Studying Several Virulence Factors Phenotypically and Genetically to Isolate Klebsiella Pneumonia from Diabetic Patients

Klebsiella pneumoniae is the most frequent cause of nosocomial and community-acquired infections. The virulence factors help Klebsiella pneumoniae establish an infection in the human body by contributing to its ability to survive in a variety of environmental circumstances. 42 K. pneumoniae clinical specimens were isolated from diabetic patients suffering from urinary tract infection and diabetic foot ulcer in this study. K.pneumoniae virulence factors are currently being identified using phenotypic and genotypic approaches. The K. pneumoniae isolates that were positive for several virulence factors included 33 (78.6%) capsule, 17 (40%) hypermucoviscosity development, 14 (33.3%) proteases, and none were positive for hemolysis . K. pneumoniae isolates had a virulence gene that was 15% wcaG, 60% entB, and negative for rmpA.

K. pneumoniae pathogenicity is mostly due to different virulence factors that allow it to overcome innate host immunity and maintain infection in a mammalian host (Bengoechea and Sa Pessoa,2019). These virulence factors aid in its survival in different environments and hence aid in establishment the disease in the human organism (Paczosa and Mecsas, 2016). K. pneumoniae pathogenicity is influenced by a number of virulence factors, such as fimbrial adhesins, lipopolysaccharides, capsule and siderophores, biofilm growth, lipase, lecithinase, haemagglutination, protease, gelatinase, hemolysis, and hypermucoviscosity. All of these virulence factors have the ability to infect both hospitalized patients and the general public with a wide spectrum of diseases (Piperaki et al., 2017;Ramakrishnan et al., 2019). Additionally, it creates hemolysin protein, a exotoxin that lyses blood cells, allowing bacteria to proliferate (Esmaeel and Sadeq, 2018).
Classical K. pneumoniae (cKp) infections are more common (Esmaeel and Sadeq, 2018). These bacteria survive in hospital settings and cause illnesses in elderly individuals. These cKp strains are not the same as hypervirulent K.pneumoniae (hvKp) strains (.Choby et al.,2020).

Bacteria:
Different enrichment, differential, and selective media were employed to culture bacteria from diabetic patients with diabetic foot ulcers and urinary tract infections who visited the diabetes center at AL-Sader Hospital in Al Najaf Province. A biochemical test was conducted to identify the bacterium after it had grown in the medium Virulence Factors Detection: Hemolysis: At 37°C, Hemolysis was seen after K. pneumonia colonies were inoculated on standard sheep blood agar. (Ramakrishnan et al.,2019).

Capsule Identification:
A K. pneumonia colony that had been incubated overnight was placed on a clean slide, stained for two minutes with methylene blue, and then rinsed with tap water. Observed a capsule encircling the organism (Collee et al., 1996). Klebsiella pneumoniae isolate was inoculated on regular sheep blood agar media and incubated at 37°C for 24 hours using the

Hyper Mucoviscosity Detection by String Test:
The string test was then demonstrated using a normal inoculation loop. When the produced string stretched longer than 5 mm, it indicated an HMV phenotype (Li et al., 2014). Protease Detection: K. pneumonia was injected onto freshly made milk agar and incubated for 72 hours at 37°C. hours, resulting in the formation of hallo surrounding the colonies, indicating protease synthesis (Piperaki et al., 2017).

Genomic DNA Extraction:
Boiling was used to process the DNA of the isolated bacterium. In 300 microliters of sterile TE buffer, many bacterial colonies were added and cooked for 15 minutes in a water bath at 100 degrees Celsius, quickly cooled to -20 degrees Celsius for 15 minutes, centrifuged, and the supernatant was saved for later amplification operations (Shah et al., 2017). Gene Amplification: The following three primers were used to amplify the genes of bacterial isolate DNA extracts: wcaG, entB, and rmpA. 8 L of master mix, 5 L of DNA template, 1.5 L for each primer, and 4 L of deionized water for PCR were added. The protocol was followed in accordance with the manufacturer's instructions for Promega Biosystem. The following thermal cycling conditions were used for amplification: Pre-denaturation at 95°C for 5 minutes, followed by 30 cycles of 1 minute at 94°C, 1 minute at 58°C, 1 minute at 72°C, and 10 minutes of final elongation at 72°C.

RESULTS AND DISCUSSION Detection of several K. pneumonia virulence factors using phenotypic techniques
In this investigation, we discovered that 33 (78.6%) of K. pneumoniae contain capsule (Table 2 and Fig. 1). Our findings contradict those of (Hullur et al., 2022), who found that the percentage of capsule generation by Klebsiella pneumonia was 100%. The capsule is a significant virulence factor that participates in at least two harmful pathways, including direct inhibition of the host immune response and phagocytosis resistance. (Kang et al., 2015). Our hypermucoviscosity analysis revealed that 17 (40%) of the Klebsiella tested positive for the string test, creating a viscous thread-like string of more than 5 mm in length and so identifying positive for the hmv phenotype (Fig. 2). Our findings were consistent with (Cerón et al., 2022), who found the ratio to be 53.3%, and agree with (Hullur et al,2022;Osama et al.,2023), who found the ratio to be 44% and 42%, respectively, and disagree with (Liu et al,2019), who found 16% of K. pneumonia to be positive. Surface polysaccharide synthesis is thought to protect K. pneumonia against phagocytosis and fatal serum immune factors (Russo and Marr,2019).

Fig. 2: Hypermucoviscosity test by K. pneumonia.
The ability of K. pneumoniae to make protease was studied by streaking on nutritional agar with skim milk and found that only 14 (33.3%) of K. pneumoniae could produce protease (Fig. 3). Our findings agreed with those of (Ramakrishnan et al.,2019), who found that 44% of K. pneumonia was positive for protease. However, our findings disagreed with those of (Hullur et al.,2022), who found that 90% of K. pneumonia was positive for protease production. Microbial proteases operate as virulence factors in many diseases caused by bacteria (protein breakdown). These enzymes are known to cause damage to the host organism's defensive proteins. Protease has been shown to antimicrobial peptide (Anbu, 2016).
Our result showed that all K. pneumonia isolates were negative for hemolysis test on blood agar

Detection of Several K. Pneumonia Virulence Factors Using Genotypic Techniques:
In the current investigation, we chose 20 isolated K. pneumoniae and employed three genes of virulence factors. Among the three identified virulence genes, the siderophore gene entB (enterobactin) was the most common (12%) (Fig. 4). Although enterobactin is the principal siderophore employed by K. pneumoniae, our findings contradict those of ( Remya et al.,2020) who found that 90% of K. pneumoniae isolates had the entB gene. The wcaG gene was found in 3 (15%) of the K. pneumonia samples (Fig. 5). Our findings were corroborated by (Soltan et al.,2020), who discovered wcaG in 27.9% of the isolates. Resistance to phagocytosis is promoted by capsule genes (wabG) (Cortés et al., 2002).
While rmp A gene was absent in all isolates. This result disagreement with (Soltan et al.,2020) who showed that 21.3% of K. pneumoniae isolates were positive for rmpA.