Quorum Sensing Mediated Pathogenicity, Virulence Genes and Class 1 Integron in Carbapenem-Resistant Clinical Pseudomonas Aeruginosa Isolates

Ceren Başkan (  ceren.yavuz@amasya.edu.tr ) Amasya University: Amasya Universitesi https://orcid.org/0000-0001-7849-4459 Belgin Sırıken Ondokuz Mayis University: Ondokuz Mayis Universitesi Enis Fuat Tüfekci Kastamonu University: Kastamonu Universitesi Çetin Kılınç Kastamonu University: Kastamonu Universitesi Ömer Ertürk Ordu University: Ordu Universitesi İrfan Erol Eastern Mediterranean University: Dogu Akdeniz Universitesi


Introduction
Carbapenems (imipenem, ertapenem, meropenem and doripenem) are β-lactam, antibiotic class members and one of the most effective antipseudomonal drugs for curing infections commonly caused by P. aeruginosa. However, recent study results have shown that P. aeruginosa is becoming increasingly resistant against carbapenems (Tacconelli et al., 2018;Hu et al., 2019). According to the World Health Organization reports, carbapenem-resistant clinical P. aeruginosa (CRPsA) ranks second based on most criteria for bacteria among 20 antidrug-resistant bacterial species (Tacconelli et al., 2018). There are several major causes of carbapenem resistance, such as extensive clinical use of this antibiotic, overexpression of e ux systems, alterations or losses in outer membrane porin D (OprD) and carbapenemase production. The most common and globally reported carbapenemase genes, known as metallo-β-lactamases (MBLs) are transported from mobile genetic elements, speci cally via integrons (Rojo-Bezares et al., 2016). In addition to these antibiotic resistance mechanisms of P. aeruginosa, its ability to produce bio lms and its success in escaping from the host immune system are related to the infection formation that is di cult to treat. Because bacterial bio lms have multiple tolerance mechanisms for antibiotic therapy, they cause bio lm infections to persist despite antibiotic exposure (Radovanovic et al., 2020). This result is owing to extracellular polymeric substances (EPS), which are detected around bio lm-forming bacteria and form a barrier against antimicrobial agents and host immune system elements (Brindhadevia et al., 2020;Chakraborty et al., 2020).
Bacteria work together to carry out cell-to-cell communication by secreting small signaling molecules (autoinducers, AIs). When the population number reaches the threshold level, they start to form a bio lm due to EPS. This communication procedure is referred to as QS. The QS system of P. aeruginosa contains two major AI signaling molecules (AHL:3-oxo-C 12 -HSL and C 4 -HSL). Each system (Las and Rhl) is encoded by two components: AI synthesis (lasI and rhlI genes) and their cognate transcriptional activating protein (lasR and rhlR genes). There is also a third system in P. aeruginosa known as the Pseudomonas quinolone signal, which is induced by 2-heptyl-3hydroxy-4-quinolone and controlled by the las and rhl systems (Brindhadevia et al., 2020;Sırıken et al., 2020;Elnegery et al., 2021). These systems in the QS mechanism cause the bacteria to have a successful role in pathogenicity by adjusting the population densities and activating the relevant virulence factors. P. aeruginosa has many virulence factors. Some virulence factor-encoding genes, such as elastase (lasB), toxin A (toxA), and alkaline protease (aprA), are governed by the las and rhl synthesis genes (lasI/lasR and rhlI/rhlR) (Attiah et al., 2021). These virulence genes have been shown to host many proteins hydrolyses, causing host tissue damage, disrupting immune response, and supporting in ammation (Iiyama et al., 2017;Haghi et al., 2019;Everett and Davies, 2021). For instance, alginate is responsible for bio lm formation and has a great role in the structural preservation and stability of bio lms (Gholami et al., 2017).
The oprL gene encodes the structural membrane lipoprotein of P. aeruginosa. The presence of this speci c outer membrane protein also has a key role in the hereditary resistance of P. aeruginosa to many antibiotics (e ux transport system or membrane selectivity). The gene is also employed for the determination of P. aeruginosa in clinical and other samples by polymerase chain reaction (PCR) at the species level or by reverse transcription (RT)-PCR assay (De Vos et al., 1997).
Due to the above-mentioned ndings, understanding the QS system involved in pathogenicity and the regulatory mechanisms that regulate the virulence genes expression is critical for the development of alternative curing approaches for the monitoring and prevention of pseudomonal infections. Therefore, this study aimed to analyze (i) the presence of las and rhl QS genes and their relation to the role of regulation of virulence factors, (ii) the presence of lasB, algD, toxA and aprA virulence genes, (iii) the presence of Cls 1integron, and (iv) bio lm formation.

