pISSN 2093-2340   eISSN 2092-6448
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Open Access, Peer-reviewed
    Infect Chemother. 2023 Mar;55(1):90-98. English.
    Published online Mar 17, 2023.
    https://doi.org/10.3947/ic.2022.0160
    Copyright © 2023 by The Korean Society of Infectious Diseases, Korean Society for Antimicrobial Therapy, and The Korean Society for AIDS
    Original Article

    High Frequency of blaOXA-48like Producing Klebsiella pneumoniae Isolated from Nosocomial Infection in Azerbaijan, Iran

    Reza Ghotaslou,1,2 Behnaz Salahi,3 Ghazal Naderi,4 and Naser Alizadeh5
      • 1Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
      • 2Central Laboratory of the Province, Tabriz University of Medical Sciences, Tabriz, Iran.
      • 3Razi Hospital, Tabriz University of Medical Sciences, Tabriz, Iran.
      • 4Division of Microbiology, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
      • 5Zanjan Applied Pharmacology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran .
    • Corresponding Author: Naser Alizadeh, Assistant Professor. Zanjan Applied Pharmacology Research Center, Zanjan University of Medical Sciences, Gavazang road, Zanjan, Iran. Tel: +98-24-3314-0232, Email: alizadeh.n@zums.ac.ir
    Received November 12, 2022; Accepted January 17, 2023.

    This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Abstract

    Background

    Klebsiella pneumoniae is one of the significant agents of hospital-acquired infections. In recent years, carbapenem-resistant K. pneumoniae (CRKP) isolates have been found in numerous epidemics of nosocomial infections. This study aimed to determine carbapenem resistance mechanisms and molecular epidemiological of CRKP infections in Azerbaijan, Iran.

    Materials and Methods

    A total of 50 non-duplicated CRKP from January 2020 to December 2020 were isolated form Sina and Imam Reza Hospitals in Tabriz, Iran. Antimicrobial susceptibility testing was performed by the disk-diffusion method. The carbapenem resistance mechanisms were determined by the phenotypic and PCR procedures. CRKP isolates were typed by the Random Amplified Polymorphic DNA PCR (RAPD-PCR) technique.

    Results

    Amikacin was the most effective antibiotics against CRKP isolates. AmpC overproduction was observed in five CRKP isolates. Efflux pump activity was found in one isolate by the phenotypic method. Carba NP test could find carbapenemases genes in 96% of isolates. The most common carbapenemases gene in CRKP isolates were blaOXA-48-like (76%) followed by blaNDM (50%), blaIMP (22%), blaVIM (10%), and blaKPC (10%). The outer membrane protein genes (OmpK36 and OmpK35) were identified in 76% and 82% of CRKP isolates, respectively. RAPD-PCR analysis yielded 37 distinct RAPD-types. Most blaOXA-48-like positive CRKP isolates were obtained from patients hospitalized in intensive care unit (ICU) wards with urinary tract infections.

    Conclusion

    The blaOXA-48-like is the main carbapenemase among CRKP isolates in this area. Most blaOXA-48-like producer CRKP strains were collected from the ICU ward and urine samples. To control infections due to CRKP, a strict control program in hospital settings is required.

    Graphical Abstract

    Keywords
    Carbapenemase; Klebsiella pneumoniae; Random Amplified Polymorphic DNA PCR

    Introduction

    Klebsiella pneumoniae is an important opportunistic pathogen and a member of the Enterococcus faecium, Staphylococcus aureus, K. pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (ESKAPE) pathogens that have experienced the greatest challenges in the world [1, 2]. They are due to a wide range of infections in humans, including bloodstream infections, liver abscesses, types of pneumonia, and urinary tract infections (UTIs) [3]. Infections caused by these isolates in clinical settings have been associated with morbidity and mortality rates higher than 40 - 50% [4]. Management of hospital-acquired isolates of multidrug-resistant K. pneumoniae is a major challenge and therapeutic options of these infections become limited [5, 6]. Unfortunately, the appropriate treatment option for carbapenem-resistant K. pneumoniae (CRKP) infections until now, not well recognized. Combination antibiotic therapies with tigecycline, colistin, aminoglycosides, and fosfomycin have been commonly used, while the treatment results were usually unacceptable [7, 8].

