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Antibiotic Resistance Properties of Pseudomonas aeruginosa Isolated From Cases of Superficial Infections at the Emergency Unit

Background Pseudomonas aeruginosa, a ubiquitous opportunistic pathogen, is one of the main causative agents of human superficial infections. Infections due to these bacteria are difficult to heal and cause serious economic issues. Objectives The present study was carried out to investigate the antibiotic resistance pattern of P. aeruginosa isolated from cases of superficial infections referred to the emergency health care units of Iranian Hospitals. Materials and Methods Three hundred swab samples were collected from patients with superficial infections. Samples were cultured and those that were P. aeruginosa positive were analyzed by the disk diffusion method. Results One hundred and seventy-two out of 300 swab samples (57.3%) were positive for P. aeruginosa. The results of the culture technique were also confirmed using the polymerase chain reaction (PCR). Females had a higher prevalence of P. aeruginosa than males, patients older than 70 years were the most infected age group and finally burn infections had the highest prevalence of bacteria. P. aeruginosa strains had the highest levels of resistance against ampicillin (93%), gentamycin (89.5%), ciprofloxacin (82.5%) and amikacin (77.3%). The most effective drugs were meropenem (2.3%, imipenem (2.9%), polymyxin B (21.5%) and cotrimoxazole (31.9%). Conclusions It is logical to primarily prescribe meropenem, imipenem, polymyxin B and cotrimoxazole in the cases of superficial infections caused by P. aeruginosa. Medical practitioners should be aware of the presence of such levels of antibiotic resistance in cases of superficial infections in Iran.


Background
Superficial infections such as burn, wound and postsurgical site infections are important causes of emergency health care-associated problems all around the world. Superficial infections cause longer hospital stays, more expensive hospitalizations and increased mortality (1). The annual superficial infection care products market is projected to reach $15.3 billion by 2010 (1). Pseudomonas aeruginosa are non-fermentative, aerobic, gram-negative rod shape bacteria, which substantially contribute to wound-related morbidity and mortality worldwide. They are widely distributed, mostly in hospital environments and are one of the most important agents of hospital-acquired superficial infections, ecthyma gangrenosum and black necrotic lesions (2,3). Superficial infections caused by P. aeruginosa are one of the most prevalent causes of hospitalization and emergency health care references all around the world (2)(3)(4)(5)(6).
Treatment of superficial infections caused by P. aerugi-nosa often requires antibiotic therapy yet the levels of antibiotic resistance in the rough strains of these bacteria have increased over time (7)(8)(9)(10)(11). Therefore, it is essential to study the levels of antibiotic resistance in the P. aeruginosa isolates of each region and even each hospital.
In the recent years, the growing incidence of P. aeruginosa has been of particular concern. The incidence of P. aeruginosa in superficial and wound infections is becoming more serious in developing countries like Iran (12,13). This issue is of higher importance for females and elders, due to their relatively lower levels of immune system.

Objectives
The present study was carried out in order to study the antibiotic resistance pattern of P. aeruginosa isolated from cases of superficial infections referred to the emergency health care units of Iranian Hospitals.

Ethical Considerations
Ethical committees of the educational hospitals approved the general principles and framework of the present investigation. Written informed consent was obtained from all of the study patients or their parents. Personal information of all patients remained confidential.

Sample Collection
From June 2014 to October 2015, a total of 300 swab samples were taken from patients with superficial infections referred to the emergency health care units of Iranian hospitals. Swab samples were taken from various types of superficial infections including wound (n = 110), burn (n = 90) and post-surgical site (n = 100) infections. Personal information like age and gender were recorded for each sample and all samples were transferred to the laboratory in a cooler with an ice pack.

Pseudomonas aeruginosa Isolation
Swab samples were inoculated on blood, MacConkey (Merck, Germany) and nutrient agar (Merck, Germany) and incubated at 37°C for 24 hours. Colonies that produced pyoverdin, pyocyanin and pyorubin pigments were transferred to nutrient agar and subcultured more than one time to obtain pure cultures. The isolates were identified using conventional biochemical tests such as motility, oxidase, catalase, citrate utilization, gelatinase liquefaction, urease production, nitrate reduction, alkaline protease production, triple sugar iron agar, oxidative-fermentative, indole, lecithinase production and hemolysin production.

DNA Extraction From the P. aeruginosa Isolates
Total genomic DNA was extracted from the bacterial colonies. A single colony was inoculated on 5 mL of brain heart infusion broth and incubated over night at 37ºC. Then 1.5 mL of a saturated culture was harvested with centrifugation for five minutes at 14,000 rpm. The cell pellet was resuspended and lysed in 200 µL of lysis buffer (40 mM Tris-acetate pH 7.8, 20 mM sodium-acetate, 1 mM EDTA, 1% SDS) by vigorous pipetting. To remove most proteins and cell debris, 66 µL of 5M NaCl solution was added and mixed well, and then the viscous mixture was centrifuged at 12,000 rpm for 10 minutes at 4ºC. After transferring the clear supernatant to a new eppendorf tube, an equal volume of chloroform was added, and the tube was gently inverted at least 50 times when a milky solution was completely formed. Following centrifugation at 14,000 rpm for five minutes, the supernatant was then removed to another eppendorf tube and double volume of 100% ethanol was added. The tubes were gently inverted five to six times, then centrifuged at 10000 rpm for five minutes. The supernatant was discarded and 1 mL of ethanol (70%) was added to the pellet, and tubes were centrifuged at 10000 rpm for five minutes. Finally, the supernatant was discarded and the pellet was dried for 10 minutes at room temperature and was then resuspended in 100 µL H 2 O. The stock was kept at -20ºC until use. The DNA concentration was determined by measuring absorbance of the sample at 260 nm, using a spectrophotometer (15).

