Prevalence of Class 1 Integron , Resistance Gene Cassettes and Antimicrobial Susceptibility Profiles among Isolates of Pseudomonas aeruginosa in Iran

Pseudomonas aeruginosa is one of the most important opportunistic human pathogens worldwide. High prevalence of Multi Drug Resistant P. aeruginosa (MDRPa) in Iran is a serious problem for antimicrobial therapy. Several studies have reported the MDRPa in Europe and Asia. Due to the use of broad-spectrum antibiotics, bacterial resistance is increasing in Iran, located in Middle East. The present cross-sectional study was designed to investigate the prevalence of class1 integron, resistance gene cassettes and antimicrobial susceptibility profiles among isolates of P. aeruginosa in Al-Zahra Hospital, Isfahan City, central part of Iran from Jan-Sep 2014. The aim of this study was to determine the antimicrobial susceptibility, the prevalence of Class1 integron, resistance gene a measuring in Iran. A total of 231 P. aeruginosa isolates were collected from clinical specimens including urine (50.6%), tracheal tube (25.5%), wound (13.4%), blood (6.1%), catheter (2.2%), cerebrospinal fluid (1.7%) and sputum (0.4%) isolates from hospitalized patients (mean age: 50.27 ± 24.12 years).The majority of patients (68%) were male. Isolates were collected from different parts of the hospital as follows: ICU, Internal Medicine, Emergency care, Pediatrics, Nephrology, Transplant Center, General surgery and Infectious. Revealed data show a high rate of MDR P. aeruginosa isolates in the studied area; also, the result signifies the spread of aadA6 among clinical isolates in hospitalized patients.


Introduction
Pseudomonas aeruginosa is one of the most important opportunistic human pathogens responsible for 10% -15 % of the nosocomial infections worldwide [1] [2].This pathogen is naturally resistant to various antimicrobial agents, and also it has a distinctive valence through multiple mechanisms to become resistant to almost all the commercial antibiotics [3] [4].High prevalence of Multi Drug Resistant (MDR) P. aeruginosa in Iran is a serious problem for antimicrobial therapy [5]- [10].Infections caused by aforementioned drug resistant pathogens are dependent with notable increase in morbidity and mortality [7].
Plasmids, transposons and integrons are mobile genetic elements that they resort of earning resistance mechanisms cooperating to P. aeruginosa multidrug resistance [9].The integron is potentially a major element in the spreading of multidrug resistance among gram-negative bacteria, particularly in genus Pseudomonas [4].Integrons are one of the genetic elements have been found to be able to elevate absorption and expression of drug resistance genes located within gene cassettes [10]- [12].Out of all sequenced bacterial genomes, integrons have been found in approximately 9% [11].Various investigations revealed the presence of class 1 integron in 40% -70% of gram-negativepathogens isolated from clinical setting.It is obvious that integrons have major antibiotic resistance roles, causing difficulties in bacterial infection control; so, it is important to understand the origin of these elements for exploring the means by which bacterial lateral gene transfer can seriously impact on, and be impacted by, human activities and for practical control of antibiotic resistance [11]- [19].Class 1 itegron is made of a functional platform (5'CS), which contains the integrase gene (intI), the recombination site (attI) and a promoter region (P ant), and a (3'CS), which normally includes the qacED1and sulI genes [6]- [24].The gene cassettes among integrons are variable in these structures, small, mobilegenetic elements, containing of a single gene (or sporadically two genes) in addition a recombination site named the 59-be [24]- [31].Several resistance gene cassettes carried by class 1 integrons in P. aeruginosa encodes multiple resistance mechanisms such as resistance to beta-lactams, aminoglycosides and other antimicrobial agents [9]- [13].Several studies have reported the multiple drug resistance in P. aeruginosa clinical strains in European and Asian countries [26] [27].Due to the use of broad-spectrum antibiotics, bacterial resistance is increasing in Iran, bordered with European and Asian countries.The present study was designed to investigate the prevalence of class 1 integron, resistance gene cassettes and antimicrobial susceptibility profiles among isolates of P. aeruginosa in Al-Zahra Hospital, Isfahan City, central part of Iran.
At first, primary isolation was performed by phenotypic characteristics on blood agar and McConkey(Merck, Germany), then the isolates identification was carried out and confirmed to the species level by standard biochemical tests including Gram stain, catalase and oxidase test, O/F (Oxidation Fermentation) test (Merck, Germany), pyocyanin pigment production and growth at 42˚C [8].

