Original Article

Prevalence of Metallo-β-Lactamase Genes in Clinical Isolates of Klebsiella pneumoniae in Health Care Centers in Mazandaran Province

Background

Klebsiella pneumoniae is a clinically significant pathogen that causes urinary tract infections (UTIs), sepsis, pneumonia, and other infections (1). The bacterium is a Gram-negative, immobile, encapsulated, lactose-fermenting, and facultative anaerobic bacillus (2). Klebsiella pneumoniae can asymptomatically colonize the skin, mouth, respiratory tract, and gastrointestinal tract, which may serve as a reservoir for human infections (3). The bacterium uses a variety of strategies to protect itself against antibiotics, one of which is the production of β-lactamase enzymes. This enzyme causes resistance by hydrolyzing and inactivating the β-lactam ring of beta-lactam antibiotics. Treatment of disease caused by K. pneumoniae has become a challenge because many strains are resistant to all β-lactam antibiotics through the production of carbapenemases (4). According to the Ambler classification, beta-lactamases are grouped into classes A, B, C, and D based on sequence similarity (5). Class B beta-lactamases require at least one or two zinc ions at their active site (6). Various beta-lactamases, including Verona integron-encoded metallo-β-lactamase (VIM), imipenemase (IMP), New Dehli metallo-β-lactamase (NDM-1), Sao Paulo metallo β-lactamase (SPM), and Seol imipenemase metallo-β-lactamase (SIM), have been detected in K. pneumoniae strains (7). Metallo-β-lactamases cause resistance to various antibiotics, including penicillins, cephalosporins, and carbapenems, except for monobactam (8,9). IMP is a carbapenemase that is resistant to almost all β-lactam antibiotics. One of the most common types of IMP is IMP-4, which was first identified in Acinetobacter spp. in Hong Kong in 2001 (10). VIM metallo-β-lactamases were first identified in Europe (11). The first case of VIM-4 in K. pneumoniae was reported in a recent study conducted in Tunisia (12). In 2009, NDM was first detected in a Swedish patient hospitalized in New Delhi, India (9). The genes encoding these carbapenemases are reported to be located on chromosomes or motile genetic elements such as plasmids, integrons, and transposons (13). Based on several reports on the presence of Metallo-β-lactamases genes in K. pneumoniae strains, the aim of the present study was to determine the prevalence of bla_IMP, bla_VIM, and bla_NDM genes in clinical isolates.

Methods

Isolation and Detection of Klebsiella pneumoniae

Clinical specimens were collected from wounds, burns, urine, and sputum. The study was conducted for seven months from June 2018 to December 2019. In total, 40 isolates of K. pneumoniae were transferred from the laboratories of Mazandaran province to the Microbiology Laboratory of Islamic Azad University, Babol branch. Isolates were inoculated on MacConkey agar plates and were incubated overnight at 37°C (Merck, Germany). Unique colonies of K. pneumoniae (lactose-fermenting mucoid colonies ) were confirmed by standard biochemical tests, including fermentation of sugars in Triple Sugar Iron Agar medium (TSI) (Merck, Germany), fermentation of acidic or alcoholic in MRVP (Methyl Red, Voges–Proskauer) broth (Merck, Germany), growth in Simmons citrate medium (Merck, Germany), urea hydrolysis in urea medium, production of indole and hydrogen sulfide, and motility in sulfide indole motility (SIM) medium (Merck, Germany). According to biochemical tests, K. pneumoniae isolates were confirmed as indole negative, MR negative, VP positive, citrate positive, urease positive, and motility negative (14). All K. pneumoniae isolates were cultured in trypticase soy broth (TSB) (Merck, Germany). Then, 800 μL of bacterial suspension and 200 μL of glycerol were mixed in a 1.5 mL tube and were stored at -20°C for further studies (15,16).

Antibiotic Susceptibility Test

According to the Clinical and Laboratory Standards Institute (CLSI) recommendation, antibiotic susceptibility testing was performed by the disk diffusion method. A bacterial suspension with turbidity equal to 0.5 McFarland was cultured on the surface of Müller-Hinton agar medium. Then, the imipenem disc (10 μg) (Padtan Teb Co-Iran) was placed on the culture medium and was incubated at 37°C for 18 hours. The diameter of the growth inhibition zone was measured and the results were recorded as resistant, intermediate, and sensitive. Isolates with growth inhibition zones of ≥22 mm, 20-22 mm, and ≤19 mm were determined as sensitive, intermediate, and resistant, respectively (17).

