Molecular Study of Two Virulence Genes of Pseudomonas aeruginosa, The Oxa 10 and Tox a with The Comparisons of The Relevant Sequences

Amir Hani Raziq
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Keywords : Pseudomonas aeruginosa, oxa 10, tox A, sequencing, phylogenetic tree.
Medical Journal of Babylon  14:1 , 2017 doi:1812-156X-14-1
Published :16 July 2017

Abstract

Pseudomonas aeruginosa is one of the leading nosocomial pathogens worldwide. Fifty pre-identified local isolates of P. aeruginosa were collected from major hospitals in Duhok and Erbil during the period from April 2015 to September 2015. The isolates were identified by classical biochemical methods and antibiotic sensitivity profiles were also obtained. Molecular investigation started with DNA extraction, confirmatory identification by the detection of 16S rRNA; PCR amplifications were applied to detect the presence of oxa 10 and toxA genes and then sequencing of the resulted PCR products was also performed. The results showed that all the fifty isolates had biochemical profiles characteristic of P. aeruginosa as it was also confirmed by the amplification band of 956 bp relevant to 16S rRNA; and that 50 % of the isolates revealed resistance to imipenem while 98 % of the isolates were resistant to cefepime. Furthermore, oxa10 gene was successfully detected by PCR in 92 % of the isolates with products bands of 760 bp while toxA gene was detected in 84 % of the isolates with an amplification bands of 396 bp. Then the PCR products of randomly selected ten samples (five for oxa 10 gene (designated A1through A5) and another five for tox A gene (designated B1 through B5) were purified by using a commercial PCR product purification kit and sequenced. The results of sequencing were analyzed in the University of Duhok/Scientific Research Center using DNASTAR/Laser Gene software. Regarding oxa10 gene, the results revealed that strains A4 and A5 are much more related when compared with the other strains. While the result of sequencing tox A gene indicated that isolates B2 and B5 are much more related in comparison with the other three isolates and isolate B1 was much more related to the positive control when all the isolates were compared.

Introduction

Pseudomonas aeruginosa is a widely distributed ubiquitous nosocomial pathogens [1]. It is widely recognized as an opportunistic pathogen, almost related to infections of immunosuppressed patients [2]. This bacteria is intrinsically resistant to multiple antibiotics including penicillins and cephalosporins, ciprofloxacins, chloramphenicol, tetracycline, erythromycin, trimethoprim–sulfamethoxazole and rifampin, this resistance might be explained by the ability to express efflux pumps and AmpC ?-lactamase constitutively, in addition to a decreased permeability of the outer membrane [3]. The simultaneous expression of a combination of multiple antibiotic resistance mechanisms is a major factor of conferring multidrug resistance (MDR) phenotype specifically noticed in nosocomial strains of P. aeruginosa which stand for the large proportion of clinical strains [4]. The ?-lactamases of the OXA-type are so termed because of their ability to hydrolyze oxacillin. These enzymes hydrolyze cloxacillin and oxacillin in a rate greater 50% than that for benzylpenicillin [5]. Oxacillinases (OXA type enzymes) grouped to molecular class D and functional category 2d. Classical OXA enzymes (OXA-1, OXA-2, OXA-10) specify resistance to ureidopenicillins and carboxypenicillins but not to ceftazidime. Moreover, the resistance to piperacillin and ticarcillin attributed to OXA-2 enzymes production is lower than the resistance shown when OXA-1 and OXA-10oxacillinasesare synthesized [6]. Five distinct categories of oxacillinases enzymes have been described in Pseudomonas aeruginosa [7]. Pseudomonas aeruginosa synthesize two different ADP-ribosyltransferase toxins: Exotoxin A (ExoA, toxA) andexoenzyme S. Exoenzyme S results in profound tissue destruction in lungs, burn and wounds infections. The extremely toxic ExoA is produced by almost every strain of Pseudomonas aeruginosa; it can impair cellular protein synthesis by inactivating polypeptide chain elongation factor 2 [8]. Exotoxin A is an important pathogenicity determinant of Pseudomonas spp., with a molecular weight of 66 kD; the action of which is to that of diphtheria toxin. The gene encoding for Exotoxin A is present in 90-95 % of P. aeruginosa strains whereas other Pseudomonas spp. and GC-rich microorganisms did not show evidence of tox A gene presence [9]. ETA contains two fragments or subunits; catalytic subunit A, and subunit B responsible for interacting with target cell surface receptors. Exotoxin Ahas considerable cytotoxicity to several mammalian cell types [10]. It is an extremely potent toxin, showing LD50 of 2.5 mg/kg in mice, and it is evidenced that mutants lacking tox A gene are less virulent than wild type variants, as well as immunization against exotoxin A results in partial immunity to P. aeruginosa infection in animal models [11]. The aims of the present work were to identify pathogenic isolates of P. aeruginosa, PCR-amplify two important virulence factors encoding genes namely, oxa 10 and tox A genes responsible for antibiotic resistance and production of Exotoxin A, respectively; and to sequence the relevant PCR products of the two genes.

