In the recent years, one of the most issues of concern among healthcare systems is resistance to carbapenems among Enterobacteriaceae due to increasing morbidity and mortality among hospitalized patients.1 Carbapenems are considered as the last-line treatment of Enterobacteriaceae infections and resistance to them is a great challenge for clinicians.1,2 Polymyxins are effective alternatives for carbapenem-resistant isolates; however, because of their nephrotoxic effect, they are not recommended for treatment.2
One of the most important emerging carbapenem-resistant bacteria is Klebsiella pneumoniae (K. pneumoniae)2 which is an opportunistic pathogen which accounts for many of nosocomial infections including urinary tract infections, pneumonia, and septicemia.3 Three main classes of carbapenemases were introduced including class A or serine protease, particularly the K. pneumoniae carbapenemase (KPC) that was mostly identified in K. pneumoniae.4 KPC is able to hydrolyze penicillins, carbapenems, cephalosporins and aztreonam.5 Class B metallo-beta-lactamase (MBL) is sensitive to metalic ion chelator like ethylenediaminetetraacetic acid (EDTA) because it is zinc dependent.6 New Delhi metallo-β-lactamase (NDM), imipenemase (IMP) and Verona integrin-encoded metallo-β-lactamase (VIM) are the most important enzymes in this class.6,7 Recently, NDM-1 has spread widely in the world.4 Class D, oxacillinase (OXA), has different variants by deletions and substitutions in a few amino acids in these types of enzymes which cause variations in hydrolyzing penicillins and carbapenems.8
Due to increasing risk of infections caused by carbapenem-resistant K. pneumoniae strains and lack of comprehensive local information, studying the frequency of resistant genes and identifying appropriate antibiotics are essential for the control of such infections. The present study aimed to investigate the antibiotic susceptibility pattern of K. pneumoniae isolates and determine the frequency of carbapenemase producing K. pneumoniae obtained from Iranian hospitalized patients by phenotypic and genotypic methods.
2.1. Study design and bacterial isolates
This cross-sectional study was performed at two major teaching Hospitals (Nemazee and Faghihi), in Shiraz, Iran during one year from March 2014 to 2015. The study was approved by regional Ethics Committee and was in accordance with the declaration of Helsinki. A total of 211 K. pneumoniae were isolated from different clinical samples. The bacteria were identified and confirmed using standard microbiologic methods. The samples were cultured on MacConkey agar (Merck, Germany) and the plates were incubated at 37 °C for 24 h. Up to five lactose-fermenting colonies were selected separately and subjected to routine biochemical tests; after that, the isolates were approved by API 20E (bioMérieux, La Balme-les-Grottes, France).
2.2. Antibiotic susceptibility tests
Antibiotic susceptibility testing was performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines using disk diffusion method.9 Antibiotic disks were (MAST, United Kingdom) ceftazidime (CAZ: 30 μg), ciprofloxacin (CIP: 5 μg), cefepime (CPM: 30 μg), piperacillin (PRL: 100 μg), ampicillin (A: 25 μg), polymyxin B (PB: 300 U), tigecycline (TGC: 15 μg) piperacillin/tazobactam (PTZ: 110 μg), aztreonam (ATM: 30 μg), gentamicin (GM: 10 μg), amikacin (AMK: 30 μg) amoxicillin/clavulanic acid (AUG: 30 μg), imipenem (IMP: 10 μg), and meropenem (MEM; 10 μg) which were used for antibiotic susceptibility tests. The CLSI interpretive criteria for Pseudomonas aeruginosa were applied to determine the susceptibility to polymyxin B. Escherichia coli ATCC 25922 was used as the quality control strain. The minimum inhibitory concentration (MIC) for imipenem was determined by E-test (Liofilchem, Italy) method.
2.3. Carbapenemase screening
Modified Hodge test (MHT) was performed to identify the carbapenemase producing K. pneumoniae isolates. Briefly, according to the CLSI recommendation, E. coli 25922 was streaked in Muller-Hinton agar, and then the meropenem disk was located in the center of a plate and test strain was streaked on a line around the meropenem disk; the result was observed after 18–24 h. To detect MBL (metallo-beta-lactamase) producing isolates phenotypically, we did double disk synergy test (DDST). Therefore, 0.5 M EDTA solution was prepared by dissolving 186.1 g of disodium EDTA. 2H2O in 1000 mL distilled water, and the pH was adjusted to 8 by adding NaOH. Then, 930 μg of the prepared solution was added to the imipenem disk, and dried in an incubator. The prepared EDTA-imipenem disk and the imipenem disk itself were placed in a plate containing Muller-Hinton agar with cultured K. pneumoniae. After 16–18 h of incubation at 37 °C, the result was considered.
