MOLECULAR CHARACTERIZATION AND EPIDEMIOLOGICAL TYPING OF

CLINICAL AND COMMUNITY ISOLATES OF STAPHYLOCOCCUS AUREUS

FROM

CHENNAI, SOUTH INDIA.

Nagarajan Abimanyu, M.Sc.

Department of Microbiology,
Dr. ALM PG Institute of Basic Medical Sciences,
University of Madras,
Chennai, Tamil Nadu, India.

INTRODUCTION
2

Staphylococcus aureus
Colonizer
Pathogen.
transiently

persistently

hospitalized patients

healthy individuals

Skin and soft tissue infections

Post operative wound infections
Intra-vascular medical device
associated infection
Sepsis

Colonization Sites

Infections

MRSA A GLOBAL BURDEN

CDC, ECDC and NHS has guidelines for healthcare
workers and the public to prevent MRSA.

due to pbp2a
Escape binding by methicillin

Encoded by mecA carried on SCCmec

1. High Resistance
2. Treatment challenges
3. Increased treatment cost
4. Increased Morbidity
and Mortality

CA MRSA

1st report- 1990s

HA MRSA

1st reported in 1961

Highly resistant

Nature

Highly pathogenic

Epidemiology

Epidemic outbreaks
communities

SCCmec

SCCmec IV VI

SCCmec I III, VII -XI/

Virulence

PVL +ve

PVL ve

Susceptible
groups

Closed communities Crowded

Hospitalized patients (after 72hrs)

Infections

Skin and soft tissue infections

Post operative wound infections

Antibiotic
resistance

Sensitive to other antibiotics

Multidrug resistant
Well explored and understood

Pathology

Wrongly diagnosed as spider bites,

tumor, etc
Eg; USA300, USA400, EMRSA-15,
16

Eg; USA100, 200, Hungarian clone,

N315, Mu3

in

healthy

Endemic /epidemic outbreaks in

hospitals

STRUCTURE AND TYPES OF SCCMEC ELEMENTS

According to International Working Group on the Staphylococcal Cassette Chromosome

elements (IWG-SCC),

A total of 11 SCCmec types have been identified and numbered as SCCmec type I - XI.

Classification is based on the combination of

Cassette chromosome recombinase (ccr) complex type and mec complex class

SCCmec type I through SCCmec type III and SCCmec type VII to XI were associated with HAMRSA,

SCCmec type IV to VI were reported from CA-MRSA.

orfX

IS1272

IS431

J1
ccr

pUB110
mecA

mecR1

mecA complex (A, B or C)

ccr complex (1, 2, 3, 4 or 5)

EPIDEMIOLOGICAL TYPING OF STAPHYLOCOCCUS AUREUS

Conventional
methods

Molecular
methods

Phage typing

PFGE

Antibiogram
typing

SCCmec
typing

Multilocus
enzyme
typing

Agr typing

PCR-RFLP

RAPD

Sequencing
based
methods

Other typing
Methods

MLST

Toxic gene
profile
typing

Spa
typing
Coag gene
typing

DNA sequencing methods including

MLST, SLST involving sequencing of
various genes and SCCmec typing are
rapid, affordable and high-throughput8
systems for typing of MRSA strains.

Global Prevalence of MRSA

Europe & UK- 20 -50%
Predominant clones
CAMRSA- EMRSA 15, 16
HAMRSA ST239, EMRSA 2

USA 30 -60%
Predominant clones
CAMRSA- USA300, USA4000
HAMRSA USA100, USA200

China -78%
Predominant clones
CAMRSA- ST-22
HAMRSA ST239

Japan -30 -50%

Predominant clones
CAMRSA- ST-5
HAMRSA N315, Mu3, Mu5

South Asia 40-70%

Predominant clones
CAMRSA- EMRSA 16, ST772
HAMRSA ST239

South Africa 40%

Predominant clones
CAMRSA- ST -8 -IV
HAMRSA ST239

Australia 30 -40%
Predominant clones
CAMRSA- EMRSA 16
HAMRSA ST239
South Africa 39-40%
Predominant clones
CAMRSA- EMRSA 2,6
HAMRSA ST239

PREVALENCE OF MRSA IN INDIA (INSAR - STUDY)

North India
Prevalence up to 68%
Predominant clones
CA MRSA ST22, ST772

South India
Prevalence 60%
Predominant clone
CA MRSA ST22, ST772
HA MRSA ST239

Chennai
Prevalence up to 52%
Predominant clones - ?

