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

Background: Klebsiella pneumoniae is one of the most important causes of opportunistic infections, including lobar pneumonia, urinary tract infection (UTIs), and wound infection. Treatment of disease caused by this bacterium has also become a challenge, as many strains are resistant to all β-lactam antibiotics. Metallo-βlactamases hydrolyze most β-lactam antibiotics, especially carbapenems, but cannot inactivate monobactams. This study aimed to determine the prevalence of Metallo-β-lactamase genes (blaIMP, blaVIM, and blaNDM) in K. pneumoniae isolated from clinical specimens. Methods: Clinical samples were collected from hospitals of Mazandaran province. Among 500 clinical samples collected, only 40 Klebsiella pneumoniae isolates were detected by culture and biochemical tests. Antimicrobial susceptibility testing was performed on all isolates by the Kirby-Bauer method. The polymerase chain reaction (PCR) technique was used for the identification of blaIMP, blaVIM, and blaNDM genes. Results: Antibiogram by disk diffusion method showed that 21 (52%) and 19 (48%) isolates were classified as imipenem resistant and sensitive, respectively. Of all the samples, 30 (75%), 7 (17.5%), and 36 (90%) contained blaVIM, blaIMP, and blaNDM genes, respectively. The co-existence of the blaVIM and blaNDM genes was observed in 22 (55%) isolates. The presence of both blaIMP and blaNDM genes was confirmed in 2 (5%) of the isolates. Four isolates (10%) had blaNDM, blaIMP, and blaVIM genes simultaneously, but none of these genes were present in one isolate (2.5%). Conclusions: This study showed that the prevalence of metallo-β-lactamase genes in K. pneumoniae isolated from clinical specimens is very high, so it is recommended that physicians treat patients based on phenotypic and genotypic characteristics.


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).
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 and bla IMP as well as bla NDM and bla VIM genes was detected in 2 (5%) and 22 (55%) isolates, respectively. The presence of both bla IMP and bla 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 (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 isolates were 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 NDM and bla IMP as well as bla NDM and bla VIM genes was observed in 5% and 55% of the isolates, respectively. In a study conducted by Urmi et al (26), multiple Metallo-betalactamase 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-betalactamase 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.