* Corresponding author: Angham J. M. Ali, College of Health and Medical 
Techniques, Al-Furat Al-Awsat Technical University, Kufa – Iraq 

E-mail addresses: angham.ali@atu.edu.iq    
 

DOI: https://doi.org/10.48112/bcs.v1i1.80  

Copyright © 2021 Biomedicine and Chemical Sciences 
 

 

How to cite:  
Ali, A. J. M., Alkhudhairy, M. K., & Tobal, D. D. (2022). Immunological Study of Klebsiella Pneumoniae Isolated from Pneumoniae Infection Patients. Biomedicine and 
Chemical Sciences, 1(1), 11–19. https://doi.org/10.48112/bcs.v1i1.80  

Biomedicine and Chemical Sciences 1(1) (2022) 11-19  

 

 

 

  
Immunological Study of Klebsiella Pneumoniae Isolated 

from Pneumoniae Infection Patients 

Angham J. M. Alia*, Miaad K. Alkhudhairyb , Dhabaa D. Tobalc 

a,b,c College of Health and Medical Techniques, Al-Furat Al-Awsat Technical University, Kufa – Iraq 
 

A R T I C L E  I N F O  A B S T R A C T 

Article history:  
This study aimed for isolation and identification of Klebsiella pneumoniae was an 
opportunistic pathogen responsible for a wide range of clinical syndromes such 

as pneumonia in both hospital and community settings. Assessment some 
immune parameter this study was carried out in Al-Diwaniya Teaching Hospital 
in Al-Diwaniya Province during the period from December 2018 to February 
2019. A total of 272 individuals in both sex: 139 males and 133 females, 

including 210 sputum and blood specimens were collected from patients with 
pneumonia and 62 blood specimens were collected from healthy persons as a 
control group. The result of Microbiological tests of sputum was found 
120(57.2%) specimens as Gram negative bacteria. From these specimens found 
62(51.7%) have been appeared as positive result for K. pneumoniae and 
represented a major cause for pneumonia in this study, 37(59.7%) of them were 
males and 25(40.3%) female. 59(95.16%) of K. pneumoniae isolates have capsule 

when stained negatively with Indian ink. While, 60 (96.77%) isolates were 
appeared hypermucoviscosity (HVM) phenotype. The level of interferon gamma 
(IFN-γ) and toll like receptor (TLR4) concentration was measured by using 
enzyme-linked immunosorbent assay (ELIZA) and was found that IFN-γ is 
significantly (p ˂0.05) raised in all age groups of patients in comparison to the 

healthy control groups. The level of IFN-γ in age group (1-10) years recorded high 
percentage (293.123) compared with other age groups.. On the other hand, the 
level of TLR4 concentration was found that is significantly (p˂0.05) raised in all 

age groups of patients in comparison to the healthy control group. The level of 
TLR4 in age group (51-60) years recorded high percentage (12.993) compared to 
other age groups. 

Copyright © 2021 Biomedicine and Chemical Sciences. Published by International Research and 

Publishing Academy – Pakistan, Co-published by Al-Furat Al-Awsat Technical University – Iraq. This is an 

open access article licensed under CC BY:  

 (https://creativecommons.org/licenses/by/4.0)  

Received on: 12 November 2021 

Revised on: 15 November 2021 
Accepted on: 22 November 2021 

Published on: 01 January 2022 

 

  

Keywords:  

ELIZA 

IFN-γ 

K. pneumoniae 
TLR4 

 

 

 

1. Introduction 

Pneumonia can be transmitted by airborne germs from 
an infected individual are inhaled by someone else. 
Anyway, most instances of pneumonia are attributable to 
self-infection with one or more types of microbes that 

originate in the nose and mouth. In healthy people, 
typical upper airway bacterial residents such as 
"Streptococcus pneumoniae" and "Haemophilus influenze" 

are the most common bacteria causing community-
acquired pneumonia (CAP) (Hanada et al., 2018). 

