Detection of VIM-1, VIM-2 and IMP-1 metallo- β-lactamase genes in Klebsiella pneumoniae isolated from clinical samples in Sanandaj, Kurdistan, west of Iran.

Background and Objectives
Klebsiella pneumoniae is an important cause of serious nosocomial infections among Gram-negative bacteria. The aim of this study was evaluating the prevalence of VIM-1, VIM-2, and IMP-1 metallo-β-lactamase genes in clinical specimens at two teaching hospitals in Sanandaj, Kurdistan west of Iran.


Materials and Methods
Four hundred different clinical specimens were collected from hospitalized patients or referred to hospitals from May 2013 to March 2014 in Sanandaj, Kurdistan, Iran. MBLs - producing K. pneumoniae detected by Double Disk Synergy Test. The MBL positive isolates were examined for the presence of VIM-1, VIM-2 and IMP-1 genes using PCR technique.


Results
Of four hundred clinical specimens, 114 K. pneumoniae isolates were identified. Twenty-eight (24.6%) isolates were resistant to imipenem and 15 strains (53.6%) were positive for MBL enzymes production. PCR results showed VIM-1 and IMP-1 genes frequencies are 4 (26.7%) and 1 (6.7%). Only one strain of K. pneumoniae was found to be MBL producer among the outpatients.


Conclusion
The study results exhibited a high level of resistance to most of the antibiotics tested and high prevalence of MBLs producing in K. pneumoniae at two hospitals. Thus, the infection control methods and the implementation of antibiotic agents should be taken into account.


INTRODUCTION
Klebsiella pneumoniae is a Gram-negative bacillus belonging to the Enterobacteriaceae family, which is the major cause of nosocomial and opportunistic infections namely pneumonia, urinary tract infections, bacteremia, and wound infections across the world, with significant morbidity and mortality , bla , rates (1). High prevalence of antibiotic resistance in Enterobacteriaceae, especially in K. pneumoniae has been reported in Iran which is challenging for hospitals. The carbapenems such as imipenem, meropenem are used to treat infections caused by Gram-negative bacteria when there is resistance to other antibacterial agents. The presence of carbapenem-resistance in Gram-negative bacteria in both the community constitutes and hospital environments are a prominent development in the field of infectious disease management and control (2).
One of the most important ways to become resistant to imipenem is production of metallo-beta-lactamases (MBL). These enzymes belong to group 3 of Bush classification based on their substrate and inhibitor profiles and to Ambler class B beta-lactamases according to their amino acid sequence homology (3,4). Several types of MBL have been identified in Gram-negative bacteria, which are geographically widespread including Verona integron-encoded metallo-β-lactamase (VIM), New Delhi metallo-β-lactamase (NDM) and imipenemase (IMP). The first reports on MBLs were VIM-1 in Italy and IMP-1 in Japan (5). During the last few years incidence rates of MBL production of the members of Gram-negative organisms especially, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacteriaceae (mainly E. coli and K. pneumoniae) has been significantly increased and are the cause of life-threatening infections among human (6). Recently, there are several reports on the emergence of VIM-producing K. pneumoniae (VPKP) isolates across the world. VIM types (e.g. VIMI,VIM2) MBLs in Southern Europe and the Far East, Australia, North and South America, India and Iran, and IMP-type MBLs in Southeast Asia are predominant (7). There are several phenotypic methods for detection of MBL-producing bacteria. The ability of inhibitors (EDTA, Thiol compounds, and Dipicolinic acid) to control the activity of these enzymes are the foundation of these methods such as double-disk synergy test (DDST) using EDTA with IPM or ceftazidime (CAZ) (8), the Hodge test (9) and the MBL E-test (10). For identification of Gram-negative bacteria MBLs, PCR assay was described in 1996 (11).
Thus, the knowledge about the antimicrobial susceptibility patterns and MBLs-producing K. pneu-and IMP-1 genes of MBLs-producing in K. pneumoniae isolates in hospitalized patients or referred to Hospitals in Sanandaj, Kurdistan west of Iran.

