==== Front Ann Saudi MedAnn Saudi MedAnnals of Saudi Medicine0256-49470975-4466King Faisal Specialist Hospital and Research Centre 2665722610.5144/0256-4947.2015.435asm-6-435Original ArticlePhenotypic and genotypic characterization of extended-spectrum β-lactamases producing Escherichia coli and Klebsiella pneumoniae in a tertiary care hospital in Riyadh, Saudi Arabia Somily Ali M. aArshad Muhammad Z. aGaraween Ghada A. bSenok Abiola C. b a College of Medicine, King Khalid University Hospital and King Saud University, King Saud University Medical City, Riyadh, Saudi Arabia b Department of Microbiology and Immunology, College of Medicine, Alfaisal University, Riyadh, Saudi ArabiaCorrespondence: Dr. Ali Mohammed Somily, Department of Pathology and Laboratory Medicine, College of Medicine, King Khaled University Hospital and King Saud University, PO Box 2925, Riyadh 11461, Saudi Arabia, T: 966-532-703-322, F: 966-11-467-9162, ali.somily@gmail.comNov-Dec 2015 35 6 435 439 Copyright © 2015, Annals of Saudi Medicine2015This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.BACKGROUND AND OBJECTIVES Extended-spectrum beta-lactamase (ESBL)-producing pathogens remain a public health concern, with limited data on the molecular characterization of isolates. We aimed to determine the molecular characterization of ESBL-producers circulating in our setting and correlate the molecular types with the minimal inhibitory concentration (MIC) to third-generation cephalosporins. DESIGN AND SETTING Retrospective study conducted during the period from January to June 2013 at King Khalid University hospital, a tertiary-care hospital in Riyadh, Saudi Arabia. MATERIALS AND METHODS All Escherichia coli and Klebsiella pneumoniae confirmed to be ESBL producers were included. The MICs of ceftriaxone and ceftazidime were determined by the E-test. Molecular characterization of ESBL-genes was performed using the Check-MDR-CT102 DNA microarray. RESULT Of 77 isolates comprising 50 (65%) E coli and 27 (35%) K pneumoniae, the majority (n=63; 81%) were from urine. Most isolates were blaCTX-M gene positive (n=72/77; 93.5%) comprising blaCTX-M1 (n=62), blaCTX-M9 (n=9) and blaCTX-M25 (n=1). Two or more ESBL genes were present in 45% of isolates with blaSHV predominating in K pneumoniae and blaTEM in E coli. Two isolates were positive for blaOXA-48 carried in combination with blaCTX-M9 and blaTEM in E coli and blaCTX-M1/CTX-M9 in K pneumoniae. Ceftriaxone MIC50 and MIC90 of ≥256 μg/mL were seen in E coli and K pneumoniae harboring blaCTX-M alone or in combination with blaSHV or blaTEM. For ceftazidime the highest MIC50 and MIC90 was seen in K pneumoniae harboring blaCTX-M+blaSHV and E coli with blaCTX-M+blaTEM combinations. CONCLUSION A preponderance of blaCTX-M suggests dissemination of the gene in our setting. The MIC for ceftriaxone and ceftazidime correlate well with molecular characterization of ESBL-producing Enterobacteriaceae. ==== Body The main mechanism of acquired resistance to extended-spectrum cephalosporins among the Enterobacteriaceae is the production of plasmid-mediated extended-spectrum β-lactamases (ESBLs) and/or AmpC β-lactamases (pAmpCs).1 The blaSHV-type and blaTEM-type ESBLs were the most prevalent enzymes in the world a decade ago, but now the epidemiology of dominant ESBL types has shifted to the blaCTX-M -type β-lactamases.2 The blaCTX-M family, first described in 1992,3 is known to be the most dominant non-blaTEM and non-blaSHV ESBL among Enterobacteriaceae and is recognized as a rapidly growing family of ESBLs that selectively prefer to hydrolyze cefotaxime rather than ceftazidime.4 Clinical laboratories have to accurately screen isolates suspected of harboring ESBLs. The Clinical and Laboratory Standards Institute (CLSI) recommends screening of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis isolates for ESBL production by the use of cefpodoxime, ceftazidime, aztreonam, cefotaxime, or ceftriaxone, followed by phenotypic confirmation with