    Extensive use of carbapenems in the treatment of extended-spectrum β-lactamase (ESBL) infections has led to the appearance and spread of CRKP isolates [1, 5]. Some resistance mechanisms to carbapenem are found in K. pneumoniae, as well as co-resistance to β-lactams antibiotics have been reported [1]. The major carbapenemase genes in K. pneumoniae are the metallo-β-lactamases such as VIM, IMP, and NDM types, the KPCs (KPC-2 to KPC-13), and the OXA-type enzymes [9]. In addition, CRKP can be developed via up-regulation of efflux pumps (AcrAB–TolC multidrug efflux pump) and loss and alteration of outer membrane porins (such as OmpK35 and OmpK36), and over-production of ESBL enzymes or AmpC β-lactamases [10].

    Notably, K. pneumoniae readily colonizes human mucosal surfaces, including the surface of the oropharynx and gastrointestinal tract. In clinical settings, there are numerous possible sources of K. pneumoniae [11]. The main source of expansion is person-to-person interaction (especially health staff to patients); contaminated surfaces and tools [1, 11]. There are several procedures to assess the genetic diversity and clonal lineage of Enterobacteriaceae including pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), multiple-locus variable-number tandem repeat analysis (MLVA), enterobacterial repetitive intergenic consensus sequences PCR (ERIC-PCR), and random amplified polymorphic DNA (RAPD) [12]. RAPD-PCR is an easy, fast, and cost-effective method that can be done for the evaluation of clonality [13]. Molecular-based approaches for carbapenem-resistant Enterobacteriaceae detection usually detect the presence of one or more carbapenemase genes as well as appropriate for screening a broad spectrum of carbapenemase genes, including at least the five most common families, and overcome most of the limitations of culture-based procedures.

    In this study, we aimed to characterize CRKP isolated from patients with nosocomial infections. We also used the RAPD-PCR-based dendrogram to assess the clonality CRKP isolates.

    Methods and materials

    1. Sample collection and isolates identification

    This study was done on 87 consecutive non-duplicate carbapenem-resistant Enterobacteriaceae isolated from inpatients during a 9-month period from January 2020 to December 2020 at Sina and Imam Reza Hospitals in Tabriz, Iran. These isolates were initially recognized via standard bacteriological tests. The inclusion criteria for CRKP were a decline in vitro susceptibility to as a minimum one carbapenem antibiotic (imipenem, meropenem, and ertapenem) agreeing to the Clinical and Laboratory Standards Institute (CLSI) guideline.

    2. Ethics statement

    This study was approved by the Ethical Committee of Tabriz University of Medical Sciences, Tabriz (IR.TBZMED.VCR.REC.1397.03). We received patient informed consent by the Ethical Committee of Tabriz University of Medical Sciences, Tabriz, Iran.

    3. Antimicrobial susceptibility patterns

    The disk-diffusion technique was used for performing antimicrobial susceptibility testing of the K. pneumoniae isolates to antibiotics according to CLSI guidelines [14]. The following antibiotic disks (Mast Ltd., Manchester, England) were used imipenem (10 µg), meropenem (10 µg), ertapenem (10 µg), aztreonam (30 µg), cefixime (5 µg), ceftazidime (30 µg), cefotaxime (30 µg), cefepime (30 µg), cefazolin (30 µg), tobramycin (10 µg), gentamicin (10 µg), amikacin (30 µg), tetracycline (30 µg), trimethoprim/sulfamethoxazole (1.25/23.75 μg) and piperacillin/tazobactam (100/10 µg). The results of antibiotics sensitivity and resistance patterns were corroborated via the American Type Culture Collection (ATCC) quality control strain Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 (Iranian Research Organization for Science and Technology, Tehran, Iran).

    4. Phenotypic detection of CRKP

    AmpC hyper-production was performed consistent with a technique defined by Martínez et al [15]. When there was a dilution difference of at least twofold between the MICs of imipenem and imipenem plus cloxacillin, the isolates were considered to be AmpC overproducers. To detect efflux pump activity according to micro broth dilution method, MICs of meropenem in combination with CCCP as an inhibitor of RND pumps K. pneumoniae was performed. Finally, CRKP was confirmed by the Carba NP test (bioMerieux, Shanghai, China) as described previously [16].

