Frequency of ant(2")-I and aac(6')-II Genes in the Clinical Isolates of Pseudomonas aeruginosa from Shahid Sadoughi Hospital in Yazd, Iran

Pseudomonas aeruginosa (P. aeruginosa) is a common gram-negative pathogenic bacteria linked to nosocomial infections responsible for 10-15% of nosocomial infections all over the world (1-2). Antibiotic resistance is a worldwide emerging issue, and among P. aeruginosa strains, the extensive use of antibiotics may be the main cause of the increase in multidrug resistance. Aminoglycosides are among the very effective series of antibiotics against a wide range of gram-negative and gram-positive aerobic bacteria. In spite of the popular use and clinical prosperity of aminoglycosides, the administration of these antibiotics to control bacterial A-R-T-I-C-L-EI-N-F-O A-B-S-T-R-A-C-T

infections has been challenging due to the emergence of resistant strains (3)(4). The enzymatic change, efflux pumps activity (MexXY-OprM), impermeability, ndvB dependent biofilm formation, PhoP-PhoQ system, as well as the activity of 16S rRNA methylases, make resistance to aminoglycosides (5)(6). Among these, the inactivation of medications through plasmid or modifying enzymes encoded by chromosomes is the most common mechanism.
The number of P. aeruginosa strains resistant to aminoglycosides is increasing (8).
The aim of the current widespread study was to examine the incidence of aminoglycoside resistance, as well as the prevalence of the resistance-modifying enzyme genes aac(6')-II and ant (2")-I, in P. aeruginosa isolates from the clinical samples of hospitalized patients in Yazd, Iran.

Sample collection and isolation
This descriptive study was carried out on 144 clinical isolates of P. aeruginosa collected from Shahid Sadoughi Hospital in Yazd during March 2016 to March 2017. A questionnaire was used for recording patients' demographic data, such as name, age, gender, type of sample, and ward. The bacterial strains were isolated from different clinical specimens, such as blood, bronchial fluid, urine, cerebrospinal fluid, catheter, pleural fluid, ear swap, sputum, and wound, consequently inoculated on sheep blood agar (Merck, Germany) and EMB agar media (Merck, Germany) incubated at 37°C for 24 h. All P. aeruginosa isolates were identified using standard biochemical tests, such as growth on Cetrimide agar (Liofilchem, Italy), growth at 42°C, oxidase test, gram staining, pigment production, and Oxidative/fermentation test.

Antibiotic susceptibility testing
Antibiotic susceptibility tests were performed using the disk diffusion method according to Clinical and Laboratory Standards Institute (CLSI) guidelines for four aminoglycosides (i.e., gentamicin [10 μg], amikacin [30 μg], kanamycin [5 μg], and tobramycin [10 μg]). All antibiotic disks were purchased from Mast Company (UK). E-test method (Liofilchem, Italy) was used to determine the minimum inhibitory concentration (MIC) of gentamicin. Based on CLSI guideline, MIC > 16 was considered resistant isolates to gentamicin (9). P. aeruginosa ATCC 27853 was used as a control in this study.

Genomic DNA extraction
In this study, genomic DNA was extracted by the salting-out method (10). The quality and quantity of obtained DNA were measured by 0.7% agarose gel electrophoresis and nanodrop, respectively. The DNA samples were stored at -20°C until the examination day.

Polymerase Chain Reaction
Polymerase chain reaction (PCR) was carried out for the detection of ant(2'')-I and aac(6')-II genes on a thermal cycler (Eppendorf, Germany). Primers were developed for each gene using Primer3 (version 0.4.0). The primer pair sequences used in this study and PCR conditions are detailed in Table 1 (11). The

