorded 96% sensitivity.As for the results of gyrB gene, about 21 isolates 84% harbored gyrB gene this finding may suggest that the gyrB gene is a good candidate for P. aeruginosa iden tification besides 16S rRNA [22][23][24] .
The sequences of the gyrB gene in P. aeruginosa and other species of Pseudomonas exhibited a larger divergence than the 16S rRNA genes, despite the fact that it is the most common gene for genetic identification and characterization of bacteria.The rationale is that the rate of gyrB molecular evolution is higher than that of 16S rRNA and that it is transmitted horizontally 21 .The gyrB, as housekeeping genes have already been used in bacterial phylogenetic analysis and identification, is a better candidate target than 16S rRNA for the identification of the Pseudomonas species 25 .
P.aeruginsa with its exceptional ability to adhere to many surfaces by forming a biofilm, sets the biofilm to be one of the most important virulence factors of the bacteria.A quantitative approach was regarded as the gold standard to assess the development of biofilms.In the current investigation, biofilm formation was observed for 18 out of 21 P. aeruginosa isolates obtained from clinical samples 85.7%.Previous researchers found that biofilm development occurred in 93.4%, 77.5%, 83.7%, and 100% of the cases studied, Baghdad Science Journal respectively [26][27][28][29] .However, they varied in their capacity to form biofilms.This is also applying to local studies, where researchers observed biofilm formation ability in 70%, and 100% respectively of the local clinical P.aeruginsa isolates 30,31 .Of the 18 total isolates, 47.6% had a strong capacity to do so, while significant isolates displayed a moderate 23.8% and weak 14.3% capacity.Similar data was reported 27,29,32 In 2017, P. aeruginosa was recognized by WHO as a priority pathogen for the development of new antimicrobial agents 33 .The current results of antimicrobial susceptibility test demonstrate a high degree of resistance to Aminoglycosides (Gentamicin 80.9%, Tobramycin 78.1% and Amikacin 66.6%, this result agreed with the researchers that recorded higher resistance to the Aminoglycosides 34,35 .Additionally, the findings demonstrate that the isolates were beta-lactam antibiotics resistant, particularly Meropenem 52.4% and Imipenem 47.6%, which are Carbapenems.These findings line up with the data provided by Ratajczak et al 29 .Imipenem-resistant strains were 52.72 % of P. aeruginosa isolated from infections, according to research done by the International Nosocomial Infection Control Consortium (INICC) 36 .A Globule rise in the number of Carbapenem resistance among Gram-negative bacilli 29 .
Overall, these findings are consistent with local studies, which illustrated a high rate of resistance to Ceftazidime, Amikacin, Ciprofloxacin, Piperacillin, Levofloxacin, and Gentamycin, with the ability to form biofilms.Which become substantially more likely to have the resistance phenotype MDR, with 13/18 (72.2%) of the isolates acquiring this phenotype 21,37,38  Similar results have been reported by other studies, which found a link between the MDR phenotype and P. aeruginosa isolates' capacity to produce a biofilm 39,40 .Another study in Iraq revealed a high ability (90.7%) to form biofilm in clinical MDR P. aeruginosa isolates, and the biofilm producers were classified into three categories (strong, moderate, and weak) according to the biofilm analysis 38 .Unlike other studies, which found that 100% of the isolates with MDR phenotype were strong biofilm producers 29 ?While in Brazil, a group of researchers discovered that 51.6% of P. aeruginosa isolates produced biofilms that were non-MDR, and 48.4% were MDR 27 .When P. aeruginosa is the etiological component in an illness, multiple drug resistance and biofilm development may make therapy challenging 29 .
The expression of genes such as cup cluster, quorum sensing systems (LasR-LasI system and RhlR-RhlI system) play a significant role among the regulatory systems of virulence factors for bacterial pathogenicity, including biofilm formation 41 .The expression of rhlR gene in Table .4was high (7.15fold), as this gene is required for biofilm formation, whereas, it showed down-regulated (< 1.0 fold) in the weak and non-biofilm producer isolates.Previous studies suggested that these isolates' inability to build biofilm was caused by conformational changes in quorum sensing (RhlR) proteins mediated by mutations in the rhlI/rhlR system 27,42 .However, the present results go with the results of another investigation which showed a strong connection between the ability to create biofilms and the presence of pertinent genes 2,43 .
Based on the current study's findings, P. aeruginosa isolates both contained and expressed the gene related to biofilm formation and were thought to have significant levels of expression with a significant compatible level of gene folding with strength of the biofilm.The expression of the cupB gene was low (1.7fold) in the non-biofilm producer isolates, compared to the reference strain employed as a positive control.The cupB gene was discovered in all biofilm producer isolates >2.0 fold, including strong 7.5 fold, moderate 4.4 fold, and weak 2.6 fold.These results are consistent with a study that revealed that the existence of biofilm genes without biofilm production might be caused by chromosomal changes in various regulatory systems, which would influence the synthesis of functional biofilm-related proteins 44 , it was also mentioned that 31.03% of P. aeruginosa isolates had the biofilm gene (pslA) while none of these isolates tested positive phenotypically for the production of biofilms in Congo red agar and microtiter plate experiments 44 .According to the present study's findings, MDR P. aeruginosa (n =  3).This may lead to the initial misconception that biofilm development is unrelated to antibiotic resistance for the isolates that generated biofilm and harbored the cupB gene but weren't MDR.It's important to note that all of the isolates used in current investigation were tested for antimicrobial susceptibility as planktonic cells rather than as biofilms.Thus, quorum sensing, which confers the MDR phenotype, and several mechanisms of biofilm and its architectural features such as glycocalyx matrix, outer membrane structure and growth rate, persistent cell formation, genetic adaptation, and stress responses, were not involved in the test 45 .
The current study demonstrated the upregulation in quorum sensing genes linked to potential virulence and the rise in biofilm biomass, as a risk factor for the emergence of extremely multi-resistant bacteria.The results of RT-qPCR showed that pvcA and pvcB, genes were presenting an over-expression (>2.0 fold) in three groups (strong, moderate and weak biofilm forming isolates).While pvcC and pvcD were downregulated <0.5 fold in all the isolates.These results are non-compatible with other research results which reported that a deletion mutation of the pvcA gene reduces the expression of cupB cupC about 17-fold and 5-fold respectively, compared to cupA gene which was only reduced by 2.2-fold 7 .This demonstrates the huge effect of pvcA on cupB gene clusters.
According to present study findings which showed different pattern of gene expression level, a new question arises, that is the pvc Genes Operon consist of One or Two operons?With their Effects on Biofilm Formation Genes in P. aeruginosa Clinical Isolates A previous study suggested that the pvc genes form one operon 12 , while a computer analysis proposed that the pvc genes consist of pvcAB operon and pvcCD operon (data not shown) 7 .This was determined by measuring the levels of gene expression for all pvc genes simultaneously.The levels of expression for pvc genes were then compared to the levels of cupB expression, which encodes fimbrial structure which aids in the biofilm formation, and the rhlR gene expression quorum sensing gene that regulates the biofilm production in P.aeruginosa.Since operons are a feature of prokaryotic genomes and allow for the functionally related genes to be regulated, transcribed and translated simultaneously 46,47 .The drastic difference in the expression of pvc genes was highly support the possibility that pvc genes form two operons pvcAB and pvcCD.The results of this study conflict with previous results that presented by other authors who have also tested this possibility 7 .
The sequence can form a stem-loop structure in the 51-bp intergenic region between pvcB and pvcC, therefore it is possible that there are two operons rather than one 10 .This sequence is most likely to have an attenuating effect on the expression of the pvcC and pvcD genes from a promoter upstream of pvcA 10 .The following model Fig. 4 is proposed to explain current findings, on the basis of the presented results, the current study suggests that pvcA alongside pvcB enhances the biofilm formation by enhancing the expression of the cup gene cluster