Otherwise, hospital acquired pneumonia (HAP) is 

usually caused by more resistant bacteria, such as "K. 
pneumoniae", "Staphylococcus aureus", "Pseudomonas 
aeruginosa", and "Escherichia coli". Individuals with a 
serious impairment of their immune system become 

susceptible to pneumonia caused by so-called 

Contents lists available at:  
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Biomedicine and Chemical Sciences 
 

Journal homepage: https://journals.irapa.org/index.php/BCS   
 

3-BCS-192-80 

mailto:angham.ali@atu.edu.iq
https://doi.org/10.48112/bcs.v1i1.80
https://journals.irapa.org/index.php/BCS/index
https://doi.org/10.48112/bcs.v1i1.80
https://journals.irapa.org/index.php/BCS/index
https://irapa.org/
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https://creativecommons.org/licenses/by/4.0
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Ali, Alkhudhairyb & Tobalc        Biomedicine and Chemical Sciences 1(1) (2022) 11-19  
 

12 

opportunistic germs, such as certain viruses, fungi, and 
bacterial related to tuberculosis (mycobacteria), which 

wouldn't ordinarily cause illness in normal individuals. 
To beat with its constant exposure to potentially 
infectious microbes, the lung depends on a hierarchy of 
defense mechanisms (Paczosa & Mecsas, 2016). 

Klebsiella pneumoniae considered as one of the most 
important pathogens but the mechanism by which this 
bacterium causes diseases is still not fully understood 
and approximately most studies have limited facts about 

this mechanism because of not wide ranges of virulence 
factors investigated, and few studies exist at the present 
time to study the relationship between virulence factors 
and resistance of K. pneumoniae to antibiotics (Paczosa & 

Mecsas, 2016). 

Successful defense against infections requires a 
coordinated action of multiple immune cell subsets. In 
this context, it is widely appreciated that interferon γ 

(IFNγ) and toll like receptors 4 TLR4 decisively coordinate 
immune responses by modulating cell-autonomous 
immunity and inflammatory responses, and by dictating 
immune cell-to-cell communications. While type I IFNγ 

are the major effector cytokines of the host defense 
response against bacterial infections. Type I IFNγ protect 
against the progression of a localized K. pneumoniae lung 

infection to invasive disease (Ivin et al., 2017). 

Toll-like receptors 4 recognize pathogens, resulting in 
onset of the inflammatory response. TLR4 are expressed 
in both cells of hematopoietic origin and stromal cells e.g. 
lung epithelium. When K. pneumoniae enters the lung, 
bacterium specific TLR4 are activated, triggering the 
release of cytokine and chemokine that attract and 
activate neutrophils. TLR4 the most important TLR for 
the recognition of K. pneumoniae by virtue of its capacity 
to sense lipopolysaccharide (LPS) present in the outer 
membrane of this Gram-negative pathogen (Sender & 

Stamme, 2014). 

2. Materials & Methods 

Table 1 
Commercial kits in the current study 

Kit Company Origin 

API-20 E bioMerieux® USA 
Human IFNγ ELIZA 

Human TLR4 ELIZA 

Elabscience 

 

European 

 

 
Table 2 
Culture media in the current study 

Medium Abbreviation Company Origin 

Blood agar BA Himedia India 

CHROM agar CHROM agar 
Becto and 
Dickinson 

France 

Eosin methylene blue 

agar 
EMB agar 

Himedia India MacConkey agar MCA agar 

Xylose lysine 

deoxycholate agar 
XDL agar 

2.1. Patients and Clinical Samples 

A total of 272 individuals in different gender and age 
groups (139 males and 133 females), 210 clinical samples 
of patients which from respiratory tract and 62 samples 

from healthy as a control group, between first December 
2018 and first February 2019 in Al-Diwaniya Teaching 
Hospital in Al-Diwaniya Province clinical specimens 

(blood and sputum) were collected at the same day of 
consultation as in follow: 

2.2. Samples Collection 

Samples were collected by using swabs from sputum. 
The sterile cotton swabs were immersed in BHI agar 
tubes and transferred to the laboratory (Collee et al., 

6991) , and three ml of fresh blood were collected. Blood 
was obtained by vein puncture after disinfect the skin 
with 70% alcohol to avoid contamination. The blood 
samples allowed to clot for about 1 hour at room 

temperature, the clot loosed gently by a wooden stick. 
Then, samples were centrifuged for 10 minutes at 3000 
revolutions per minute (rpm), the serum separated and 
transferred to other tubes for storage at -20 ° C (Lewis et 

al., 2001). 