Detection of Metallo -β-Lactamase Genes

The polymerase chain reaction (PCR) technique was used for the identification of metallo-beta-lactamase genes including bla_NDM, bla_IMP, and bla_VIM in K. pneumoniae isolates. DNA purification was performed by boiling method. The two colonies were transferred into a test tube containing 1 mL of distilled water, boiled in a water bath for 10 minutes, and centrifuged at 1000 rpm for 5 minutes. Then, 3 μL of supernatant was used in the PCR master mix (18). PCR amplification was performed in a final volume of 25 μL containing 12.5 μL of Master mix (CinnaGen Company, Iran), 3 μL of supernatant containing bacterial DNA, 1 μL of each primer (20 pmol), and 7.5 μL of sterile distilled water. All PCR assays were performed using an Eppendorf 5331 MasterCycler Gradient Thermal Cycler (Hamburg, Germany). The primers used for detection of these genes were listed in Table 1.

Thermal cycles of PCR were performed as follows. A cycle of initial denaturation was performed at 94°C for 3 minutes. Then, 35 cycles, including 94°C for 1 minute, annealing at a suitable temperature for 1 minute according to Table 1, and polymerization at 72°C for 1 minute were carried out. Final polymerization was done at 72°C for 10 minutes (19,20). In the PCR reaction mixture, distilled water was used as the negative control. Finally, PCR products were electrophoresed in 2% agarose gel at 95 V for 100 minutes and were observed by UV transilluminator (Vilber Lourmat, Collégien, France).

Statistical Analysis

The results of this study were analyzed using SPSS (Statistical Package for Social Sciences) version 21.0.

Results

A total of 40 K. pneumoniae isolates were identified from clinical specimens. K. pneumoniae strains were isolated from 11 sputum, 21 urine, 3 blood, and 5 wound samples. The results of antimicrobial susceptibility testing performed on 40 isolates of K. pneumoniae showed that 21 (52%) and 19 (48%) isolates were resistant and sensitive to imipenem, respectively, and none of the samples were intermediate (Figure 1).

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Figure 1.

Antibiogram of Klebsiella Pneumoniae Strains to Imipenem by the Disk Diffusion Method R: resistant; S: sensitive

Figure 1.

Antibiogram of Klebsiella Pneumoniae Strains to Imipenem by the Disk Diffusion Method R: resistant; S: sensitive

PCR technique was used to identify metallo-β-lactamase resistance genes. Agarose gel electrophoresis of PCR products produced special bands of 236, 587, and 383 bp for bla_NDM, bla_IMP, and bla_VIM, respectively (Figures 2, 3, and 4).

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Figure 2.

Agarose Gel Electrophoresis of the PCR Amplicon of bla_NDM. Ladder: (50 bp); Lanes 2,3,8 and 11: positive samples; Lanes c^-: negative control; Lane 1, 4, 6, 7, 9, and 10: negative samples.

Figure 2.

Agarose Gel Electrophoresis of the PCR Amplicon of bla_NDM. Ladder: (50 bp); Lanes 2,3,8 and 11: positive samples; Lanes c^-: negative control; Lane 1, 4, 6, 7, 9, and 10: negative samples.

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Figure 3.

Agarose Gel Electrophoresis of the PCR Amplicon of bla_IMP. Ladder: (50 bp); Lanes 10: positive samples; Other lanes: negative samples; c^-: negative control.

Figure 3.

Agarose Gel Electrophoresis of the PCR Amplicon of bla_IMP. Ladder: (50 bp); Lanes 10: positive samples; Other lanes: negative samples; c^-: negative control.

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Figure 4.

Agarose Gel Electrophoresis of the PCR Amplicon of bla_VIM. Ladder: (50 bp); Other lanes: positive samples; Lanes 6: negative samples; c^-: negative control.

Figure 4.

Agarose Gel Electrophoresis of the PCR Amplicon of bla_VIM. Ladder: (50 bp); Other lanes: positive samples; Lanes 6: negative samples; c^-: negative control.

