phase 1 final

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.docx


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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.docx


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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

phase 1 final

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