10

These studies were done for few clinical isolates only

RATIONALE FOR THE STUDY

Methicillin resistant Staphylococcus aureus continues to

be the leading cause of nosocomial infections accounting
for 20% to 68% incidence in India.

In the community setting, incidence of MRSA causing

skin and soft tissue infections are increasing (10 20%).

Community acquired MRSA has been recognized as a

substantial public health threat owing to their high
virulence and rapid spread in the community.

However, there is scarcity of data on the molecular

epidemiology of S. aureus in Indian settings.
11

SCOPE AND PLAN OF THE STUDY

Which are the clones circulating in our part of the country?

Are the clones of HA MRSA different from CA MRSA?

What is the prevalence of virulence genes?

Is PVL a marker for CA MRSA?

Are there any novel genotype circulating in our part of the

country?

What are the clones responsible for HIV associated infections?

Is there emergence of clindamycin, mupirocin and fusidic acid

resistance among S. aureus isolates?
12

OVERALL STUDY
Part I
Part II
Part III

Part IV

Part - V

Detection of MRSA from various

groups
Detection of antibiotic

resistance profile and

determinants

Detection of virulence

determinants

Genotyping (epidemiological

typing) by various methods

Characterization of MRSA clonal

complexes based on MLST

13

ETHICAL CLEARANCE

Ethical clearance was obtained for the study from

IEC of Dr. ALM PGIBMS
IRB of Madras Medical College and Hospital, Chennai
IRB of Govt. hospital of thoracic medicine, Tambaram

Informed consent was obtained from the study

participants

14

STUDY GROUPS
Total number of S. aureus
included for the study
Hospital Inpatient
setting
(n=251)

HIV infected
Patients
(n=70)

n= 769
Hospital Outpatient
setting (n=225)

Group
I

Group
II

Group
III

Group
IV
Healthy
communities at risk
15
of CAMRSA
infection (n=223)

INCLUSION CRITERIA
FOR

HOSPITAL SETTINGS
S. aureus isolates collected from the inpatient and outpatients settings of
2 tertiary care centre (HIV, non-HIV ) in Chennai, South India.

Inpatients

settings

(Hospital

Outpatients settings (Community

acquired isolates)

acquired isolates)

S. aureus isolates collected from

S.

hospitalized

patients with wound, skin and soft

patients

with

post

aureus

isolates

infections

collected

attending

from

operative wound, skin and soft

tissue

the

tissue infections after 72 hrs of

outpatient facility of tertiary care

hospitalization.

centre and from hospitalized patients

within 72 hrs of hospitalization. Should

Repeat isolate from

same
patient
was
excluded from the study

not have history of hospitalization 16

in
the previous six months

CLINICAL SAMPLES
Pus and pus swabs from various
pyogenic infections

Samples
collected
using
standard
methods

Wound infection
Ear infections
Post-operative wound
Abscesses
Cellulitis
Folliculitis
Carbunculosis
Necrotizing fascititis
Gangrene
Impetigo and
Scalded skin syndrome

ABSCESSES
Extremities
Gluteal
Breast
Axillary
Groin

The patient details including name, age, sex, date and hours of hospital
admission, underlying clinical condition, previous medical history and antibiotic
treatment if any were noted using a sample request form cum questionnaire.

17

18

INCLUSION CRITERIA
FOR

COMMUNITY SETTINGS
S. aureus isolates collected from healthy
individuals from various closed communities

Sports teams

Orphanages

Old age homes

Specimen: Anterior nasal swab.

Demographic details of the individuals were recorded.

Exclusion Criteria
Individuals with history of hospitalization within past six months.

19

COLLECTION OF NASAL SWABS FROM COMMUNITY SETTINGS

Old age
Home

Orphanage

Old age home

20

ISOLATION AND IDENTIFICATION OF S. AUREUS

Nasal swabs

Pus and pus swabs

Enrichment with
7.5%NaCl
Nutrient broth

Transported to
laboratory at 4C

Initial isolation - blood agar

Identification of S. aureus was done using

standard methods
21

ISOLATION AND IDENTIFICATION OF S. AUREUS

Salt nutrient broth

Tube coagulase test

Isolation on blood agar

DNase test

Slide Coagulase test

Mannitol salt agar

22

SOURCE OF S. AUREUS ISOLATES INCLUDED IN THIS STUDY

23

Out of 852 healthy individuals from various communities, 223 (26.17%) were found to be carriers of S. aureus.

