Comparitive Phenotypic Characterization Of Virulence Factors And ESBL Producers Among Classic And

Hypermucoviscous Kleblsiella pneumoniae In Clinical Isolates

M.Anandhi karthikeyan1, P.Suganthi* 1, P. Rubini2, Aneesha.S 1, Swathi.M1,

V. Latha 1

*1Department of Microbiology, Dr.ALMPG IBMS, University of Madras, Chennai,

India 600113

Abstract

Background

Klebsiella pneumonia is a ubiquitous Gram-negative bacterium and also an opportunistic

pathogen can express a multitude of virulence factors and also causing emergence of

antimicrobial-resistant and hyper virulent lineages.

Materias and Methods

A total of 20 clinical isolates were collected from different tertiary care hospital in

chennai. Classic klebsiella pneumonia(cKP) and Hypermucoviscous K. Pneumonia (hmKP)were

determined by using string test. Antimicrobial susceptibility testing was performed by Kirby

Bauer disk diffusion method. Phenotypic virulence factors were performed to compare the

manifestation in cKP and hmKP.

Result

In this comparative study, AST shows 85% Cephalosporin, 80% of Quinoline, 75%

AMP β-lactam and 60% Aminoglycosides resistance.Out of 20 isolates 17 ESBL positive in

that, 8 cKP and 9 hmKP. 10 isolates shows string test positive.In capsule staining heavy capsule

content in hmKP than cKP, 20% cKP and 30% hmKP were positive to gelatinase test. In

protease test 40% cKP positive and 10% hmKP were positive, lecithinase test showed positive

to 40% cKP and 20% hmKP, lipase activity showed positive to 40% cKP and 80% hmKP, only

10% of hmKP shows positive to blood hemolysis test. 90% cKP and hmKP is Mannose-resistant

type-3 fimbriae 10% of cKP and hmKP shows Mannose-sensitive type-1 fimbriae in direct

haemagglutination. In biofilm detection assay, the 40% cKP and 60% hmKP were strong,10%

cKP and 30% hmKP were moderate and 50% cKP and 10% hmKP were weak.

Conclusion

Majority of our isolates showed presence of multiple virulence factors. Among

Klebsiella pneumoniae isolates haemagglutination and biofilm formation were found to be the

predominant virulence factor in that hypermucoviscous Klebsiella pneumonia is more virulent

than classic Klebsiella pneumonia.

key words: hypermucoviscous Klebsiella pneumonia, classic Klebsiella pneumoni,

phenotypic characterization, virulence factors.

Introduction

Klebsiella pneumoniae is a Gram-negative pathogenic bacterium that has a mucoid

phenotype which is conferred by polysaccharide capsule attached to bacterial outer membrane.

also considered as one of the opportunistic pathogens causing broad spectra of diseases such as

urinary tract infections, cystitis, pneumonia, surgical wound infections, endocarditis ,septicemia,

pyogenic liver abscesses, necrotizing pneumonia and endogenous endophthalmitis(Alyssa S.

Shon et al., 2013; Paczosa MK et al., 2016). Klebsiella pneumoniae can be broadly classified

into two subtypes; classic Klebsiella pneumoniae (cKP) and non-classic Klebsiella pneumoniae

(ncKP). These strains are notorious with antimicrobial resistance profile and virulence profiles.

Several clones of ncKP leads to causing severe and difficult to treat infections due to their

continuous mutation and acquisition of plasmids and transposons that carries resistant and

virulent genes.

There are “classic” and “hypermucoviscous” strains of Klebsiella pneumoniae. “Classic”

are virulent strains that are usually associated with urinary tract, pneumonia and nosocomial

infections(Bei Yao et al., 2015). Classic Klebsiella strains have recently gained notoriety due to

their propensity to acquire antimicrobial resistance(Marisa B. De Jesus et al., 2015). Within last

few decades, extended-spectrum β-lactamase (ESBL) positive classic K. pneumoniae isolates

have been discovered world-wide.

