Comparison of two disk diffusion and E-test methods in determining antibiotic susceptibility of Pseudomonas aeruginosa strains in isolated burn infections of Ahvaz City

Original article: Comparison of two disk diffusion and E-test methods in determining antibiotic susceptibility of Pseudomonas aeruginosa strains in isolated burn infections of Ahvaz City Changiz Ahmadizadeh1 ,Mehrdad Pashazadeh2* Abstract: Background: Pseudomonas aeruginosa with widely distributed in nature, for human beings is considered an opportunistic pathogen that causes infections of broad-spectrum, including administrative, respiratory, septicemia and bacteremia and sepsis in patients with the burning city of Ahvaz. Method and Material: A total of 95isolates of Pseudomonas aeruginosa isolated from burn patients from January 2015 assemble and biochemical identification test, then they are antibiotic resistance in E. test and disk diffusion method were compared. Findings: From 95 different clinical isolates of Pseudomonas aeruginosa isolated from E. test with the highest sensitivity to the antibiotic ceftazidime , 70(68/73%) and ciprofloxacin 50 (63/52%) and gentamicin 48 (52 /50 %) and the antibiotic imipenem 44 (31/46%) were sensitive and disk diffusion method antibiotic ceftazidime, 67(52/70%) and the antibiotic Ciprofloxacin 51(68/53%) and safety antibiotic imipenem 49(57/51%) and gentamicin 48(52/50%) were sensitive. Conclusion: Statistically significant differences between E. test and disk diffusion antimicrobial susceptibility of there (p<0.05) and disk diffusion method can replace E. test, and also the most sensitive antibiotics, the antibiotics used The study of the isolated Pseudomonas is ceftazidime.


Introduction
Pseudomonas bacteria that put into gram-negative and aerobic organisms and can be found in water and soil, move through of two or three polar flagella. Pseudomonas aeruginosa which previously named Bacillus Piocianic, usually with the low number of fluorine construct human skin and gut 2 . Hospitalacquired infections are one of the most challenging problems in advanced and developing countries. After Staphylococcus aureus and Escherichia coli, pseudomonas aeruginosa is the third reason of hospitalacquired infections 1 . This gram-negative bacillus leads to infecting tissues from bones and joints to stomach and gut and also causes systemic disease by producing poisons in soft tissues of immune system deficiency patients; this condition arouses especially in HIV, cancer and severe burn. As these bacteria have low needing's to grow, it can easily survive in environment and affect susceptible (sensitive) patients 4 . One of the most effective reasons for its priority as a pathogen, is its high natural resistance in comparison with most antibiotics. Therefore mortality rate caused by pseudomonas aeruginosa is reported by up to 50%. Deficiency in immune system and also resistance against several antibiotics are among impressive factors 3 . As a result of serious grounded disease, destruction of protecting membrane and skin barriers caused by using invasive medical devices and long hospitalizing, the risk of obtaining hospital infections is high especially in hospitalized patients 5 .
Encountering with different antimicrobial factors may complicate this hospitalizing and create favorable conditions through resisting and surviving in respect to host of bacterial fluorine and or transferred strains from hospital environment. This bacteria shows more opposition against antimicrobial factors. Serious infections made by common antimicrobial-resistant anti pseudomonas are major problems 8 . Pseudomonas aeruginosa shows very high resistance to antimicrobial drugs. These features and characteristics by using selective pressure of mutation in genes and chromosomes, leads to over expression ofampC gene, restraint or inactivation of oprD, and over expression of effluent pumps in drugs 10 . Moreover it can receive drug resistant factors by horizon gene transfer through coded mobile genetic elements to work with class B penmases (also called metallo-beta-lactamase, that hydrolyze all beta-lactams except aztreonam). Strains of XDRPa with extreme drug resistance against pseudomonas aeruginosa emerged in hospitals which become a real threat to national health 9 . Such infections caused by resistant microorganisms limit drug selections. Consequently these resistant strains in hospital infections are accompanied by increase in mortality and long hospitalizing 11 . The best way to decrease such mortalities is to prevent these infections. Antibiotic resistance is an increasingly clinical dilemma and major threat of national health. That is an international problem, however all countries are involved with this problem, but this extension in developing countries is unknown 12 . Considering bacteria resistance and pathogenesis against antibiotics, it is essential to find precise and effective method for evaluating sensitivity and resistance of antibiotics. Among several testing antibiotic sensitivity methods, two methods are selected, the former is disk diffusion and the latter is E-test 6 . The purpose of this study is to determine antibiotic resistance of pseudomonas aeruginosa strains in burn infections of AhvazCity to select suitable treatments, increase treatment effects, decrease considerable economic costs, and reduce hospitalization time.