clavulanate.5 However, multiple resistance mechanisms may obscure the ability to detect ESBLs, as the new classes of non-ESBL enzymes that are emerging may result in resistance patterns that overlap those caused by the ESBLs, leading to the failure of conventional clavulanate-based ESBL detection. Our aim was to determine the epidemiology as well as genotypic and phenotypic characterization of ESBL-producing isolates including carbapenem-resistant Enterobacteriaceae (CRE) and to correlate the molecular types of ESBLs with the minimal inhibitory concentration (MIC) of ceftriaxone and ceftazidime. MATERIALS AND METHODS This study was carried out at King Khalid University Hospital, Riyadh, Saudi Arabia. The study included 77 non-duplicate, consecutive, phenotypically confirmed E coli and K pneumoniae ESBL-producing isolates identified between January to June 2013. These isolates were from various specimen types including, blood, wound swabs, endotracheal secretions, sputum, body fluids, and urine, which were routinely cultured in the bacteriology laboratory. Identification of the organisms and susceptibility testing were carried out according to our laboratory policy by Vitek 2 (Biomerieux, Marcy l’Etoile) for blood and sterile body fluids samples and by MicroScan Walkaway 96 plus System (Siemens Healthcare Diagnostic Inc.) for other samples. We used MicroScan as a backup automated identification system in case the isolate was not identified or results were equivocal by Vitek 2. The isolates flagged as ESBL by Microscan or Vitek-2 were confirmed using the E-test method as recommended by CLSI. Only the first representative isolate per patient was included and all other repeat isolates were excluded. Antimicrobial susceptibility testing Forty-eight of 77 isolates were tested for AmpC production using the Kirby-Bauer method on Mueller Hinton agar using commercially available discs Mastdiscs ID inhibitor combination disks (MDI) (Mast Diagnostics company, Bootle, Liverpool, UK) in accordance with manufacturer’s guidelines. This methodology utilizes three discs: disc A, containing cefpodoxime 10 μg + AmpC inducer; disc B, consisting of cefpodoxime 10 μg + AmpC inducer + ESBL inhibitor; and disc C, consisting of cefpodoxime 10 μg + AmpC inducer + ESBL inhibitor + AmpC inhibitor. Interpretation of the test requires comparison of the zone of inhibition of disc C with the inhibition zones of each disc A and B. If disc C shows a zone difference of ≥5 mm from discs A and B this is indicative of AmpC production. The minimum inhibitory concentration for ceftriaxone and ceftazidime was determined by E-test (AB Biodisk, Solna, Sweden) following the manufacturer’s instructions and results were interpreted as per CLSI guidelines.5E coli ATCC 25922, K pneumoniae ATCC 700603 and P aeruginosa ATCC 27853 were used for quality control. Genotypic testing for ESBL and Carbapenemases genes Testing was carried out using the Check-MDR CT102 DNA microarray (CheckPoints BV, Wageningen, Netherlands) at the Antimicrobial Resistance Research Laboratory, Alfaisal University, Riyadh, Saudi Arabia. At the time of genotypic testing, the research laboratory was blinded to the findings of the phenotypic assays. The Check-MDR CT102 combines ligation-mediated amplification with detection of amplified products on a microarray to detect ESBL genes blaCTX-M, blaTEM, and blaSHV as well as carbapenemase genes including KPC, NDM, VIM, IMP, OXA-48. Whole-cell DNAs were extracted from overnight bacterial cultures using the Mo Bio UltraClean Microbial DNA Isolation Kit. Microarray assays were performed according to manufacturer instructions with provided tubes and reagents. Briefly, 10 μL of purified genomic DNA (5–50 ng/μL) was added to 5 μL of proprietary mix containing ligation probes and thermostable DNA ligase. The sample was run in T100 thermal cycler (Bio-Rad) for 3 min at 95°C, followed by 24 cycles of 0.5 min at 95°C and 5 min at 65°C, and final denaturation at 98°C for 2 min. For the DNA