Phenotypic Detection of Virulence Factors of Uropathogenic Enterobacteriaceae

Members of the family Enterobacteriaceae are the major cause of nosocomial infections, including approximately 70% of gastrointestinal infections, 60% to 70% of urinary tract infections (UTIs), and septicemias. Since 1990, members of Enterobacteriaceae have emerged as predominant pathogens causing UTIs. In this study, we performed phenotypic testing of the virulence factors in uropathogens. A total of 118 uropathogens belonging to the family Enterobacteriaceae were isolated from suspected UTI cases in patients aged > 18 years. Uropathogens were tested for virulence factors, such as hemolysin, phospholipase, gelatinase, and lipase production, as well as for mannose resistance, mannose-sensitive hemagglutination (MRHA, MSHA), serum resistance, and cell surface hydrophobicity. Among 118 uropathogenic Enterobacteriaceae isolates, E. coli was the most prevalent (62,52.5%), followed by Klebsiella (28,23.7%), Citrobacter (23,19.5%), and, Proteus species (5, 4.2%). Serum resistance was the most common virulence factor and was seen in 54(87%) of E. coli , 24 (85.7%) of Klebsiella species, 22 (95.7%) of Citrobacter species, and 5(100%) of Proteus species isolates. Furthermore, 10 (16.1%) isolates of E. coli showed mannose resistance hemagglutination and 7(11.3%) showed mannose-sensitive hemagglutination. Two (8.7%) isolates of Citrobacter species showed mannose resistance and mannose-sensitive hemagglutination. Hemolysis was observed in 33(53.2%) isolates of E. coli , 11(39.3%) of Klebsiella species, 15(65.2%) Citrobacter species, and 1(20%) Proteus isolate. Gelatinase production was observed in 14(22.6%) of E. coli , 6(21.4%) of Klebsiella species, 7(30.4%) of Citrobacter species, and 5(100%) of Proteus isolates. All E. coli, Klebsiella species, and Citrobacter species isolates tested negative for phospholipase. This study showed that the Enterobacteriaceae family plays a role in UTIs by evading the host immune response through the production of various virulence factors.


INTRODUCTION
Urinary Tract Infections (UTIs) form one of the most common bacterial infections in both community and hospital settings,and there has been an increase in morbidity and mortality due to UTIs in the recent past worldwide. 1,2pproximately 10% of humans develop UTIs in their lives at some time.The prevalence of UTIs is age-and sex-dependent.During the first year of life, UTIs occur around 2% of males and females; however, in the latter half, the UTI infection increases in females. 3rolonged hospital stay due to medical or surgical ailments and catheterization of the urinary bladder are risk factors for both sexes in older age.
Among microbial agents that cause UTIs, bacteria are more common than viral or fungal agents.Gram-negative organisms constitute 90% of UTI cases, and the remaining 10% are Grampositive organisms amongst the bacterial etiology.About 65%-90% of the most frequently isolated uropathogens remain Escherichia coli (E.coli). 4,5he most common uropathogens in the community and healthcare facilities belong to the Enterobacteriaceae family.Many of these bacteria are significant human pathogens that cause various infections. 6Some of the members which belong to this family are E. coli, Klebsiella species, Citrobacter species, Proteus species, Enterobacter species, etc., are found in the normal gut flora of mammals and can be isolated from the environment.
Certain virulence factors are expressed by uropathogens to combat the host's immune system or to colonize the organ, which is either a part of the microorganism or secreted by them.
These can be divided into two categories 7 : The following virulence factors were described using uropathogenic E. coli as a prototype.[     The first step in pathogenicity is the adhesion of the pathogens through adhesins such as fimbriae or pili, which help them adhere to the host cell and play a role in evading the host's immune response.Type P fimbriae play a key role in pyelonephritis,and type 1 fimbriae (mannose-sensitive adhesins) promote the development of cystitis 9,10 .Capsular polysaccharide (K antigen) blocks the alternate complement pathway and impairs the classical complement pathway, especially by K1, to protect the bacteria from serum killing; the degree of serum resistance is proportional to the amount of capsular material present, and smooth strains are more serum resistant than rough strains. 11,12Hemolysin, a cytolytic protein secreted by pathogenic strains, lyses erythrocytes, leading to the release of iron and other nutrients that are beneficial to bacteria.They are pro-inflammatory and contribute to inflammation, tissue injury, and impairment of chemotaxis and host defenses. 13his study aimed to identify the phenotypic characteristics of uropathogen virulence factors of the Enterobacteriaceae family in adults aged> 18 years.

Aims and objectives
1. Phenotypic detection of virulence factors of the uropathogenic Enterobacteriaceae.

MATERIALS AND METHODS
Type of Study: a prospective study Place: Tertiary care hospital

Inclusion criteria
• Non-repetitive urine samples were collected from patients aged > 18 years with clinical

Exclusion criteria
• Patients on antibiotics, diuretics, and those with chronic kidney disease.• Polymicrobial growth in culture.
In this study, all significant uropathogens of the family Enterobacteriaceae were initially identified by conventional biochemical reactions (Indole, Triple sugar iron, mannitol motility media, urease production by Christensen's urease agar, and citrate utilization by Simmons Citrate Agar) and later screened for virulence factors, namely hemolysin, lipase, gelatinase, lecithinase (phospholipase) production, hemagglutination inhibition assay, serum resistance, and cell surface hydrophobicity.