2.3. Identification of Klebsiella Pneumoniae Isolates 

Isolates were identified depending on the morphological 
features on culture medium and biochemical tests 

according to the classification of MacFaddin (2000).  

2.4. Cultural Characteristics  

All samples were inoculated on to BA, MCA, EMB, XLD 
and CHROM Agar and incubated at 37˚C for 24 hours 

under aerobic condition. Colonies were purified by sub-
culturing for further identification. Color, shape, size, 
edge, type of haemolysis and lactose fermenter were 
observed (Harley & Prescott, 1996). 

2.5. Virulence Factors Detection 
2.5.1. Capsule 

The negative staining bacteria of Indian ink was 
inquired to detect the presence of bacterial capsule, 

according to Atlas et al.,  (1995). 

2.5.2. Hypermucoviscosity Test (HMV) 

Mucoid isolates of K. pneumoniae were identified by 

inoculating bacteria on MCA and incubated at 37 ºC for 
24 hours, after incubation the colony touched by a loop 
and lifted vertically away from surface of MCA, colonies 
formed a string ≥ 5 mm in length show HMV isolates 

(Macfaddin, 2000). 

2.6. ELIZA for Determination of IFN-γ and TLR4 
Concentration  

2.6.1. Principle of ELZA Assay  

The assay of ELIZA was performed according to the 
method mentioned by the manufacturing company. The 
OD was measured spectrophotometrically at a wavelength 
of 450 nm ± 2 nm. The concentration of human IFN-γ was 

calculated in the samples by comparing the OD of the 
samples to the standard curve. 

3. Results & Discussion 
3.1. Clinical samples Collection 

The result of Microbiological tests of 210 sputum was 
found 74(35.2%) specimens as Gram positive bacteria 

and 120(57.2%) specimens as Gram negative bacteria, 
while 61(7.1%) of rest specimens wasn’t show any growth. 
The results of isolation were summarized in Figure 1. 



Ali, Alkhudhairyb & Tobalc        Biomedicine and Chemical Sciences 1(1) (2022) 11-19  
 

13 

The most general results in this study evidenced that 
the majority of patients with pneumonia showed the 

presence of Gram-negative bacteria more than Gram 
positive bacteria. Similar observations have been made by 
other investigators in the world. For instance, in the 

United States, Barazile and Nepal infection with Gram 
negative bacteria account for about 50-70% of nosocomial 

infections, and similar data have been reported from 
other parts of the world (Parajuli et al., 2017). 

 

 
Fig. 1. The percentage of bacterial isolation from the lower respiratory tract infections 

 

3.2. Identification of Klebsiella Pneumoniae Isolates  

In the current study, from 120 sputum specimens that 

found as Gram negative bacteria, 62(51.7%) specimens 
have been appeared as positive result for K. pneumoniae 
and represented a major cause for pneumonia while 

58(48.3%) of specimens were represented other Gram-
negative bacteria (Figure 2).  Many different results of 
nosocomial K. pneumoniae isolations were detected in all 
over the world such as: in Iraq, Ali & Ismael (2017) were 
isolated K. pneumoniae in low percentage (8.1%) from 
sputum in Erbil City. 

 

 
Fig. 2. Percentage of isolation of 120 isolates of K. pneumonia and other Gram-negative bacteria depending on Microbiological tests 

 

Sixty tow isolates were belonging to K. pneumoniae 
positively from respiratory tract infections, 37(59.7%) of 
them were males and 25(40 .3%) female (Table 3). 

Table 3 
Distribution of K. pneumoniae isolates among both sex 

Type of sex K. pneumoniae Type of sex 

Male 37 59.7 
Female 25 40.3 

Most of the K. pneumoniae isolates in this study were 
collected from male patients and this result similar to 
Nirwati et al. (2019) study who found that K. pneumoniae 

isolation rate was 64.07% in male and was 35.93% in 
female patients.  