MATERIALS AND METHODS
Bacterial isolates and identification. In this study, four hundred clinical isolates which were collected from different clinical specimens in hospitalized patients or referred (outpatients) to Beasat and Toohid hospitals during May 2013 to March 2014 in Sanandaj, Kurdistan, Iran. The included samples were as urine (n=208), wound (n=52), blood (n=50), tracheal aspiration (n=48) and sputum (n=42). The isolated bacteria were identified according to standard methods including colonial morphology, Gram staining, catalase, oxidase, urease and IMVIC tests (12). Detection and final approval of testing bacterial isolates was based on the use of API20E kit (Bio-Merieux, Marcy-l'Etoile, France).

Detection of MBLs.
The presence of MBL production was screened by the double-disk synergy test (DDST) using imipenem (IPM) disk and imipenem in combination with EDTA (6). After overnight incubation, the difference in ≥7mm between the inhibition zone diameter of the IPM-EDTA disk and IPM the only disk was considered to be a positive for the presence of MBLs interpreted as being DDS test. In this study, K. pneumoniae ATCC 700603 was used as positive control.
moniae is helpful for the treatment of infections Molecular identification of bla VIM1

VIM2
caused by these strains. The purpose of this study bla IMP1 genes. DNA was extracted using boiling was to determine the prevalence of VIM-1, VIM-2 method (15). Briefly, 0.5 ml of distilled water with 1 loop of bacteria was removed from an overnight culture (Trypticase soy agar, Merck, Germany) and the cell suspension was heated for 15 min at 100°C, and then centrifuged at 13,000 g for 5 min. The supernatant obtained was used as template for amplification. Polymerase chain reaction (PCR) for the detection of pertaining to the type of wards shown in Table 2.
The frequency of K. pneumoniae isolates in outpatients included; urine 25 (67.6%), wound 9 (24.3%) and sputum 3 (8.1%). The most and the least number of K. pneumoniae isolates belonged to ICU (28.9%) and CCU (5.3%), respectively. The study population bla IMP-1 , bla VIM-1 , and bla VIM-2 genes was carried out included 54% male and 46% female patients with an on a thermal cycler (Bio-Rad, USA) (16). PCR products were electrophoresed on 1.5% agarose gel and photographed by UV trans-illuminator. Primers and conditions of polymerase chain reaction are shown in Table 1.  (Fig. 1). K. pneumoniae resistant to antibacterial agents in hospitalized patients and outpatients are shown in Table 3.

RESULTS
Upon examining the frequency of MBL enzymes pertaining to K. pneumoniae isolates, 28 of 114 isolates (24.6%) were resistant to imipenem and 15 cases (53.6%) were positive by Double Disk Synergy Test (imipenem disk alone and imipenem with-EDTA). MIC (E-test) by IPM strips was performed for all isolates. All VIM-1 and IMP-1 producing isolates of K. pneumoniae had MIC > 4 µg/ml. The VIM-1 produces were resistant to 4 antibiotics, cefotaxime, cefazoline, ceftazidime, and kanamycin. PCR results showed 4 isolates (26.7%) were positive for (32.5%) isolates belonged to outpatients. The distribution of K. pneumoniae in hospitalized patients bla bla VIM-1 IMP-1 (Fig. 2) and 1 isolate (6.7%) were positive for (Fig. 3), none of tested isolates were positive     (27) for bla VIM-2 gene. Higher prevalence of VIM-1 producing isolates of K. pneumoniae was seen in urine samples (20%). Only one VIM-1 producing K. pneumoniae isolates found in outpatient urine was detected by PCR. Distribution of metallo-β -lactamases in clinical samples are shown in Table 4.