Statistical analysis
The correlation between the genes and resistance to aminoglycosides were analyzed using the Chi-square test by SPSS software (version 16.0).
Totally, 144 strains of P. aeruginosa were isolated from the patients within the age range of 7 months to 80 years who were referred to Shahid Sadoughi Hospital of Yazd. The mean age of the subjects was 22.7±34.9 years. The majority of the isolates were collected from the patients hospitalized in burn ward (n=68; 47.2%) and intensive care unit (ICU) (n=27; 18.8%) followed by surgery (n=24; 16.7%), infectious disease (n=18; 12.5%), and neurology (n=7; 4.9%) wards. Most of the strains were isolated from the male subjects with 34 years of age.
The results of the present study showed that frequencies of aac(6')-II and ant(2'')-I genes in the studied isolates were 93 (64.6%) and 114 (79.2%), respectively (Figure 1). A significant correlation was observed between the presence of aac(6')-II gene and resistance against    Antibiotic therapies are extensively used for the treatment of infectious diseases (10). According to the literature, it has been revealed that P. aeruginosa is the most common bacterial isolate in burn wards in Tehran, Iran (11)(12). Results of multiple studies have shown that P. aeruginosa is highly prevalent in Iranian hospitals. The findings of this study revealed that most of the P. aeruginosa isolates were reported in the burn ward (n=68; 47.2%). The obtained results of the present study are in line with those of other studies .
In the present study, the highest frequency of P. aeruginosa isolation after the burn ward was related to ICU (n=27; 18.8%). The results of a study by Shakib et al. in 2014 carried out in the ICU of Besat Hospital in Sanandaj, Iran, confirmed the results of the present study (14). P. aeruginosa is resistant to a spectrum of antibiotics, such as macrolides, beta-lactams, and aminoglycosides (13). Aminoglycoside resistance in P. aeruginosa is often associated with the development of different enzymes (8). This resistance has become a worldwide issue (14), particularly in Asia (15).
Based on the disk diffusion findings, 81.9% and 55.6% of the isolates were resistant to kanamycin and tobramycin, respectively. This shows that the rate of resistance has increased, compared to those in other studies (11)(12)(13)(14)(15). Constraints in phenotypic methods made researchers prove their phenotypic results by means of molecular methods. It was shown in PCR results that 114 (79.2%) and 93 (64.6%) isolates harbored ant(2")-I and aac(6')-II genes, respectively. The prevalence of genes in the present study suggested an increasing trend of resistance to antibiotics in comparison to that of the previous years.

Discussion
In a study conducted in South Korea, the prevalence rate of ant(2")-I gene was 43.6% (21). Therefore, controlling and treating the infections caused by P. aeruginosa is complex. It is essential to revise the treatment protocols in order to prevent from transferring resistant genes to the clinical isolates. In this study, the expression of aac(6')-II and ant(2")-I genes has a very high prevalence in comparison to that of other studies.
Considering the fact that most of the samples were collected from the burn ward, the probability of harboring aac(6')-II and ant (2")-I genes is also higher due to the high aminoglycoside resistance of these strains. Of course, it should be noted that the prevalence of these enzymes is geographically diverse, leading to distinct prevalence among countries. Most of the isolates with phenotypic resistance to aminoglycosides (80%) have ant(2′′)-I gene, and the presence of this enzyme is one of the most common mechanisms of resistance to aminoglycosides.
It is worth noting that since there was no gene concerning these enzymes in the phenotypic sensitive isolates, the significance of these genes in inducing the resistance of P. aeruginosa can be noted against aminoglycosides. In this study, there was a significant relationship between antibiotic resistance and ant(2′′)-I gene. Due to the high prevalence of ant(2′′)-I gene observed in the experiments of this study and its effect on increasing the resistance to aminoglycosides, this correlation seems argumentative.
Another important point to be noted is that, in addition to the mechanism of aminoglycoside-modifying enzymes, which is the main subject of the present study, other mechanisms, such as impenetrable and efflux pump, in P. aeruginosa are prevalent. In fact, if the isolates without the gene modifying enzymes are phenotypically resistant, they are probably resistant to aminoglycosides through other mechanisms (8).
Resistance to aminoglycosides continues to be a major concern in Iran. Since these aminoglycoside resistance genes are transmissible by genetic elements, they can be spread and disseminated among other bacteria. Aminoglycoside resistance among clinical isolates of Pseudomonas aeruginosa is indicated to be a major significance in the future; therefore, the surveillance of aminoglycoside resistance genes and use of aminoglycosides with stronger affinity to their targets and resistance to these modifying enzymes are crucial.