Distribution of spa Types, Integrons and Associated Gene Cassettes in Staphylococcus aureus Strains Isolated From Intensive Care Units of Hospitals in Tehran, Iran

1. Background

Staphylococcus aureus (S. aureus) is the major cause of infection in either hospitals or within communities across the world (1) causing a variety of illnesses that can range from mild skin infections and wound infections to endocarditis, pneumonia, bacteremia and life-threatening diseases. Staphylococcus aureus, as one of the most prevalent pathogens in hospitals, can easily be transmitted by direct contact (including contaminated hands or droplet transmission) and indirect contact (such as environment or hospital air) between patients and medical staff (2). The most important factor contributing to the successful extensive distribution of this nosocomial pathogen is stated to be its remarkable ability to acquire resistance to new antimicrobial agents (3).

Shortly after the introduction of penicillin as a first therapeutic option for the treatment of infections caused by penicillin-resistant S. aureus, Methicillin-Resistant Staphylococcus aureus (MRSA) emerged in the 1960s, and since the 1980s, MRSA strains have become endemic in hospitals worldwide with associated significant patient morbidity and mortality. To the extent that MRSA have emerged as a major public health concern (4). Methicillin resistance primarily results from the presence of mecA gene, which encodes a modified penicillin-binding protein (PBP2a) and has low affinity for β-lactams. During the past several decades, in spite of introducing a variety of therapeutic measures including antibiotic therapy, MRSA strains have shown a remarkable ability for rapid development of multi-drug resistance (MDR) (5).

The widespread emergence of MDR S. aureus, as a common cause of nosocomial infections, is becoming a serious concern in global public health. The increase of resistance does not only lead to increase of economic burden but it also may cause serious therapeutic problems as well as exacerbates infection control in hospitals. Although the mechanisms of resistance among bacteria are very diverse, horizontal gene transfer has already been proved as the most important mechanism for the dissemination of antimicrobial resistance in microbial populations. Also, the role of integrons as a genetic element in horizontal transfer of antibiotic resistance has been well established (6).

Integrons are a vital element in the spread of MDR, particularly in gram-negative pathogens. They are normally motionless but can be transferred through mobile genetic element, e.g., plasmids and transposons. The basic structure of integrons is composed of 5’and 3’-conserved segments with gene cassettes containing antibiotic resistance genes. Essential components of the 5’ region consist of an integrase gene (intI) encoding an integrase, a recombination site (attI), and a strong promoter gene. To date, several classes of integrons have been described based on the integrase genes (7). Class 1 integrons are distributed in both gram-positive and especially in gram-negative bacteria isolated from clinical samples. They are often associated with lateral transfer of antibacterial resistance genes. Class 2 integrons are less common compared to class 1 integrons and have frequently been reported in gram-negative bacteria. Reports on other classes of integrons are scarce (8). Although the role of integrons in the development of MDR in gram-negative bacteria is definite, relatively little data exists on the prevalence of integrons in gram-positive bacteria, especially in S. aureus isolates.

2. Objectives

The present study was an attempt to understand the possible presence and thus dissemination of different classes of integrons and associated gene cassettes among S. aureus isolates recovered from hospitalized patients in intensive care units (ICUs) of hospitals in Tehran, Iran. We also intended to detect different molecular types of integron-bearing S. aureus strains by spa typing.

3. Methods

3.1. Sampling and Data Collection

During a five-month period, 1st of April to 31st of August 2015, a total of 80 S. aureus isolates were recovered from various clinical specimens of hospitalized patients in ICU wards in five hospitals of Tehran, Iran: one in northern Tehran (A, general and governmental hospital), one in central Tehran (B, specialized and sub specialized private hospital), one in southern Tehran (C, general and governmental hospital), one in eastern Tehran (D, general and private hospital), and one in western Tehran (D, specialized and sub specialized governmental hospital). In the present study, all of the hospitalized patients in the ICU, who were found to have S. aureus infection were included. Duplicate isolates and patients who had a history of previous use of antibiotics before sampling were excluded from the study. According to the inclusion and exclusion criteria, 290 clinical specimens obtained from hospitalized patients at the ICU were included and 65 samples were excluded from our study. The research was approved by the Ethics Committee of Shahid Beheshti University of Medical Sciences, Tehran [94-1293]. Written informed consent was obtained from the patients to use their samples for research purposes. It is important to note that all the equipment and kits in this research were calibrated and certificated by the manufacturer. All the obtained biological samples were transported to the laboratory within two hours of collection and were processed immediately. Identification of S. aureus isolates was performed based on standard microbiological procedures such as colony morphology, gram staining, growth on mannitol salt agar, and production of catalase, coagulase and DNase. All the isolates were also evaluated for the presence of the femA and nucA genes via the polymerase chain reaction (PCR) (9).