amplification step, 30 μL of manufacturer provided mix containing PCR primers, deoxynucleoside triphosphates, and thermostable polymerase were added and the sample was heated for 10 min at 95°C followed by 30 cycles of 0.5 min at 95°C, 0.5 min at 55°C, 0.5 min at 72°C, and a final denaturation step of 2 min at 98°C using the Veriti thermal cycler (Applied Biosystem). The ligation and DNA amplification steps were carried out in separate rooms in accordance with manufacturer guidelines. DNA hybridization of the amplified ligation products was carried out in customized Array Tubes (3 samples per tube). Hybridization was performed using 10 μL of each amplified reaction product with 300 μL of preheated hybridization buffer for 30 min at 50°C under rotational shaking (400 rpm) in a thermo-mixer (Eppendorf, Hamburg, Germany). Unbound DNA was washed away using two 5-min incubation steps with 300-μL blocking buffer under the same conditions. Then 150-μL freshly prepared conjugate solution was added to each array tube and incubated for 15 min at 30°C at 400 rpm on the thermomixer. Excess conjugate was washed away by two 5 min incubation steps using 300-μL detection buffer under the same conditions. Finally, 150 μL of the staining solution was added and incubated for 15 min at room TEMperature. After 15 min, tubes were inserted in the single-channel ATR03 array tube reader, the images were acquired and interpreted with the manufacturer provided software controls for assessing the success of each critical step in the procedure, including ligation specificity and efficiency, PCR amplification, hybridization efficiency, label detection and label quality, which are inbuilt in the array analysis. RESULTS The study included 77 isolates from various body sites, comprising 50 (65%) E coli and 27 (35%) K pneumoniae, which were phenotypically confirmed to be ESBLs producers. The majority of the isolates (n=60; 77.9%) were isolated from urine samples. Other specimen sources were wound swabs (n=8; 10.4%), blood (n=3; 3.9%), sputum (n=3; 3.9%) and sterile body fluids (n=3; 3.9%). The isolates were mostly from inpatients (n=40; 52%) with a predominance of adult patients (n/N: 62/77; 81%). Thirty-four isolates were from males and 43 from females. Figure 1 shows the demographic characteristics of the patient population. The blaCTX-M gene was harbored by majority of the isolates (n=72/77; 93.5%) and comprised of three blaCTX-M groups namely blaCTX-M1 (n=62), blaCTX-M9 (n=9) and blaCTX-M25 (n=1). The blaCTX-M gene was of comparable predominance in both E coli (n=48/50; 96%) and K pneumoniae (n=23/27; 85%), However, almost half of the isolates harbored at least two or more ESBL genes (n= 35/77; 45%) (Table 1) with blaSHV occurring most commonly in K pneumoniae (n=16/27; 59%). Although blaTEM was detected in 26% (n= 13/50) of E coli only a single K pneumoniae isolate was positive for this gene. Two isolates were positive for blaOXA-48 which was carried in combination with blaCTX-M (Group 9) and blaTEM in one E coli isolate and blaCTX_M (Groups 1 and 9) in a K pneumoniae isolate. No AmpC producer was identified. There was 100% concordance between phenotypic detection of ESBL and molecular characterization using the Checkpoint MDR system. The MIC50 and MIC90 for ceftriaxone in E coli harboring blaCTX-M genes alone or in combination with blaSHV or blaTEM were ≥256 μg/mL. In contrast, ceftriaxone MIC90 ≥256 μg/mL was only observed for K pneumoniae isolates harboring blaCTX-M alone or blaCTX-M + blaTEM genes (Table 2). For ceftazidime, in K pneumoniae harboring blaCTX-M + blaSHV only the MIC50 and MIC90 was much higher compared to isolates carrying other gene combinations, although the small sample size precluded a statistical analysis (Table 2). In E coli, the highest ceftazidime MIC50 and MIC90 was seen in isolates harboring a combination of blaCTX-M + blaTEM genes (Table 2). The E coli isolate harboring bla- OXA-48 showed an MIC of >256 for ceftriaxone and 1.5 for ceftazidime