The prevalence of metallo-β-lactamase genes is shown in Table 2.

Of the 40 K. pneumoniae isolates studied, the most common resistance gene was the blaNDM gene with a frequency of 36 (90%), but the bla_IMP gene had the lowest prevalence (17.5%). The prevalence of the bla_VIM gene was 75%. The coexistence of the bla_NDM, bla_IMP, and bla_VIM was observed in 4 (10%) isolates of K. pneumoniae. The coexistence of bla_NDM andbla_IMP as well as bla_NDM andbla_VIM genes was detected in 2 (5%) and 22 (55%) isolates, respectively. The presence of both bla_IMP andbla_VIM genes was not detected in any of the isolates. Moreover, 2.5% of the isolates lacked beta-lactamase resistance genes.

Discussion

Klebsiella pneumoniae plays a clinically significant role in hospitalized infections. The bacterium causes many infections, such as UTIs, liver abscesses, pneumonia, and bacteremia (21). In our study, bla_NDM gene was detected in 90% of the K. pneumoniae isolates, which differs from studies conducted in Northwest Pakistan (22.7%) (22), India (19%) (23), China (14.9%) (24), Egypt (26.32%) (25), and Bangladesh (22.4%) (26). In a study conducted by Saremi et al in Iran, the prevalence of bla_NDM-1 gene was 1.92% (7). In a study conducted in Peru, 85% of Klebsiella pneumoniae isolateswere positive for bla_NDM gene, which is consistent with our study (27). The prevalence of the bla_NDM gene varies in different parts of the world depending on the level of health and overuse of antibiotics. In the current study, the bla_IMP gene was observed in 17.5% of the isolates, which is not consistent with studies conducted in South India (8%) (28), China (36.5%) (29), and Egypt (15.2%) (30). In the present study, the prevalence of the bla_VIM gene was 75%. In a study conducted by Ripabelli et al (31), bla_VIM gene was detected in 76.5% of the isolates, which is consistent with our study. In our study, the coexistence of bla_NDMandbla_IMP as well as bla_NDMandbla_VIM genes was observed in 5% and 55% of the isolates, respectively. In a study conducted by Urmi et al (26), multiple Metallo-beta-lactamase genes were present simultaneously in one K. pneumoniae isolate. The results of a study conducted by Hussein showed the coexistence of both bla_NDM-1 and bla_IMP genes in only three strains of K. pneumoniae (32). These studies show that the simultaneous presence of resistance genes varies in different geographical areas. In our study, 1 (2.5%) isolate did not contain any of the metallo-beta-lactamase resistance genes. In an investigation carried out by Pragasam et al (33), 4 isolates were negative for all the genes tested, which is consistent with our study. In the genotyping method, 97% of K. pneumoniae isolates contained Metallo-beta-lactamase resistance genes, but in the phenotyping method, 52% of isolates showed resistance to imipenem. Different phenotyping and genotyping results can be due to mutations in the promoter or coding sequence of metallo-beta-lactamase genes, which in turn prevent gene transcription and enzyme inefficiency, respectively. It is recommended that physicians use the results of this study to select the appropriate antibiotics to treat patients. Carbapenem-resistant K. pneumoniae strains are usually resistant to most beta-lactam antibiotics. Therefore, treatment options are limited to polymyxins, tigecycline, and aminoglycosides (34).

Conclusions

Due to the high prevalence of metallo-β-lactamase genes in this study and their roles in treatment failure, rapid detection of these strains in laboratories plays a more influential role in controlling the spread of these strains. Therefore, appropriate policies are needed to regulate the use of penicillins, cephalosporins, and carbapenems in patients. We recommend regular monitoring and screening for the emergence of K. pneumoniae strains containing the metallo-beta-lactamase genes.

Acknowledgments

The authors would like to thank Molecular Biology Laboratory of Islamic Azad University, Babol Branch.

Authors’ Contribution

Majid Alipour designed the study, analyzed data, wrote the manuscript, and carried out the interpretation of the results. Rougayeh Alizadehand Fatereh Rezaee performed experimental tests and prepared the samples. All authors read and approved the final manuscript.

Conflict of Interests

There is no conflict of interest for authors.

Ethical Approval

There are no ethical issues for this article.

Funding/Support

None.

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