3.2. Antimicrobial Susceptibility Testing

In vitro susceptibility testing was performed using a panel of 16 antibiotic disks for all the isolates by the Kirby-Bauer disk diffusion procedure, according to the guidelines of the clinical and laboratory standards institute (CLSI) (10). The following antimicrobial drugs were used in the present survey: penicillin (PG 10 µg), ampicillin (AP 10 µg), teicoplanin (TEC 30 µg), ceftriaxone (CRO 30 µg), gentamicin (GM 10 µg), kanamycin (K 30 µg), amikacin (AK 30 µg), tobramycin (TN 10 µg), linezolid (LZD 30 µg), erythromycin (E 15 µg), gatifloxacin (GAT 5 µg), clindamycin (CD 2 µg), levofloxacin (LEV 5 µg), ciprofloxacin (CIP 5 µg), and trimetoprim-sulfamethoxazole (TS 25 µg). The minimum inhibitory concentration (MIC) for vancomycin was determined with E-test strips (AB Biodisk, Sweden), according to the manufacturer’s instructions. For all the isolates, intermediate sensitivity was scored as resistance. Multidrug Resistance (MDR) was defined as resistance to three or more unique antibiotic classes in addition to resistance to beta-lactams (11). All antibiotic disks used in the current research were obtained from Mast co., UK. A standard reference strain, S. aureus ATCC25923, was used as a quality control strain in every test run. Staphylococcus aureus isolates were preserved in Tryptic Soy Broth (TSB; Merck co., Germany) containing 20% glycerol at -70°C until further molecular analysis.

3.3. Methicillin-Resistant Staphylococcus aureus Screening

Methicillin resistance was detected using a cefoxitin disc (30 µg) and an oxacillin disc (1 µg) on Mueller Hinton agar plates supplemented with 4% NaCl in accordance to the clinical and laboratory standards institute (CLSI) guidelines (10). Isolates with phenotypic resistance to oxacillin were also tested for the presence of the mecA gene using PCR.

3.4. Extraction of Plasmid and Genomic DNA

Genomic DNA of the strains was extracted using the commercial kit InstaGene Matrix (BioRad, Hercules co., CA, USA) along with the addition of lysostaphin (Sigma-Aldrich co., USA) to a final concentration of 15 µg/mL. The Qiagen Plasmid Midi Kit was used for plasmid DNA extraction, according to the manufacturer’s instructions.

3.5. spa Typing

Spa typing was performed as described by Harmsen et al. for all MRSA isolates. For spa typing, the polymorphic X region of the protein A gene (spa) was amplified by PCR (12). The PCR products were subjected to DNA sequence analysis, and their nucleotide sequences on both strands were determined using an ABI Prism 377 automated sequencer (Applied Biosystems, Perkin-Elmer co., Foster City, CA). Sequence editing was done using Chromas software (version 1.45, Australia). Edited sequences were assigned to particular spa types according to the guidelines described by a Ridom SpaServer database (http://www.spaserver.ridom.de).

3.6. Detection of Integrons

The presence of class 1 and 2 integrons was investigated using PCR with degenerate primers, described by Moura et al. (13). Polymerase chain reaction conditions for amplification of 280 bp fragment of the int1 and 232 bp fragments of the int2 by thermocycler (Eppendorf co., Hamburg, Germany) were as follows: initial denaturation for five minutes at 94°C, 35 cycles of denaturation at 94°C for 45 seconds, annealing at 58°C for 45 seconds, and extension at 72ºC for one minute. The final extension was carried out at 72°C for five minutes.