PART I
DETECTION OF MRSA
24

DETECTION OF METHICILLIN RESISTANCE

Methicillin resistance was screened by using cefoxitin (30g)
Himedia along with other antibiotics.

MRSA was confirmed by using two sets of multiplex PCR

Novel Triplex PCR

targets
mecA
femA
Pvl

Published
primers
from
previous studies
were selected

McClure et al., 2006

targeting
16S rRNA
mecA

Standardizing in Gradient
Master cycler using the
positive
controls
and
negative controls

pvl

25

All the S. aureus isolates included in this study were tested by both the methods

The prevalence of MRSA among all S. aureus isolates included in this

study was found to be 368/769 (47.85%).

81.67%
7.17%

58.22%

22.85%

26

27

28

PART II

ANTIBIOTIC RESISTANCE

29

There are reports on the significant difference in antibiotic

resistance pattern between CA-MRSA and HA-MRSA.

1. Antibiotic resistance profile

a.
b.

disc diffusion to various antibiotics

MIC studies oxa, mup, clinda, erythro, vanco, linezolid

2. PCRs to characterize the drug-resistance determinants of

a.
b.

Mupirocin
Fusidic acid

30

SCREENING FOR ANTIBIOTIC RESISTANCE

AST was done by Kirby Bauer's disc diffusion test for the following groups
of antibiotics.
Aminoglycosides

Inducible clindamycin resistance was

detected by D- test method.

Cephalosporins
High-level mupirocin resistance
was detected by mupirocin
(200g) disc diffusion

Fluroquinolones
Glycopeptides
Cotrimoxazole

Mupirocin
Tetracycline
Linezolid
Rifampicin
Erythromycin
INDUCIBLE

Clindamycin
Fusidic acid.

CLINDAMYCIN
RESISTANCE

31

DETERMINATION OF MINIMUM INHIBITORY CONCENTRATION (MIC)

MIC for the following antibiotic was done by agar dilution
method

Oxacillin (Himedia)
Cefoxitin (Himedia)
Vancomycin (Himedia)

Mupirocin (Himedia)
Clindamycin (Himedia)
Erythromycin (Himedia)

Linezolid (Himedia)
32

DETECTION OF HIGH-LEVEL MUPIROCIN RESISTANCE

High-level mupirocin resistance in S. aureus due to the presence of IleS2
carried on the large conjugative plasmid.
Also called as mupA gene
It was detected by PCR (Yun et al., 2003).

DETECTION OF ACQUIRED FUSIDIC ACID RESISTANCE DETERMINANTS

Fusidic acid resistance is due to mutation in EF-G and acquired fusidic acid resistance genes.
The detection of acquired fusidic acid resistance determinants
fusB,
fusC &
fusD
was done using multiplex PCR method described by Castanheira et al., 2010.

All the isolates included were tested for the mupirocin and fusidic
resistance by above PCR methods.

33

ANTIBIOTIC RESISTANCE AMONG S. AUREUS ISOLATES

All the tested S. aureus isolates were found to be susceptible to

linezolid and vancomycin.

MRSA isolates showed highest resistance irrespective of the settings.

Constitutive clindamycin resistance (cMLSB)

Group I - 6 /205 (2.92%) MRSA
Group II - 4/131 (4.05%) MRSA

Inducible clindamycin resistance (iMLSB) isolates

51 from group I,
14 from group II,
2 from group III and
3 from group IV

Four MSSA showed resistance to fusidic acid which included 3/94

(3.19%) of group II and 1/46 (2.17%) of group I.

