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Evaluation of Virulence Factors and Antibiotic Resistance Patterns in Clinical Urine Isolates of Klebsiella Pneumoniae in Semnan, Iran

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Evaluation of Virulence Factors and Antibiotic Resistance Patterns in Clinical Urine Isolates of Klebsiella Pneumoniae in Semnan, Iran View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by eprints Iran University of Medical Sciences Jundishapur J Microbiol. 2018 July; 11(7):e63637. doi: 10.5812/jjm.63637. Published online 2018 June 9. Research Article Evaluation of Virulence Factors and Antibiotic Resistance Patterns in Clinical Urine Isolates of Klebsiella pneumoniae in Semnan, Iran Ali Jazayeri Moghadas,1 Farzaneh Kalantari,2 Mohammad Sarfi,3 Soroush Shahhoseini,3 and Shiva Mirkalantari4, * 1Bacteriology and Virology Department, Medicine Faculty, Semnan University of Medical Sciences, Semnan, Iran 2Laboratory Technical Officer, Shafa Hospital, Semnan, Iran 3Student Research Committee, Semnan University of Medical Sciences, Semnan, Iran 4Department of Microbiology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran *Corresponding author: Shiva Mirkalantari, Department of Microbiology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran. E-mail: [email protected] Received 2017 November 04; Revised 2018 April 19; Accepted 2018 April 29. Abstract Background: Klebsiella pneumoniae as an opportunistic pathogen can be the cause of a range of nosocomial and community - ac- quired infections. Many virulence factors help these bacteria overcome an immune system and cause various diseases. K1 and K2 capsular antigens, also magA, wcaG, and rmpA are well - known K. pneumoniae virulence factors. Klebsiella pneumoniae has been re- vealed to have the ability to acquire resistance to many antibiotics, which cause treatment failure. Objectives: This study aimed at determining the prevalence of magA, wcaG, rmpA, Capsular type K1, Capsular type K2, TEM, and SHV in K. pneumoniae isolates. Methods: A total of 173 non - duplicate K. pneumoniae isolates were collected from two different hospitals in Semnan, Iran, from urine specimens. Klebsiella pneumoniae was identified by conventional bacteriological tests. Disk diffusion test was performed according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). Detection of virulence factors, TEM, and SHV gene was performed by specific primers. Results: Frequency of virulence factors was as follow: capsular type K2: 32.9%, rmpA: 20.2%, capsular type K1: 6.9%, and wcaG: 16.2%. Also, the SHV and TEM were observed in 46.8% and 33.5%, respectively. Antibiotics resistance rates were as follow, imipenem: 7.5%, ciprofloxacin: 16.1%, levofloxacin: 17.3%, amoxicillin - clavulanic acid: 30%, trimethoprim - sulfamethoxazole: 32.9%, cefepime: 34.1%, nitrofurantoin: 35.8%, amikacin: 36.4%, aztreonam: 39.3%, ceftazidime: 42.7%. Conclusions: Frequency of some virulence factors including capsular type K2, rmpA, wcaG, and also resistant rate to imipenem, amikacin, and ceftazidime were significantly higher than similar studies. Presence of virulence factors accompanied by drug resis- tance should make bacteria an infectious agent and lead to treatment failure. Keywords: Klebsiella pneumoniae, Virulence Factors, Antibiotic Resistance 1. Background capsule is one of the most important virulence determi- nants, which helps achieve biofilm formation and leads to Klebsiella pneumoniae, as an opportunistic pathogen, protection against phagocytosis, antimicrobial peptides, can be the cause of a range of nosocomial and commu- and serum bactericidal activity. There are more than 77 nity - acquired infections (1-3). Pathogenic K. pneumoniae types of capsular antigens in K. pneumoniae strains, of strains are responsible for urinary tract, respiratory, and which K1 and K2 serotypes are well known (9, 10). blood infections and are associated with mortality and Klebsiella pneumoniae also carries virulence - associated morbidity in patients (4-6). Pathogenicity of K. pneumo- genes, which may encode capsules (magA, K2A, WcaG) and niae is the result of production of many virulence factors a capsular regulator gene (regulator of mucoid pheno- that help these bacteria overcome the immune system and type (rmpA). Mucoviscosity - associated gene A (magA) and cause various diseases. Different virulence factors, such as WcGA genes located within the gene clusters are responsi- lipopolysaccharide (o - antigen), capsular polysaccharides ble for encoding capsular polymerase and capsular biosyn- (K antigen), fimbriae and siderophores contribute to the thesis, respectively (11-13). Capsular biogenesis by magA is pathogenicity of Klebsiella (7,8). Among these factors, the achieved through conversion of manose to fucose, which Copyright © 2018, Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited Jazayeri Moghadas A et al. may enhance the ability of the bacteria to evade phagocy- stain was performed for suspected colonies. Biochemi- tosis by macrophage. The regulator of mucoid phenotype cal tests, including lactose fermentation, gas production A(rmpA) gene located on the plasmid increases capsular from glucose, FeS production, motility, indole production, polysaccharide production and is found in the hypermu- sodium citrate utilization, and urea utilization were used coviscous phenotype. Moreover, two genes, including the for K. pneumoniae identification (21). magA and rmpA, were initially associated with invasive in- fections (14, 15). 