Agarose Gel Electrophoresis
Fifteen microliters of PCR products were resolved on a 1.5% agarose gel containing 0.5 mg/mL of SYBR Green in trisborate EDTA buffer at 90 V for 40 minutes, also using suitable molecular weight markers. The products were examined under ultraviolet illumination.

Statistical Analysis
The results were transferred to a microsoft excel spread- sheet (Microsoft Corp., Redmond, WA) for analysis. Statistical analysis was performed using the SPSS/16.0 software (SPSS Inc., Chicago, IL) for significant relationships between incidences of antibiotic resistance of P. aeruginosa isolated from the samples of superficial infections. The chi-square test and Fisher's exact 2-tailed test analysis were performed in this study. Statistical significance was regarded at a P < 0.05.

Results
The present investigation was carried out to study the prevalence of antibiotic resistance of P. aeruginosa isolated from various types of superficial infections. Table 1 shows the total distribution of P. aeruginosa in the swab samples taken from various types of superficial infections. Of the 300 studied swabs, 172 (57.3) samples were found to be contaminated with P. aeruginosa. The results of the culture technique were also confirmed using the PCR method (Figure 1). Swab samples, which were taken from female cases (64.2%), patients older than 70 years (68.5%) and cases of burn infections (66.6%), had the highest prevalence of P. aeruginosa. Statistically significant differences were seen in the prevalence of P. aeruginosa between male and female cases (P = 0.039), younger than 10-years-old and older than 70-years-old patients (P = 0.016) and cases of burn infections and wound infections (P = 0.041). Table 2 shows the antibiotic resistance pattern of P. aeruginosa isolated from various types of superficial infections. We found that the P. aeruginosa strains of superficial infections harbored the highest levels of resistance against ampicillin (93%), gentamycin (89.5%), ciprofloxacin (82.5%) and amikacin (77.3%), and also the lowest levels of resistance against meropenem (2.3%), imipenem (2.9%), polymyxin B (21.5%) and cotrimoxazole (31.9%). P. aeruginosa strains of males had a higher prevalence of antibiotic resistance than females (P = 0.026). Statistically significant differences were seen between the type of infection and prevalence of antibiotic resistance (P = 0.044), and also between the age of patients and prevalence of antibiotic resistance (P = 0.032).

Discussion
The results of the present study showed that P. aeruginosa has a higher prevalence in various types of superficial infections. Overall, 62.6% of the swab samples were positive for P. aeruginosa. To the best of our knowledge, this finding is the highest prevalence of P. aeruginosa in swab samples of superficial infections. Lower prevalence rate of P. aeruginosa in human superficial infections have been reported previously by Ranjan  High prevalence of P. aeruginosa in the clinical samples of our study maybe due to the fact that the type of samples (swabs samples of the site of infection) and health care managements is different with those of other investigations. In fact, the presence of environmental pollution, especially in the hospital environment as well as contaminated and lack of optimal disinfection of instruments and equipment of hospitals are the main reasons for the high prevalence of P. aeruginosa (62%) in post-surgical site infections of our study. Low levels of healthcare management in Iranian healthcare units and hospitals have been recognized from the results of our study and the results of various previous Iranian investigations (13,18,19). Higher sensitivities of female skin are a reason for the higher prevalence of P. aeruginosa in their superfi-  7) reported a higher prevalence of P. aeruginosa clinical infections in males than females, which were different to our results. Their reason for the high prevalence of bacteria in males is that they are more in contact with the polluted outside home environment. Also they do exhausting and hard work outside the home. Therefore, they are more prone to get superficial infections.
Aging, decrease in the levels of keratin skin cells and reduction in the level of immunity are reasonable factors for the higher prevalence of P. aeruginosa in older than 70-years-old patients. High prevalence of P. aeruginosa in old patients has been reported previously (23)(24)(25). In despite of the results of a previous investigation, which showed a high prevalence of P. aeruginosa in children (26), the results of our study showed that less than ten years old patients had a lower prevalence of bacteria. One possible explanation for this finding is that the age range of younger than ten-year-old patients of our study was eight to ten years. On the other hand, there were no younger than eight-year-old pediatrics in our study population.
Irregular and unethical antibiotic prescription and even self-treatment by strong antibiotics cause such high levels of resistance in the P. aeruginosa strains of our investigation. Differences in the idea of medical practitioners in antibiotic prescription cause variations in the levels of antibiotic resistance against different antibiotics. In addition, the differences in the bactericidal activities of antibiotics and also difference in difficulty in developing resistance against various antibiotics are two other reasons for differences in the levels of antibiotic resistance.
The present study was one of the most extensive prevalence reports of P. aeruginosa and its antibiotic resistance pattern in the burn, post-surgical site and wound infection samples of emergency health care units of Iranian Hospitals. Our results showed that resistant strains of P. aeruginosa had a high prevalence in patients older than 70-years-old and especially in the samples taken from the site of burn infections. In keeping with this, the prevalence of these bacteria in cases of wound and post-surgical infections and also other studied groups were considerable. Hence, judicious use of antibiotics is required by clinicians. Also, because of the variation of resistance pattern in each hospital, it is important for each region and even hospital to formulate their own antibiotic policy, according to their local resistance pattern. We recommend the initial prescription of meropenem, imipenem and polymyxin B antibiotics for treatment of the cases of superficial infections in Iran.