Klebsiella pneumoniae is a Gram-negative bacterium, which is known as normal constituent of the human gastrointestinal tract. K. pneumoniaeis currently emerging as a significant nosocomial pathogen.¹Carbapenem drugs such as meropenem, imipenem and ertapenem are used for treatment of infections due to drug-resistant K. pneumoniae.²

Resistance to carbapenem is predominantly associated with the production of carbapenemase enzymes. Based on the antibiotic hydrolytic mechanism at the active site, carbapenemase enzymes were divided into class A and class B.³ The class A enzymes contain a serine in the active site and their functions are inhibited by beta-lactamase inhibitors, such as clavulanic acid, tazobactam, and sulbactam. The class B metallo-beta-lactamases (MBL), such as bla_IMP and bla_VIMare another class of carbapenemases.⁴ These are zinc-dependent beta-lactam-hydrolyzing enzymes with board spectrum activity, which hydrolyze carbapenems and other beta-lactam antibiotics, except for aztreonam.⁵The MBLs are not inhibited by beta-lactamase inhibitors but their functions are blocked by zinc chelating agents (EDTA). In most cases, the coding genes for MBLs are located in integrons and placed on chromosomes and/or plasmids.⁵In the first reports, bla_IMP and bla_VIMwere detected in Japan and Italy, respectively.⁶ Predominantly, MBLs have been described in non-fermenting Gram-negative bacteria. But now the MBLs have been reported from several Gram-negative bacteria including K. pneumoniae. Specifically, carbapenem-resistant K. pneumoniae is currently known as a major concern in recent years.⁷

In this report, we describe the antibiotic resistance profiles and prevalence of MBL genes including bla_VIM-1,bla_VIM-2, and bla_IMP-1 in K. pneumoniaeisolates recovered from inpatients from Hamadan province, west of Iran.

Collection of specimens

From November 2015 to February 2016, one hundred K.pneumoniae isolates were collected from different infection specimens of inpatients and outpatients, which were referred to three teaching and treatment university hospitals in Hamadan, Iran. Only one isolate per patient was included in the sample. Isolated strains were identified by routine conventional tests including indole, methyl red, Voges-Proskauer, lactose fermentation, etc.⁸ and confirmed by API 20E strips (bioMérieux, Marcy l’Étoile, France). Also, isolated strains were confirmed as K. pneumoniae by PCR. For this purpose, PCR primers (ureD) were used for specific identification of K. pneumoniae (Table 1).

Table 1. The primers used in this study

Antimicrobial susceptibility testing

Antibiotic susceptibilities for clinical isolates were defined by both disk diffusion and E-test methods. The antibiotic susceptibility profiles of K. pneumoniae according to CLSI were assessed for gentamicin (10 µg), aztreonam (30 µg), cefepime (30 µg), cefotaxime (30 µg), cefixime (10 µg), ceftazidime (30 µg), ceftizoxime (30 µg), ciprofloxacin (5 µg), ceftriaxone (30 µg), imipenem (10 µg), and meropenem (30 µg) (Mast, London, UK), determined by the disk diffusion method.¹³ MDR isolates were described as resistant to 3 or more antibiotics.

Also, the minimum inhibitory concentration (MIC) value of imipenem for clinical isolates of K. pneumoniae was evaluated by using the E-test method. According to CLSI, 0.5 McFarland of bacterial inoculum was prepared and cultured on Mueller-Hinton agar (Himedia, Delhi, India) plates, and then disks or strips were placed on the medium. Subsequently, the plates were incubated for 16-18 h at 35°C.¹³

Detection of MBL production

Phenotypic MBL production was assessed by both double disk synergy (DDS) test and E-test (AB Biodisk, Solna, Sweden).¹⁴For this purpose in the DDS test, the inhibition zone of imipenem and imipenem-EDTA containing disks was measured. According to the manufacturer’s protocol, the inhibition zone diameter for imipenem and imipenem combined with EDTA was 13 mm and 14.5 mm, respectively. After overnight incubation, the presence of an enlarged inhibition zone was interpreted as being DDS test positive and any synergistic inhibition zone was interpreted to be positive by the MBL screening test.¹⁴

MBL E-test strips (AB Biodisk) using imipenem and imipenem-EDTA were used according to the manufacturer's instructions. The E-test MBL strips consisted of a wide range of imipenem MICs (4-128 µg/mL) overlaid with a constant concentration of EDTA.

Detection of bla_VIM-1, bla_VIM-2, bla_IMP-1

The bla_VIM-1, bla_VIM-2, and bla_IMP-1 genes detection was carried out by PCR with specific primers (Yekta Tajhiz Azma, Iran) for all 100 isolates. DNA was extracted using the boiling method. Briefly, a cell pellet was homogenized in 500 μL of TE (10 mM Tris, 1 mM EDTA, pH

The primers used in this study are listed in Table 1. The PCR was performed according to a previous study.¹⁵ The PCR products were observed on 2% agarose gel under constant voltage at 85V. In PCR amplification, Pseudomonas aeruginosa ATCC 27853 was used as negative control.

Statistical analysis

Data were entered into a database using SPSS 23 (IBM Corp., Armonk, NY, USA). Differences between proportions were analyzed using the Chi-square test; p<0.05 was considered significant.

Bacterial collection

During the study period, one hundred K. pneumoniae strains were isolated. Bacterial strains were isolated from different clinical specimens including blood culture (32), urine (18), stool (13), sputum (13), wound swabs (12), diabetic foot pus (5), skin and soft tissue abscesses (4), and tracheal aspirate (3) (Table 2).

