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7/30/2019 Phase 1 Final
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Cephalosporin and Carbapenem resistance in
gram negative bacteria causing Neonatal Sepsis
Guide:Dr.Sulagna Basu
Scientist C
Dept. of Bacteriology,
National Institute of Cholera andEnteric Diseases,(ICMR Kolkata).
Internal Guide:Dr.Winkins Santosh
Assistant Professor,
Dept of Biotechnology,
SRM University.
Presented byPoulami Dutta
Regn no:1641110013
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Neonatal Sepsis
Infection of blood by bacteria or any other virulentpathogen is referred to as septicemia.
Sepsis that develops within 28 days of life is known as
neonatal sepsis or sepsis neonatorum.
Neonatal sepsis is of 3 types:
Congenital neonatal sepsis
Early onset sepsis(EOS)
Late onset sepsis(LOS)
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Each year, globally around 3.6 million babies die within the first 28 days of life (the neonatal period) among which 98% deaths
occur from developing countries (Zupan, 2005).
In India there is an estimated 1.84 million under five children death and of these approximately 41% happen in the neonatalperiod (Lahariya et al., 2010).
In India the major causes of neonatal deaths are sepsis and pneumonia 30.4%, birth asphyxia 19.5% and pre-maturity 16.8
Baqui AH, Darmstadt GL, Williams EK, Kumar V, Kiran TU, Panwar D.et al. 2006. Rates, timingand causes of neonatal deaths in rural India: implications for neonatal health programs. Bull WorldHealth Organ.84:706713.
Zupan J. 2005. Perinatal mortality in developing countries.N Engl Med. 352:2047-2048
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Treatment prescribed by WHO:
Gentamicin
Ampicillin
Other antibiotics which are administered:
Cephalosporin
Carbapenem
Aminoglycoside
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REVIEW OF LITERATURE Study of the pattern of colonization of neonatal gut by gram
negative bacilli and evaluation of the association between gutcolonization and sepsis.(S .Dasgupta et al., 2011)
The study of a cluster of septicaemic newborns with imepenemresistantE.coli in the blood.(P. Mukherjee,S. Basu et al., 2010)
Identification of CTX-M-14 like and CTX-M-27 like ESBLsbelonging to the CTX-M-9 group in gram negative bacteria.(S.Basu,A.K Singh et al.,2011)
Study of the MDR gram negative bacilli causing early onset sepsisin neonates in India.(R.Viswanathan,A.K Singh et al.,2011)
Carbapenemase has been analyzed as the versatile betalactamase.(Anne Quineen and Karen Bush 2008)
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OBJECTIVES
Identification of carbapenem and
cephalosporin resistant determinants in gram
negative bacteria causing neonatal sepsis by
phenotypic and genotypic analysis
Analysis of the coexistence of other beta-
lactamases,16srRNA methylase genes and
integrons
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Work Plan & Methods used Detection of the micro-organism using a few
biochemical assays
Phenotypic analysis
1.Antibiotic susceptibility patterns
Disc diffusion test
MIC determination
2.Detection of ESBLs and MBLs Combination disk test
3. Detection of Carbapenemase
Modified Hodge test
http://localhost/var/www/apps/conversion/tmp/scratch_5/MODIFIED%20HODGE%20TEST.docxhttp://localhost/var/www/apps/conversion/tmp/scratch_5/MODIFIED%20HODGE%20TEST.docx7/30/2019 Phase 1 Final
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Contd.
Genotypic analysis
1.PCR followed by agarose gel electrophoresis
2. Transmissibility of carbapenem and
cephalosporin resistance determinants byconjugation expts
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Results of the biochemical tests 2 different types of bacteria has been identified-
Gram negative bacteria (Klebsiella and E.coli)
Gram positive bacteria (Staphylococcus aureas)
More number of the samples contained gram negative bacteria than grampositive bacteria
Most prevalent organism found- Klebsiella
Results of Identification using API 20E strips
It is confirmed that the identified organisms are E.coli andKlebsiella pneumoniae
Work in progressCollection of samples from Birbhum State General Hospital (Rural part of Bengal) -
Identification of the most prevalent causative organism
Comparison of the urban and rural samples
More no. of the organisms screened till now seem to be
Acinetobacter spp.