 

 

3.3. Cultural and Morphological Characteristics  



Ali, Alkhudhairyb & Tobalc        Biomedicine and Chemical Sciences 1(1) (2022) 11-19  
 

14 

Isolates of K. pneumoniae were detected based on 
cultural, Morphological, microscopical and biochemical 

characteristics, according to MacFaddin (2000). Colonies 
of K. pneumoniae were observed, rounded, smooth, 
convex and Gamma hemolytic when cultured and 
streaked on BAB. While the colonies on MCA and EMB 

agar which appeared typically large, mucoid, with pink to 
red pigment, usually diffusing into the surrounding agar, 

indicating fermentation of lactose and acid reduction. On 
the other hand, colonies found with yellow color on XLD 

agar as indicator for saccharide fermentation (Figure 3). 
The confirmatory cultured tests were performed by 
growing all 62 isolates of K. pneumoniae given greenish 
blue colonies on Chrome agar media (Figure 3) 

. 

 
Fig.3. Colonies of K. pneumoniae growing on different culture media after 24 hrs of incubation under aerobic condition .  

 

(A): Blood agar,; (B): MacConkey (C): Xylose lysine 

deoxycholate agar, (D): Eosin methylene blue agar 

 
Fig 4. Greenish blue colonies of K. pneumoniae on chrome agar surface 
after incubation for 24 hru at 37oC under aerobic condition 

3.4. Microscopically Diagnosis 

The results of light microscopic examination under oil 
immersion lens and the magnification power (1000 X). 
Cells of K. pneumoniae isolates were appeared Gram 
negative rod and non-spore forming. 

3.5. Biochemical Tests 

All Gram-negative isolates that grown on MCA, EMB 
agar, XLD agar undergo biochemical tests in order to 
distinguish K. pneumoniae isolates from other members 

of related lactose and sucrose fermented bacteria. All 
isolates of K. pneumoniae were appeared lactose and 
sucrose fermenters. The biochemical tests that mentioned 

in Table (4) have been carried out for all (62) K. 
pneumoniae isolates under study according to MacFaddin 
(2000). Isolates were showed positive results for catalase 
test, while negative results for oxidase, lipase and 

protease test. 

Table 4 
Biochemical tests for detecting K. pneumoniae isolates under study 

Biochemical Test Result 

Lactose 

Sucrose 
Catalase 

Oxidase 

Lipase 
Protease 

+ve 

+ve 
+ve 

-ve 

-ve 
-ve 



Ali, Alkhudhairyb & Tobalc        Biomedicine and Chemical Sciences 1(1) (2022) 11-19  
 

15 

+ve: Positive result, - ve: Negative result. 

Finally, the last confirmatory step for detecting K. 
pneumoniae under study was done by using API 20 E. 
The API 20 E was one of accurate assay consists of a set 
of biochemical tests that were placed on a single strip.  
It’s easy in preparation, low cost and rapid to diagnosis of 

bacterial species. For these reasons all bacterial isolates 
under study were examined via API 20 E (Figure 4.6). The 
results showed that 62(51.7%) belong to the K. 

pneumoniae of total Gram-negative bacterial isolates. 
Several studies have recommended the necessity of using 
(Api 20 E) to diagnose detect of Klebsiella spp. because 
this method has a high degree of accuracy, confidence. In 
addition, it is easy and fast, and it reduces effort and time 
(Sakkas et al., 2019). 

.  

 

 
Fig. 6. The API 20 E positive result for K. pneumoniae isolate no. 45 

 

3.6. Capsule and Hypermucoviscosity 

All 62 K. pneumoniae isolates under study were 
examined to inquire a capsule, using negative staining of 
Indian ink and examined to detected HMV phenotype 

among these isolates by HMV test. Formation a 
mucoviscous line more than 5 mm between an 
inoculation loop and the colony of bacteria considered as 
a positive result for HMV production. The result of direct 

examination showing that 59(95.16%) of 62 K. 
pneumoniae isolates have capsule. While, 60 (96.77%) 
isolates were appear to produce HVM phenotype (Table 

5). 

Table 5 
Virulence factors among 62 K. pneumoniae isolates 

Virulence Factors Result 

Capsule 

Hypermucoviscosity (HVM) 

59(95.2%) 

60(96.8%) 

Previous reports have relationship between capsulated 

isolates and HMV phenotype (Gharrah et al., 2017). 
Confirmed that HMV production depend on the type of 
capsule. Vuotto et al. (2014), also summarized the 
relationship between many virulence factors like capsular 

polysaccharides, siderophores, aggregative adhesion, and 
fimbriae play a major role in the severity level of K. 
pneumoniae infections. 