Material And Methods
Isolation, identi cation, carbapenem-resistance pro le of P. aeruginosa A total of 52 carbapenem-resistant P. aeruginosa isolates obtained from various clinical samples [tracheal aspirate (n=37), blood (n=8), urine (n=5) and wound (n=2)] at the Clinical Microbiology Laboratory of Kastamonu Training and Research Hospital in Turkey, were employed as a material. The isolation and identi cation of the isolates were performed using a conventional method (determination of Gram and oxidase reaction, beta-hemolytic activity on sheep blood agar, colony morphology, pigment production, growth at 42°C) and the VITEK 2 compact system, and the carbapenem-resistance pro le of the isolates was detected by the VITEK 2 compact system (BioMéreux, France) (Nakasone et al., 2007). Molecular con rmation of the isolates was applied using PCR assay based on P. aeruginosa species-speci c oprL gene region detection according to Ahmadi and Roodsari (Ahmadi and Roodsari, 2016).

Slime production
Slime production was determined by cultivation of the isolates on CRA (including 37 g/L BHI broth, 10 g/L agar base, 50 g/L sucrose, 1 L water and 0.8 g/L Congo Red indicator) plates, as described by Freeman et al (Freeman et al., 1989).
The isolates were kept in nutrient broth with 15% glycerol at -86 °C for further analysis. P. aeruginosa ATCC 15692 was employed as a positive reference strain for detection of the oprL gene region, two QS systems and four virulence genes, and slime production tested.

DNA extraction
The isolates were sub-cultured on Trypticase Soya Agar (TSA) and a maximum of ve colonies grown on TSA were collected. DNA extraction of the isolates was carried out using boiling method according to Katvoravutthichai et al. (Katvoravutthichai et al., 2016). The sequence, product size of the primers and ampli cation program (TurboCycler Lite 9020, Blue-Ray, Biotech) for the PCR assay utilized in this study are depicted in Table 1.

Detection of QS system genes
For this purpose, four gene regions (lasI/R, rhlI/R) associated with the las and rhl QS system were analyzed in the CRPsA isolates using PCR assay, previously described by Schaber et al. (Schaber et al., 2004).

Detection of QS-related virulence factors and class 1 integron
For this purpose, lasB, algD, toxA, and aprA virulence gene regions were determined using single target PCR assay with minor modi cations to the protocol proposed by Martins (Martins et al., 2014). The total PCR mixture was 25 µL, including 1XPCR Buffer, 2.5 mM of MgCl 2 , 0.2 mM of dNTPs, 0.2 µM of each primer, 1U of Taq polymerase, and 1 µL of template DNA.
Detection of Cls1 integron: For the presence of Cls1 integron, the integrase gene (intI1) was determined using the PCR assay previously described by Bass (Bass et al., 1999).

Statistical analysis
Fisher's exact test was performed to analyze the relationship among the las and rhl QS systems, virulence gene distribution and bio lm formation. A statistical analysis was carried out using SPSS/20.0 software.