2.4. DNA extraction and molecular assays
DNA extraction from the studied isolates was done by boiling method, as described previously.4 PCR amplification for detection of blaKPC, blaOXA-48-like,blaNDM, blaVIM, and blaIMP genes was carried out on a Veriti 96-well thermal cycler instrument (Applied Biosystems at Life Technologies, Foster City, CA), as described previously.10 The primers were used at concentrations of 0.3–0.4 μM. They were provided by Takapoo zist Co, Tehran, Iran. The primer sequences and amplicon sizes are listed in Table 1. Amplification reaction was performed in a final volume of 25 μL containing 200 μM concentrations of dNTPs, 1.5 mM MgCl2, 2 U taq polymerase (CinnaGen, Iran) and 3 μL DNA templates. Positive controls for targeted genes were kindly obtained from Pasteur Institute, Tehran, Iran. The PCR program consisted of an initial denaturation step at 95 °C for 5 min, followed by 35 cycles of DNA denaturation at 95 °C for 45 s, primer annealing for 45 s(Temperature was depending on the sequences of primers), and primer extension at 72 °C for 1 min, followed by a final extension at 72 °C for 8 min. The PCR products were analyzed by electrophoresis on 1.5% agarose gels in 0.5 × Tris- Acetate-EDTA (TAE) buffer. The gels were stained with KBC stain (CinnaGen, Iran) and the PCR products were visualized with UV light.
2.5. DNA sequencing
The purified PCR products of blaNDM positive isolates were sequenced using the ABI capillary system (Macrogen Research, Seoul, Korea). Then, the sequences were compared using online BLAST software (http://www.ncbi.nlm. nih.gov/BLAST/), and confirmed as NDM-1 variant. So far, these sequences in the GenBank nucleotide database under accession numbers: KU577461.1, KU577460.1, KU577459.1, KU577458.1, KU570060.1, KU570059.1, KU570058.1, KU543692.1, KU543691.1, KU543690.1, KU341526.1, KU341525.1, KU341524.1, KU248755.1, KU198639.1, KU198638.1, KU198637.1, KU198636.1, KU162940.1, KT365396.1, KT365395.1, KT365394.1, KT365393.1, KT365392.1, KT347222.1 are available on the Internet at the National Center of Biotechnology Information website (http://www.ncbi.nlm.nih.gov).
2.6. Statistical analysis
Analysis was performed using SPSS™ software, version 21.0 (IBM Corp., USA). The results are presented as descriptive statistics in terms of relative frequency. Values were expressed as the percentages of the group (categorical variables). Chi–square or Fisher's exact tests were used to determine the significance of the differences. A difference was considered statistically significant if the p value was less than 0.05.
Totally, of 211 confirmed K. pneumoniae isolates, 104 (49.3%) were obtained from female and 107 (50.7%) from male subjects. Meanwhile, 103 (48.18%) isolates were obtained from hospitalized patients in the intensive care units (ICUs). The results of antibiotic susceptibility showed that all the isolates were resistant to ampicillin, and then mostly resistant to piperacillin and ceftazidime with 76.3% and 66.8%, respectively. On the other hand, the highest sensitivity was toward polymyxin B, followed by carbapenem. The full results of antibiotic resistance pattern of K. pneumoniae isolates are presented in Table 2. Despite the higher frequency of carbapenem-resistant isolates in ICUs, compared to other wards, the differences were not statistically significant. Moreover, there was no statistical association between the source of infections and rate of carbapenem-resistance.
Of 29 carbapenem-resistant K. pneumoniae isolates, all were high-level imipenem-resistant (MIC ≥ 4), except for 4 isolates. The lowest and highest level of imipenem MIC for carbapenem-resistant isolates were estimated 1.5 mg/L and >32 mg/L, respectively. DDST results showed that 27/29 (93.1%) carbapenem-resistant isolates were MBL producing ones. The results of MHT revealed that 27 of 29 carbapenem-resistant isolates were carbapenemase producing ones. Meanwhile, the two phenotypic negative carbapenemase and MBL producing isolates were different.
In PCR assay, none of the isolates contained blaKPC and blaIMP genes. The presence of blaOXA-48-like gene was detected in 2 (0.9%) isolates and confirmed by sequencing of amplicons. Two blaOXA-48-like positive isolates were carbapenem-resistant K. pneumoniae. First, one was taken from the sputum of a 55 year old man in the ICU in his seventh day of hospitalization and the second one was taken from blood culture of a 51 year old man in his fourth day of hospitalization in ICU.