 

3.7. Interferon gamma (IFN-γ) Detection    

Interferon gamma, the only member of type II 
interferon, it played an important role in immune 
systems. 12 patients positive pneumonia caused by K. 
pneumoniae and 62 controls were enrolled in the current 

study. Controls were matched with the cases in age and 
gender. The age groups of patients span from 1 to 60 
years old. 

The level of IFN-γ concentration in this study was 

measured by using ELIZA assay and was found that is 
significantly (p ˂0.05) raised in all groups of patients in 
comparison to the healthy control group. The level of in 
age group (1-10) years recorded higher percentage 

(293.123) (pg/ml) IFN-γ compared with other age group 
followed by age group (˂60), (11-20), (51-60), (41-50), (31-
40) and (21-30) years (293.123, 278.234, 267.165, 
256.234, 190.34, 167.234 and 134.234, respectively), 
while the level of IFN-γ concentration (pg/ml) among 

healthy control groups were arranged descending 
according to previous age groups in patients age group: 

(48.123, 42.134, 42.122, 41.234, 39.123, 35.123 and  
25.122, respectively). All results of IFN-γ concentration 
levels are summarized in Figure (7). 

The study groups in the current study, included 37 

males and 25 females, it was observed that there were 
significant differences between males and females at a 
significant level (p ˂0.05), when measuring the IFN-γ 
concentration, it was observed that the level of 

concentration IFN-γ in female patients and controls was 
295.234 pg/ml and 46.234 pg/ml, respectively which is 
higher than the level of IFN-γ concentration in the male 



Ali, Alkhudhairyb & Tobalc        Biomedicine and Chemical Sciences 1(1) (2022) 11-19  
 

16 

patients groups, it was 245.342 and in the control groups 
it was 42.345. The results are summarized in the Figure 

(8). 

 
Fig. 7. Interferon gamma concentration (pg/ml) distributed according to the age groups for both patients and control groups 

 

 

 
Fig. 8. The levels of IFN-γ (pg/ml) concentration according to patients with pneumonia and control sex groups 
 

 

It was also observed that the mean for the 
concentration of IFN-γ in female patients groups was 
295.234, and in the control groups it was 46.234, which 
is higher than the mean for the concentration of IFN-γ in 

the male patients groups, as it was 245.342 and in the 
control groups it was 42.345. The means and the 
standard deviations were calculated at a significant level 

p ˂0.05 .IFN-γ is capable of organizing many protective 
functions to heighten immune responses in infections. It 
can exhibit its immunomodulatory effects by enhancing 
Ags and presentation and processing, increasing 

leukocyte trafficking, boosting the antimicrobial functions 
and affecting cellular proliferation and apoptosis.  



Ali, Alkhudhairyb & Tobalc        Biomedicine and Chemical Sciences 1(1) (2022) 11-19  
 

17 

This study, was found relationship between action of 
type I IFN and lung infection with K. pneumoniae, a 

pathogen with one of the highest emergences of antibiotic 
resistant strains, MDR and have more sever virulence 
factors such as (capsulated and mucoid phenotype). The 
results of the current study confirm the role of IFN-γ in 
fighting bacteria, especially K. pneumoniae, and 
defending the body through many mechanisms. There are 
many studies that show how IFN-γ works, such as the 
Xiong et al. (2016) found that research over the last 

twenty years demonstrates that activation of early 
inflammatory responses such as IFN-γ, is essential to 
clear K. pneumoniae infections.  

In this study, significant differences were found for 
levels of IFN-γ concentration between age groups, and it 
is interesting that the age group (1-10) years showed the 
highest level for this IFN-γ and was followed by the age 

group (˂60) years followed by (11-20), (51-60) years and 
the rest of the groups were somewhat close to each other. 
The reason for these results may be due to that the level 
of IFN-γ rises to counter infections caused by K. 
pneumoniae, especially in age groups that have a 
somewhat weak immunity such as infants, children and 
the elderly.  

