K. pneumoniae, IMP-1

1. Third Semester
Molecular Biology
UPB
2019
Daniela Pérez Valencia

2. Introduction
Klebsiella Pneumonie is a Gram-negative, non-motile,
encapsulated, lactose fermenting, facultative anaerobic, rod
shaped bacterium found in the normal flora of the intestines. It is
clinically the most important member of the Klebsiella genus of
Enterobacteriaceae. It naturally occurs in the soil and about 30%
of strains can fix nitrogen in anaerobic condition.
o Pneumonia
o Urinary tracts infections
o Chronic pulmonary disease enteric pathogenicity
o Nasal mucosa atrophy
o Rhinoscleroma
o Feces are the most significant source of patient infection,
followed by contact with contaminated instruments

3. Introduction
IMP 1 Metallo B lactamase
is a type of enzyme that makes bacteria resistant to a large
number of beta-lactam antibiotics. Among them are those of
the carbapenem family, the main weapon against bacterial
strains resistant to antibiotics.
This enzyme is usually found in Gram-negative bacterias such
as Klebsiella pneumoniae and Escherichia coli, although it can
reach other strains by horizontal gene transfer.

4. Objective
Determine the molecular and phenotypical
characteristics of carbapenemase producing
Klebsiella pneumoniae and identified the risks factors
underlying its acquisition.

5. Methods
PCR:
Is used in molecular biology to make many copies of
(amplify) small sections of DNA or a gene.
Using PCR it is possible to generate thousands to millions of
copies of a particular section of DNA from a very small
amount of DNA.
It is used in the early stages of processing DNA
for sequencing, for detecting the presence or absence of a
gene to help identify pathogens during infection, and when
generating forensic DNA profiles from tiny samples of DNA.

6. Methods
MLST
Multilocus sequence Typing is a technique in molecular
biology for the typing of multiple loci. The procedure
characterizes isolates of microbial species using the DNA
sequences of internal fragments of multiple housekeeping genes.
Approximately 450-500 bp internal fragments of each gene are
used, as these can be accurately sequenced on both strands
using an automated DNA sequencer. For each housekeeping
gene, the different sequences present within a bacterial species
are assigned as distinct alleles and, for each isolate, the alleles at
each of the loci define the allelic profile or sequence type

7. Methods
PFGE
Pulsed-field gel electrophoresis (PFGE) is a laboratory technique used by
scientists to produce a DNA fingerprint for a bacterial isolate. A bacterial
isolate is a group of the same type of bacteria
Advantages of PFGE
•High concordance with epidemiological relatedness
•Can be applied as a universal generic subtyping method for many different
bacteria with only the choice of the restriction enzyme and electrophoresis
conditions optimized for each species
•Stable and reproducible DNA restriction patterns
•More discriminating than methods such as ribotyping or multi-locus for many
bacteria”
.
https://www.cdc.gov/pulsenet/pathogens/pfge.html

8. • bacterial cells from an agar
plate.
• mixes bacterial cells with
melted agarose and pours into
a plug mold.
• The bacterial cells are broken
open with biochemicals, or
lysed, so that the DNA is free in
the agarose plugs.
• Load the DNA gelatin plug into
a gel, and places it in an electric
field that separates DNA
fragments according to their
size.
• The gel is stained, so that DNA
can be seen under ultraviolet
(UV) light. A digital camera
takes a photograph of the gel
and stores the picture in the
computer.

9. Results
Table 1
2 1 3 5 4

10. Results: PFGE
Fig 1
100% 80%

11. Results
Table 2

12. Results
Table 3

13. Discussion
Author Discussion Yes or not
Fukigai S, Alba J,
Kimura S, Lida T,
Nishikura N, Ishii
Y
“We demonstrated that IMP-1 MBL was the unique carbapenames detected in K.
pneumoniae in our hospital, with an incidence of only 1.2% among all K. Pneumoniae
isolated during the study period. However, several transmissions in the hospital. Were
suspected because accumulation was observed in three specific wards, and a
dendrogram indicated a close relationship between strains isolated in the same Ward.
The first outbreak of K. pneumoniae producing IMP-1 in Japan was reported from a
community hospital in 2007””
Salomao MC,
Guimaraes T,
Duailibi DF,
Perondi MBM
“A number of risk factors for CRE cabapenemase-producing Enterobacteriaceae (CPE)
acquisition have been identified including previous antibiotic use, severity of illness,
invasive/indwelling devices, previous or prolonged hospitaliazation, surgery, liver
disease, and organ/stem cell transplantation”
Pitout Jd,
Nordmann O,
Poirel L
“Specific STs such as ST258 and ST147 are reportedly high –risk clones associated with
the international spread of carbapenemase producin K. Pneumoniae”

14. Conclusions
1. Faecal matter is the most common route of
infection in patients, it is observed in 8 of 16
patients in the same hospital. That means that 50%
of the patients presented fecal colonization
2. Klebsiella pneumoniae is especially susceptible in
patients with cirrhosis, there were 9 cases in which
3 patients were alcoholics