DNA Extraction and PCR Amplification
DNA extraction was carried out on MDR P. aeruginosa by boiling method.The template DNA stored at −20˚C until polymerase chain reaction (PCR) amplification was performed.MDR P. aeruginosa isolates were screened for the presence of class 1 integron by using 4 specific primers located on intI1 gene, qacED, sulI and gene cassette regions.Master mix component was as follow: 10× PCR buffer in a final concentration of 1×, MgCl2 (50 mM) in a final concentration of 1.5 mM, 10 mM dNTPs Mix in a final concentration of 0.2 mM, Forward and Reverse primers in a final concentration of 0.4 µM.PCR amplification was performed in a total volume of 25 µl (24 µl of PCR master mix plus 1 µl of template DNA).Amplification was carried out in a thermocycler (Eppendorf Master cyclers, MA) using the published paper programs [6] [7]: The primers (obtained from Takapouzist Company, Iran) used for the analysis and PCR conditions are summarized in Table 1.Class1 integron variable regions were amplified using primers Int1, In5'cs and In3'cs (Table 1).After performing PCR reaction, amplification products were identified using 1.5% agarose gel electrophoresis stained with 0.5 µg/ml ethidium bromide solution.Then, the results visualized using UV trans-illuminator on the UV gel document (Ingenius, Syngene).The size of specific bands was compared with 100 bp DNA ladder (Bioneer, Korea).

Antimicrobial Resistance Pattern
The resistance pattern of P. aeruginosa isolates in this study revealed that 192 (83.1%) were Multi Drug Resis-   1).

Detection and Characterization of Integrons
PCR assay was performed to detect integrin integrase genes (intI1), qacED, and sulI genes, and gene cassette regions (5CS/3CS) among 192 clinical MDR P. aeruginosa isolates.Out of 192 isolates, 146 (76%) were positive for class 1 integron by amplifying the intI1 gene.Nucleotide sequence analysis of the class 1 integron variable regions revealed the presence of 3 different fragment sizes of approximately 0.8, 1.2 and 2.5 kb (Figure 2).120  The isolation sites and samples found in this study have been summarized in Table 3; also, the percentage rate of antibiotic resistance and presence of class 1 integrons among 192 MDR P. aeruginosa strains isolated from patients hospitalized in this study has been summarized in Table 4, Figure 1 and

Discussion
Multi Drug Resistant P. aeruginosa is a serious challenge for treatment of nosocomial contagion, and a suitable antibiotic choice to initiate remedy is necessary for optimizing the clinical consequence [17].As the results revealed, the most of isolates were resistant to several antimicrobial agents tested in this investigation and most of prevalence of class 1 integrons found in P. aeruginosa isolates consistent with those previously reported from    Iran [1]- [21].The high spread of MDRP.aeruginosa has become serious challenge in clinical settings.192 isolates (83.1%) were considered as MDRPa (Multi Drug Resistant P. aeruginosa) which may lead to problems in treatment of P. aeruginosa infection.The special high rate of MDRPa found in this work could be due to the repeated and unsuitable use of antibiotics in Iran.The results of the present study indicated that the frequency of MDRPa was 83.1%; also, 88 (38.1%) isolates of P. aeruginosa were resistant to all the classes of antibiotics tested as well as strong relevancy intense was observed between integron presence and multiresistance.
The results of the present investigation showed thatthe screening for integrons in P. aeruginosa clinical isolates (192 MR and 39 Non-MR) from the Al-Zahra hospital; and146/231 isolates carry class 1 integrons.Out of these 192 MR isolates, 146 (76%) were positive for the presence of class 1 integrons.Several reports have revealed the presence of aminoglycoside resistance genes associated with integrons found in gram-negative bacteria [14]- [16] and also the present study revealed that aminoglycoside resistance was spread more than in the majority of isolates, including those of integron-positive.According to results, a significant difference was shown in distribution and frequency of class 1 integrons among MR and non-MR clinical strains of P. aeruginosa from Al-Zahra hospital, since only MR isolates (48.4%) harboured class 1 integrons carrying gene cassettes (P < 0.001).Among MDR isolates, 146 (76%) showed class 1 integrons.The rate of class 1 integrons observed in this study is higher than previously reported rates of 57.4% from Nigeria [9], 41.5% from Brazil [6], 45.8% from patients in southern china [4], 82% from Thai hospital in Thailand [7] and 69.2% from burn patients in Guilan, Iran [10].In the current study a notable relevance was found between the presence of integrons and resistance against gentamicin, amikacin, imipenem, meropenem, ceftazidime, cefepime, tobramycin, norfloxacin, aztreonam and ciprofloxacin (P < 0.001).
The high resistance level against aminoglycoside antibiotics (gentamicin, amikacin and tobramycin) was observed in this study.Resistance to this class of antibiotics in P. aeruginosa is usually associated with the production of aminoglycoside adenylyltransferase which leads to resistance to aforementioned antibiotics [12].Class 1 integrons have been dependent with different antibiotic resistance genes and organized important role in the expansion of antimicrobial resistance [7].
High resistance among P. aeruginosa strains against the following antibiotics: gentamicin, ciprofloxacin, tobramycin, norfloxacin, ceftazidime, cefepime, imipenem, meropenem, amikacin, aztreonam isolated from sputum, tracheal tube, urine and wounds was observed.The resistance rate of P. aeruginosa isolates from sputum, tracheal tube, urine, wound, blood, catheter andcerebrospinal fluid was reported as 100%, 89.8%, 83.8%, 80.6%, 64.3%, 60% and 50% respectively.In the present study, piperacillin/tazobactam and colistin was the most efficient antibiotics against P. aeruginosa and the antibiotic susceptibility test results showed 43.3% susceptible to piperacillin-tazobactam and 41.1% susceptible to colistin compared with other studies carried out on P. aeruginosa isolated from burn patients in Tehran in which reported 87.2% resistance to piperacillin-tazobactam [5].This investigation reports 93 (48.4%)P. aeruginosa isolates containing class1 integron carrying resistance gene cassettes.Variable region genes of class 1 integrons found in our isolates were as follows: aadB gene (about 0.8 kb), aadA6, orfD (about 1.2 kb) and aacA7, aacA8, bla oxa2 , bla NDM-1 gene (about 2.5 kb) and 120 clinical isolate have an aadA6-orfD array (about 1.2 kb).The aadA6-orfD had the maximum gene cassette in this investigation as well as the most prevalent gene cassette array in P. aeruginosa in Thai hospital in Thailand [7].
Genes giving resistance to aminoglycosides and ß-lactams are frequently found in integrons from P. aeruginosa isolates and the most popular aminoglycoside resistance gene cassettes related to aad and aac families [15]- [17].The first report of identification and sequencing of a streptomycin/spectinomycinadenylyl transferase gene (aadA) was carried out in Escherichia coliand recently the crystal structure of an aminoglycoside adenylyl transferase (AadA) has been reported [22].Aminoglycosides are a relatively large class of antibiotics and AAC (3)-II determines resistance to gentamicin, tobramycin and netilmicin [30]; also, AAC(6′)-II determines resistance to netilmicin, gentamicin and tobramycin.AAC(3)-I determines resistance to gentamicin and AAC(6′)-I determines resistance to tobramycin, netilmicin and amikacin.Finally, ANT (2′)-I determines resistance to gentamicin and tobramycin [31] [32].This is noticeable that orfD has unknown functions and can potentially translate as a polypeptide chain.An ORF (open reading frame) is a portion of a DNA molecule that contains no stop codons, when translated into amino acids [25].Twenty three of 192 (12%) clinical isolates have aacA7, aacA8, bla oxa2 , bla NDM-1 (2.5 kb).bla oxa2 belongs to class D extendeds pectrum b-lactamases and hydrolyzes b-lactam antibiotics such as ceftazidime.
Class D β-lactamases (oxacillinases) (OXA type enzymes) belongs to functional group 2d and molecular class D [29].OXA-1, OXA-2 and OXA-10 enzymes determine resistance to ureidopenicillins and carboxypenicillins but not to ceftazidime.Resistance, resulting from production of piperacillin and ticarcillin OXA-2 enzymes is lower than the resistance that develops when OXA-10 and OXA-1 oxacillinases are produced [29].Study done among clinical isolates of Pseudomonas aeruginosa in 5hospitals Iran, Tehran reported class 1 integron containing aadB, aadA6-orfD, aacA4 and bla oxa10 [13].Another study on gene cassette of Class 1 integrons in P. aeruginosa isolates from clinical settings in Amazon region, Brazil shoed that the most frequent gene cassette found was from the aacA family [6].These our data indicate thatbla NDM-1 gene is the first report among class 1 integrons found in P. aeruginosa isolates from clinical settings in Iran.A member of a large gene family that encodes beta-lactamase enzymes called carbapenemases is NDM-1 gene [23].