The presence of blaNDM gene was identified in 27 (10.9%) isolates, 23 of which were carbapenem-resistant K. pneumoniae. All of 4 blaNDM positive and carbapenem-susceptible isolates originated from newborns with an age range of 2 days–7 months old who were hospitalized in neonatal intensive care units (NICUs). Sequencing results of the isolates harboring blaNDM confirmed all of them as NDM-1 variant. The detailed results of 29 carbapenem-resistant isolates are shown in Table 3.
In the present study, we described the occurrence of carbapenem producing K. pneumoniae isolates with phenotypic and genotypic methods. One of the most important finding of the present study is the high prevalence of blaNDM-1 gene in the collected isolates. Previously, it has been shown that the majority of the blaNDM-1 harboring isolates were from Asian countries, mostly India, Pakistan and China.11 To the best of our knowledge, our study is the first report of K. pneumoniae harboring blaNDM-1 in the Southwestern Iran. In the present study of 29 carbapenem-resistant isolates, 79.3% were blaNDM-1 positive. In the recent years, blaNDM-1 has been identified in different countries such as Iran as well. Shahcheraghi et al. from Tehran (North of Iran) in 2013 reported the first detection of blaNDM-1 containing K. pneumoniae isolate.12 Also, Nobari et al. in Tehran 2009–2012, identified 3 blaNDM positive in 42 carbapenem-resistant K. pneumoniae isolates.4 In the recent years, Fazeli et al. from Isfahan 2012–2013, among 49 carbapenem-resistant K. pneumoniae isolates, detected 6 blaNDM-1 positive.13 One general concept from previous studies and our results is the increasing trend of blaNDM-1 harboring K. pneumoniae strains in Iranian hospitals, which can be a serious concern. Recently, closest to our finding, from our neighboring countries in Persian Gulf Cooperation Council (Saudi Arabia, United Arab Emirates, Oman, Kuwait, Qatar, and Bahrain), the most common carbapenemases were reported OXA-48 (35 isolates) and NDM (16 isolates) types, and no KPC-type or IMP-type were detected.14 One of the important findings in this study was that 4 isolates were sensitive to imipenem and meropenem, MHT and DDST were negative too, but the existence of blaNDM-1 was positive by PCR, and after sequencing it was confirmed. This can be due to the fact that beta-lactamase gene expression can be controlled by several mechanisms.15–17 Of course, as one of our limitations those isolates need to be evaluated by real time PCR; then, we may have a better understanding about the reason of loss of beta-lactamase expression.
In this study, blaOXA-48-like gene was detected in two isolates by PCR assay and then verified by sequencing; these two isolates were positive in MHT and unlike NDM positive isolates, their DDST was negative. Their antibiotic susceptibility profile was similar to other carbapenem-resistant isolates and they were resistant to all the tested antibiotics, except for one isolate which was sensitive to polymyxin B. blaOXA-48-like belongs to class D carbapenemase (oxacillinase) that has six variants which differ in few amino acids deletion or substitutions, and OXA-48 is the most common variant.18 Previously, the only report of blaOXA-48 gene in Iran was by Azimi et al. from Tehran 2014, in 27 carbapenem-resistant K. pneumoniae isolates recovered from burn patients.19 The widespread prevalence of OXA-48-positive carbapenem-resistant K. pneumoniae strains has been reported in several Asian and European countries including Turkey, Saudi Arabia, Taiwan, China, Russia and France which has become an expanding problem.20–25
In our findings, we cannot find any of the targeted genes in 4 carbapenem-resistant isolates, so their resistance to carbapenems may be due to other mechanisms including production of extended-spectrum beta-lactamases (ESBLs), AmpC beta-lactamases, decreased permeability of outer membrane or efflux pumps activity, or probably the presence of other genes that were not studied in this research.26,27 In the present study, the rate of antibiotic resistance with some variation which may arise from the source of infections and geographical distribution was comparable with the previous reported studies in Iran.3,12,14,28
This is the first identification of blaNDM-1 and blaOXA-48-like in K. pneumoniae in Southwestern Iran and the highest reported prevalence of blaNDM in this bacterium in Iran. Since carbapenem-resistant isolates containing NDM-1 were almost resistant to all the tested antibiotics, the resistance due to this gene may be increased in the near future as a potential health threat. Therefore, due to the increased prevalence of blaNDM, as determined in our study, we recommend restricted carbapenem prescription and effective infection control polices for preventing the spread of resistant strains.
We thank all personals at the Nemazee hospital Microbiology laboratory for their friendly cooperation. This study was supported by Shiraz University of Medical Sciences, grant No. 93–7139. This article extracted from the MSc thesis by Ms. Zahra Hosseinzadeh under the supervision of Dr. M. Motamedifar. The authors wish to thank Dr. Nasrin Shokrpour at the Research Consolation Centre (RCC) at Shiraz University of Medical Sciences for her invaluable assistance in editing this manuscript.
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