    5. Molecular detection of carbapenemases genes and porin coding genes

    A loopful of bacteria was suspended in 3 ml of TE buffer (10 mM Tris-HCL, 1 mM EDTA, pH 8.0) and placed at 80 °C for 20 min to kill the bacteria. DNA was extracted by CTAB method. PCR tests were done using specific primers for the blaKPC,blaVIM,blaIMP,blaNDM, and blaOXA-48-like carbapenemases coding genes, as well as OmpK36 and OmpK35 as porin coding genes [13]. Gel electrophoresis was performed for 40 min on a 1.5% agarose gel at 80 V and after staining with safe stain visualized under UV light. P. aeruginosa (blaVIM and blaIMP,) and K. pneumoniae (blaKPC,blaNDM, and blaOXA-48-like) were used as controls in this study.

    6. Random Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD-PCR)

    Genomic DNA of K. pneumoniae isolates was extracted using a DNA extraction kit following the manufacturer’s instructions. A DNA template with primer (5-AAGACGCCGT-3) was used for RAPD-PCR [13].

    7. Statistical analyses

    The results were investigated by the SPSS software for Windows (version 23.0, IBM Corporation, Seattle, Washington DC, USA). P-value <0.05 was reflected as statistically significant.

    Results

    Of 87 carbapenem-resistant Enterobacteriaceae isolates that were resistant to as a minimum one of the imipenem, ertapenem, and meropenem antibiotics established on standard bacteriological tests, 50 CRKP isolates (58%) were identified. The mean age of patients was 58 ± 20 years and 30 males and 20 females were involved in the study. Urine was the most common specimen source (42%) followed by wound (28%), blood (22%), and fluids (8%). The frequency of CRKP isolates recovered from hospital wards was as follows ICU (42%) and infectious (34%), burn (14%), internal medicine (8%), and emergency (2%). The antimicrobial susceptibility patterns of these isolates are shown in Figure1. A high rate of resistance was found to trimethoprim/sulfamethoxazole (86%), while 43 (86%) of isolates were susceptible to amikacin and followed by tobramycin (52%).

    Figure 1
    Antimicrobial resistance patterns of Klebsiella pneumoniae isolates.
    TOB, tobramycin; TMP/SMX, trimethoprim/sulfamethoxazole; TET, tetracycline; TZP, piperacillin/tazobactam; CFZ, cefazolin; FEP, cefepime; CTX, cefotaxime; CAZ, ceftazidime; CFM, cefixime; AZT, aztreonam; GEN, gentamicin; AMK, amikacin; IPM, imipenem/cilastatin; MEM, meropenem; ETP, ertapenem.

    The overproduction of AmpC in CRKP isolates was found in five isolates (10%). These isolates had an overproduction of AmpC by carbapenem activity with a drop of MIC up to double. Carba NP test (bioMerieux, Shanghai, China) detected carbapenemases in 48 isolates (96%). We found only one CRKP isolate (2%) with the efflux pump inhibitor presence (CCCP) revealed below a threefold decline in the MIC of meropenem among 50 CRKP isolates. The most frequently carbapenemase genes were blaOXA-48-like (n = 38; 76%) followed by blaNDM (n = 25; 50%), blaIMP (n = 11; 22%), blaVIM (n = 5; 10%), and blaKPC (n = 5; 10%). Of 50 CRKP isolates, the genes coding of OmpK35 and OmpK36 were identified in 38 (76%) and 41 (82%) CRKP isolates, respectively. The genes coding of OmpK35 and OmpK36 were detected in all CRKP recovered from blood, wound, and urine samples.

    According to the dendrogram, the CRKP isolates were distributed into 37 different RAPD types (Fig. 2), each comprising one to forty-one isolates. All isolates were typed by RAPD-PCR, but two isolate was not typed. Related isolates (similarity ≥80%) could be identified within the 5 clusters (A-E) and one major clone (Table 1) in two hospital. Most of the carbapenemases producer isolates were included in cluster A (Table 2). According to the statistical analysis tests, the RAPD-PCR genotyping did not correlate with antibiotics patterns and hospital wards. Cluster A had high frequency in ICU (n = 18), followed by infectious (n = 13) and burn (n = 6) wards. In addition, the blaOXA-48-like gene (n = 33) and urine specimen (n = 16) were most prevalent in cluster A.