Results and discussion
CRPsA infections are one of the most serious healthcare-related infections because they are most commonly utilized for the last-choice antibiotic to cure P. aeruginosa infections (Morita el al., 2014). Many researchers stated that CRPsA's infections cause problems such as increased mortality, longer hospital stay duration, and increased medical costs. According to available data from Turkey and other countries, the maximum human clinical CRPsA ratio is 60% ( It is well known that the P. aeruginosa's virulence genes expression is a highly complex procedure that is generally governed by las and rhl QS system genes. Kumar et al. stated that QS system-de cient strains that fail to create successful infection were associated with a decrease in virulence factors expression (Kumar et al., 2009). In the present study, two QS system genes were detected in almost all isolates (98.7%) and compared with the presence of the two QS system genes, the percentage of las system genes (98.07%) were higher than that of rhl system genes (90.38%). In addition, there was a positive correlation between two QS system genes (p < .10). Ellappan et al. reported that lasR genes and rhlR genes were identi ed in 81% and 84%, respectively, of the clinical CRPsA isolates (Ellappan et al., 2019). The current study results are higher [lasR (94.23%) and rhlR (82.69%)] than Ellappan et. Al.'s study results but lower than El-Mahdy and El-Kannishy's study ndings, in which QS lasR and rhlR genes were detected in all of the isolates (El-Mahdy, El-Kannishy, 2019).
Bacteria with QS systems that govern virulence factors and bio lm formation are more resistant to most treatment agents, such as carbapenems and next-generation antibiotics (Tanveer et al., 2020). The present study results supported this viewpoint to some extent. Hence, four virulence genes were identi ed at a ratio between 46.15% and 88.46% for 52 CRPsA isolates. When considering two QS system genes and slime production (67.30%) with four virulence genes, it can be concluded that the CRPsA isolates are highly virulent.
Pathogenesis of P. aeruginosa involves many stressful conditions (interferon, IFN), etc.) created by the host immune system, and to ensure that bacteria overcome many stressful factors and survive, a wide range of virulence genes are expressed, particularly by the las and rhl QS systems (Gonçalves et al., 2017). For instance, IFNproduced by T-cells coordinates many different immunological responses (Schroder et al., 2004). IFN-binds to P. aeruginosa outer membrane protein E (OprF). When the binding step occurs, the rhl QS system activates for the production of some virulence factors, such as lecA (encodes lecA, which are cytotoxic and adhesive factors) and pyocyanin. Afterward, the rhl QS system induces cytotoxic exoproducts such as exotoxin A to enter the host cell and then cause bio lm formation (Laughlin et al., 2000). Therefore, toxA gene is an important virulence factor in encoding exotoxin A (exoA). Our result of 86.53% is consistent with the results of Gonçalves et al. ( As an important virulence factor, the algD gene has a crucial role, especially in chronic lung infections and alginate biosynthesis. During infection and antibiotic therapy, the bacteria are transformed from the nonmucoid phenotype into mucoid-producing bacteria and start to produce alginate (Dogget, 1969). In the late stage of infection, mucoidproducing bacteria are dominant and cause deterioration, leading to a high mortality rate (Davis et al., 1980). In the current study, the algD gene was detected in 46.15% of the isolates. The results indicate that nearly half of the isolates have the mucoid-producing property and that there were signi cant correlations between algD and the rhl system (p .05), between algD and slime production (p .01), between algD and lasB (p .05) and between algD and toxA (p .05). Bogiel  In our study, the CRPsA isolates were capable of bio lm formation due to algD gene (46.15%) and slime production (67.30%). These co-existing properties were present in 22 (42.30%) of the CRPsA isolates. From 37 tracheal origin isolates, 72.97% and 51.33% of the CRPsA isolates were capable of slime production and carried the algD gene, respectively. Thus, the majority of the tracheal origin CRPsA isolates have the potential of bio lm formation. According to Bogiel et al.'s study results, there was a positive correlation between toxA genes and algD genes (p<0.05). Our study supports their results in terms of a positive correlation between toxA genes and algD genes (Bogiel et al., 2021).
There is limited research on bio lm formation of CRPsA isolates (Kumar et al., 2009). In El-Mahdy and El-Kannishy's study, bio lm formation was detected in 65.2% and 94.1% of carbapenem-sensitive strains and carbapenem-resistant strains, respectively, and lasR and rhlR genes were identi ed in all CRPsA isolates (El-Mahdy and El-Kannishy, 2019). The authors concluded that bio lm formation was signi cantly related to carbapenemresistant isolates. Kumar et al.'s study results support this conclusion. Similarly, in our study, slime production was determined in 67.30% of the isolates, and there was a positive correlation between the rhl QS system and slime production. A signi cant correlation between slime production and algD, as well as lasB genes (Table 3), was also detected (Kumar et al., 2009).
As a protease enzyme, elastase B (lasB) (pseudolysin) is encoded by the lasB gene. LasB is associated with cystic brosis due to elastinolytic activation and with vascular in ammation due to elastin ber's disorganization in vascular tissue due to protease degradation by lasB (Schultz and Miller, 1974). Similar to aprA, lasB also degrades some proteins, such as In conclusion, based on the ndings of the current study, there is a signi cant positive correlation between las-rhl system, and between the QS system and four virulence genes and slime production. Cls 1 integron is common in the tested CRPsA isolates. Therefore, the CRPsA isolates are highly virulent and QS systems have a signi cant role in pathogenesis. Carbapenemase gene can be transferred among bacteria. All of the results indicate that CRPsA isolates are great concerns in terms of clinical aspects and to control of spread of the carbapenemase gene.

