Molecular characterization of Multi Drug Resistance Klebsiella pneumoniae

in Hospitalize patients

Name of Student: SAMMAN

Roll No.: L1F21MSMR0004
Class: MS RESEARCH
Department: MICROBIOLOGY
Faculty: FACULITY OF SCIENCE
Name of Supervisor: DR SABA
Name of co-supervisor : DR FARHAN
Introduction
Klebsiella pneumoniae, belonging to the Enterobacteriaceae family, is a natural inhabitant of the
gastrointestinal tract microbiome of healthy human and animals. It is a common opportunistic hospital-
associated pathogen, accounting for about one third of all Gram-negative infections overall. It is involved
in extra-intestinal infections including urinary tract infections, cystitis, pneumoniae, surgical wound
infections and life-threatening infections, such as endocarditis and septicemia.
(Podschun and Ullmann 1998).
Klebsiella pneumoniae is an important multidrug-resistant (MDR) pathogen affecting humans and a major
source for hospital infections associated with high morbidity and mortality due to limited treatment options.
Klebsiella pneumoniae infections occur in humans of all ages, however the highest risk groups appear to
be infants, the elderly and the immunocompromised. One or more virulence factors may contribute to
pathogenicity in humans. In this article we review three factors that may mediate virulence: cell wall
receptors, capsular polysaccharide, and endotoxin. First, the presence of cell wall receptors enables K.
pneumoniae to attach to the host cell, thereby altering the bacterial surface so that phagocytosis by
polymorphonuclear leukocytes and macrophages is impaired and invasion of the non-phagocytic host cell
is facilitated. Second, invasion of the host cell is also facilitated by the large polysaccharide capsule
surrounding the bacterial cell. Third, K. pneumoniae produces an endotoxin that appears to be independent
of factors that determine receptors and capsular characteristics.
Bacteria belonging to the genus Klebsiella frequently cause human nosocomial infections. In particular, the
medically most important Klebsiella species, Klebsiella pneumoniae, accounts for a significant proportion
of hospital-acquired urinary tract infections, pneumonia, septicemias, and soft tissue infections. Klebsiella
pneumoniae is an opportunistic pathogen, which mostly affects those with weakened immune systems and
tends to cause nosocomial infections It is also an important cause of serious community-onset infections
such as necrotizing pneumonia, pyogenic liver abscesses and endogenous endophthalmitis.

Objective
 To check the prevelance Klebsiella pneumonia among hospitalize patients
 To check the antibiotics suspectibility pattern of Klebsiella pneumonia strains against high
generation of antibiotics for emergence of Multi drug resistance
 Molecular determination of Klebsiella pneumonia of drug resistance

Literature review
Klebsiella pneumoniae has been associated with different types of infections and one of the most important
aspects of Klebsiella is the emergence of multi-drug resistant strains particularly those involved in
nosocomial diseases. Klebsiella pneumoniae plays a major role in the worldwide burden of antibiotic
resistance. This burden is mainly hospital associated, and involves HiR epidemic strains that possess a super
resistome and cause infections with limited treatment options, leading to increased complications, higher
mortality rates and costs. Being a hospital-associated pathogen, Klebsiella is continuously exposed to
multiple antibiotics resulting in constant selective pressure, which in turn leads to additional mutations that
are positively selected.
Methodology
Sample
We have collected standard and clinical isolates of K.pneumoniae from hospitals and utilized them for their
antibiotic resistant ability.
Media

 MacConkey agar
 Blood Agar medium
Biochemical Test
Oxidase test
Detect cytochrome c oxidase gram + ve bacteria. deep purple / blue colour change in 10/30 seconds. Gram
–ve not produce catalase no reactive, after adding 3% hydrogenperoxide .
Catalase test
Pour 1-2 ml of hydrogen peroxide solution into a test tube. Using a sterile wooden stick or a glass rod, take
several colonies of the 18 to 24 hours test organism and immerse in the hydrogen peroxide solution. Observe
for immediate bubbling.
TSI
With a straight inoculation needle, touch the top of a well-isolated colony. Inoculate TSI by first stabbing
through the center of the medium to the bottom of the tube and then streaking the surface of the agar slant.
Leave the cap on loosely and incubate the tube at 35°-37°C in ambient air for 18 to 24 hours.
Citrate
Streak the slant back and forth with a light inoculum picked from the center of a well-isolated colony.
Incubate aerobically at 35 to 37C for up to 4-7 days. Observe a color change from green to blue along the
slant.
Indole
Take a sterilized test tubes containing 4 ml of tryptophan broth. Inoculate the tube aseptically by taking the
growth from 18 to 24 hrs culture. Incubate the tube at 37°C for 24-28 hours. Add 0.5 ml of Kovac's reagent
to the broth culture. Observe for the presence or absence of ring
Coagulase
If 'positive', macroscopic clumping would be observed in the plasma within 10 seconds, with no clumping
in the saline drop. If 'negative', no clumping will be observed.
Antibiotics
Antibiotic sensitivity of clinical K.pneumoniae isolates was done by Bauer’s and Kirby’s disc diffusion
method. Antibiotic sensitivity of clinical K.pneumoniae isolates was done by Bauer’s and Kirby’s disc
diffusion method. Organisms were grown in BHI broth and inoculated on Mueller Hinton agar plates by
sterile swabs and then antibiotic discs were placed on media and pressed gently followed by overnight
incubation.
DNA Extraction
DNA extraction was carried out by boiling method. Briefly, K. pneumoniae strains were grown overnight
at 35 °C on MacConkey agar. Two or three colonies of each culture were harvested from the surface of the
agar plates and re -suspended in 200 μl of sterile distilled water. The cell suspension was heated for 15 min
at 100 °C and then centrifuged at 10,000 g for 10 min and used supernatant as a source of template DNA
for bla-KPC gene amplification by polymerase chain reaction (PCR).
PCR
The primer will be selected and design according to antibiotics sensitivity pattern of Klebsiella pneumonia
strains.
References
Arnold RS, Thom KA, Sharma S, Phillips M, Johnson JK, et al. Emergence of Klebsiellapneumoniae
Carbapenemase (KPC) - Producing Bacteria. NIH Public Access J. 2011;104(1):40–45.
Fader, RC, Avots-Avotins, AE, Davis, CP: Evidence for pili-mediated adherence of Klebsiella
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J.R.Graybill, L.W. Marshall, P. Charache, C.K. Wallace and V.K. Melwin “Nosocomial pneumonia: A
continuing major problem”, ,Am. Rev.Respir. Dis., 1973, 108:1130-1140.
Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing
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Li, B., Zhao, Y., Liu, C., Chen, Z., & Zhou, D. (2014). Molecular pathogenesis of Klebsiella
pneumoniae. Future microbiology, 9(9), 1071-1081.