Genetic Detection of IMP-1 Gene and its Relationship with Biofilm Formation in Klebsiella pneumonia

Background: Klebsiella pneumoniae were considered as normal flora of skin, and intestine. It can cause damage to human lungs; the danger of this bacterium is related to exposure to the hospital surroundings. materials and methods: the detection of Klebsiella pneumoniae on morphological and biochemical tests and then assured with VITEK 2 system. Resistance to antibiotics was determined by Kirby-Baeur method. And genotyping of IMP-1 in isolates was done by PCR technique, then biofilm formation was identified by Micro titer plate method. Results: The present study included a collecting of 50 specimens from different clinical specimens, (blood 40%, urine 30%, sputum 20%, wound infection 10%); 10 isolates were identified as Klebsiella pneumoniae . All isolates, under study, developed high resistance toward Cefitrixon, Ampicillin, Amoxicillin, Ticarcillin, Ticarcillin+Clavulanic acid, and Ceftazidim estimated by disc diffusion method. All isolates characterized by harboring the highest resistant in a percentage reached 100% against antibiotics, under study. This study determined the Minimal Inhibitory Concentration were detected by eight E-test strips for isolates. As well as the isolates were strong biofilm production for three isolates, while three were moderate of biofilm formation and other isolates were weak former; at the value of (P≤0.05) was considered as a significant. Genotype detection of Metalo-beta lactamase ( IMP-1) by PCR technique in Klebsiella pneumoniae . Upon using PCR technique exposed only three isolates;30% of isolates (two from urine, one from blood) samples harbored IMP-1 gene. The study was also found relationship between IMP-1 and biofilm formation in isolates which were harboring these genes, when ( P ≤ 0.05). Conclusions: K. pneumoniae were isolated from different sources. All isolates were resistant to most antibiotics used in this study. The isolates have Metallo-beta lactamse. PCR was showed K. pneumoniae have IMP-1 gene,. This study also found there was relationship between biofilm formation and IMP-1 gene in K. pneumoniae ( P ≤0.05).

giving also resistance to aminoglycosides, beta lactams, but also to other groups of antibiotics [4].
On the other hand, Klebsiella can also cause urinary tract and wound infections, alsocan produce The prevalence of its must be controlled by using newly antibiotics [9].

Isolation of bacteria:
During the period from September to December 2019, fifty specimens from different clinical specimens (blood, urine, wound infection, sputum) from hospitalized patients in Baghdad, ten isolates were identified as Klebsiella pneumoniae. Then they were cultured onto MaCconkey agar, blood agar, and incubated at 37°C for 24hrs. [10].

Identification of bacteria:
Klebsiella pneumoniae were identified depending on the morphological and microscope features. The plates of MaCconkey agar were streaked with a pure colony of tested bacteria and then incubated at 37°C for 24 hrs. Then confirmed identification with VITEK 2 system [10].

The sensitivity test with antibiotics discs:
The plates of Mueller-Hinton agar were inoculated by dipping a sterile swab into the inoculums culture with 1.5 × 10 8  The products were run for 120 min at 90 V. The bands were observed after staining with ethidium bromide by using an ultraviolet-light [12].  The method identified in [13] was followed as the standard test for biofilm formation detection. Briefly, only the bacterial cultures of the diluents (200 μL), and another with 50 μL, it was then a negative control which added 200 μL of Brain heart infusion broth eight wells with no further additions. For 18-24hrs, the micro titer plate was incubated at 37 C, then, was washed five times with distilled water, and was left in dry air for 15 min. The plate was stained with 0.1% Crystal Violet 200 μL for 15 min, and was washed with distilled water. So 200μl of 95 % methanol was added for 10 min. to each well. The amount of crystal violet collected by the ethanol in each well was quantified using an ELISA reader to calculate the OD 580nm.Statistical analysis was expressed as Mean ±SD between the control and each of bacteria with Excel software. Cut off value (ODc) can provide isolates as shows in [

Statistical Analysis
Data presented as a frequency and percentage, while Mean ±SD was used to analyze the data in this study. (P ≤ 0.05) considered statistically by using program SPSS Statistics (2012) [14].