70(19.23%) MRSA

34

35

36

37

Fig; Inducible Clindamycin Resistance

MRSA

MSSA

100%

100%

90%

90%

80%

80%

70%

70%

60%

60%

iMLSB -ve

50%

iMLSB+ve

iMLSB -ve

50%

iMLSB+ve

40%

40%

30%

30%

20%

20%
10%

10%

0%

0%
Group I

Group II

Group III Group IV

Group I

Group II

Group III Group IV

Inducible clindamycin resistance was found to be high among MRSA and MSSA
from Hospital Associated Infections

38

39

PART III
DETECTION OF VIRULENCE FACTORS
40

S. aureus is a well armed pathogen with an array of virulence factors

that promote their colonization, invasion and pathogenesis.
Earlier studies report that virulence factors of community-associated
infections differ from hospital-associated infections.

In the present study, multiplex PCRs for the detection of

virulence determinants
1. Exotoxins
2. Innate Immune Evasions
3. Surface adhesins
were carried out to compare their prevalence among S.
aureus isolates from community and hospital.

41

Exotoxins
A total of 27 exotoxins including
Enteroxins A B C D E G H I J K L M N O P
Exfoliative toxins ETA, ETB, ETD

Hemolysins HLA, HLB, HLD, HLG & HLG2

Leucocidins LukD-E, LukM

Toxic shock syndrome toxins-1

42

Tested for all the isolates included in the study.

ENTEROTOXINS SEA TO SEE, SEG TO SEO

Thirteen enterotoxin genes were detected using the modified methods of
Shukla et al. (2010).
Three multiplex PCR were employed which included

MPCR
ENT-1

seb, sec, sed, and sej

MPCR
ENT-2

sea, seg, seh, and sei

MPCR
ENT-3

sek, sel, sem, sen and seo.

43

EXOTOXINS CONTINUED

Exfoliative toxins - eta, etb and etd

Detected by multiplex PCR as described by Rucikova
et al., 2005.

Hemolysins - hla, hlb, hld, hlg & hlgV

Detected by multiplex PCR standardized using the
primers described by Jarruad et al., 2000.

Leucocidins - lukD, lukE and lukM

Detected by triplex PCR standardized using the
primers of Jarruad et al., 2000 & Shukla et al., 2010.

Toxic shock syndrome toxin - tst-1

Detected by PCR method by Jhonson et al., 1993

44

A novel multiplex PCR was designed and standardized for the

detection of innate immune evasions (IEC)

Innate immune evasions - sak, scn and chps

Enterotoxins - sea and sep

Beta hemolysin complete

The primer sequences were designed by using pick primers of

NCBI database.

Standardized using Gradient Thermal Cycler

45

S. aureus RN6390, S. aureus N315 and S. aureus MW2 were used as

positive controls.

MSCRAMMs
(microbial surface components recognizing adhesive matrix molecules)

Nine MSCRAMMs were detected by using two multiplex PCR as

described by Tristan et al. (2003).

M PCR-1
5. Fibrinogen binding protein (fib)

6. Fibronectin binding protein A (fnbA)

1. Collagen binding protein (cna),

7. Fibronectin binding protein B (fnbB),

2. Enolase (eno),

8. Clumping factors A (clfA)

3. Elastin binding protein (ebpS)

9. Clumping factor B (clfB)

4. Bone sialoprotein binding protein

46

(bbp)

M PCR-2

Except leukocidin M (lukM) and exfoliative toxin D (etd),

all other virulence genes were detected.

47

48

49

50

38%

53%
37%
57%

24%
37%

59%

75%
94%

51

52

53

54

55

56

57

PART IV

EPIDEMIOLOGICAL TYPING OF S. AUREUS

58

PCR based molecular techniques such as agr typing, SCCmec

typing and sequencing based typing methods has been found
to be tool for epidemiological typing of MRSA
To detect the epidemiology of the MRSA in Chennai

SCCmec typing
PCR based
methods

Sequence
based
methods

agr typing
spa typing
Spa type >10 isolates

MLST typing
59

Note:
Proof reading DNA polymerase (AmpliTaq gold) was
the PCR reactions involving sequencing.

used

for

SCCMEC TYPING

1
All isolates were tested
for SCCmec
type
using the method of
Boye et al., 2007.

A simple multiplex
PCR detects SCCmec
types I to V.

Two methods

SCCmec types was done for selected isolates

using the method of Kondo et al., 2007.

A complex method involves ccr typing and

mec class typing

Can be used to detect new SCCmec types

60

AGR TYPING

Multiplex PCR as described by Lina et al., 2006, was carried out for
all the isolates included in this study.
Multiplex PCR uses
universal forward primer
4 different reverse primers.