Methods
This research was done from autumn 2015up to spring 2016, so we collected 95 isolated pseudomonas aeruginosa patients from Taleghani burn center of Ahvaz,which include injury, blood, urine, mucus, cornea, tracheal tube, and biopsy, and separated those using recognition and purification tests. The tests involve differential ones such as oxidase, oxidative fermentative (OF), triple sugar iron agar (TSI), and citrate. So sensitivity test is done in two ways, agar disk diffusion (Kirby-Bauer) and E-test, utilize four antibiotics including ceftazidime (30/ug), imipenem (10/ug), ciprofloxacin (5/ug), gentamicin (10/ug) to get antibiotic resistance pattern of isolated strains. We bought antibiotic disks from Padtan teb Company and E-test strips used in this study were Italian. In this method, after providing hemogenic solutions from bacteria, first we planet all samples on muellur-hinton agar growth media with sterile swab, then place antibiotic disks on agar surface using pence, finally we put plates in incubator for 24 hours with 37 0 c temperature. After heating, the inhibition zone diameter resulted from antibiogram was measured according to clinical and laboratory standards institute (CLSL) instruction.

Fig1: Positive result of tested bacteria in disk diffusion method
In the second method to determine minimum inhibitory concentration (MIC) with E-test, first we sample sterile swaps from bacterial suspension of pseudomonas aeruginosa that is equivalent to 0/5 McFarland standard then steady spread them on special MIC determination media with E-test method. After that carefully weplace one E-test strip on media with aseptic condition then put the plate in incubator for 24 hours with 37 0 c temperature. When surveying the results, the number opposite to last inhibition zone diameter and beside strip was MIC with mcg/ml measurement unit. The result of both disk diffusion test and E-test registered in data collection form. Finally all experimental results done with SPSS17 software and sensitivity or insensitivity of each strain with separate code entered in to the program. In this study p values lower than 0.05 considered meaningful (P<0.05).

Findings
In this study 118 sample suspicious to pseudomonas was collected since autumn 2015 to spring 2016.

Discussion
Proper specification of hospital-acquired infections, type of microorganisms and their resistance pattern, and correct use of antibiotics are among important points that should be observed to reduce drug resistance 7 . Pseudomonas aeruginosa bacteria is the most important factor in hospital-acquired infections especially in immunodeficiency patients such as cancer and burn affected more one 2 . Treatment condition in patients with pseudomonas aeruginosa infection is problematic, especially when this organism inherently is resistant to several antibiotics and could obtain resistance against all antimicrobial drugs 11 . Mostly determination of antibiotic resistance pattern is one of the substantial elements in successful treatment of bacterial infections 13  with E-test method and 70/52% with disk diffusion method, so among other antibiotics ceftazidime is the best option for fighting against pseudomonas aeruginosa bacteria in burn infections. According to this research findings, the comparison between two methods of E-test and disk diffusion shows that this two antibiogram methods are relatively equal, hence it is suggested that in vitro disk diffusion method is suitable substitution for E-test, because E-test strips are expensive and using this method is not cost effective. Moreover it is necessary to conduct similar researches in other places of country to be informed about resistant strains' prevalence. Thus using this information and establishing control and evaluation systems that mostly managed by hospitalacquired infection committee, we can decrease these resistances in country.