http://localhost/var/www/apps/conversion/tmp/scratch_5/API%2020E%20strips.docxhttp://localhost/var/www/apps/conversion/tmp/scratch_5/API%2020E%20strips.docx7/30/2019 Phase 1 Final
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Results of the Disc Diffusion tests
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11CAZ CT
X
CR
O
CL FEP PIP AK GM CIP ME
M
GA
T
MI TGC TZP
1 R R R R R R R R R R R S R R
2 R R R S R R R R R R R S R R
3 R R R R R R R R R R R S R R
4 R R R R I R R R R R R R R R
5 R R R S R R R R R R R R R R
6 S R R R R R R R R R S R S R
7 R R R R R I R R R R S R R R
8 R R R R R S R R R R R S S R
9 R R R S R R S R R R S S S R
10 S R R R R R R R R R R S S R
11 R R R R I R I R R R S S S R
12 R R R S R R I R R R R S R R
13 S R R S R R R R R R I R R R
14 R R R R R R R R R R S S R R
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CA
Z
CTX CR
O
CL FEP PIP AK GM CIP ME
M
GA
T
MI TG
C
TZP
15 R R R R R R R R R R S S R R
16 R R R R R R R R R R S S S R
17 R R R S R R S R R R R R S R18 S R R R S S R R R R R R R R
19 R R R R I R R R R R I S R R
20 R R R R I R R R R R S S R R
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Fig: Kirby -Bauer disc diffusion method of determining antibiotic susceptibility pattern
of some representative test isolates (E.coli)
Zone of
clearance
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Graphical representation of the antibiotic susceptibility patterns
of 20 test isolates
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Interpretation
Majority of the test isolates are either
resistant or shows intermediate resistance
against Cephalosporin and Carbapenem
antibiotics.
It can be assumed that large number of
isolates contain cephalosporin resistant
genes(ESBLs) and Carbapenem resistantgenes.
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Results of the Combination disk tests
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Fig: Combination disk method of a few representative test isolates (E.coli)
Zone of
clearance
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ESBL+ve
(CTX+C/CTXCC ) - CTX 5 (CTX+C/CTXCC)CTXCX 5
AmpC +ve
(CTXCX/CTXCC)- CTX 5
(CTXCX/CTXCC)- CTX+C 5 (MRP+BO/MRP+CL)- MRP 5
(MRP+DP)-MRP 3
KPC+ve
(MRP+DP/MRP+CL)MRP 3 (MRP+BO)MRP 5
MBL+ve
(MRP +DP)-MRP 5
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Interpretation
It can be interpreted that the test isolates
may contain genes like
OXA1,TEM,IMP,VIM,SIM,SPM,GIM,NDM ,CTX-
M ,SHV-1 and AmpC
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Genotypic analysis-PCR results..
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Screening of the CTX-M gene
CTX-M gene
+ve
control
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Screening of the NDM gene
-C +C L1 L2 L3 M
NDM gene
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Screening of the AmpC gene
M -C +C L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12
AmpC gene
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Future work
Further tests will be carried out to confirm thepresence of the resistant determinants.
Phenotypic tests like MIC determination tests
and Modified Hodge tests. Genotypic analysis-screening of other genes
like TEM,IMP,VIM,SIM,SHV-1,OXA.
Conjugation expts.
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References
Das. P,Singh.AK, Dasgupta. S,Ramamurthy. T,and Basu.S. Colonization of the gutwith gram negative bacilli, its association with neonatal sepsis and its clinicalrelevance in a developing country. J. Med. Microbiol.2011; 60: 1651-1660
Mukherjee. R,Das.P and Basu.S.Aetiology and Antimicrobial resistance ofNeonatal Sepsis at a tertiary care centre in Eastern India.Indian J
Pediatr.2011;58(32)
Roy. S, Mukherjee.S, Singh .AK, and Basu.S.CTX-M-9 group extended-spectrum -lactamases in neonatal stool isolates: emergence in India. Indian J. Med.Microbiol.2011;29: 305-308.
Viswanathan R,Singh AK,Ghosh.C and Basu.S.Profile of Neonatal Septicemia at adistrict level sick Newborn care unit.Indian J Pediatr.2010;43
Viswanathan R,Singh AK, Basu.S,Chatterjee.S,Sardar.S and Isaacs.D.Multi-drugresistant gram negetive bacilli causing early neonatal sepsis in India.Indian JPediatr.2011;76
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THANK YOU