Molecular Investigation of Outer Membrane Channel Genes Among Multidrug Resistance Clinical Pseudomonas Aeruginosa Isolates

Background: Multidrug resistance Pseudomonas aeruginosa (MDRPA) is most important issue in healthcare setting. It can secrete many virulence effector proteins via its secretion system type (T1SS-T6SS). They are using them as conductor for delivering the effector proteins outside to begins harmful effect on host cell increasing pathogenicity, competition against other microorganism and nutrient acquisition. Methods: The study include investigation of 50 isolates of MDRPA for transport secretion system and resistance for antibiotics. Molecular diagnosis using P. aeruginosa specific primer pairs, investigation of AprF, HasF, XcpQ, HxcQ, PscC, CdrB, CupB3, and Hcp using specific primer pairs by PCR were also performed. Results: The results revealed high resistance to beta lactam antibiotics (78% for ceftazidime, 78% for cefepime and 46% for piperacillin) can indicate possessing of isolates for beta lactamases and this confirmed by dropping resistance to piperacillin to 16% when combined with tazobactam. Also, the results shown the ability of MDRPA for pyocyanin biosynthesis using the system of genes. Conclusions: The current study conclude that all isolates of P. aeruginosa were highly virulent due to their possessing of all transport secretion system to deliver different effector proteins with possible harmful effects of these proteins.


Introduction
Pseudomonas aeruginosa (P. aeruginosa) is a nosocomial opportunistic pathogen that can dominate all niches (1). The transport of proteins from the cytoplasm into different compartments of the cell, the environment, and/or other bacteria or eukaryotic cells is an important function of prokaryotic cells, a process known as protein secretion. Protein secretion systems are required for bacterial development and are used in a variety of functions. Bacterial pathogens use secretion systems to influence their hosts and generate a replicative niche (2). Exoenzymes secreted by P. aeruginosa cause damage to host tissue by disrupting normal cytoskeletal structure, depolymerization of actin filaments, and cleavage of immunoglobulin G (IgG) and A (IgA), and all these processes lead to invasion, dissemination, and the development of chronic infections (3). Small molecules, proteins, and DNA are transported into the extracellular space or target cells via the secretion system. The structural and molecular characteristics of Gram-negative bacteria's six secretion systems (types I-VI) (4,5). Proteins can be secreted using one of two methods. One method is a one-step technique in which proteins from bacteria's cytoplasm are transported and supplied straight into the host cell through the cell membrane. Another involves a two-step process in which proteins are first carried out of the inner cell membrane, then into the periplasm, and finally into the host cell through the outer cell membrane (6).
Pseudomonas aeruginosa has many secretion systems. There are two types of T1SS in p. aeruginosa, T1SS-Apr and T1SS-Has (7,8). The secretion system of T1SS-Apr called AprDEF with outer membrane protein called AprF (PA1248) which act as outer channel for secretion system who exploit to secret important alkaline protease called AprA(PA1249) and AprX(PA1245). T1SS-Has secretion system called HasDEF with HasF (PA3404) as outer channel used to secret HasAp (PA3403) (a Heme acquisition Protein) (9,10). P. aeruginosa strain PAO1 possesses two complete and non-redundant T2SS, referred to as the Xcp and Hxc systems (11). Xcp T2SS plays an important role in bacterial virulence by its capacity to deliver a large panel of toxins and degradative enzymes into the surrounding environment (12). T2SS-Hxc secretion system called HxcP-HxcZ, with HxcQ (PA0685) as outer channel used to secret LapA (PA0688) (Low-molecular weight alkaline phosphatase) (13,14). The type III secretion system (T3SS) is a bacterial nanomachine that resembles a syringe on the bacterial surface. The T3SS 'needle' delivers translocon proteins into eukaryotic cell membranes, subsequently allowing injection of bacterial effectors into the cytosol (15). T3SS-PscC (PA1716) as outer channel secretion system, used to secret ExoS (PA3841) (GTPase activating protein (GAP) domain and adenosine diphosphate ribosyltransferase domain (ADPRT)) and ExoT (PA0044) (GTPase activating protein (GAP) domain and adenosine diphosphate ribosyl transferase domain (ADPRT)) and ExoY (PA2191) (Adenylate cyclase) (16) and ExoU (PA14_51530) (Patatin-like, phospholipase) (17). Type IV pili (T4P) filamentous surface appendages, sophisticated biological nanomachines (18). The T4SSs functionally encompass two major subfamilies, the conjugation systems, and the effector translocators. The conjugation systems are responsible for interbacterial transfer of antibiotic resistance genes, virulence determinants, and genes encoding other traits of potential benefit to the bacterial host. The effector translocators are used by many Gramnegative pathogens for delivery of potentially hundreds of virulence proteins termed effectors to eukaryotic cells during infection (19). T5SS secretion system called CdrB with CdrB (PA4624) as outer channel used to secret CdrA (PA4625) (Adhesin) (20) and P-usher secretion system called CupB3 with CupB3 (PA4084) as outer membrane channel used to secret CupB5 (PA4082) (Putative adhesin) which involved in the assembly of fimbriae at the bacterial cell surface (21). Hemolysin coregulated protein (Hcp), a ring-shaped hexamer secreted by all characterized T6SSs, binds specifically to cognate effector molecules (22). Hcp protein is a core component of the T6SS tail tube and acts as an exported receptor and a chaperone of effectors (23). Resistance to antibiotics may be either intrinsic or acquired (24,25). The prominence of multidrug resistant P. aeruginosa is growing in the world, limiting the therapeutic options (26,27). Despite the emergence of modern antibacterial agents with anti-Pseudomonal activity, P. aeruginosa continues to be the leading cause of lifethreatening infections in hospitals (28,29). Even though increase of multi-drug resistant Pseudomonas aeruginosa strains, which are intractable to be treated, some available antibiotics still able to dominate pseudomonal infections with a reasonable percentage of success, for example, colistin sulfate and quinolones (ciprofloxacin and levofloxacin) (30,31). The current study was conducted to investigate outer membrane genes for transport secretion system among multidrug resistance P. aeruginosa isolates.