Targets the variable region (BCD) of operon agrABCD

agr II

agr
I

Agr
types

agr
III
61

agr
IV

SPA TYPING

Spa typing was done by the method of Koreen et al., 2003. for all the S. aureus
isolates included in the study.
DNA by
boiling
lysing
method

PCR was
carried out

Individual
spa types
were checked
for relativity
using BURP
analysis

Sequences
were checked
and analyzed
for spa types
using
Bionumerics
v 7 software

Spa typing
Sequencing
was done by
using ABI
prism BigDye
Terminator v3.0

Sent to
sequencing

Checked with
Agarose gel
electrophoresis

Purified using
Qiagen spin
columns

Spa gene
product
and primer
premix

62

MLST TYPING
Multilocus sequence typing was done as described by Enright et al., 2000 using
7 house keeping genes.
Spa types having more than 10 isolates were considered for the MLST.

Isolates for MLST typing were selected randomly.

arc

Carbamate kinase

aro

Shikimate dehydrogenase

glp

Glycerol kinase

gmk

Guanylate kinase

pta

phosphate acetyltransferase

tpi

triosephosphate isomerase

yqi

Coenzyme A acetyltransferase

63

Alleles of multiple
gene sequences
were designated
using the free
online software
available at www.
mlst.net
Sequence were
trimmed to
specified
length by
aligning with
reference
sequence
Sequence
homology was
checked with
the reference
sequences
available at
mlst.net

DNA by
boiling
lysing
method

PCR was carried

out for the
individual
housekeeping
genes

Checked with
Agarose gel
electrophoresis

MLST

Sequencing
was done by
using ABI
prism BigDye
Terminator
v3.0

Purified using
Qiagen spin
columns

Sent to
sequencing

Individual
gene
product
and primer
premix
64

51.2%
71.7%

40.9%

5.8%

28.2%

62.5%
81.2%
31.2%
12.5%

A total of 10 carrier isolates of MSSA (mecA negative) were found to be

positive for the SCCmec elements.

Of the 10 MSSA with SCCmec elements, 9 were type I and one was type IV.

65

66

AGR TYPING

All four types of accessory gene regulator subtypes

were detected among the S. aureus isolates
included in the study.
Agr types

The prevalence of agr types

among various groups
agr type II

(n=417)

agr type I

(n=208)

agr type III

(n=105)

agr type IV

(n=33)

agr I (27.0%)
agr II (55.5%)
Significantly
among MRSA

agr III (13.6%)

agr IV (2.0%)
high

Predominant
among MSSA

agr IV associated with exfoliative toxins

67

13 isolates were found to be negative for the agr

100bp ladder
agr type IV (625bp)
agr type I (441bp)
agr type II (325bp)

68

SPA TYPING
A total of 78 different spa types were obtained, which include

t002, t003, t005, t008, t015, t021, t031, t037, t064, t084, t091, t114, t1154,
t1223, t127, t1309, t1458, t1515, t159, t1598, t164, t1754, t1835, t189,
t1931, t2078, t209, t213, t2194, t2393, t2494, t2526, t2663, t267, t2700,
t272, t273, t2815, t3087, t3092, t310, t311, t3169, t3204, t345, t3554, t359,
t3596, t360, t3841, t3924, t3937, t4104, t425, t442, t448, t4615, t4665,
t4685, t487, t4897, t4936, t5122, t521, t5594, t6099, t616, t657, t701, t7200,
t852, t901, t9036, t9037, t937, t939 and t985.
t657 (n=159) was the predominant spa type among MRSA
t3841 (n=24) was the predominant spa type among MSSA isolates.

t9036

CA MRSA skin infection (n=1)

t9037

Carrier MSSA isolates (n=7)

2 novel spa
types

A total of 19 spa types were found to be have more than 10 isolates.

69

100bp ladder

Spa gene product

350bp to
450bp

Representative
Pictures of spa types
Obtained by using
Bionumeric software

70

MSSA
MRSA

t005

t037

t015

t064

t021

t1154
t1223
t159
t164
t1931
t209

t272

71

MSSA
MRSA

t3204
t345
t3841
t425
t442
t4615

t657
t852
t9037

72

BASED ON REPEAT PATTERN (BURP) ANALYSIS

73

MLST TYPING

The major spa types (n 10) in this study were typed for sequence
type by using MLST.

