First report of TEM-104-, SHV-99-, SHV-108-, and SHV-110-producing Klebsiella pneumoniae from Iran

Introduction: Extended-spectrum beta-lactamases (ESBLs) are bacterial enzymes capable of hydrolyzing beta-lactams. The aim of this study was to describe the prevalence of TEMand SHV-type ESBL-producing Klebsiella pneumoniae strains in Zahedan, Southeast Iran. Methods: A total of 170 non-repetitive K. pneumoniae strains were collected from patients referred to three teaching hospitals of Zahedan. Antibiotic susceptibility testing was determined for 17 antibiotics using the Kirby-Bauer disc diffusion method. The frequency of ESBL-producing strains was calculated, and minimum inhibitory concentrations of ESBLproducing strains were determined for cefotaxime, ceftazidime, ceftriaxone, and cefpodoxime. The presence of blaTEM and blaSHV genes was tested in all ESBL-producing strains using polymerase chain reaction and DNA sequencing. Results: Among the 170 K. pneumoniae clinical isolates, 55 (32.4%) were ESBL producers; 92.7% (n=51) and 72.7% (n=40) of the isolates carried the blaSHV and blaTEM genes, respectively, and 67.3% (n=37) carried both genes. The sequencing results showed that all blaTEM types were blaTEM-1, except for two isolates that were blaTEM-104. The blaSHV types were blaSHV-1, blaSHV-11, blaSHV-12, blaSHV-99, blaSHV-108, and blaSHV-110. Conclusions: The percentage of blaTEM and blaSHV among ESBL-producing K. pneumoniae isolates from Zahedan is relatively high, indicating the need for further surveillance and consideration in antibiotic use. To the best of our knowledge, this is the fi rst report of TEM-104-, SHV-99-, SHV-108-, and SHV-110-type ESBLs among clinical isolates of K. pneumoniae from Iran, and TEM-1, SHV-1, SHV-11, and SHV-12 appear to be the dominant ESBLs in this region.


INTRODUCTION
Klebsiella pneumoniae is an important opportunistic nosocomial pathogen responsible for a variety of infections, including urinary tract infections, pneumonia, septicemia, wound infections, and infections in intensive care units (1) .This bacterium is one of the leading causes of community-acquired and nosocomial infections and is associated with high mortality rates (2) .The prevalence of K. pneumoniae with multi-drug resistance, which commonly leads to systemic infections and death, has been increasing in recent years (3) .The emergence of infections caused by these multidrug-resistant strains is a clinical challenge, with limited and complex treatment options (4) .Beta-lactam antibiotics (including penicillins, cephalosporins, carbapenems, or monobactams) have been widely applied in the treatment of serious infections caused by Gram-negative bacteria since the 1980s, contributing to the rapid spread of bacteria resistant to these antibiotics worldwide (5) .The main mechanism of resistance is the production of beta-lactamases, which can protect bacteria against the lethal effect of betalactam antibiotics on cell wall synthesis (6) .Hydrolysis of betalactam antibiotics by beta-lactamases is the most common mechanism of resistance for this class of antibacterial agents in clinically important Gram-negative bacteria (7) .Extended spectrum beta-lactamases (ESBLs) were fi rst described in 1983, which are capable of hydrolyzing oxyimino-cephalosporins (e.g., cefotaxime, ceftazidime, and ceftriaxone) and monobactams (e.g., aztreonam), but not cephamycins or carbapenems (8) .Betalactamase inhibitors (such as clavulanic acid, sulbactam, and tazobactam) prevent the action of ESBLs (9) (10) .Most of the genes encoding ESBLs are plasmid-borne and are often located in the transposons and integrons, which facilitates their mobilization with other resistance determinants.Thus, the genes encoding ESBLs may be easily transferred between bacteria.The most prevalent ESBLs are included in three groups: temoneira (TEM), sulfhydryl variable (SHV), and cefotaximase (CTX-M).The SHV enzymes are named after the thiol variable active site and are commonly associated with K. pneumoniae.The TEM enzyme was named after the patient from whom it was isolated, Temoneira, and was fi rst discovered in Escherichia coli in Greece.In addition, chromosomal-related CTX-Mtype ESBL was reported in Kluyvera ascorbata (11) .Detection of ESBL production is important, because ESBL-positive strains are associated with increased mortality as compared to ESBL-negative strains.One major concern is the spread of ESBL-positive bacteria within hospitals, which may lead to outbreaks or to endemic occurrence.Another concern is the failure to treat infections caused by ESBL-positive organisms, as therapeutic choices are currently limited (1) .Detection of ESBLs by conventional antimicrobial susceptibility methods and delay in the detection and reporting of ESBL production by Gramnegative bacilli are associated with prolonged hospital stays, and increased morbidity, mortality, and healthcare costs (9) .Relatively little research on ESBL-producing bacteria has been conducted in the Southeastern region of Iran, and the resistance patterns of bacterial strains, especially the ESBL-producing mechanism, has not been determined in this region.Therefore, the aim of the present study was to evaluate the prevalence of TEM-and SHVtype ESBL-producing K. pneumoniae in Zahedan, Southeast Iran, using phenotypic and genotypic methods.