This led to emergence of strains such as hypermucoviscous Klebsiella pneumoniae

(hmKP). This strain was first identified in Eastern part of Asia and has spread Worldwide. These

hypermucoviscous strains are non-resistant to most of the commonly used antimicrobials in the

early years of treatment(Marisa B. De Jesus et al., 2015). Recently Carbapenem-resistant

hypermucoviscous Klebsiella pneumoniae strains has been reported.

Virulence factors comprise many mechanisms and allowing the bacteris to cause various

infections. K. pneumoniae employs many strategies to grow and protect itself from the host

immune response. Numerous factors contribute to K. pneumoniae virulence, such as the ability

to produce siderophores, fimbriae, lipopolysaccharide (LPS), and extracellular polysaccharide

capsule (Kalaivani R et al., 2019). Hypervirulence is associated with these factors, particularly

with the overproduction of capsular polysaccharide. coating more tough than the classical

capsule. Both classical and hypermucoviscous strains contain specific K antigens. Classical K.

pneumoniae contain K1-K78 capsule types and hypermucoviscous K. pneumoniae contain K1

&K2 capsule types. Klebsiella pneumoniae strains with K1 and K2 capsular type shows invasive

ability of causing community acquired infections like pyogenic liver abscesses (Paczosa MK et

al., 2016). Type 1 and type 3 fimbriae were most important adhesive structure that have

characterized as virulence factor in Klebsiella pneumonia (Schroll et al., 2010; Paczosa MK et

al., 2016). The type 3 fimbriae are expressed during biofilm formation on catheters. Biofilm

formation in K. pneumoniae is affected by mutations in genes responsible for capsule

biosynthesis and surface EPSs (Balestrino et al., 2008; Boddicker et al., 2006), genes for the

synthesis of cell surface type 3 fimbriae (Di Martino et al., 2003; Langstraat et al., 2001). This

comparative study aims to gains the virulence characteristics of ESBL producing Classic and

hypermucoviscous Klebsiella pneumonaie

Materials and Methods

Twenty Klebsiella pneumoniae clinical isolates were collected from different tertiary care

hospitals Chennai obtained from various clinical samples of Pus (9), Urine (7) and Endotracheal

aspirates (4) shown in fig1. All isolates were confirmed biochemically using standard

biochemical standards Antimicrobial sensitivity testing was performed by Kirby Bauer disk

diffusion method for different classes of antibiotics as per CLSI. Klebsiella pneumoniae isolates

were studied for their presence of ESBL producers and various virulence factors phenotypically.

Detection of ESBL Producing Isolates

For each pure isolate antimicrobial sensitivity testing was performed by disk diffusion technique

and ESBL production is detected using standard disc diffusion susceptibility test with

Cefotaxime-Clavulanic acid (30/10µg) on Muller-Hinton Agar. The presence of ESBL was then

confirmed by Modified Double Disc Synergy Test (MDDST) by CLSI.

Phenotypic detection of virulence factors of K. pneumoniae isolates

1. Phenotypic detection of hypermucoviscosity

Hypermucoviscosity of the 20 isolates were tested by evaluating the formation of a mucoviscous

string of >10 mm, when using an inoculation loop to stretch a colony grown on blood agar plate.

(Mustafa Muhammad Gharrah et al., 2017, H.C. LEE et al., 2006)

2. Capsule detection staining

The presence of capsule is detected by using nigrosin staining on K. pneumoniae isolates. Place

a small drop of negative stain (India ink or Nigrosin) on the clean glass slide. Using a sterile

loop add a loopful of bacterial culture on the stain and smear it using another clean glass slide.

The thin smear is allowed to air dry. Later flood the smear with Saffranin for about 1 minute.

Drain the safranin by tilting the slide at 45 degree angle and let stain run off until it air dries.

Examine the smear microscopically under oil immersion 100X ( Sagar Aryal 2018).

