Evaluation of Status of Pulsed Field Gel Electrophoresis as a Molecular Typing

Technique in Indian S. Typhi Strains

Meenakshi, Randhawa V S, Rao B, Dutta R, Kaur R, Deptt. of Microbiology, Lady Hardinge Medical College,

New Delhi Lady Hardinge Medical College,New Delhi

Introduction• Enteric fever - an important public health problem in developing

countries.

• Typing of S.Typhi is useful in outbreak investigations.

• National Salmonella Phage Typing Centre in Lady Hardinge Medical College carry out essential typing of Salmonella by Phage Typing Method from all over India since 1961

• Phage typing – phenotypic method of subtyping of bacteria based on susceptibility patterns of lysis by a set of phages with different specificity.

• A large number of strain belong to single E1 phage type reducing its typeablity.

• Genotyping technique – chromosomal Based and Plasmid based.

• PFGE- Chromosomal based • Principle based on restriction digestion of whole

genome of bacteria by rare restriction enzymes and electrophoresis the DNA by alternate pulse of electric current at a given angle.

• Gold standard since it was adapted in 1991 .

Methodology• One hundred and thirty seven strains received at the National

Salmonella Phage Typing Centre, LHMC, New Delhi included in this study.

• All isolates subjected to serotyping, biotyping, phage typing and antimicrobial susceptibility testing by CLSI disk diffusion (CLSI) method - Ciprofloxacin, Cefotaxime, Ampicillin, Chloramphenicol, Trimethoprim-Sulfamethoxazole and Tetracycline.

• Subsequently, MIC of the isolates was determined by E-test.

• Pulsed Field Gel Electrophoresis (CHEF DR-III Bio-Rad) was performed using Pulsnet protocol from CDC, USA using XbaI restriction enzyme.

• Reference strain Salmonella. ser. Braenderup ATCC -H9812 from CDC used as control.

• Results analyzed using Gel Compare II software (Applied Maths).

Results

North Zone Central Zone South Zone

1. L.H.M.C, Delhi

2.C.R.I Kasauli

3. U.C.M.S, Delhi

4. Batra Hospital, Delhi

5. C.M.C, Ludhiana, Punjab

6. D.M.C, Ludhiana, Punjab

7. Sardar Patel Medical College, Rajasthan

1. Kasturba Medical College, Mumbai

2.MGIMS,Sevagram, Maharashtra

3.Government Medical College, Nagpur

4. G.M.C, Mumbai

5. A.F.M.C, Pune

6. K.E.M, Pune

7. Goa Medical College ,Goa

1.Pondicherry Institute of Medical Sciences, Pondicherry

2. C.M.C ,Vellore

3. King Institute of Preventive Medicine, Chennai

4. Madurai Medical College, Madurai

5. Nazism’s Institute of Medical Sciences, Andhra Pradesh

6.Sundram Medical Foundation, Chennai

7.Bose Clinical Lab, Madurai

8.Government Medical College Kerala

9.I.G.I.C.H, Bangalore

List of Collaboration Centres

Antibiotic Susceptibility Testing

• Multi drug resistance S.typhi (MDRST) 5.5%.• Ciprofloxacin 91.2% are from immediate

susceptibility .• 98.9% are Nalidixic Acid resistant.• Cefotaxime all are sensitive.

Phage Typing Results

Geographical Zone

Total no- strains

Profiles Obtained

North 51 3Central 21 4South 65 4

• Typeability of phage typing = 76.4%

• A large percentage of the strains belonged in Vi-Negative category, which are negative for Vi antigen and hence cannot be typed by available phage typing scheme.

• E1 Phage type - 49.08%. • Vi negative strains - 18.6%. • UVS (Untypeable for Vi strains)-5%

PFGE Patterns

M: Reference strain Salmonella. ser. Braenderup-H9812

1-11 :Test strains

PFGE Typing Results

Geographical Zone

Total no- strains

profiles obtained

North 51 28Central 21 12South 65 23

• The Commonest PFGE Type Is SX3 in north and central region but SX5 and SX15 in South Region.

In the north zone, 28 different PFGE profiles were obtained which were grouped in to 6 different groups.

In the central region 28 different profiles were obtained which were grouped in 4 different groups.

In the south region 35 different profiles were obtained which belonged to 10 different groups.

Comparison of Phage Typing with PFGE

Technique Typeability= No of strains assigned Types/total no of strains

Discriminatory Index sD = 1 - ----------- Σ nj (nj -1) N (N - 1) j =1

Phage Typing 76.4% 0.09

PFGE typing 100% 0.78

Discussion & Conclusion

• PFGE represents a good typing tool depicting more typeability than Phage Typing and should be used to see molecular heterogeneity amongst S. Typhi strains.

• A similar study from Kolkata also found PFGE more discriminatory in Indian strains than any other phenotypic method (Dutta S et al 2014).

References

• Dutta S, Das S, Mitra S, Jain P et . Antimicrobial resistance virulence profiles and molecular subtypes of S.enterica Serovars Typhi and Paratyphi A. Blood isolates from kolkata India during 2009-2013. 2014 PLoS ONE (8): e101347.