Conclusion
In conclusion, our data show that high rate resistant to multiple drugs among P. aeruginosa signifies the spreads of aadA6 among clinical isolates in hospitalized patients in Al-Zahra hospital.There is a significant relationship to antibiotic resistance and class 1 integron and mobile genetic elements play a major role in the development of a resistance gene.

Figure 2 .
Figure 2. PCR amplification products of class 1 integron variable regions using primers In5' and In3' CS.L: 100 bp DNA ladder (Bioneer, Korea); lanes 1-11, integron profiles 1 -11 respectively among 192 MDR P. aeruginosa isolates in Al-Zahra Hospital in Isfahan, Iran.isolates (62.5%) carried class 1 integron with sizes of approximately 1.2 kb, twenty three isolates (12%) with sizes of approximately 2.5 kb and three isolate (1.5) with sizes of approximately 0.8 kb.The isolation sites and samples found in this study have been summarized in Table3; also, the percentage rate of antibiotic resistance and presence of class 1 integrons among 192 MDR P. aeruginosa strains isolated from patients hospitalized in this study has been summarized in Table4, Figure1and Figure3.

Figure 3 .
Figure 3.The percentage rate of antibiotic resistance and presence of class 1 integrons among 192 MDR P. aeruginosa isolates from hospitalized patients in Al-Zahra Hospital in Isfahan, Iran.

Table 1 .
Primer sets and PCR conditions used in this study.

Table 2 .
Factors associated with class 1 integron among Pseudomonas aeruginosa isolates.

Table 3 .
Distribution of Pseudomonas aeruginosa isolates according to hospitals wards and sample types.

Table 4 .
The percentage rate of antibiotic resistance of class 1 integrons positive isolates among 192 MDR P. aeruginosa types.