LINK BETWEEN SOME VIRULNCE FACTORS GENES AND ANTIBACTERIAL RESISTANCE OF PSEUDOMONAS AERUGINOSA

The research was aimed to demonstrate the frequency of virulence factors genes ( exoenzyme S and exotoxin A ) and to express their relationship to antibacterial resistance among Pseudomonas aeruginosa isolated from patients suffering from otitis externa. The results were revealed that only twenty one (32%) out of 65 clinical ear swabs were Pseudomonas aeruginosa . According to PCR amplification, 18 (85.7%) bacterial isolates were expressed both toxins ( exoenzyme S and exotoxin A). The data get by real time experiments were revealed that the isolates were give higher percentage of resistance were seen against Ceftazidime (90.5%) and Gentamicin (88.5%).Only thirteen isolates (61,90%) of Multidrug resistance (MDR) – Pseudomonas aeruginosa isolates were detected, and two (9.5%) isolates were resist to all 8 classes of antibiotics and considered as PDR, and three (14.2%) bacterial isolates resist seven classes of antibiotics which considered as XDR.


INTRODUCTION
Otitis externa is as an acute, chronic, and necrotizing inflammation of the external ear canal (1). Inflammation takes various types which includes: acute, chronic, and may be developed into tumor, circumscribed otitis externa (2). In contrast to acute otitis externa, chronic otitis externa is generally caused by allergies or underlying inflammatory dermatologi conditions (3) Pseudomonas aeruginosa (PA) is the most common cause of acute external otitis, accounting for more than 70 percent of cases in one series. It's (an opportunistic pathogen) can cause a higher percentage of acute or chronic infections with higher frequencies of mortality and morbidity (4,5). A wide range of infections caused by PA including: endocarditis, septicemia, pneumonia, wounds, burn, otitis and bacterial keratitis. It is also noticed in a cystic fibrosisfrequently, immunosuppressed and /or nosocomial infections (6,7).These infections are very difficult to eliminate due to the presence of (numerous virulence factors) such as flagella, pili, proteases, elastase, exopolysaccharides, iron chelators, lipases, and a variety of various toxins, including : exotoxin A, and the Type III Secretion System (T3SS) toxins ExoS, ExoT, ExoU, ExoY, in addition to chemical compound and pyocyanin (8). Two members of virulence factors genes namely ( Exoenzyme S and Exotoxin A) catalyzes transporting of the Adenosine di phosphate (ADP) -ribose moiety of nicotinamide di phosphate ( NAD) to the eukaryotic cells proteins ( 9,10)., several lines of studies were suggested that Exoenzyme S may be play a distinct role in the pathogenicity of PA. The role of exoenzyme in the past experiments were showed to be unselective in choice of substrate proteins, however, now has noticed that it preferentially ADP-ribosylates several of the low-molecular weight GTPbinding proteins. In addition to, there is higher similarity with cholera toxin, exoenzyme S also needed a protein of eukaryotic cells for their enzymic activity (11). Exotoxin A catalyzes the ADP-ribosylation of elongation factor 2 (EF-2), leading to inhibition and disruption of synthesis of proteins (7).

MATERIALS AND METHODS
Samples collection: During the period extended from February to August of the year of 2020. Sixty five of clinical ear swabs were collected from patients who suffering from ear infections and attended into out-patients at Teaching Hospitals in Baghdad city. Bacterial isolation: All the clinical ear swabs were cultured on Blood, MacConkey, Brain heart infusion agar medium, and the selective media of PA Cetrimide agar, and all cultured media were incubated for 18 -24 hrs. at 37 ºC. The suspected bacterial isolates which possibly belong to PA inoculated on Cetrimide agar to demonstrate the characteristic traits of this bacteria for ex: blue-greenish color, and the presence of fruity odor. The other diagnostic tests were included the detection of phenotypic and microscopic traits in cooperated with biochemical diagnostic tests. Which were done according to Forbes et al (12).

Bacterial identification
Colonial and phenotypic morphology on blood, MacConkey, Brain heart infusion, and Cetrimide agar medium were based principally to detect bacterial isolates of PA, and their colonial shape, texture, color and edges were noticed. The macroscopic examination were cooperated by microscopic examination of a Gram stained of bacterial smear which examined under a light microscope with special regard towards cell shape and arrangement. All suspected bacterial isolates of PA were subjected to many biochemical tests such as oxidase, catalase, and triple sugar Iron test etc.

