Post on 24-Sep-2020
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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