RESULTS AND DISSCUTIONS:
Identification of bacteria by biochemical tests: Ten isolates of K. pneumoniae were isolated from different clinical specimens (40% of blood,30% of urine, 20% of sputum, 10% of wound infection) as shows in [ fig.1].

Figure (1): Distribution of K. pneumoniae in clinical specimens
K. pneumoniae were cultured on MacConkey agar, the bacteria were lactose fermenter so that appeared pink colonies. And also were cultured on Simmon Citrate agar the bacteria can utilize the citrate; therefore, the media color was changed to blue. Indole test was negative for K. pneumoniae. On blood agar bacteria gave gamma hemolysis [10]. The confirmation of identification was done by VITEK 2 system.

Sensitivity test for antibiotics:
The sensitivity of K. pneumoniae isolates were tested to a number of antibiotics used to treat some of the infections caused by this species in humans. [ fig.2  In the other hand, other study was showed ESBL producing K. pneumoniae isolates were resisted to ampicillin, and third-generation cephalosporins. And these isolates were sensitive to meropenem, amikacin, and ciprofloxacin [16]. A study with disc diffusion method was found, that K.
pneumoniae were sensitive to, Cefotaxime, Imipenem /Cilactin, Sparfloxacin, and Norfloxacin antibiotics [17 and 18]; these results agree with result of this study, K. pneumoniae were resisted to most antibiotics used in this study.

MIC of E-test strips against K. pneumoniae:
The E-test was used in this study to determine the Minimum Inhibitory Concentration (MIC) for antibiotics by using diffusion methods. The result showed as elliptical inhibition zone around the strips. In this study used Ampicillin, Cefoxitin, Cefperazone strips for isolates of K. pneumoniae, and they were not giving MIC value for isolates because they resistant to them.

Identification of Metallo-beta lactamase genes by PCR:
From ten isolates of K. pneumoniae, results of PCR were showed only three isolates possess IMP-1 gene (No. 5,9,10), while the other isolates (1,2,3,4,6,7,8) don't have this gene. The isolates (No.5,9,10) were clinical isolates (two from urine, one from blood), on the other hand, all isolates were not possessed NDM-1 gene; the results showed in [ fig. 3] about 30% of isolates were carried the gene of IMP-1. In the United States, a study found that PCR was performed for genes of MBLs, including VIM, IMP, molecular result showed that VIM-1 and IMP-1 genes are 15.6 and 6.4%, respectively. But in this study all three IMP-1 producing K. pneumoniae isolates and this agrees with this study result of found IMP-1 gene in K. pneumoniae [19]. Another study in Japan found that a KPC-producing organism to become endemic in Japan is currently of great interest in Klebsiella pneumoniae ST258 isolated from a Japanese patient [20]. And this study does not agree with Egyptian study which mentioned that positive for bla KPC, bla VIM, bla NDM, blaOXA-48-and none was bla IMP-positive [21]. ‫م‬   The three isolates contained the IMP-1 gene, and they also formed a strong biofilm, and these isolates were more resistant to antibiotics. The results showed that the bacteria used in this study were strong biofilm production for three isolates, while three were moderate of biofilm formation; while other isolates were weak producer. The value of (P≤0.05) was considered as a significant.
The results as show in [table 4 and 5].  The results of this study agreed with another study which found a significant correlation between the ability to form biofilms and the isolates of K. pneumoniae [19]. It concluded that raising the carbapenem resistance of strains with biofilm producing K. pneumoniae and this agrees with the results of this study [22].

CONCLUSION:
K. pneumoniae were isolated from different sources. All isolates were resistant to most antibiotics used in this study. The isolates have Metallo-beta lactamse ;(class B) carbpemenase.
PCR showed that K. pneumoniae have IMP-1 gene in a clinical isolate. This study also found that there was relationship between biofilm formation and IMP-1 gene in K. pneumoniae (P≤0.05).

Conflict of interests.
There are non-conflicts of interest.