Isolation of bacteria
Klebsiella pneumoniae isolates were obtained from 170 non-repetitive clinical samples, including urine, tracheal tube secretions, blood, wound secretions, urine catheter secretions, synovial fl uid, and pharyngeal secretions, collected from patients referred to three teaching hospitals (Khatam al Anbiya, Ali ibn Abi Talib, and Buali) of Zahedan, Southeast Iran from January to December 2012.Collected samples were cultured on blood agar and MacConkey agar (Merck, Darmstadt, Germany) media.K. pneumoniae strains were identifi ed based on standard microbiology methods such as Gram staining, oxidase and catalase tests, as well as biochemical tests with reference to standard tables (12) .

Ethical considerations
This study was evaluated and approved by the Ethics Committee of Zahedan University of Medical Sciences (project No. 90-17).

Screening for ESBL-producing isolates
Based on the CLSI recommended method for screening of ESBL-producing isolates, the phenotypic confi rmatory test was conducted with the disc diffusion method using discs containing cefotaxime (30µg) and cefotaxime/clavulanic acid (30/10µg), as well as those containing ceftazidime (30µg) and ceftazidime/ clavulanic acid (30/10µg) (14) .

Measurement of the minimum inhibitory concentration of the isolates
The minimum inhibitory concentration (MIC) values of four antibiotics, cefotaxime, ceftazidime, ceftriaxone, and cefpodoxime, were determined for isolates with an ESBLpositive phenotype using the E-Test method (Liofi lchem MIC test strips, Italy).The concentration gradient for cefotaxime was 0.002-32µg/ml, and that for ceftazidime, ceftriaxone, and cefpodoxime was 0.016-256µg/ml.The interpretative criteria were applied following the E-test manufacturer's guidelines and according to CLSI recommendations.E. coli ATCC 25922 was used as a quality control for the MIC test strips (14) (15) .

DNA extraction and polymerase chain reaction amplifi cation
After performing the MIC measurements and determining the resistant and ESBL-producing samples, the samples deemed resistant in the phenotypic confi rmatory test were selected to evaluate the presence of the beta-lactamase genes bla TEM and bla SHV using polymerase chain reaction (PCR).The plasmid deoxyribonucleic acid (DNA) of ESBLs-positive isolates, which was extracted with AccuPrep Plasmid Nano-Plus Plasmid Mini Extraction Kit Cat.No.: K-3112 (Bioneer, Daejeon, South Korea), was used for detection of the bla TEM gene.In addition, the chromosomal DNA of isolates with an ESBLs-positive phenotype was isolated with the Molecular Biological System Transfer (MBST) DNA extraction kit (Iran), and used for detection of the bla SHV gene.The primers and PCR conditions described by Nasehi et al. (16) were used to amplify the bla TEM and bla SHV genes (16) .

Sequencing of PCR products
To confi rm the accuracy of the test as well to determine the subtypes of the bla genes detected, samples testing positive for the bla TEM and bla SHV genes were sequenced by Bioneer Company, and the results were analyzed using the BLAST program (www.ncbi.nlm.-nih.gov5BLAST).

Statistical analysis
The data were qualitatively described (percentage and frequency) using SPSS Software (version 18).
The phenotypic confi rmatory test showed that among the 170 isolates, 55 (32.4%) were ESBL producers.According to the CLSI criteria, if the MIC of cefpodoxime is ≥8 or if the MICs of cefotaxime, ceftazidime, and ceftriaxone are ≥2 for a bacterial strain, it can be considered as an ESBL producer (14) .Using these criteria, all 55 isolates were identifi ed as ESBL producers to the four antibiotics mentioned above according to the MIC results.
The PCR assay of the ESBL-producing isolates indicated that 40 (72.7%)isolates were positive for the bla TEM gene, and 51 (92.7%) isolates had the bla SHV gene.Moreover, 37 (67.3%) of the isolates had both the bla TEM and bla SHV .Figure1 and Figure 2 shows the electrophoretic patterns of the bla TEM and bla SHV genes, respectively.
Overall, 91 positive PCR products, including 40 samples related to the bla TEM gene and 51 samples related to the bla SHV gene, were sequenced and analyzed.The majority of those positive for the bla TEM gene were of the bla TEM-1 subtype, with only two samples showing a different subtype, bla TEM-104 .Among the samples positive for the bla SHV gene, 17 samples were bla SHV-11 subtype, 16 samples were bla SHV-1 subtype, 14 samples were bla SHV-12 subtype, 2 samples were bla SHV-110 subtype, 1 sample was bla SHV-99 subtype, and 1 sample was bla SHV-108 subtype.