Production of Hemolysin
Zone of hemolysis was observed on 5% sheep blood agar after overnight incubation at 37°C as described by Mittal S et al. 14 [Figure 1]

Property of Serum Resistance
The viable count of the inoculum was compared after 3 h of incubation with serum to their original count, as described by Mittal S et al. 14

Cell Surface Hydrophobicity (CSH)
Determined by salt aggregation test (SAT) as described by Mittal S et al.

Hemagglutination
Agglutination of RBC by bacteria was performed using the slide method as described by R Hassan et al. 15 [Figure 3] • For full fimbriation, the isolates were incubated in nutrient broth at 37°C for 48 h.• On a slide, a drop of the bacterial suspension and a drop of 3% RBC suspension were added and incubated for 5 min at room temperature.• If agglutination was seen, the bacterial suspension was further tested with the addition of 2% w/v D-mannose.If clumping was seen, they were considered mannoseresistant hemagglutination (MRHA).

Detection of Lipase
Production of lipase by the isolates was determined by spot inoculating on Tween 80 medium and incubating for a week at 37°C and observing for a zone of opaque halo around the spot as performed by R Hassan et al. 15 [Figure 4]

Detection of phospholipase (Lecithinase)
Production of Lecithinase by the isolates was determined by spot inoculating on Egg yolk agar medium and incubating them at 37÷C for 24-48 as described by Pramodhini S et al. 16 and observed for the appearance of opaque (precipitation) halo around the colony.[Figure 5]

Detection of Gelatinase
This was performed as described by Pramodhini S et al. 16 by the stab method wherein a heavy inoculum of the isolate was stabbed 3-4 times in the nutrient gelatin and incubated at 37°C for 48 h following which the tubes were kept in the refrigerator for 30 min and looked for free flow of the medium indicating the liquefaction of gelatin by the production of gelatinase.[Figure 6]

RESULTS
A total of 118 isolates belonging to the Enterobacteriaceae family were tested for virulence factors.

Escherichia coli (n=62)
• Hemolysis was observed in 53.2% of the isolates.Studies conducted by Mittal et al., 14 Raksha et al., 20 Tabasi M et al., 21 and Shah C et al. 22 reported hemolysis in 47.4%, 41.36%, 34%, and 32.3% of the isolates, respectively, which are comparable to the results obtained in this study.• Studies by Raksha et al., 20 and Vagarali et al. 23 reported MRHA in 30.9% and 25% of isolates, respectively.In this study, it was observed that 16.1% of isolates demonstrated MRHA which was comparable to the results obtained by Vagarali et al. 23 However, MSHA was observed only in 11.3% of isolates.• A study by Hasan R et al. 15 noted MSHA in 28% of isolates.A study conducted by Tabasi M et al. 21observed that 62.8% of isolates showed MSHA and 37.2% of isolates showed MRHA.Shah C et al. 22 observed that 5.7% of isolates showed MSHA and 52.3% showed MRHA.• Serum resistance in the present study was noted to be 87.1%, which was comparable to that reported by Vaish et al. 24 (95.38%).• Cell surface hydrophobicity was observed in 14.5% of isolates, similar to a study done by Raksha et al. 20 where it was noted to be 26.36%.• In the present study, 9.7% of the isolates exhibited lipase activity; however, in a study conducted by Bhattacharyya et al. 25 lipase activity was noted in 100% of the isolates.
In the present study, serum resistance was noted in a majority of isolates (87.1%), followed by hemolysis (53.2%), which could play a role in disease pathogenesis by evading the immune response and causing inflammation, thus predisposing to severe disease.

Klebsiella species (n=28)
There are limited studies on the phenotypic detection of virulence factors in uropathogenic Klebsiella.
• In the present study, 39.3% of Klebsiella  15 and Chaudhary N et al. 26 reported it to be 7% and 43.75%, respectively.• Serum resistance was noted in 85.7% of isolates, which was higher than that reported by El-Naggar et al.In the present study, 3 of the patients had urosepsis (2 E. coli and 1 Citrobacter species) and had hemolysin, serum resistance, and gelatinase properties.The other virulence factors observed in these cases were cell surface hydrophobicity and mannose-sensitive hemagglutination.
In cases of pyelonephritis, renal calculi, and post-infectious glomerulonephritis, the most common virulence factors observed in this study were hemolysin and serum resistance, followed by cell surface hydrophobicity and gelatinase.

CONCLUSION
In the present study, the most common uropathogens isolated were E. coli and Klebsiella pneumoniae.Among the virulence factors, serum resistance followed by hemolysin production was the most common, followed by hemagglutination and gelatinase production.Serum resistance and hemolysin properties set the pace for the pathogenesis of renal disease by facilitating the secretion of interleukins, evading the host immune response, and promoting ascending infection.Other virulence factors, such as lipase and phospholipase (lecithinase), digest the extracellular matrix, thereby facilitating the entry of the organism into the host.

Future scope
Effective development of a vaccine against these virulence factors may decrease the number and severity of UTIs that are still in the trial phase.

Figure 2 .Figure 3 .Figure 4 .
Figure 2. Serum resistance [Figure 2] • A mixture was prepared with a bacterial suspension made in Hank's balanced salt solution from an overnight culture on blood agar and an equal volume of serum and incubated at 37°C for 3 h.• Following this, 10 µL of this mixture was streaked on a blood agar medium and incubated overnight at 37°C.• The uropathogen was considered sensitive if the viable count fell to <1% of the original count and serum resistant if > 90% of organisms survived.

Figure 8 .
Figure 8. Distribution of virulence factors tested in the Enterobacteriaceae family

Figure 9 .
Figure 9. Virulence factors of Uropathogens tested in the Enterobacteriaceae family

Table 2 .
Detection of virulence factors among the uropathogens of the Enterobacteriaceae family Organism