    Figure 2
    Dendrogram of 48 carbapenemase-producing strains based on RAPD-PCR profiles with isolates’ characteristics. Strains 13, 14, 15, 16, 17, 24, 25, 35, 38, 41, and 47 belong to Imam Reza Hospital.
    RAPD-PCR, random amplified polymorphic DNA; ICU, intensive care unit; NDM, New Delhi metallo-β-lactamase; VIM, verona imiopenemase; IMP, imiopenemase; KPC, Klebsiella pneumoniae carbapenemase.

    Table 1
    The frequency of Klebsiella pneumoniae clusters based on wards and specimens by the RAPD-PCR method

    Table 2
    The frequency of carbapenemases genes in clusters based on RAPD-PCR profiles

    Discussion

    The CRKP isolates have been recognized as a cause of nosocomial infections and carry a multiplicity of antibiotic resistance genes [17]. Also, it is responsible for various infections including pneumonia, UTIs, bacteremia, and liver abscesses [18]. In the current study, a statistical relationship of carbapenem-resistant mechanisms, molecular typing of CRKP, and demographic characteristics was considered. For the reason that the effect of geographical characteristics on antibiotic susceptibility testing of CRKP isolates, the outcomes of this study can be useful and appropriate in the antibiotic therapy of CRKP isolates from Azerbaijan, Iran.

    In our study, the prevalence of CRKP in nosocomial infections was 58%. According to some study, reported by Paczosa et al. [19], and Lin et al. [20], more prevalence of CRKP has been described. In the current study, the frequency of these isolates in urine, wound, blood, and fluids specimens were 42%, 28%, 22%, and 8%, respectively. This finding has been reported by other studies [21] that UTIs and blood infections were the high prevalent infection caused by CRKP isolates. ICU is the main ward with cases of CRKP isolates (42%), followed by infectious (34%) and burn (14%) wards, because in this ward mechanical ventilation is more used, and is closely related to the antibiotic usage; invasive procedure and operation, and low immunity status of patients [22].

    According to the disk-diffusion test, resistance to imipenem, ertapenem, meropenem, and co-trimoxzaole were 100%, 100%, 96%, and 82% respectively, while the CRKP isolates were susceptible to amikacin and tobramycin with 14% and 52%, respectively. This finding is in agreement with previous studies [23, 24]. Consider CRKP isolates are high prevalence and high virulent, causes a wide range of nosocomial infections, multi-drug resistance; and rapid spread of the resistance to other bacteria. So, CRKP isolates possess the maximum risk to public health [25]. Nowadays, we have limited therapeutic options for the management of infections caused by CRKP [25]. The differences in the distribution of these isolates and their antibiotic susceptibility patterns might be due to the diverse carbapenemases genes, different geographical locations, and poor infection control in health care settings [9].

    In this research, AmpC over-production was detected by phenotypic methods in 10%, while the efflux pump activity with inhibitor (CCCP) was found in one isolate. In contrast, some of the studies such as Mendes et al. [26] reported a low prevalence of overexpression of the intrinsic chromosomal ampC gene, while Hoek et al. [27] shown that an AmpC-phenotype reported in 68% of isolates. The difference could be caused by the difference in the study population or different geography and patterns of antibiotic usage [28]. Related finding beforehand designated by Pottathil et al. [29] indicated that Carba NP test (bioMerieux, China) had good specificity and sensitivity in the recognition of carbapenemases-producing isolates. We detected carbapenemases-producing isolates by the Carba NP test (bioMerieux, China) in 96%. Therefore, the Carba NP test (bioMerieux, China) is a sensitive indicator for the detection of carbapenemases activity in clinical settings. This finding was also reported previously [30].