DISCUSSION
Resistance in Klebsiella pneumoniae strains to a wide spectrum of antibiotics is rapidly increasing.Therefore, awareness toward the prevalence and extent of resistant strains to antibiotics can help to determine the effectiveness of different antibiotics for the treatment of infections.The prevalence rate of infections caused by organisms that are resistant to betalactam antibiotics is increasing.Therefore, identifi cation of ESBL-producing strains is a great necessity in both hospitals and the community (17) .In this study, the rates of resistance in K. pneumoniae isolates to ceftazidime, cefotaxime, ceftriaxone, cefpodoxime, and aztreonam antibiotics were higher than those reported by Ghafourian et al (18) , from isolates in Milad Hospital of Tehran (the capital of Iran), which were 65.8%, 46.4%, 60.4%, 52%, and 51%, respectively, indicating a trend toward increasing resistance to these antibiotics in Iran.Furthermore, we identifi ed an overall frequency of 32.4% ESBL producers, which is less than those reported in Turkey (69%) (10) , Taiwan (97%) (11) , India (97%) (19) , Saudi Arabia (55%) (20) , and Nepal (62.7%) (21) , but higher than those reported in Lebanon (20%) (22) , the United States (7.6%), and Canada (4.9%) (23) .Al-Agamy showed that the rates of resistance of ESBL-producing isolates to cefotaxime, ceftazidime, aztreonam, cefepime, gentamicin, amikacin, and ciprofl oxacin were 97.7%, 95.5%, 97.7%, 47.8%, 88.9%, 77.3%, and 11.4%, respectively, which supports our fi ndings for cefotaxime, but indicates a higher resistance rate to the other antibiotics.Furthermore, Al-Agamy identifi ed that all ESBL-producing isolates were susceptible to imipenem (20) , whereas in our study, 5.5% of the ESBL-producing strains were resistant to imipenem.Although this is currently a small proportion, this fi nding can be considered a warning for the future, highlighting the necessity of further surveillance.In the present study, 100% of the ESBL-positive strains were resistant to cefotaxime and cefpodoxime, 98.2% were resistant to ceftriaxone, 81.8% were resistant to aztreonam, and 78.2% were resistant to ceftazidime, which is indicative of the overall high rate of antibiotic resistance in this region.Therefore, to prevent a further increase in the resistance to these antibiotics, prescriptions must be carefully considered in each case.According to our study, colistin sulfate, imipenem, amikacin, and chloramphenicol are more appropriate choices for the treatment of infections induced by ESBL-producing isolates, regardless of their side effects.Since these drugs are more expensive and can cause more complications, the accurate control and logical prescription of cephalosporins might result in better outcomes.The present study further showed that the bla SHV gene is highly (92.7%) prevalent in clinical ESBL-producing strains, and the most frequent subtypes were SHV-11, SHV-1, and SHV-12.
In 2007, Al-Agamy et al. (20) showed that 214 (97.3%) of 220 strains isolated in Saudi Arabia carried the bla SHV gene, which is higher than that found in our study (20) .A study conducted by Ghafourian et al. (24) in 2011 in Iran determined that 94% of K. pneumoniae strains carried the bla SHV gene, which is in parallel with our results (24) .In recent years, the high relative prevalence of the bla SHV gene in ESBL-producing K. pneumoniae strains has been reported worldwide.The prevalence of ESBL-producing E. coli and K. pneumoniae isolates (TEM-and SHV-type ESBLs) was less than 1% in 1997 in the Netherlands, which is a country known to employ antibiotics minimally; however, more recent studies indicate not only an increasing rate of resistance but also the emergence of new types such as SHV-31 in K. pneumoniae (25) .In the present study, 72.7% of the strains carried the bla TEM gene, and the major subtype was TEM-1.These results are similar to those reported in India and Turkey, with a prevalence of 73% and 73.4%, respectively (10) (26) .Feizabadi et al. conducted a similar study on K. pneumoniae strains in Hospitals of Tehran in 2007, and also found that TEM-1, SHV-12, and SHV-1 were the most prevalent subtypes (27) .In a subsequent study, Feizabadi et al. (27) further identifi ed that all of the K. pneumoniae isolates from a Labbafi nejad Hospital that carried the bla TEM gene were the TEM-1 subtype, and TEM-1, SHV-11, SHV-5, and SHV-12 were reported as the dominant ESBLs in resistant K. pneumoniae strains in Iran (28) .Overall, it seems that the majority of ESBLs in K. pneumoniae strains of Zahedan are derived from the bla SHV and bla TEM genes, with SHV-1, SHV-11, SHV-12, and TEM-1 as the most common subtypes.Moreover, the TEM-104, SHV-99, SHV-108, and SHV-110 subtypes were reported for the fi rst time in Iran in this study.In conclusion, the prevalence of ESBLs is relatively high in the Southeast region in Iran, indicating that further studies are warranted to collect comprehensive epidemiological data of resistant strains.