Phenotypic Detection of Some Virulence Factors among Hypervirulent Klebsiella pneumoniae and Classic Klebsiella pneumoniae Associated with Urinary Tract Infections

in larger quantities

The present study aimed to investigate prevalence and some virulence factors among hyper virulent Klebsiella pneumoniae (HVKP) and classic Klebsiella pneumoniae (CKP) isolated from urinary tract infections.Three hundred urine samples collected from patients with urinary tract infections were cultured on MacConkey agar for primary isolation of K. pneumoniae isolates.Vitek 2 Compact System was used for identification of K. pneumoniae.HVKP and CKP were detected by string test.Some virulence factors (biofilm formation, haemolysin, and urease production) possessed by the bacterial isolates were investigated phenotypically.
Sixty bacterial isolates belonged to K. Pneumoniae where 15 (25%) isolates belonged to HVKP as viscous string measuring >5 mm in length and 45 (75%) isolates belonged to CKP where viscous string measuring <5 mm in length.The results of haemolysis test showed that 60% of HVKP isolates gave β-haemolysis, while 51.1% of CKP isolates gave β-haemolysis.All bacterial isolates (100%) of HVKP and CKP showed the ability to produce urease.Phenotypically, all HVKP and CKP isolates showed the ability to form biofilm while only 33.3% and 11.1% isolates showed high ability to form biofilm respectively by Tissue Culture Plates methods (TCP).
polysaccharide is synthesized by the rmpA and rmpA2 genes, which is screened for by a string assay (Tang et al., 2021).
K. pneumoniae possess several virulence factors that increase their pathogenicity, including capsule formation, hemolysin, urease production, and biofilm formation (Dadi et al., 2020).capsule consists of a capsular polysaccharide is the most important virulence factor for K. pneumoniae, as it prevents phagocytosis by the host cells (Mantin and Bachman, 2018).More than 70 serotypes of capsules were recorded.K1 and K2 serotypes were associated with HVKP are responsible for human respiratory tract infections, while K8, K9, K10, and K24 serotypes are responsible for urinary tract infections (Shon et al., 2013;Paczosa and Mecsas, 2016).Hemolysin is a type of toxin secreted by some bacterial species that destroys the cell membrane of red blood cells, leading to their lysis (Mare et al., 2020).K. pneumoniae have the ability to produce urease, which breaks down urea into two parts of ammonia and one part of carbon dioxide (Meknonnen et al., 2020).Ammonia combines with magnesium and phosphate ions, forming a precipitate of triglycerides, causing infection of the urinary tract and stones (Duran et al., 2022).Biofilm is an assembly of microorganisms with their secretions, as the bacteria show new characteristics after the formation of the biofilm that they did not show during free growth because their growth within the biofilms leads to a change in their gene expression on surface markers and changes in nutrient metabolism (Siddique et al., 2020).Biofilm provides bacterial cells with a way to hide from the body's immune system and protection from the immune response as well as protection from toxins, antibiotics, and inappropriate environmental conditions such as drought and ultraviolet radiation (Crabbé et al., 2019).
It is important to detect HVKP due to its high mortality in comparison to multidrug-resistant and CKP infections, where coexisting hepatobiliary disease is a potential risk factor for these infections (Parrott et al., 2021).Also, most of the K. pneumoniae infections were caused by CKP strains that cause infections in immunocompromised patients, and occurrence of multiple antibiotic resistance K. pneumoniae strains was the reason why urinary tract infections became difficult to treat and these bacteria became life-threatening (Kuehn., 2013) so that this study aimed to investigate the prevalence of HVKP and CKP associated with UTI and detect some of their virulence factors.

MATERIALS AND METHODS Isolation and Identification of K. Pneumoniae:
Vitek 2 Compact System (ID/GN) was used to identify K. pneumoniae that isolated from 300 urine specimens collected from patients with UTI.

Detection of HVKP and CKP:
String test described previously (Chang and Ong, 2022) was followed up to differentiate HVKP from CKP.The test is considered positive if a viscous string measuring >5 mm in length was obtained by stretching bacterial colonies on blood agar base and MacConkey agar plates with a bacteriology inoculation loop or needle which referred to HVKP, while it was considered negative if a viscous string measuring <5 mm in length which referred to CKP.

Detection of Haemolysin and Urease Production:
Blood agar base plates (supplemented with 5% human blood) were inoculated with young bacterial colonies by streaking methods.Hemolysis pattern was detected by appearance of either complete blood hemolysis known as ß-hemolysis or partial hemolysis (greenish zone around colonies) known as α-hemolusis or nonhemolysis pattern.Tubes of urea agar base supplement with 40% sterile urea were inoculated with fresh bacterial colonies and incubated for 6-48 hrs. at 37ᵒC.Changing the colour of media from yellow to red referred to the ability of isolates to produce urease (MacFaddin, 2000).