Materials and methods

N/A

Results

The results of the present study showed that all the selected isolates showed phenotypic criteria characteristics of P. aeruginosa. Antibiotic resistance profiles of the isolates ranged from the highest to Cefepime shown by 98% of the isolates to the lowest to imipenem revealed by 50 % of the isolates (Table 3).

Discussions

although considerable improvement in the survival of burn victims has been attained, complications after primary infection still represent the primary etiology of morbidity and mortality [20]. burn wound infection due to pseudomonas aeruginosa creates an important danger in terms of sepsis, graft loss, prolonged durations of hospital admission, and increased mortality [21]. infections by p. aeruginosa are hardly treated because of the intrinsic ability of the bacteria to resist many classes of antibiotics and the capability to acquire resistance trait through several mechanisms. all the documented antibiotic resistance strategies might be noticed in this pathogen[adaptive, acquired, and intrinsic] many times all within an individual isolate and the resistance rates are exaggerating despite the usage of combination drug therapies [22]. as few recent medications are now in use to counteract infections caused p. aeruginosa, there has been an approach to re-use older agents such as polymyxins that had originally rendered out of use due to extensive information of deletingrious drawbacks [23]. nosocomial outbreaks due to multidrug resistant bacteria (mdr) had been almost recognized as a result of infections caused by bacterial colonization of burn lesions. [24]. multidrug resistant strains of p. aeruginosa (resistant to at least three of the following antibiotics cefotaxime, imipenem, gentamicin and ciprofloxacin) are usually isolated from persons affected by nosocomial infections [4, 25]. the major source of this type of resistance is the release of ?-lactamase enzyme. the synthesis of the enzyme might be elicited to an elevated expression rate resulting in a quantity high enough to confer resistance [26]. in the current research, antibiotic sensitivity study revealed resistance trait to the generally prescribed antibacterialssuch as gentamycin, cefepime and meropenem which are usually indiscriminately given as primary therapeutic approach for prolonged duration. other researchers also noted this higher rate of resistance to the above mentioned agents [27]. also, resistance to tobromycin was higher than that reported by others [25]. the results of the present study showed that imipenem was the most effective antibacterial against pseudomonas aeruginosa was with 50 % of the studied isolates being sensitive while the higher resistance (98%) was displayed by cefipime which in turn can be justified by the prolonged and continuous prescription of this antibiotic for the treatment of pseudomonas infection in our locality. p. aeruginosa has multiple pathogenicity determinants that may participate to its disease causing ability [28]. exotoxin a is an extracellular toxin that is released by the majority of clinical strains of pseudomonas aeruginosa. it contributes to the pathogenicity of the bacterium by inhibiting the synthesis of proteins, direct cellular effects, and alteration of the immune functions of the targets [29]. as this toxin is usually produced by this pathogen, it can be a reliable candidate for rapid identification and differentiation of p. aeruginosa in clinical settings [30]. the results of the present work were not correlated with these of other investigators [31]who claimed that tox a gene was successfully amplified in 100 % of their isolates, however, when the high expression of the gene is taken into consideration, both studies confirmed the importance of exo a as far as invasion in concerned. in a study conducted by a group of researchers [32], they stated that exo a gene was properly identified in 26% of their isolates and this markedly disagreed with the results obtained in the current study, the discrepancy might be explained by the fact that different isolation source of the microbe dictate different genetic ability to express a gene of interest, as invasion in cases of otitis is not a priority for the pathogen compared to burn infections [33]. among p. aeruginosa isolates, research indicated that there is a conserved set of 5021 genes, but between two different isolates of p. aeruginosa, a substantial variation can be detected [34]. it is generally accepted that, the greater the number of the sequenced genomes the higher the variation among pseudomonas isolates [35]. it was postulated that the huge and complex genome of p. aeruginosa reflect evolutionary adaptations allowing it to survive in different ecological sites. p. aeruginosa has well known abilities to transport, metabolize and grow on organic materials, variety of iron-siderophore intake systems, and the improved characteristic to produce molecules [e.x., enzymes]. p. aeruginosa also has the highest number of genes devoted to command and control systems noticed within microorganism. the proposed role of theses regulatory genes is the alteration of the transcriptional activity and biochemical characteristics in response to a change in environmental settings. infections due to pseudomonas spp. are difficult to manage because of intrinsic antibiotic resistance. indeed, the unusual regulatory features may offer greater flexibility for manipulative drug resistance by gene activity modulation than found in other bacterial spp. collectively, the divergent metabolic ability, transport mechanisms and regulatory adaptations empower this bacterium to survive and compete with other microorganisms. therefore, sequencing of genes provides an interesting data for the discovery and usage of new antibacterial agents, and an avenue for the invention of efficient approaches to combat the serious nosocomial conditions attributed to this pathogen [36].

Conclusions

The majority of the studied isolates constitutively expressed oxa 10 and toxA genes and the relevant sequences of the two genes revealed striking similarity indicating that these genes are conserved among isolates and their diversity is a matter of simple change that implies no functional alterations.

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