Results And Discussion
CRPsA infections are one of the most serious healthcare-related infections because they are most commonly utilized for the last-choice antibiotic to cure P. aeruginosa infections (Morita el al., 2014). Many researchers stated that CRPsA's infections cause problems such as increased mortality, longer hospital stay duration, and increased medical costs. According to available data from Turkey and other countries, the maximum human clinical CRPsA ratio is 60% ( It is well known that the P. aeruginosa's virulence genes expression is a highly complex procedure that is generally governed by las and rhl QS system genes. Kumar et al. stated that QS system-de cient strains that fail to create successful infection were associated with a decrease in virulence factors expression (Kumar et al., 2009). In the present study, two QS system genes were detected in almost all isolates (98.7%) and compared with the presence of the two QS system genes, the percentage of las system genes (98.07%) were higher than that of rhl system genes (90.38%). In addition, there was a positive correlation between two QS system genes (p < .10). Ellappan  Bacteria with QS systems that govern virulence factors and bio lm formation are more resistant to most treatment agents, such as carbapenems and next-generation antibiotics (Tanveer et al., 2020). The present study results supported this viewpoint to some extent. Hence, four virulence genes were identi ed at a ratio between 46.15% and 88.46% for 52 CRPsA isolates. When considering two QS system genes and slime production (67.30%) with four virulence genes, it can be concluded that the CRPsA isolates are highly virulent.
Pathogenesis of P. aeruginosa involves many stressful conditions (interferon, IFN), etc.) created by the host immune system, and to ensure that bacteria overcome many stressful factors and survive, a wide range of virulence genes are expressed, particularly by the las and rhl QS systems (Gonçalves et al., 2017). For instance, IFNproduced by T-cells coordinates many different immunological responses (Schroder et al., 2004). IFN-binds to P. aeruginosa outer membrane protein E (OprF). When the binding step occurs, the rhl QS system activates for the production of some virulence factors, such as lecA (encodes lecA, which are cytotoxic and adhesive factors) and pyocyanin. Afterward, the rhl QS system induces cytotoxic exoproducts such as exotoxin A to enter the host cell and then cause bio lm formation (Laughlin et al., 2000). Therefore, toxA gene is an important virulence factor in encoding exotoxin A (exoA). Our result of 86.53% is consistent with the results of Gonçalves et al. (Gonçalves et al., 2017). (87.3%) and Bogiel et al (93.9%). These ndings indicate that the toxA gene is highly common among CRPsA strains (Bogiel et al., 2021). In addition, according to some researchers there is a positive correlation among rhl system, exoA and bio lm formation, and the results of the present study also show agreement with the results (Laughlin et al., 2000;Diggle et al., 2006).
Another virulence factor is AprA, which also has a role in P. aeruginosa pathogenesis due to the degradation of wide proteins and destroys the host defense system (Hoge et al., 2010). In our study, the aprA gene was detected in 88.48% of the isolates, whereas Rojo-Bezares et al. and  As an important virulence factor, the algD gene has a crucial role, especially in chronic lung infections and alginate biosynthesis. During infection and antibiotic therapy, the bacteria are transformed from the nonmucoid phenotype into mucoid-producing bacteria and start to produce alginate (Dogget, 1969). In the late stage of infection, mucoidproducing bacteria are dominant and cause deterioration, leading to a high mortality rate (Davis et al., 1980). In the current study, the algD gene was detected in 46.15% of the isolates. The results indicate that nearly half of the isolates have the mucoid-producing property and that there were signi cant correlations between algD and the rhl system (p .05), between algD and slime production (p .01), between algD and lasB (p .05) and between algD and toxA (p .05). Bogiel et al. and Ellappan et al. detected the algD gene in 92.5% and 93%, respectively, of the CRPsA isolates. The results of both studies are higher than our study results (Ellappan et al., 2018;Bogiel et al., 2021).
