Molecular epidemiology of VIM and IMP-producing Klebsiella isolates from urinary tract infection patients in an educational hospital in Shahrekord, Iran

Background and aim: Klebsiella is an opportunistic organism that is the cause of many nosocomial infections. The present study was designed to investigate the molecular epidemiology of Verona integron-encoded metallo-βlactamase (VIM) and Imipenemase (IMP)-producing Klebsiella isolates in patients with urinary tract infection (UTI) in an educational hospital in Shahrekord, Iran. Methods: In a cross-sectional study, from 234 urine samples, 80 isolates of Klebsiella were identified with biochemical tests. In order to determine the production of Metallo-β-lactamases (MBLs), Modified Hodge Test (MHT), EDTA Disc Synergy (EDS) test and Ampicilin C (AmpC) disc test were performed. The frequency of VIM and IMP genes was determined after DNA-amplification with PCR by electrophoresis technique. As an internal control in PCTR, 16SrRNA was considered. Results: Phenotypic tests showed that out of the 80 isolates, 18 (22. 5%), 18 (22. 5%) and 10 (12.5%) isolates were positive for MHT, EDS and AmpC disc test, respectively. Following DNA amplification by PCR, the genes of interest were analyzed by electrophoresis technique. The findings were as follows: 22 isolates (27.5%) carried the VIM gene, but the IMP gene was not found in any of the isolates. Conclusions: Expansion of Klebsiella strains that produce MBLs is a severe threat to health centers and public health. The findings of this study showed that Klebsiella may produce MBLs. These enzymes can in turn degrade carbapenem antibiotics, which are considered as a last resort in the treatment of multidrug-resistant (MDR) infections.


Introduction
Urinary tract infection (UTI), affecting 150 million people yearly, are an important threat to human health. Between 30 to 40 percent of UTI are due to nosocomial infections (1,2). Klebsiella pneumoniae and Escherichia coli are known to cause more hospital infections in comparison to other bacteria of the Enterobacteriaceae family (3).
Klebsiella is an opportunistic organism, which may lead to the development of severe diseases such as pneumonia, septicemia and UTI (4). Klebsiella can be present in hospitals, persist on environmental surfaces and colonize diverse parts of the human body.
Therefore, the patients are at high risk of being infected by Klebsiella during their hospital stay. The frequent use of microbial agents and thereby the continuous exposure of this bacterium to many antibiotics has contributed to the spread of the multidrug-resistant strains, which has made it challenging for healthcare settings to control infections with this bacterium (5,6). This bacterium is a dangerous pathogen affecting humans and acts as the primary source for hospital-related infections. Due to the limited treatment options, infections with this bacterium are associated with high morbidity and mortality (7). Similar to other countries in the world, antibiotic resistance and nosocomial infections are also major problems in Iran (8).
Antibiotic resistance has made treating UTI more challenging. There are more than 12,000 deaths per year from UTI in USA (9), with a prevalence of 4.92% among Iranian children (10) and 14.96% in Northern Iran (11). The carbapenems consist of Ertapenem, Imipenem, Doripenem and Meropenem.
The most essential resistant isolates of Klebsiella pneumoniae are carbapenem and cephalosporinresistant strains (12)(13)(14).  (17)(18)(19)(20). Molecular epidemiological studies are important to identify the source of infections so that the healthcare settings come up with innovative, preventative strategies to control the transmission of bacteria between patients and even among wards. This study aimed to explore the epidemiology of VIM and IMP genes in UTI patients in Shahrekord city, in the center of Iran.

Phenotypic detection of MBLs enzymes
Phenotypic tests, including Modified Hodge

Test (MHT), EDTA Disc Synergy (EDS) test and
AmpC disc test were performed to detect the MBLs produced by the isolates.

Modified Hodge Test
In directions. After having taken these actions, the plates were maintained at 37 C° overnight. Then, appeared clover leaf shape in the inhibition site of bacterial growth resulted from the production of MBL by the tested isolates (21,22).

EDTA Disc Synergy test (EDS)
In this test, a 0.5 M EDTA solution and a meropenem disc (10 µg) were used. In order to perform this test, suspension samples equivalent to 0.5 McFarland were passaged on Mueller-Hinton agar. A meropenem disc (10μg) was placed on the plate. The blank disc (containing 10 ml of a 0.5 M EDTA solution) was placed at a 10 mm distance from the meropenem disc. The plate was incubated overnight at 37 C°. Increased growth inhibition zone between two discs is an indicator of MBL production (22).

AmpC disc test
The AmpC disc test method was used to identify AmpC β-lactamase. In this setting, 0.5 M of a McFarland suspension of E. coli ATCC 25922 was passaged on Mueller-Hinton culture medium. Besides, a cefoxitin disc (30 μg) was placed on the plate near the blank disc, which was exposed to normal saline.
Then the intended colony were added to the blank disc.
To be able to assess whether there was AmpC βlactamase production, the growth area of the cefoxitin disc in the vicinity of the blank disc was observed (23, 24). The availability of a dentin in the growth are of the cefoxitin disc was an indication of AmpC βlactamase production.

Detection of VIM and IMP genes
In order to perform PCR, DNA was extracted by the conventional boiling method. In this method, 1 ml of sterile distilled water was poured in a 1.5 ml microtube. Then 4 to 5 fresh bacterial colonies were dissolved, after which the microtubes were placed on a heated plate at 95 C° for 15 minutes to boil. The samples were centrifuged at 14000 rpm for 5 minutes.
The transparent liquid containing bacterial DNA was The polyacrylamide gel was stained by the silver nitrate method (Figure 2). All the specimens contained 16SrRNA as internal control genes, which confirmed the presence of this genotype gene for Klebsiella.

Statistical analysis
Descriptive statistics (i.e. frequency, percentage, and the mean and standard deviation) and inferential statistics (Fisher's exact test) were performed in order to analyze the data. Hereby, P < 0.05 was considered as α.

Resistance to Meropenem and Imipenem
After incubating the plates containing the meropenem and imipenem discs, the diameters of the The presence of a notch in the side of the disc reflects the production of AmpC β-lactamase ( Figure   1). In this test, 10 (12.5%) isolates had an β-lactamase.
Results of the phenotypic tests, which were used to detect MBL enzymes, are presented in Table 2.

Discussion
Our findings showed that 24 isolates were resistant to Meropenem and Imipenem. In addition, PCR showed that 22 isolates, carried the VIM gene, but the IMP gene was not found in any isolate.
Nowadays, bacterial resistance against several antibiotics has become a global concern.
Genes encoding for MBL's are found more frequently on integrins, particularly class 1 integrins, which has the genetic mobility of these elements, promoting the  Also, positive phenotypic tests may be due to the presence of other MBLs genes such as NDM, GIM, SIM, SPM. Therefore, it is suggested that the presence of these genes be investigated in future studies.

Conclusion
Klebsiella, with resistance to Meropenem and Imipenem, is considered as a substantial public health challenge to patients. This need to direct more attention to antimicrobial resistance by monitoring and surveillance. Furthermore, it would be essential that antibiotics be prescribed appropriately to prevent the emergence and spread of bacterial strains with resistance to carbapenemase in hospitals and the community.

Conflict of Interests
None.