3.7. Detection of Gene Cassettes Inserted in the Variable Regions and Sequencing

Amplification of the variable region between class 1 and 2 integrons was performed using primer pairs introduced by Moura et al. (13). The PCR products of variable regions were purified by the QIAquick Gel Extraction kit (Qiagen co., Hilden, Germany). Purified PCR products were subjected to sequencing with an ABI Prism 377 automated sequencer (Applied Biosystems, Perkin-Elmer co., Foster City, CA) in both directions. The sequences were assembled making use of the SeqMan program within the Lasergene suite version 7 (DNAstar Inc., Madison, WI, USA). The BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) against GenBank database and the Integron Database INTEGRALL (http://integrall.bio.ua.pt/) were performed repeatedly for sequence comparison and annotation.

3.8. Statistical Analysis

Statistical analysis was carried out using the SPSS, version 18.0 software (SPSS Inc., Chicago, IL). Chi-square was run to determine the P value. A P value of less than 0.05 was considered statically significant.

4. Results

During the study, a total of 80 S. aureus isolates were obtained from 290 clinical specimens isolated from hospitalized patients in ICU wards of five hospitals in Tehran. The average age of the participants was 39 (median 41.8, ranging from 9 months to 69 years of age). In the present investigation, 72.5% of the patients were female and 27.5% male; the M:F ratio was 0.37. The age distribution was 10% for patients aged equal or less than 18 years, 77.5% for 19 to 59 years, and 12.5% for equal or above 60 years. Clinical specimens included wound (n = 28, 35%), blood (n = 15; 18.8%), sputum (n = 8; 10%), ear secretions (n = 7; 8.7%), catheter (n = 7; 8.7%), body fluids (bronchoalveolar lavage and cerebrospinal fluid) (n = 6; 7.5%), urine (n = 5; 6.3%) and pus (n = 4; 5%).

4.1. Antimicrobial Resistance Phenotypes

All S. aureus isolates were subjected to antimicrobial susceptibility testing. The result of antimicrobial susceptibility test of 80 S. aureus clinical isolates revealed the rates of resistance to the majority of tested antibiotics ranging between 37.5% and 80 %. All the isolates were 100% susceptible to vancomycin, teicoplanin and linezolid, and 100% resistant to penicillin and ampicillin. Based on the results obtained in the present study, the lowest levels of resistance (37.5%) were related to levofloxacin. None of the observed strains were sensitive to the antimicrobial agents. Also, it was found that all the isolates were MDR. In particular, 25 (31.3%) of the isolates were resistant to nine antibiotics, 19 (23.8%) were resistant to ten antibiotics, 14 (17.5%) were resistant to eight antibiotics, and 11 (13.8%) were resistant to eleven antibiotics. Simultaneous resistance to five, seven, four, twelve and six antibiotics was seen in four, three, two and one isolates, respectively. The results of antimicrobial susceptibility testing of isolates are shown in Table 1. Overall, 69 (86.2 %) isolates were resistant to methicillin and were confirmed as MRSA based on the detection of the mecA gene.

4.2. Detection of Class 1 and Class 2 Integrons and Characterization of the Variable Region

The class 1 and 2 integrons were commonly found in 45 (56.3%) and 15 (18.7%) isolates, respectively. The existence of class 3 integron was not confirmed in any of the MDR strains. Co-existence of class 1 and 2 integron was detected in eight isolates (10%). The frequencies of different classes of integrons among 80 S. aureus isolated from ICU wards are presented in Table 2. The results indicated that the relationship between resistance to ceftriaxone, gentamicin, kanamycin, tobramycin, and ciprofloxacin and the presence of integron was statistically significant. The correlation between these two factors is given in Table 1. Out of 45 isolates carrying class 1 integrons, 38 (84.4%) were located on plasmids and seven (15.6%) on chromosomes while out of fifteen isolates carrying class 2 integrons, ten (66.7%) were located on plasmids and five (33.3%) on chromosomes. Accordingly, the positive rate of both classes 1 and 2 integrons in plasmid DNA was higher than that of genomic DNA. All the integron-bearing S. aureus strains were screened for variable regions. Different sizes of the variable region of integron were subjected to sequencing. The results of sequencing of the integron cassette region revealed six different gene cassettes (aadA2, aadB, blaoxa, aacA4, cmlA6 and catB) in class 1 integrons and three different gene cassettes (dfrA1, aadA1, and sat2) in class 2 integrons. The numbers of cassette genes in class 2 integrons were much more limited compared with those of class 1 integrons. In three (37.5%) isolates harboring both class 1 and 2 integrons simultaneously, PCR could not amplify the variable region as well as three (20%) isolates harboring class 2 integrons. All the S. aureus isolates were spa typed and five different spa types were discriminated (t790 in 26 strains; t030 in 18 strains; t969 in 14 strains; t7580 in 12 strains; t1425 in 10 strains). Genotypic characteristics of 80 integron-bearing S. aureus strains are summarized in Table 3.