DISCUSSION
K. pneumoniae is a member of the Enterobacteriaceae family. The quantity of reports shows that the isolation of species of this genus from both hospital and community samples has increased (17). In the current study, from total clinical samples, prevalence of K. pneumoniae was 28.5%, which is close to the study in Brazil (26.2%) (18), but it was Fig. 3. PCR analysis for the bla IMP -1gene in K. pneumonihigher in comparison with the study in Iran (25.3%) (19), and a study in India (25%) (20). In this study, K. pneumoniae showed varying levels ranging from   (23). Our results proved that meropenem and imipenem remain effective drugs against K. pneumoniae isolates. In the present study, more than half of K. pneumoniae isolates were resistant to other important antimicrobial agents such as kanamycin (61.4%), cefotaxime (59.6%), cefazoline (56.1%) and ceftazidime (52.6%) which matched the results achieved in the north of Iran in 2016 (21). Extensive variation in the prevalence and antibiotic resistance is probably due to the variation in risk factors, the widespread use of antibiotics for the treatment of infections and differences in geographical locations. Some of the important reasons for increasing drug resistance in Iran are the lack of communication between physicians and microbiologists, limited infection control programs and poor sanitation. The results of this study proved comparatively low resistance to the majority of K. pneumoniae isolates in the outpatients when compared to the patterns from hospitalized patients. These findings may imply that these drugs still be useful for treating uncomplicated cases where the organisms are indicated as etiologic agents of diseases in the hospital. In the present study, the highest number of K. pneumoniae were isolated from urine samples in hospitalized patients which admitted to ICU, and also maximum resistance to imipenem was observed in urine samples, which were accordance with studies of Rai in 2011 (17). ICU has been known as a source of creating, spreading and enhancing antimicrobial resistance and patients are exposed to a number of potential risk factors for colonization/infection such as high invasive procedures, elderly people, longterm hospitalization and misuse of suitable antibac-terial agents, and Klebsiella spp. is one of the most common pathogens isolated in ICUs (24).
In the past few years, MBLs have been identified via clinical isolating by increasing frequency. The enzymes produced by these strains (K. pneumoniae) have been responsible for prolonged nosocomial outbreaks and a serious threat to public health worldwide (25).
In regards to MBL enzymes in this study (53.5%) was in line with a study in India in 2012 (26), but it was higher compared to a study in Malaysia in 2015 (27). The results of this study showed that the majority of MBL producing K. pneumoniae (14/15) were extracted from hospitalized patients and only one isolate (1/15) was MBL producer in outpatients by DDST. The presence of MBL-producing bacteria in the outpatients portends serious health risk because the organisms that produce MBL are notably resistant to carbapenems including imipenem and meropenem, which are antibiotics reserved for serious bacterial infections (28). In this study, VIM-1 gene prevalence was 26.6% (4/15) which matched with the results achieved in India in 2013 (29), but it was higher than the study published in Iran in 2018 (19), and in India in 2014 (20), and it was lower compared to studies in Nepal 2014 (6). By DDST and PCR analysis, we determined one IMP-1 producing K. pneumoniae (6.7%) in urine samples in hospitalized patients, which was similar to findings of Khodadadian study in 2018 (19), but it was lower than study reported by Jin Young in 2016 (30). It seems that the difference in the prevalence of MBL-producing K. pneumoniae strains in different hospitals is due to the genetic difference between species, spreads to resistance genes and affects the spread patterns of different MBLs in various countries.
In our study, the MICs (E-test) of imipenem against 4 VIM-1 and IMP-1 Metallo-β lactamase producing K. pneumonaie isolates ranged from 4 to 32 mg/l and exhibited IPM resistant phenotypes. The resistance to imipenem may also be attributed to the loss of porins. In the present study, MBL positive pneumoniae to most of the other tested antibiotics and the majority MBL production of K. pneumoniae was associated with hospitalized patients in ICU. Therefore, accurate and rapid detection of these MBL-producing strains is vital among isolates circulating in high-risk wards such as ICUs, for suitable selection of effective therapy and nosocomial infection control.