Materials and Methods
Ethical Approval informed consent was obtained from all human adult participants or parents or legal guardians of minors. The project was approved by scientific committee and Bioethics committee under project no. 325 on 29 December 2020.

Ethical approval
The study has been approved by the Ethics Committee of Kashan University of Medical Sciences, Iran and all experiments were reviewed and approved following the current European Union Directive (2010/63/EU) guideline on the protection of animals used for scientific purposes.

Bacterial isolates
Fifty P. aeruginosa isolated were obtained from different specimens and subjected for primary identification test using Pseudomonaschromogenic agar (Condalab/Spain) and confirmed using genus specific (for Pseudomonas spp.) and species specific (for P. aeruginosa) ( Table 1).

Antibiotic susceptibility Assay
It was performed using 14 antibiotics agent according to CLSI-2019 (32).

Polymerase Chain Reaction
DNA was extracted according to manufactures instructions (IntronBio/Korea). The primers were dissolved according to manufacturer instructions (Macrogen/Korea). The primer pairs and PCR conditions were demonstrated in (Table 1).
Outer membrane channels of Transport secretion systems presence were investigated via detection of AprF, HasF, XcpQ, HxcQ, PscC, CdrB, CupB, HcP genes which encode for outer membrane channels. The results revealed that 93% of p. aeruginosa isolated have T1SS-T6SS genes (Figs. 2 and 3). Table 3 shows high prevalence of coexistence secretion system channel genes among p. aeruginosa which act as conductor for delivering the effector proteins outside to begins harmful effect on host cell increasing pathogenicity and for competition against other microorganism, nutrient acquisition. If one or more channel blocked this will lead to determine and decrease the number of secreted proteins consequentially the bacterium became less virulence.

Discussion
Our results may be totally agreeing with previous studies whose found that dominance of P. aeruginosa among UTIs, RTIs and wound-burn infections (34). Implication of P. aeruginosa in UTIs may be as nosocomial pathogen resulted from placing and removing of indwelling urinary catheters (35). Admission to an intensive care unit (ICU) raises the risk of MDR P. aeruginosa infection in critically ill pneumonia patients (36). P. aeruginosa is a significant cause of nosocomial infections in burn centers, which may be due to a high frequency of antibiotic resistance and the potential to form biofilms (37). P. aeruginosa shows resistance to a wide range of antibiotics, comprises aminoglycosides, quinolones and β-lactams. The resistance may be intrinsic (low outer membrane permeability, coding for efflux pumps and the making of antibiotic-inactivating enzymes), acquired (either horizontal transport of resistance genes or mutational alteration) and adaptive (involves formation of biofilm which provide as a diffusion barrier to edge antibiotic access to the bacterial cells) resistance (38). Step 1: 95 ºC, 2 min.
Step    The results shown high resistance to beta lactams (ceftazidime (CAZ), cefepime (FEP), piperacillin (PRL) and this is mainly mediated by beta lactamases due to that when use piperacillin-tazobactam the resistance was dropped from 46% to 16%. Beta lactamases regard as intrinsic mechanism of resistance leading to inactivating of beta lactam rendering them inactive. Beta lactamase inhibitor like tazobactam (An irreversible inhibitor of a wide variety of bacterial beta-lactamases) can improve many beta lactams like piperacillin once combined with them.
Piperacillin-tazobactam is the most widely used lactamase inhibitor combination for treating P. aeruginosa infections (39,40). The results of current study revealed the ability of all P. aeruginosa isolated to produce pyocyanin making them more virulent and have great harmful consequences due to implication of pyocyanin in tissue damage, interfering with immune response and triggering proinflammatory responses (41).
Determination of different virulence genes of P. aeruginosa isolates suggest that they are associated with different levels of intrinsic virulence and pathogenicity. This may have different consequences on the outcome of infection (42). So, our result revealed that the isolate has high virulence mechanism to deliver the effector proteins affecting host cells and other bacteria in their milieu (43)(44)(45).
The current study concludes that all isolates of P. aeruginosa were highly virulent due to their possessing of all transport secretion system to deliver different effector proteins with possible harmful effects of these proteins.