Schüller et al. (2013) explained the high levels of IFN-γ 

concentration in both adults and children and believed 
that IFN-γ levels may be close despite varying age groups 
and explained that plasmacytoid dendritic cells (pDC) 
release high concentrations of type IFN-γ in response to 

TLR7 and TLR9 stimulation in adults. However, newborn 
pDC are severely limited in secreting interferon α/β upon 

exposure to different viruses, despite expressing levels of 
TLR7 and TLR9 that are similar to adults. 

3.8. Toll Like Receptor (TLR4) Detection 

Toll like receptor dependent signaling, triggers 
numerous immune pathways, leading to neutrophil 
recruitment, antimicrobial peptide production. These 

pathways are required for the host to sense a bacterial 
pathogen and to mount a protective immune response 
(van Lieshout et al., 2015). In the present study, the same 
groups of patients and control groups that were subjected 

to the measurement of TLR4, again subject for measuring 
TLR4, were as follows: 12 patients with pneumonia and 
62 controls groups. Controls were matched with the cases 
in age and gender. Also, the age groups of patients ranges 

from 1 to 60 years old. 

The level of TLR4 concentration in this study was 
measured by using ELIZA assay and was found that is 
significantly (p˂0.05) raised in all groups of patients in 

comparison to the healthy control group. The level of in 
age group (51-60) years recorded higher percentage 
(12.993) TLR4 (pg/ml) compared to other age groups 
followed by age group (˂60), (1-10), (11-20), (41-50), (31-

40) and (21-30) years (12.993, 12.559, 12.244, 11.455, 
11.011, 10.111, 9.124, respectively), while the level of 
TLR4 concentration among healthy control groups were 
arranged descending according to age groups in patients 

age group: (1.012, 1.001, 1.222, 1.667, 1.899, 1.011 and 
1.993, respectively) (Figure 9). 

 

 

 
Fig. 9. The levels of TLR4 concentration according to patients with pneumonia and control age groups 

 

 

The sex groups in this study, included 62 patients: 37 
males and 25 females infected with K. pneumoniae, it was 
observed that there were significant differences between 

males and females at a significant level (p ˂0.05), when 
measuring the TLR4 concentration by ELIZA assay and it 
was observed that the level of TLR4 concentration in male 

patients was 11.786 and 1.034 was in control, 
respectively which were close to the level of TLR4 
concentration in the female patients groups 12.043, and 
in the control groups was 1.996. (Figure 10). 



Ali, Alkhudhairyb & Tobalc        Biomedicine and Chemical Sciences 1(1) (2022) 11-19  
 

18 

 
Fig. 10. The levels of TLR4 concentration according to patients with pneumonia and control sex groups 
 

 

In the current study, it was also observed that the 
mean for the concentration of TLR4 in male patients' 
groups was 11.786, and in the control groups it was 
1.034, which is higher than the mean for the 

concentration of TLR4 in the female patients' groups, as it 
was 12.043 and in the control groups it was 1.996. The 
means and the standard deviations were calculated at a 
significant level p ˂0.05 . 

Toll-like receptors are transmembrane proteins that 
recognize infection and damaged host cells, which lead to 
the consequent inflammation reactions. TLR4 might 
influence the progress of pneumonia (Cai et al., 2015). 

Jeon et al. (2017) suggest that TLR4 is more important in 
the immune response against K. pneumoniae infection 
than TLR2. Therefore, TLR4 play cooperative roles in 
innate immune responses in the lungs that induces 
systemic inflammation during K. pneumoniae infection. 

There is a strong relationship between the production 
of TLR4 and HMV + capsulated K. pneumoniae, and since 

all the isolates under study are capsulated and HMV 
phenotype, this justifies the production of TLR4 at high 
levels compared to the control groups. This interpretation 
is based on a study conducted by Hunt et al. (2014) in 
which confirmed that the strains of K. pneumoniae 
produces LPS, which can be sensed by the innate 
immune TLR4. In disease models, TLR4 deficiency 
renders the host less able to respond to infection by many 

pathogens. However, it has in the last ten years been 
reported that HMV + capsulated K. pneumoniae are 
masked from TLR4 recognition by their capsule. 

Competing Interests 

The authors have declared that no competing interests 
exist. 

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