In our study, the CRPsA isolates were capable of bio lm formation due to algD gene (46.15%) and slime production (67.30%). These co-existing properties were present in 22 (42.30%) of the CRPsA isolates. From 37 tracheal origin isolates, 72.97% and 51.33% of the CRPsA isolates were capable of slime production and carried the algD gene, respectively. Thus, the majority of the tracheal origin CRPsA isolates have the potential of bio lm formation. According to Bogiel et al.'s study results, there was a positive correlation between toxA genes and algD genes (p<0.05). Our study supports their results in terms of a positive correlation between toxA genes and algD genes (Bogiel et al., 2021).
There is limited research on bio lm formation of CRPsA isolates (Kumar et al., 2009). In El-Mahdy and El-Kannishy's study, bio lm formation was detected in 65.2% and 94.1% of carbapenem-sensitive strains and carbapenem-resistant strains, respectively, and lasR and rhlR genes were identi ed in all CRPsA isolates (El-Mahdy and El-Kannishy, 2019). The authors concluded that bio lm formation was signi cantly related to carbapenemresistant isolates. Kumar et al.'s study results support this conclusion. Similarly, in our study, slime production was determined in 67.30% of the isolates, and there was a positive correlation between the rhl QS system and slime production. A signi cant correlation between slime production and algD, as well as lasB genes (Table 3), was also detected (Kumar et al., 2009).
As a protease enzyme, elastase B (lasB) (pseudolysin) is encoded by the lasB gene. LasB is associated with cystic brosis due to elastinolytic activation and with vascular in ammation due to elastin ber's disorganization in vascular tissue due to protease degradation by lasB (Schultz and Miller, 1974). Similar to aprA, lasB also degrades some proteins, such as INF-γ, tumor necrosis factor-α and interleukin-6 (Horvat et al., 2010). lasB has an important role in the differentiation of pseudomonal bio lms (Yu et al., 2014). Tielen et al. showed that overexpression of lasB gene was not applicable to hard bio lm, but it contributes to the altering of the extracellular polymeric substances of the bio lm structure, such as reducing the alginate content but increasing the rhamnolipids concentration (Tielen et al., 2010). In this respect, in our study, lasB was detected in 69.23% of the CRPsA isolates, and there was a positive correlation between lasB and slime production and between lasB and algD genes. In the present study, lasB was identi ed in 69.23% of the CRPsA isolates. The ratio was quite lower than the ratios indicated by Ellappan  ). Similar to this study, certain studies investigate the presence of integrons among CRPsA isolates, and a corresponding ratio between 67% and 13.6% has been obtained (Sung et al., 2009;Estepa et al In conclusion, based on the ndings of the current study, there is a signi cant positive correlation between las-rhl system, and between the QS system and four virulence genes and slime production. Cls 1 integron is common in the tested CRPsA isolates. Therefore, the CRPsA isolates are highly virulent and QS systems have a signi cant role in pathogenesis. Carbapenemase gene can be transferred among bacteria. All of the results indicate that CRPsA isolates are great concerns in terms of clinical aspects and to control of spread of the carbapenemase gene.
Declarations Author contributions C.B., BS and İ.E designed the study; E.F.T and Ç.K. collected carbapenem-resistant P. aeruginosa isolates and carried out phenotypic identi cation; C.B. and B.S. performed the molecular assay; C.B., B.S., E.F.T., Ç.K., Ö.E., and İ.E. prepared and revised the manuscript. All authors gave the nal approval of the version to be published.

Disclosure Statement
All the authors report that they have no competing interests in this work.

Financial Disclosure
There is no nancial support.