3.4. Antibiotic Susceptibility Testing Resistance of pathogenic bacteria to different antibi- otics has become a serious worldwide problem because Disks diffusion test was performed according to the of the fatal outcome of defective treatment and the dif- guidelines of the Clinical and Laboratory Standards In- ficulty to find treatment options (16). Klebsiella pneumo- stitute (22). Used disks (Rosco, Taastrup, Denmark) were niae has been revealed to have the ability to acquire re- amikacin (30 µg), ciprofloxacin (5 µg), amoxicillin - clavu- sistance to many antibiotics, especially third generation lanic acid (20/10 µg), ceftazidime (30 µg), imipenem (10 cephalosporins. Multidrug resistance of K. pneumoniae µg), cefepime (30 µg), aztreonam (30 µg), nitrofurantoin against the antibiotics used for therapy intensifies the vir- (300 µg), and trimethoprim - sulfamethoxazole (1.25/23.75 ulence potential of Klebsiella. Beta - lactam antibiotics are µg). Escherichia coli (ATCC 25922) strain was used for qual- one of the most commonly used antibiotics in the treat- ity control. ment of bacterial infections and the production of B - lacta- mase enzymes are the most common bacterial resistance 3.5. DNA Extraction mechanisms (17, 18). In the recent years, Extended Spec- trum Beta Lactamase (ESBL) producer K. pneumoniae have DNA of each K. pneumoniae isolate was extracted from increased over the world. The ESBLs are divided to several 1 mL of overnight bacterial culture. Extraction was per- groups; the main groups are TEM, CTX, and SHV derivatives formed as previously described by Kuske et al. (23). The su- (19, 20). pernatant was used as the template DNA in PCR reactions. 2. Objectives 3.6. Detection of Capsular type K1, Capsular type K2, rmpA, wcaG, TEM, and SHV The present study aimed at determining the preva- Specific primers for detection of Capsular type K1, Cap- lence of magA, wcaG, rmpA, Capsular type K1 and Capsular sular type K2, rmpA, wcaG, TEM, and SHV are shown in Ta- type K2 virulence genes along with SHV and TEM beta lac- ble 1. For capsular type K1, capsular type K2, rmpA, and tam resistance genes in K. pneumoniae isolates. wcaG amplification were performed as follow: initial de- naturation at 95°C for five minutes, followed by 35 cycles 3. Methods at 94°C for 30 seconds, 58°C for 90 seconds and 72°C for 90 seconds and a final extension at 72°C for 10 minutes (24). 3.1. Ethics Statement Polymerase Chain Reaction (PCR) conditions for TEM am- The ethics committee of the Semnan University of plification were as follow: initial denaturation at 94°C for Medical Sciences, Semnan, Iran, approved this study three minutes, 35 cycles at 94°C for 30 seconds, 45°C for one (IR.SEMUMS.REC 1394.29). minute, 72°C for one minute, final extension at 72°C for 10 minutes (25). The SHV was amplified under the following 3.2. Sampling conditions: 94°C for three minutes, 35 cycles at 94°C for 30 minutes, 60°C for one minute, 72°C for one minute, and a A cross sectional study was performed. A total of 173 final extension at 72°C for 10 minutes (25). The PCR prod- non - duplicate K. pneumoniae isolates were collected dur- ucts were analyzed by electrophoresis with 1% agarose gel ing year 2015 at two different hospitals of Semnan, Iran, in 1X Tris - Acetate - EDTA buffer. The gels were stained with from urine specimens. ethidium bromide and the PCR products were visualized under UV light (Figure 1). 3.3. Bacterial Identification The specimens were cultured on blood agar and Eosin 3.7. Statistical Analysis Methylene Blue (EMB) agar (Merck, Darmstadt, Germany), and incubated at 37°C for 24 hours. Klebsiella pneumoniae Confidence interval test was used to assess the statisti- was identified by conventional bacteriological tests. Gram cal significance with confidence level of 95% (α = 0.05). 2 Jundishapur J Microbiol. 2018; 11(7):e63637. Jazayeri Moghadas A et al. Table 1. Specific Primers for Detection of Capsular Type K1, Capsular Type K2, rmpA, wcaG, TEM, and SHV Gene Primer Sequence Product Size (bp) Reference Capsular type K1 MagA - F
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