4.3. Spa Type t790

The most predominant spa type in the present study was spa type t790, all of which were MRSA. The majority of spa type t790 (69.2%) were obtained from wound infections. All the patients infected with spa type t790 were in the age group of 19 to 59 year-olds. Amongst these t790 spa types, 18 (69.2%) carried integron class 1. None of the strains carried integron class 2. Co-existence of class 1 and 2 integrons was detected in eight isolates (30.8%). The integron cassette regions in three strains, simultaneously harboring class 1 and 2 integrons, were not amplified by PCR. The MDR pattern among spa type t790 isolates included resistance to 11 (26.9%), 10 (23.1%), 9 (42.3%) and 8 (7.7%) antibiotics. The most prevalent gene cassette among spa type t790 isolates was blaoxa gene cassette leading to resistance to beta-lactam antibiotics.

4.4. Spa Type t030

The second most common spa type identified in the current survey was spa type t030. The majority of spa type t030 (33.3%) were obtained from blood infections. The presence of class 1 and 2 integrons in strains with spa type t030 was confirmed in 50% and 44.4% of isolates and only a single isolate did not harbor an integron. Interestingly, the cassette region of class 2 integron in three strains could not be amplified by PCR. The most gene cassette type among spa type t030 isolates was aad gene cassette conferring resistance to aminoglycosides. The results showed that seven (38.9%) isolates were resistant to at least nine antibiotics, seven (38.9%) were resistant to at least eight antibiotics, two (11.1%) resistant to at least nine antibiotics, one (5.6%) resistant to eleven antibiotics, and one (5.6%) resistant to five antibiotics.

4.5. Spa Type t969

Fourteen (17.5%) patients were observed to be infected by isolates belonging to this spa type. These isolates were recovered from blood (n = 4; 28.6%), catheters (n = 4; 28.6%), wounds (n = 3; 21.4%) and pus samples (n = 3; 21.4%). The majority of the infected patients were elderly; 11.3% over 60. Nine (64.3 %) isolates were positive for class 1 integron and the remainder (35.7%) was integron-negative. Isolates with spa type t969 had diverse antibiotic resistance patterns.

4.6. Spa Type t7580

The other spa type identified in the study was spa type t7580. These isolates were isolated from ear (n = 3; 25%), urine (n = 3; 25%), wounds (n = 3; 25%), sputum (n = 2; 16.7%), and body fluids (n = 1; 8.3%). Class 1 and 2 integrons were detected in 50% and 33.3% of isolates, respectively. Two isolates (16.7%) were integron-negative. The dominant resistance profile among these spa types included resistance to 10 antibiotics (41.7%).

4.7. Spa Type t1425

This spa type was mostly isolated from sputum. The frequencies of class 1 and 2 integrons were identical (30%). Forty percent of the isolates did not harbor any integron. Ninety percent of the patients infected with this spa type were under 18 years of age. Isolates with this spa type were remarkably less antibiotic resistant.

4.8. Nucleotide Sequence Accession Numbers

The nucleotide sequence obtained in this study are available in the GenBank nucleotide database under accession numbers LC093087, LC093088, LC093089, LC093090, LC093091, LC093092, LC093093, LC093094, LC093095, LC093096 from the gene cassette of class 1 integrons and LC093990, LC093991 and LC093992 from the gene cassette of class 2 integrons

5. Discussion

Integrons, genetic elements containing gene cassettes carrying antibiotic resistance genes, are linked to MDR and subsequently constrict the therapeutic options and aggravate clinical outcomes (14). The role of class 1 and 2 integrons in the dissemination of antibiotic resistance genes in gram-negative bacteria is well documented. Nevertheless, little is known about the prevalence of class 1 and 2 integrons in gram-positive bacteria leading to MDR distribution (8). The present study concentrated on the carriage of class 1 and 2 integrons and associated gene cassettes in S. aureus strains recovered from clinical samples of patients in ICUs and also detection of different spa types in Tehran, Iran.