    Porins, including OmpK35 and OmpK36, play an essential part in the process by which antibiotics are able to penetrate cells, as well as in the susceptibility of bacteria to cephalosporins and carbapenems [31, 32]. In addition, it has been found that the absence of OmpK35 and OmpK36 plays a significant part in the virulence and infection caused by CRKP [31]. Reduced virulence and infection severity can result from deleting OmpK36 and OmpK35. Consistent with previous research by Wasfi et al. [31] and Ranjbar et al., [32] we found that OmpK35 and OmpK36, which code for a porin protein, were widely distributed among CRKP isolates.

    In agreement with additional study supported by Wasfi et al. [13] efflux pump activity by molecular methods was observed in K. pneumoniae isolates while some researchers could not find any over production of efflux pump by phenotypic technique [33]. Most of the K. pneumoniae isolates were lost the AcrAB efflux pump [34]. Limited information exists about the specific and exact role of the AcrAB efflux pump in the resistance of CRKP to carbapenem antibiotics.

    In the present study, blaOXA-48-like (78%) gene was the predominant carbapenemases genes, followed by blaNDM (48%), blaIMP (22%), blaVIM (12%), and blaKPC (8%) which is likeminded with the results stated by Candan et al. and Lee et al. [35, 36]. Furthermore, the blaOXA-48-like gene was most prevalent in ICU (80%) and urine specimens (80%). The plasmid-encoded OXA-48 (blaOXA-48-like) is found in K. pneumoniae and confers a low level of sensitivity to carbapenems [37].

    In the current study, the CRKP isolates were distributed into 37 different RAPD types, each type comprising one to forty-one isolates. Related isolates (similarity ≥80%) could be identified within the 5 clusters. According to the statistical correlation tests, the RAPD-PCR genotyping did not correlate with antibiotics patterns, hospital wards, and type of specimens. The results obtained by Wasfi et al. [13] showed that K. pneumoniae strains were classified into 18 RAPD-types and it was significantly associated with antibiotic susceptibility patterns, which were consistent with the study conducted by Panah et al. [38].

    Limitations of the present study include (1) the tested isolates were taken from a single city and two territory medical centers, (2) low sample size, (3) ampC and efflux pumps did not confirm by molecular methods. It is recommended to perform molecular typing by PFGE and MLST methods that might aid to find the source of resistant isolates in future studies. As the results, approving the antibiotic susceptibilities of CRKP is vital for treatment-related decision-making as well as early detection of blaOXA-48-like producer K. pneumoniae strains with quick and trustworthy tests like the Carba NP test (bioMerieux, China) and monitoring clonal relationships of the strains with molecular typing procedures may decrease the dissemination of infections due to CRKP.

    In conclusion, K. pneumoniae is the most common Enterobacteriaceae resistant to carbapenem. This study demonstrates that blaOXA-48-like production is the main carbapenem resistance mechanisms in CRKP isolates. Most blaOXA-48-like producer K. pneumoniae strains were recovered from the ICU ward and urine specimens. A strict infection control program in hospital settings is recommended.

    Notes

    Funding:This article was financially supported by the Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran (1397.03).

    Conflict of Interest:No conflict of interest.

    Author Contributions:

    • Conceptualization: RG, NA.

    • Data curation: RG, NA.

    • Formal analysis: RG, NA, GN.

    • Funding acquisition: RG.

    • Investigation: BS, GN.

    • Methodology: RG, NA.

    • Project administration: NA.

    • Resources: RG, BS, GN, NA.

    • Software: BS, GN.

    • Supervision: RG.

    • Validation: RG, NA.

    • Visualization: RG, NA.

    • Writing - original draft: RG, BS, GN, NA.

    • Writing - review & editing: RG, BS, GN, NA.

    ACKNOWLEDGMENTS

    This article was financially supported by the Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

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    MeSH Terms
    Amikacin
    Anti-Bacterial Agents
    Azerbaijan*
    Clergy
    Cross Infection*
    DNA
    Drug Resistance, Bacterial
    Humans
    Intensive Care Units
    Iran*
    Klebsiella pneumoniae*
    Klebsiella*
    Membrane Proteins
    Pneumonia
    Polymerase Chain Reaction
    Program
    Random Amplified Polymorphic DNA Technique
    Respect
    Urinary Tract Infections
    Metrics
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