while the K pneumoniae isolate with bla- OXA-48 showed MIC >256 for both ceftriaxone and ceftazidime. Both blaOXA-48 positive isolates were sensitive to imipenem and meropenem using CLSI breakpoints. They were resistant to amoxicillin-clavulanic acid, ceftazidime, ceftriaxone, gentamicin and piperacillin-tazobactam. DISCUSSION In recent years, data from the Arabian Peninsula has shown a high occurrence of ESBL-producing isolates with rates as high as 31.7% in Kuwait,6 41% in United Arab Emirates7 and 55% in Saudi Arabia.8 A study from Bahrain reported that 22.6% of Enterobacteriaceae at a major tertiary center were ESBL producers.9 Although current data indicate that ESBL-producing pathogens are an emerging public health concern in the region, there are limited data on the molecular characterization of local isolates. The findings in our study indicate that most of the ESBL isolates harbored blaCTX-M gene, mainly blaCTX-M-1 followed by blaSHV and blaTEM. Previous studies from Saudi Arabia have reported detection of blaCTX-M in 34% and 71% of ESBL-producing Enterobacteriaceae isolates.8,9 Our finding indicates a higher preponderance of blaCTX-M positive isolates indicating further dissemination of this resistance gene among Enterobacteriaceae isolates circulating in our setting. However, as in other studies, we also document a high prevalence of urinary isolates harboring the blaCTX-M, although in contrast to other reports, it appears that we have more isolates among inpatients. This suggests that in addition to further dissemination, these blaCTX-M-positive isolates continue to be important agents of community and nosocomial infections in our setting. Correlation of the presence of resistance genes with the MICs indicate that the blaCTX-M was more effective in driving higher MICs of ceftriaxone and ceftazidime in both K pneumoniae and E coli isolates. All ESBL producing E coli and K pneumoniae isolates had ceftriaxone MIC50 and MIC90 of ≥256 ug/mL. Ceftazidime MIC50 and MIC90 for E coli and K pneumoniae were 12, 64 and 24, ≥256 ug/mL, respectively. This data shows that both drugs are good for the detection of ESBLs while ceftriaxone is more sensitive than ceftazidime. One isolate (positive for blaTEM) had ceftriaxone MIC 1.0 while ceftazidime MIC ≥256, another isolate (positive for blaTEM and blaCTX-M) had ceftazidime MIC 1.0 while ceftriaxone MIC was 64. This means that use of both ceftriaxone and ceftazidime MICs ensures that all ESBLs would be detected even if only phenotypic methods are used. There was good correlation between the phenotypic tests for ESBL resistance and the molecular characterization using the Check MDR platform. The molecular methodology has the advantage of providing data on the precise type of resistance gene responsible for the phenotypic characteristic seen. This is of use in the clinical setting as such molecular data provides important baseline information useful for instituting effective control measures. In addition, the use of the molecular methodology is useful for the detection of isolates harboring genes encoding for carbapenemase production in particular blaOXA-48. The low MICs of carbapenems, which blaOXA-48-producing isolates often display, makes their detection difficult, thus facilitating dissemination. Current meropenem susceptibility breakpoints often fail to detect CRE isolates in areas with a high prevalence of blaOXA-48 producers.10 In addition, ertapenem resistance has been shown to have a low positive P value for the detection of carbapenemase production, in low-prevalence setting.11 Our findings reflect these challenges as both blaOXA-48 positive isolates were sensitive to meropenem using current guidelines. Without the use of molecular methodology, these CRE would have been missed and disseminated in the healthcare facility. Our findings indicate the need for specific and sensitive methods for detection of blaOXA-48 producers in clinical microbiology laboratories. Both blaOXA-48 isolates