Table 2. Antibiotic resistance rate (%) of K. pneumoniae isolates from different specimens

Sex, age and ward distribution

Of 100 patients infected by K. pneumoniae, 64 patients were women and 36 were men. The mean age of patients was 44.24 years (SD=18 years; range: 1 to 78 years). In this study, K. pneumoniae strains were recovered from the following wards: intensive care unit (ICU, 19), outpatient care (OPD, 4), coronary care unit (CCU, 10), pediatric (7), internal medicine (10), urology (4), cardiology unit (5), dialysis (17), burn unit (6), and infectious diseases (18) (Table 3).

Table 3. Antibiotic resistance rate (%) of K. pneumoniae isolates in different wards

Antibiotic susceptibility testing

The antimicrobial resistance of K. pneumoniaeisolates was determined in all 100 isolates and the results of susceptibility testing by disk diffusion method are presented in Table 2. Of 100 isolates, 95 and 88 isolates were susceptible to meropenem and imipenem, respectively. The highest resistance rate was found to cefixime (48%), followed by ceftriaxone (47%). Also, in this study 43 K. pneumoniae isolates were MDR. A higher frequency of MDR isolates was observed in strains recovered from the ICU (89.5%) and followed by burn (66.7%) and infectious diseases (55.6%) wards. Antimicrobial resistance patterns of the all K. pneumoniaestrains are shown in Tables 2 and 3.

The MICs of all isolates were investigated for imipenem by using E-test and its results were interpreted according to CLSI breakpoints.¹³ The MIC interpretive criteria for both imipenem and meropenem were <4 μg/mL. In accordance to disk diffusion, MIC results showed that twelve isolates were resistant to imipenem (4-128 μg/mL). Also, antimicrobial susceptibility testing demonstrated that five isolates were resistant to meropenem (4-64 μg/mL).

Detection of bla_VIM-1, bla_VIM-2,bla_IMP-1

PCR was performed for all 100 K. pneumoniae isolates to detect the bla_VIM-1, bla_VIM-2 and bla_IPM-1genes. The bla_VIM-1 gene was detected in 5 isolates, but the genes bla_VIM-2 and bla_IPM-1 were not found in K. pneumoniae isolates recovered in this study.

K. pneumoniae is an opportunistic pathogen, which causes different infections such as bacteremia, gastrointestinal and urinary tract infections. Rapid spread of K. pneumoniae in the hospital described this bacterium as the main cause of nosocomial infection.^10,16 Primarily, K. pneumoniae was susceptible to most β-lactams except for penicillin agents, but now resistance to the β-lactam category, especially carbapenems, has developed. The most important resistance mechanism to the carbapenem category is MBL enzymes production (Ambler class B enzymes). The MBL enzymes are able to destroy β-lactams and are associated with emerging resistance to all β-lactams including penicillins, carbapenems, extended-spectrum cephalosporins, but not aztreonam.⁶ Also, VIM and IPM are the most important MBL variants.

In our study, in accordance to other studies performed in Iran, the highest resistance in K. pneumoniae was observed towards third generation cephalosporins including cefixime, ceftriaxone, cefotaxime, and ceftazidime.^17-19 The main reasons for the high resistance of K. pneumoniae to this category are associated with overuse and empiric therapy in our hospitals.¹⁸ In a previous study, Cornaglia et al. showed that administration of β-lactams is a main risk factor for the emergence of MBL isolates in their community.²⁰ On the other hand, K. pneumoniae has the ability of rapid distribution of resistance genes among species.¹⁷

The earlier studies illustrated that carbapenems including imipenem and meropenem have the highest activity against K. pneumoniae and susceptibility rates were 100%.^23,24 But in further studies, in accordance with our results, resistance to imipenem and meropenem was observed.^19,22,25 Our result showed that resistance to imipenem was 12%, which is in accordance with the previous studies that demonstrated resistance rates that ranged from 2 to 24%.^19,22,25Also, resistance to meropenem was to 28% in Iran.¹⁹

Numerous reasons are associated with carbapenem resistance. One of the most important mechanisms is MBL production. Using the DDS test, 5 of 12 imipenem-resistant isolates were MBL positive. Probably, the resistance in imipenem-resistant strains which are MBL negative is associated with other mechanisms such as activation of efflux pumps, decreasing outer membrane permeability (porin) and high-level AmpC cephalosporinase production.²²In a previous study performed in Iran, Hashemi et al. demonstrated that 3.5% of isolates were MBL positive.²² In some studies, the isolation rates of VIM-1-producing K. pneumoniae higher than in the current study. For example, in a study performed by Daikos et al., 26.7% of K. pneumoniaeisolates from blood were VIM-1 positive.²⁶ The diversity of carbapenem resistance rate and prevalence of beta-lactamase genes in different studies may be related to geographical areas and predominant endemic bacteria, which exist in the region.¹⁹ Direct and indirect contact was defined as a main way of transmission of MBL-producing K. pneumoniae from patients to healthy persons,²⁷ thus contact precautions are useful to reduce the risk of transfer.

In summary, our result showed that the most effective antibiotic agents for the treatment of infection caused by K. pneumoniaein this region are meropenem and imipenem. The carbapenem category is the most important choice in emergency conditions. Thus, suitable prescription by doctors and lack of arbitrary and excessive use of these antibiotics by the community are critical factors to control and prevent the emergence of carbapenem resistance.

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