3. Gelatinase detection test

Nutrient agar plates with 3% gelatin were prepared and the classic and hypermucoviscous K.

pneumoniae isolates were ‘spot’ inoculated on the medium. Inoculated plates were incubated at

37˚c for 24 hours. The gelatinase producing K. pneumoniae isolates were identified by clear

zone around the colonies once the medium was flooded with Mercuric chloride solution.

Mercuric chloride solution was prepared using Mercuric chloride HgCl2 (15 g), conc.

Hydrochloric acid HCl (20ml), distilled water (100ml) Mustafa Muhammad Gharrah et.,al 2017.

4. Protease activity detection test

Skimmed milk agar plates were made using autoclaved Skim milk (with 1.5% final

concentration) into the molten Nutrient Agar medium. This were mixed thoroughly and poured

into the sterile petri dishes. The classic and hypermucoviscous Klebsiella pneumoniae were

‘spot’ inoculated on the medium and incubated at 37˚c for 48-72hrs in inverted position.

Protease production was identified by formation of clear zone around the colonies. (Kalaivani

Ramakrishnanet., 2019)

5. Lecithinase detection test

Trypticase soy agar with the addition of 0.11% of Calcium Chloride and 5% egg yolk were used

for the demonstration of lecithinase activity. The medium was autoclaved at 121˚c with 15lbs

for 15 mins. Then the isolates of K. pneumoniae were ‘spot’ inoculated on the solidified medium

in the sterile petri dishes. After incubation at 37˚c for 24 hours, the opaque zone around the

colonies were observed for positive lecithinase production. (Marie T. Esselmann and Pinghui V.

Liu, 1960)

6. Lipase activity detection test

This test was carried out by ‘spot’ inoculating the 24hrs culture of Klebsiella pneumoniae

isolates into the egg yolk agar medium and incubated at 37˚c for 24 hrs. After incubation, the

plates were flooded with 1% saturated aqueous solution of Copper sulphate (CuSo4) and allowed

to stand for 20 mins. After few minutes of drying the solution from the medium, the

interpretation were made. The greenish blue colour of opulence indicates lipase production.

(Kalaivani Ramakrishnan et al., 2019)

7. Blood hemolysis test

Blood agar plates were used containing 5% human blood. The total 20 isolates were streaked on

the blood agar plates and incubated at 37˚c for 24hrs. After incubation the plates were examined

for α, β and γ hemolysis. (Mustafa Muhammad Gharrah et al., 2017)

8. Direct haemagglutination test

3% RBC was prepared by centrifuged the whole blood at 1800 rpm for 15 mins along with

normal saline. The supernatant was removed and again normal saline was added for

centrifugation. This was repeated until the clear supernatant obtained. The pellet contain packed

cell volume of RBC, it was then made up to 3% suspension by adding the fresh saline in them.

This was used immediately or within a week when stored at 4˚c. The 20 isolates were inoculated

into fresh LB broth and incubated at 37˚c for 24-48hrs. The slide haemagglutination test carried

out by adding one drop of broth culture and one drop of 3% RBC. The slide was rotated to mix

them for 5 mins. Presence of clumping was taken as positive haemagglutination test. Later 2%

mannose solution was added to the positive haemagglutination isolates and examine for the

presence or absence of clumping. Mustafa (Muhammad Gharrah et.,al 2017)

9. Biofilm detection assay

The prevalence of producing biofilm by classic and hypermucoviscous Klebsiella pneumoniae

were detected using tube method, 96-well titer plate assay and Congo-red agar plate method.

Tube method

LB broth with 1% glucose was prepared and isolates were inoculated into 10ml of the broth in

test tubes. After 24hrs of incubation at 37˚c, the tubes are decanted, washed with the saline and

dried. Tubes were stained using 0.1% crystal violet for 20 mins. Later the tubes were washed

with saline and dried in inverted position. Tubes were examined for the stained film which lined

the wall and the bottom of the tube. The tubes were classified into strong, moderate and weak

based on the biofilm formed on the test tubes.