It has been reported that HA-MRSA from diverse geographical locations have remarkably different genetic characteristics (15-17). In the current survey, the prevalence of HA-MRSA isolates was 86.2%, which was higher than that in Taiwan (1), Nigeria (18), Hungary (17, 19), Egypt (16), Serbia (20) and Croatia (15). The increased rate of resistance to methicillin among the isolates obtained in our study could be attributed to the longer period of the study, the sample type, investigated population and different wards of the hospital.

The MRSA strains are usually resistant to macrolides, lincosamides, aminoglycoside, and approximately all currently available beta-lactam antimicrobial agents, like penicillin and cephalosporins (21-23). The susceptibility pattern revealed that vancomycin, teicoplanin, and linezolid had good activity against S. aureus infections, while penicillin and ampicillin had the lowest antibacterial effect on S. aureus isolates. The results were in agreement with previous findings from Italy (24), Croatia (15), Taiwan (1), Serbia (20), and Turkey (25).

Although previous studies have revealed the emergence of MRSA with reduced susceptibility or gradual increased resistance to vancomycin in Iran (26), the results obtained in the present study can help infer that proper antibiotic prescription protocols, extensive surveillance programs, and standard principles of infection control in health care systems may have led to a decrease in vancomycin resistance in MRSA isolates.

The results demonstrated that resistance to ciprofloxacin (80%), erythromycin (78.8%), gentamicin (75%), ceftriaxone (72.5%), kanamycin (68.8%), and clindamycin (65%) was relatively high while approximately less than half of the strains were resistant to levofloxacin (37.5%) and trimethoprim-sulfamethoxazole (47.5%). This is largely in accordance with the findings reported by Ko et al. in a study of 74 MRSA strains isolated from 12 Asian countries (27). Antimicrobial resistance patterns revealed that all the isolates (100%) were MDR, which is in accordance with the findings of Xu et al. in China (6), yet in comparison to the previous investigations in Serbia (83.9%) (20) and Taiwan (75.8%), (1) a high frequency of MDR S. aureus was observed in our study. Studies have shown that the frequency of MDR S. aureus isolates seems to vary by region (6, 15, 18, 24, 28). Increase in the frequency of MDR S. aureus is a challenging and serious public health concern especially in ICUs.

As mentioned, integrons are widely known for their role in the dissemination of antibiotic resistance, particularly among gram-negative pathogenic bacteria. Class 1 integrons are the most prevalent class among mobile integrons. The classification of different integrons is based on the relative homology of intI (7). In this survey, the presence of class 1 and 2 integrons was confirmed in 45 (56.3%) and 15 (18.7%) isolates, respectively. These findings are consistent with previous studies in which the rate of detection of integron class 1 was more than integron class 2 (6, 28, 29). In the study of Xu et al. in China, out of 30 S. auerus isolated from environment and surgical patients, 16 (53%) clinical and environmental isolates were positive for the class 1 integrase gene (6). In a study conducted by Guney et al. that investigated the presence of class 1 integron in MRSA isolated from a 1150-bed training and research hospital located at Black Sea region in Turkey, one hundred clinical MRSA isolates were investigated. The result revealed that none of the isolates harbored class 1 integron (29). The high prevalence of class 1 integron in our survey strongly demonstrated that class 1 integron might serve as a reservoir for antimicrobial resistance in Iranian S. aureus strains.