in this study were resistant to piperacillin/tazobactam. It has been suggested that piperacillin/tazobactam and temocillin can be used as highly sensitive surrogate markers for CRE.10 We conclude that MIC methods (E-test) for ceftriaxone and ceftazidime correlate well with molecular classification of ESBLs-producing Enterobacteriaceae especially for E coli. We recommend performing this test for screening purposes in clinical laboratories. Figure 1 Demographic distribution of patient population. Table 1 Distribution of ESBL genotypes (number of isolates). E coli (n=50) K pneumoniae n=27) Total (n=77) Distribution of the single ESBL gene blaCTX-M only 34 2 36 (46.75%) blaSHV only 0 4 4 (5.2%) blaTEM only 1 0 1 (1.3%) Total # of isolates with single ESBL gene 35 6 41 (53.2%) Distribution of the ESBL gene combinations blaTEM + blaCTX-M 12 1 13 (16.9%) blaSHV + blaCTX-M 1 12 13 (16.9%) blaTEM + blaSHV + blaCTX-M 0 7 7 (9.1%) blaCTX-M + blaTEM + blaOXA-48 1 0 1 (1.3%) blaCTX-M + blaOXA-48 0 1 1 (1.3%) Total # of isolates with ≥2 genes 14 21 35 (45.45%) Table 2 Comparative analysis of ceftriazone and ceftazidme MIC in isolates habouring different combinations of resistance genes. Antibiotic MIC values MIC50 MIC90 Isolates with CTX-M gene only Ceftriaxone K pneumoniae (n=2) 48 256 E coli (n=34) 256 256 Ceftazidime K pneumoniae (n=2) 1.5 32 E coli (n=34) 12 48 Isolates with CTX-M + TEM only Ceftriaxone K pneumoniae (n=1) 96 96 E coli (n=12) 256 256 Ceftazidime K pneumoniae (n=1) 1.5 1.5 E coli (n=12) 16 64 Isolates with CTX-M + SHV only Ceftriaxone K pneumoniae (n=12) 256 256 E coli (n=1) 256 256 Ceftazidine K pneumoniae (n=12) 24 256 E coli (n=1) 12 12 ==== Refs REFERENCES 1 Batchelor M Threlfall EJ Liebana E Cephalosporin resistance among animal-associated enterobacteria: a current perspective Expert Rev Anti Infect Ther 2005 3 3 403 417 15954857 2 Livermore DM Canton R Gniadkowski M Nordmann P Rossolini GM Arlet G CTX-M: changing the face of ESBLs in Europe J Antimicrob Chemother 2007 59 2 165 174 17158117 3 Bauernfeind A Casellas JM Goldberg M Holley M Jungwirth R Mangold P A new plasmidic cefotaximase from patients infected with Salmonella typhimurium Infection 1992 20 3 158 163 1644493 4 Bonnet R Growing group of extended-spectrum beta-lactamases: the CTX-M enzymes Antimicrob Agents Chemother 2004 48 1 1 14 14693512 5 Clinical and Laboratory Standards Institute Performance standards for antimicrobial disk susceptibility test 9th ed Approved standard M2–A9 and M 100 Villanova, PA Clinical and Laboratory Standards Institute 2013 6 Mokaddas EM Abdulla AA Shati S Rotimi VO The technical aspects and clinical significance of detecting extended-spectrum betalactamase-producing Enterobacteriaceae at a tertiary-care hospital in Kuwait J Chemother 2008 20 4 445 451 18676224 7 Al-Zarouni M Senok A Rashid F Al-Jesmi SM Panigrahi D Prevalence and antimicrobial susceptibility pattern of extended-spectrum beta-lactamase-producing Enterobacteriaceae in the United Arab Emirates Med Princ Prac 2008 17 1 32 36 8 Al-Agamy MH Shibl AM Tawfik AF Prevalence and molecular characterization of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in Riyadh, Saudi Arabia Ann Saudi Med 2009 29 4 253 257 19587523 9 Bindayna KM Senok AC Jamsheer AE Prevalence of extended-spectrum beta-lactamase-producing Enterobacteriaceae in Bahrain J Infect Public Health 2009 2 3 129 135 20701872 10 Huang TD Poirel L Bogaerts P Berhin C Nordmann P Glupczynski Y TEMocillin and piperacillin/tazobactam resistance by disc diffusion as antimicrobial surrogate markers for the detection of carbapenemase-producing Enterobacteriaceae in geographical areas with a high prevalence of OXA-48 producers J Antimicrob Chemother 2014 69 2 445 450 24055766 11 Cohen Stuart J Leverstein-Van Hall MA Dutch Working Party on the Detection of Highly Resistant Microorganisms Guideline for phenotypic screening and confirmation of carbapenemases in Enterobacteriaceae Int J Antimicrob Agents 2010 36 3 205 210 20598859