Antibiotic sensitivity test
The disc diffusion method represented the gold standard for confirming and determining the susceptibility of the bacteria, in this test, the isolated bacterial pure colonies were selected and suspended into nutreient media, then were standardized by using a turbidity test and this point was done by using Macferland no. o.5. The standardized suspension is then cultured and disseminated onto Muller Hinton agar plate, and the different antibiotic discs were tapped and putted on the inoculated plates Seventeen different antibacterial disc were permitted to disseminate through the solidified agar which includes : and Chloramphenicol (C) ( Table 1). All inoculated agar plates were incubated overnight at 37 °C. After that, the size of the inhibition zone formed around each disc was measured and recorded. Primers of this genes Table 2.The specific primer of Exoenzym S gene  The present result comes in fit with the previous studies which revealed that both of PA and Staphylococcus aureus were the most commonly isolated organisms (14). The isolates are ( poly-microbial ) in a significant number of cases. Other Gram-negative bacteria are uncommon. Aspergillus species and Candida species are rare fungal infections (15) . Swabs taken from the external canal should be swabbed with caution because (they may be reflect normal flora or colonizing organisms). Ear swabs should be taken only in severe cases or unresponsive. The previous researches revealed that the bacterial flora or bacterial commensals of the External Auditory Canal (EAC) is commonly composed of Grampositive bacteria. The most prevalent recognized bacteria are Staphylococcus epidermidis (38%) fallowed by Diptheroid (22.4%). Gram -negative bacteria are less common, isolated from (< 5% ) of the external auditory canal (AOE) specimens. Prolonged exposure to the water may leads to the changes of flora of the EAC, becoming dominated by Gram -negative organisms for example PA which constitutes the most common pathogenic bacteria in AOE, detected in (22-62%) of cases in series on AOE. Staphylococcus aureus (11-34% of cases) is the most important Grampositive bacteria (16,17,18). Antibiotics Resistance in PA Sensitivity and resistance phenotypic pattern were done on each of diagnosed 21 PA isolates by using agar disc diffusion method and by using seventieth different antibiotic disc. The results were interpreted according to recommendation of the National Committee for Clinical Laboratory Standard (NCCLS) (19). The Pseudomonal isolates that were found to belong to different interpretive categories, including susceptible (S), and resistant ®.
Approximately 95% of Pseudomonas strains were susceptible to colistin, Imipenem, and pipracillin followed by 90.4 % that were susceptible ciprofloxacin and 80.9 % of all isolates give sensitivity against ceftazidime, amikacin, gentamycin and levofloxacin. tetracycline were (71.4%), aztreonam and trimethoprimsulfamethoxazole were (66.6%), However, a high percentage of Pseudomonas strains were resistant to Amoxicillin (95%) followed by ampicillin and nalidixic acid (90%), Azithromycin and chloramphenicol (57.1%), the result of antibiotic sensitivity test of pseudomonal isolates against Mezlocillin was (52.3%).The frequencies of resistance phenotypic patterns to the 17 tested antibiotics were arranged in the following table 5.Totaly 21 (60 %) bacterial isolates were (resistant to three or more drug classes) were defined as MDR strains. Two (9.5%) isolates were resistant to all eight classes of antibiotics and considered as Pan drug resistance (PDR), and three (14.2%) bacterial isolate could resist seven classes of antibiotics and considered as extensive drug resistance ( XDR). Table 5. Sensitivity and resistance phenotypic pattern of twenty one P. aeruginosa isolates In the last years, the prevalence of higher risk strains of PA such as (MDR; XDR; and PDR), has constitute a major public health problem in the worldwide. Also recently there is a significant increasing in the percentage of MDR ,XDR, and PDR of PA bacterium, with a percentage between (16% and 31%) in many countries (20,21.22). Many Europian countries reported frequency of antibiotics resistance exceeded (10%) for all antibiotics classes under control and notification (23). Combination of resistance was also noticed in PA In the last seven years (the European Centers for Disease Prevention and Control) revealed that (14%) of PA strains were expressed antibiotic resistant to more than two antibacterial classes and (6.5%) to more than four antibacterial different classes under control (EARS-Net) 23 . The observation of United States, revealed that the MDR -PA is the cause of (14%) of severe health care associated infections (24). There are different and a big cause why the prevalence of (MDR), (XDR), and (PDR) PA bacteria have in recent years become issues of public health concern. The first reason, PA responsible for highly severe infections, especially in the health care units and in immunosuppressed persons. Second reasons, it has an outstanding capacity for being selected and for dissemination of antibacterial resistance in the human body (25,26,27). Third, the successful worldwide spread of the so-called "high-risk" clones of PA poses a threat to global public health that needs to be studied and managed with urgency and determination (28,29,). Quantitative analysis of Exoenzyme S and exotoxin A virulence gene The expression of Exoenzyme S and Exotoxin A genes were detected successfully by using new molecular technique which is Real time PCR (qRT-PCR) with used specific primer. The amplification accuracy of gene product was noticed by the value of cycle threshold (Ct) for the triplicate reactions and the results revealed that 18 (85.7%) of the isolates exhibited both the Exoenzyme S and Exotoxin A genes and only three bacterial isolates don't exhibited exotoxin gene ( Table 6, 7 and Figure 1).