Conclusion
Current findings indicate that gyrB gene is a good candidate for P.aeruginosa identification besides 16S rRNA and there was a relationship between the patterns of biofilm production and antibiotic susceptibility profile.The ability to form biofilms was substantially more likely to have the resistance phenotype MDR.And pvc genes constitute two operons and pvcAB operon plays the role of enhancing the expression of cup genes and thus promotes biofilm formation.The data state that pvcCD operon is down-regulated in biofilmforming isolates which denies its involvement in increasing biofilm intensity.

Figure 1 .
Figure 1.Amplified 16S rRNA products.Electrophoresed on 1.5 % agarose for 1.5 h. with 70 volt.The product size was 144 pb.Lane M: DNA Ladder; lane C: Negative control; lane 1 to 16: tested isolates Biofilm Assay:The ability of 21 P. aeruginosa isolates to generate biofilms was examined.Of the biofilmproducing phenotypes, 18 (85.7%) of the P. aeruginosa isolates were considered biofilm

Figure 2 .
Figure 2. P. aeruginosa isolates resistance to different antibiotic group.

Figure 3 .
Figure 3.The fold change gene expression of the genes used in this study for biofilm forming groups.

Figure 4 .
Figure 4. Diagram of the proposed pvc operons

Table 5 . The statistical analysis of gene expression with Biofilm grouping Biofilm forming groups Fold Change in Gene expression
** Highly significant =p≤0.01,* Significant =p≤0.05,non-significant p>0.05 Table.3.