Six different gene cassettes (aadA, aadB, blaoxa, aacA4, cmlA6 and catB) in class 1 integron and three different gene cassettes (dfrA1, aadA1 and sat2) in class 2 integron were identified. Given the previous researches and Integron Database INTEGRALL (http://integrall.bio.ua.pt/): aadA gene cassette conferred resistance to streptomycin and spectinomycin; aadB gene cassette conferred resistance to gentamicin, tobramycin, kanamycin, dibekacin and sisomicin; blaoxa gene cassette conferred resistance to beta-lactam antibiotics such as ampicillin, cephalothin, oxacillin and cloxacillin; aacA gene cassette confers resistance to amikacin, dibekacin, isepamicin, netilmicin, sisomicin and tobramycin; cmlA6 and catb gene cassettes conferred resistance to chloramphenicol; and sat2 gene cassette confers resistance to streptothricin. Overall, nine different gene cassettes were detected. In the present study, cassette genes encoding resistance to aminoglycosides (aadA, aadB) and beta lactams (blaoxa) were the most predominant cassettes in the class 1 integron followed by aacA gene cassette. The cmlA6 gene cassette was found to be rare in class 1 integron. In a study conducted by Xu et al. on nosocomial MRSA strains sampled during 2001 to 2006, it was found that seventy-six out of 179 (42.5%) of the tested strains carried class 1 integrons and four unique arrays of gene cassettes (aadA2, aacA4- cmlA1, dfrA17-aadA5 and dfrA12-orfF-aadA2) were the most frequently detected resistance genes found in clinical isolates (30). It is established that class 1 integrons are associated with a variety of resistance gene cassettes, yet aminoglycoside resistance determinant are detected frequently (6, 30). It should also be noted that this integron had been reported to be associated with Tn3 transposon family (Tn21 or Tn1696) therefore widespread distribution of class 1 integron could be attributed to the spread of integron-containing transposon (7, 8). In class 2 integron, the most predominant cassette gene was dfrA1 that conferred resistance to trimethoprim. The literature review showed that class 2 integrons are associated with the Tn7 transposon and dihydrofolate reductase enzyme encoded by the dfr gene, which is located on Tn7 (8). The frequency of class 1 and 2 integrons in plasmid DNA was higher than that of genomic DNA, which is largely in accordance with the findings of Ren et al. (31).

In the present study, we made an attempt to determine different types of spa in integron-bearing S. aureus strains. The main spa types from our survey were t790, t030, t969, t7580 and t1425 respectively. In agreement with earlier reports, we found spa type t790 with a high percentage of MDR and integron class 1 (69.2%) as the most common spa type among our isolates (21). Although t790 was previously reported as a predominant community-acquired MRSA (CA-MRSA) clone in Iran (21), Germany (32) and Ireland (33), in our study t790 was detected as the predominant spa type among the HA-MRSA isolates. It can be deduced that CA-MRSA spa types have successfully established themselves in Iranian hospitals and health care settings.

The majority of t030 strains from our study were resistant to ciprofloxacin, erythromycin, gentamicin and clindamycin. Class 1 integron was the most frequent integron among this spa type. The most common gene cassette was aad that conferred resistance to aminoglycosides. These findings are consistent with the study of Chen et al. in China in 2010. They demonstrated that spa t037, which was prevalent before year 2000, was rapidly replaced by the t030 MRSA clone, which emerged in 2000. Since then, t030 (73.3%) has become the most popular MRSA clone. Antibiotic susceptibility testing exhibited that predominant antibiotic resistance profile among t030 isolates included resistance to tetracycline, erythromycin, gentamicin, ciprofloxacin, clindamycin and rifampin. Another interesting finding was that the first patient with t030 MRSA was found at an ICU (34). Considering the high resistance of this spa type to many antibiotic agents and easy transmission, special care must be taken to avoid the spread of this particular type. In spite of high frequency of integron class 1 and diverse antibiotic resistance patterns of t969, t7580 and t1425 types, they had limited frequency in S. aureus isolates in our study. Previous studies have also confirmed that spa type t969 has low frequency compared with other spa types (35, 36).

A major strength of this study was that it was performed on S. aureus strains isolated from hospitalized patients in ICUs, in order to study the distribution of integron and associated gene cassettes in different spa types of nosocomial S. aureus; however our study had limitations, which was the modest sample size and the impossibility of using other methods such as pulsed-field gel electrophoresis (PFGE) and Multilocus sequence typing (MLST).

5.1. Conclusion

To summarize, our study revealed that a large proportion of S. aureus strains in our hospitals harbored an integron, which may lead to dissemination of multiple antibiotic resistance. This dilemma emphasizes that antibiotic resistance remains a problem and strategies to control S. aureus infections must be revised. We also confirm the presence t790, t030; t969, t7580 and t1425 with a high level of multiple antibiotic resistance in Iran. Therefore, it can be suggested that in order to understand the prevalence and epidemiology of integron in different molecular tapes of S. aureus, further studies are required.