96-well microtiter plate assay

100ml of LB broth with 1% glucose were prepared and the isolated were inoculated into the

broth (1000µl), incubate for 24hrs at 37˚c. According to 1:100 dilutions, 100µl of 24hrs culture

mixed with 1000µl of fresh LB broth with 1% glucose. This dilution was pipetted out about

100µl into the 96-well microtiter plate. The plate was incubate for 48hrs at 37˚c. After

incubation, invert the microtiter well and drain the broth. Wash the well using normal saline for

3-4 times. Dry the plate, add 0.1% crystal violet in the wells. After 15-20 mins of staining, drain

the wells. Wash with saline, 3-4 times and dry the plate. Add 30% Glacial Acetic acid into the

microtiter wells. Biofilm quantification were obtained by measuring the absorbance at 570 nm.

Biofilm values were obtained by calculating the mean of the absorbance for at least three

independent experiments and compared to negative.

Congo-red agar method

For this method, medium was prepared using Congo-red powder (0.8gm/L), Brain heart infusion

broth (37gm/L), Agar no.1 (10gm/L) and sucrose (5gm/L). First Congo red dyes were prepared

separately and autoclaved at 121˚c for 15 mins. Later it was mixed with sterilized brain heart

infusion agar with sucrose at 55˚c. The plates were made and isolates were streaked on the

medium. The biofilm formation observed by the formation of black colonies. The isolates

classified strong, moderate and weak based on the intensity of the black colonies production.

RESULTS

Total of twenty clinical isolates of classical and hypermucoviscous Klebsiella pneumoniae were

observed that 45% from pus (n=9), 35% from urine (n=7) and 20% from endotracheal aspirates

(n=4) were collected from different patients of tertiary care hospitals in Chennai it was shown in

Fig1. It was observed that out of 20 isolates, 85% shows Cephalosporin 80% Quinoline 75%

AMP β-lactam and 60% Aminoglycosides resistance. Out of 20 isolates 17 were confirmed to be

ESBL positive 8 classical K. pneumoniae and 9 hypermucoviscous K. pneumoniae. Out of 20

isolates, 10 (50%) isolates showed positive string test (≥5 mm) results which represents

hypermucoviscosity of hypermucoviscous K. pneumoniae (hmKP). The capsule staining results

showed higher capsule content in hmKP than cKP was shown in figure2. In gelatinase test

shows that the medium became opaque and the clear zone was observed around the colonies

after flooding the plates with Mercuric chloride, 20% (n=2) of isolates showed positive results in

cKP and 30% (n=3) of isolates showed positive results in hmKP.

In protease detection test the positive result opaque zone around the edges of colonies after

incubation, cKP showed 40% (n=4) and 10% hmKP (n=1). In lecithinase test positive results

showed clear zone around the cKP 40% (n=4) and hmKP shows 20% (n=2). In lipase positive

results showed oily appearance in the colonies and opaque zone around the edges colonies after

flooding the plate with 1% aqueous saturated Copper sulphate (CuSo4) after 20 mins, 80% (n=8)

of hmKP and 40% (n=4) of cKP. The blood hemolysis test shows that all cKP isolates have

gamma hemolysis but only one (n=1) hmKP isolates have β-hemolysis. The presence of

haemagglutination even after addition of mannose indicates the Mannose-resistant

haemagglutination whereas mannose-sensitive haemagglutination was detected by the absence

of haemagglutination in the test. The direct haemagglutination results showed that 90% (n=9) of

cKP and hmKP have Mannose-resistant type-3 fimbriae. Only 10% (n=1) of cKP and hmKP

showed Mannose-sensitive type-1 fimbriae. Biofilm formation were observed in three methods:

Tube method, Congo Red Agar plate method and 96-well microtiter plate assay method and

results were classified as strong, moderate and weak. The cKP isolates showed 40% (n=4)

strong, 10% (n=1) moderate and 50% (n=5) weak biofilm formation while hmKP isolates shows

60% (n=6) strong, 30% (n=3) moderate and 10% (n=1) weak biofilm formation. The

Comparitive phenotypic detection of virulence factors of both classical and hypermucoviscous

Klebsiella pneumonia are showed in Table1 and Fig 2

Pus45%

Urine35%

Endotracheal aspirates20%

CLINICAL ISOLATES

Pus

Urine

Endotracheal aspirates

Fig 1

Table1

S.n

o

Phenotypic detection of

various virulence factors

Ckp isolates

showed positive

virulence factors

(%) (n=10)

Hmkp isolates showed positive

virulence factors (%) (n=10)

1 Gelatinase 20% 30%

2 Protease 40% 10%

3 Lecithinase 40% 20%

4 Lipase 80% 40%

5 Haemagglutination MSHA 10% 10%

6 MRHA 90% 90%

7 Biofilm 40% 60%

Fig 2

String test positive ESBL positive klebsiella pneumoniae

detected by MDDST

Classic klebsiella pneumonia capsules Hypermucoviscous klebsiella pneumoniae

capsules

Positive gelatinase test Positive lecithinase test

classic k. pneumonia Hypermucoviscous k. Pneumonia classic k. pneumonia

Hypermucoviscous k. Pneumonia

Positive lipase test

Classic K.pneumoniae Hypermucovicous K.pneumoniae Blood hemolysis test

Positive Protease test for Classic and hypermucoviscous K.pneumoniae

Direct haemagglutination test

Mannose-resistant haemagglutination and Mannose-sensitive haemagglutination

Biofilm detection assay

Tube method Congo-red agar plate

method 96-well microtiter plate

assay method

DISCUSSION

Klebsiella pneumoniae possess various virulence factors and various drug resistant

mechanisms which are proven to initiate greater lethal effects. It proportionally leads to high

morbidity among infected population. ESBL are typically resistant to some or all

cephalosporins, but they exhibit some differences. ESBLs are inhibited by β -lactam inhibitors

and do not hydrolyze cephamycins. ESBLs are typically encoded by plasmid-borne genes. Since

some laboratories do not routinely identify the specific mechanism of resistance to

cephalosporins in this study out of 20 isolates 17 were confirmed to be ESBL positive out of 17

ESBL positive isolates, 8 were from classical K. pneumoniae and 9 were from

hypermucoviscous K. pneumoniae.

Detection of various virulence factors plays important tool to identify the virulence

potential of the pathogen. The direct haemagglutination test results shows that 90% (n=9) of

each cKP and hmKP have Mannose-resistant type-3 fimbriae. Only 10% of each cKP and hmKP

shows Mannose-sensitive type-1 fimbriae. Isolates with type 3(MR) fimbriae were known to

bind with the human endothelial cells, epithelial cells of genitourinary and respiratory system

which were the majority of observed results in this study isolates. Contrast, only 50% of our K.

pneumoniae isolates were found to be able to produce hypermucoviscosity in our study. Biofilm

formation was observed to be 40% Strong in cKP and 60% strong in hmKP of this study

isolates. Various fimbrial adhesins have been shown to play a role in biofilm formation . Most

K. pneumoniae isolates express type 3 fimbrial adhesin ( Bandeira et al., 2014 ). Many studies

have revealed that type 3 fimbriae (mrkD gene) are important in K. pneumoniae biofilm

formation (Jagnow and Clegg , 2003 ; Ong et al., 2010 and Bellifa et al., 2013).