RESULTS
The results of string test showed that out of 60 K. pneumoniae isolates only 15 (25%) isolates gave positive results, as a viscous string measuring >5 mm in length of colonies grew on blood agar base (BAB) and a viscous string measuring >20 mm in length of colonies grew on MacConkey agar, indicating that these isolates belong to HVKP.The results also showed that 25 (41.66%)isolates showed a medium mucoid pattern, as a viscous string measuring 5 mm in length of colonies grew on BAB, and 20 (33.33%) isolates did not show any viscous or mucoid pattern on the culture medium which indicated that these isolates were belong to CKP (Fig. 1).
The results of haemolysis test showed that 9 (60%) HVKP isolates gave β-haemolysis, while 6 (40%) HVKP isolates showed no haemolysis of blood agar base.Among CKP isolates only 23 (51.1%) isolates gave β-haemolysis, while 22 (48.9%)CKP isolates showed no haemolysis of blood agar base (Fig. 2).In addition, all isolates of HVKP and CKP showed the ability to produce urease where the colour of the urea agar base culture was change to pink colour after 24hr of incubation (Fig. 3).The results of biofilm formation by Congo red test showed that all isolates of HVKP and CKP have the ability to form biofilm by changing the colour of the medium to black.The results of the quantitative assessment of the biofilm formation showed that only 5(33.3%)HVKP isolates and 5(11.1%)CKP isolates have high ability to produce a biofilm and showed a high ability to adherence where the optical density values of the biofilm produced by these isolates ranged between 0.243 -0.395.The percentage of HVKP and CKP isolates that showed moderate ability to produce biofilm and adhesion were 60% (9 isolates) and 62.2 % (28 isolates) respectively, where the optical density values of the biofilm produced by these isolates ranged between 0.124 -0.230.Only 1(6.7%)HVKP isolates and 12(26.7%)CKP isolates showed weak or no biofilm formation (range of optical density 0.056 -0.118) (Table 2).et al., 2010).It acts as a shield to protect the bacterial cells from the process of phagocytosis by the immune cells and protects the bacterial cells from the effect of antibiotics by preventing their deposition on the surface of the bacterial cells and it also works to activate the innate immunity of the cells (Lawlor et al., 2005;Paczosa and Mecsas, 2016).Despite the virulence of these strains, some studies indicated that some strains of K. pneumoniae do not have this phenotype and that some HVKP strains may not cause purulent liver abscesses (Yu et al., 2007).
Hemolysin is considered one of virulence factors that contribute to increasing the pathogenicity of bacterial cells.It helps in providing nutrients to bacterial cells, as the lysis of blood cells leads to the provision of iron to bacterial cells, and also helps to secrete histamine and destroy white blood cells (Liu et al., 2020).The results of the current study showed the ability of some K. Pneumoniae isolates to produce hemolysin, this may be due to the occurrence of a genetic mutation in ns-h in K. Pneumoniae isolates, which leads to showing their ability to produce hemolysin.Some studies indicated that in the absence of the aerobactin system, bacterial cells resort to hemolysin production as an alternative pathway for obtaining iron (Sukhan et al., 2010).
Urease is one of the important virulence factors possessed by isolates of K. Pneumoniae bacteria.It breaks down urea into ammonia NH4 and carbonic acid H2CO3 and transforms the medium into an alkaline one (Boer and Hausinger, 2012) as the liberation of ammonia leads to an increase in the pH of the medium and thus a change in the color of the medium due to the sensitivity of the reagent in the culture medium to a change in the pH (Mishra and Agrawal, 2012).
Biofilm is an important virulence factor that bacteria possess.It contributes to enhancing the bacteria's ability to overcome host defences and play role in many bacterial infections (Clegg and Murphy, 2016).It also promotes transmission of resistance genes, increases expression of efflux pumps genes, and reduces antibiotic permeability into bacterial cells (Uruèn et al., 2021).The results of the current study showed a high percentage of K. pneumoniae isolates that produce a biofilm, which plays an important role in increasing the virulence of these isolates.The ability of the bacterial isolates to produce a biofilm is attributed to their possession of fimbria type 1 and type 3, in addition to the presence of LPS and capsule, as these are important virulence factors that help biofilm formation (Clegg and Murphy, 2016).K. pneumoniae biofilm contributes to infection and the development of severe HVKP infections not only in immunocompromised patients but also in healthy adults (Khaertynov et al., 2017).

CONCLUSION
K. pneumoniae represented one of the most causes of UTI and prevalence of CKP followed by HVKP.Distribution of virulent factors that increase the pathogenicity of HVKP and CKP, particularly biofilm formation plays an important role in spreading of antibiotic resistance among isolates.

ACKNOWLEDGMENT
We would like to thank Kufa University / Faculty of Education for Girls/ Department of Biology for using Microbiological Laboratory.REFERENCES Ashurst, J. V., and Dawson, A. (2020)

Table 1 :
used to detect the ability of bacterial isolates to form biofilm. Changing the colour of congo red media from red to black indicate positive results while the quantity of biofilm was detected by measuring the optical density of biofilm layer as mentioned in Table1.Optical density (OD) of biofilm-producing isolates(Hassan et al., 2011).