Lecithinase and phospholipase are enzymes released by bacteria which have the ability

to destroy host tissues. The enzyme lipase digests the host cellular lipid for its nutritional

demand. In lipase activity test, 80% of hmKP and only 40% of cKP shows positive result

whereas in Lecithinase production test, 40% of cKP and 20% of hmKP shows positive result, in

contrast to our study 58% were reported to be positive by Gharrah et al., Close to our findings,

recently Alam et al., from Lucknow reported 76.9% positive result for lipase formation in their

studies. Lecithinase production among Klebsiella pneumoniae was found to be directly

associated with skin and soft tissue infections and systemic infections. Enzyme gelatinase

hydrolyses gelatin and has the ability to hydrolyse collagen in subcutaneous tissue during wound

infections. In the present study gelatinase production was observed among 20% in cKP and 30%

in hmKP, protease production was observed among 40% in cKP and 10% in hmKP. Pereira et

al., reported that none of their Klebsiella isolates showed haemolytic activity. But in contrast, a

significant percentage of 10% of hmKP isolates showed haemolytic activity. Capsule is complex

of lipid polysaccharide which helps to make a biofilm formation and has antiphagocytic activity

to resist the host immune systems. Biofilm is a common virulence factor which is the mode of

growth for bacteria in natural and clinical environments. Bacterial extracellular polysaccharides

have been shown to mediate closely packed with cell-to cell and cell to surface interactions that

are required for the formation, cohesion and stabilization of bacterial biofilms. In this study 10

hypermucoviscous Klebsiella pneumonia in capsular staining results shows higher capsule

content compare to classic Klebsiella pneumonia which shows that these types of heavy capsular

polysaccharide in hmKP protects the bacterium from phagocytosis by polymorphonuclear

granulocytes and prevents killing by serum factors. Gram-negative bacterial colonization often

precedes nosocomial pneumonia.

Virulence factor in multidrug resistance Klebsiella pneumoniae isolates can cause

various life threatening infections. Majority of our isolates showed presence of multiple

virulence factors. Among Klebsiella pneumoniae isolates haemagglutination and biofilm

formation was found to be the predominant virulence factor in that hypermucoviscous Klebsiella

pneumonia is more virulent than classic Klebsiella pneumoniae. Majority of classic Klebsiella

pneumoniae showed Lipase, Lecithinase and Protease formation. Most researches are dedicated

to studying either antimicrobial resistance or virulence, though the biological effect and relation

between those factors are of particular importance. Since the third-generation cephalosporins,

like other 𝛽-lactam antibiotics, are crucial for treatment of severe hospital-onset or community-

acquired infections caused by K. Pneumonia (D. M. Livermore et., al) Data presented in this

report shows that hmKP strains are more virulent than cKP. Detection of virulence factors might

help in management process, as presence of more virulence factors increases the pathogenic

ability which leading to lots of therapeutic challenge.

CONCLUSION

In this comparative study, the differences in the prevalence of virulence factors and

ESBL among classic and hypermucoviscous Klebsiella pneumoniae were observed. This study

suggests the correlation between the ESBL production and virulence factors. According to our

study, hmKP isolates shows strong biofilm formation compared to cKP. The biofilm formation

is the main source for nosocomial infection. To prevent the spread of this one need to perform

better hospital management practices. It is necessary to analysis the virulence factors and ESBL

to increase the clinical awareness there by decreasing the catastrophe of therapeutic response in

infected individuals. Furthermore, Klebsiella pneumoniae is evolving in last few decades

becoming next superbug. Efforts should be taken to prevent the multi-drug resistant Klebsiella

pneumonia from spreading. At present, the prevalence of hypermucoviscous in the elderly,

especially ESBL- hypermucoviscous and MDR- hypermucoviscous is increasing. This present

study is to construct awareness for the importance of community acquired and hospital acquired

infection due to classic and hypermucoviscous Klebsiella pneumoniae ability to produce ESBL

production and various virulence factors and also alert infectious disease clinicians to the

possible presence of hypermucoviscous in MDR resistant colonizing isolates. Patients harboring

these isolates are at risk of developing invasive infections.

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