The sequence types of the representative spa types

ST1
ST239
MRSA

ST 368
ST1208

ST5
ST772
ST22
ST30
ST672

ST6
ST20
ST45
ST109
ST120

MSSA

74

ST772 is a single locus variant of ST1 and also called as Bengal Bay
clone.
ST239 is the predominant MRSA clone causing HA infections all
over the world.
ST120 and ST672 were the predominant MSSA clones
ST22 detected in this study is a variant of EMRSA-16, the
predominant CA-MRSA clone circulating in European countries.
ST6, ST18, ST672, ST120, ST45, ST30 were found to be MSSA
circulating in community

ST672, ST45, ST109, ST30 were found to be MSSA circulating in

hospitals.
75

PART V
CHARACTERIZATION OF CLONAL
COMPLEXES

76

The virulence and resistance determinants in S.

aureus play an important role in the severity of the
infections.

The virulence and resistance characteristics of the

clonal complexes were compared and correlated
with the severity of infection.

77

CLONAL COMPLEXES (CC) - MLST

Clonal complexes of MLST types was detected by using e

BURST analysis of sequence types.

The clonal complexes

(CC) were characterized
for their

Infections/carriers /
Group

Resistance
Virulence
Agr type

Pvl

Spa type
MRSA/
SCCmec
type
CC

78

CLONAL COMPLEXES (CC) - MLST

1. CC1
2. CC5
3. CC6
4. CC8
5. CC9

ST1, ST772
ST5
ST6
ST239, ST368, ST1208
ST109

6. CC18
7. CC20
8. CC22
9. CC30
10. CC45
11. CC121

ST18
ST20
ST22
ST30
ST45
ST120

Clonal complexes of MRSA were found to be CC1, CC22 and CC8.

CC-121 comprises the majority of the nasal carrier isolates

CC-1 covers the majority of community acquired infections, HIV associated infections

CC-8 covers the majority of the hospital associated infections.

79

80

9 CCs were found to be associated with the nasal carriage

4CCs with hospital associated infections and
5CCs with community associated infections
Shows that carrier isolates are highly diverse in nature

81

CHARACTERISTICS

OF CLONAL COMPLEXES OBTAINED IN THIS STUDY

1. CC1 ST772
Subcontinent clone or Bengal Bay Clone
CA-MRSA

2. CC8ST239, ST368,
ST1208

ST772 MRSA V (PVL +ve)

CC8 ST239

Single locus variant of ST-1 (Prevalent in

USA)

International MDR HA MRSA

Predominant spa type t657

Highly pathogenic associated with
abscess
Prevalent countries Indian sub continent
Emerging as HA acquired multidrug
resistant
Recently reported from Western countries
Other members t347 (pvl MSSA); t1931
(MSSA)
Previously reported from India by Gayathri et al.
2010 and Dzouza et al. 2010

ST239MRSA III (Hungarian clone)

Double locus variant of ST-8
Spa type t037
High level mupirocin and inducible
clindamycin resistant
High level methicillin resistant
MIC >256g/ml
2nd major MRSA among HA infections
82
Previously reported from India by Song et al. 2011

CC8 - ST368
MDR HA MRSA clone

ST368-MRSA-III
Spa type t425/ agr -I
Detected in 6% of MRSA causing HA infections (DM)
Previously reported from Sri Lanka.

CC8- ST1208
ST1208-MRSA-III/
ST1208-MRSA-V
Spa types t1223 & t064
t064 CA-MRSA middle ear, skin and soft tissue infections
t1223 HA-MRSA post operative infections
Recently reported from India

83

4. CC-22; ST22
International clone of CA MRSA

ST22-MRSA-IV
ST22- the second major (9% of total isolates)
Spa types t005/ t852
t852 pvl MRSA/t005 MSSA with mecA-ve SCCmec remnants
Well known CA MRSA causing skin and soft tissue infection
Found to be gentamycin resistant MRSA
Prevalent in Europe, India

5. CC-5; ST5
ST5-MSSA
Highly virulent -super-antigen enterotoxins, leucocidins and is resistant to
multiple antibiotics.
Pvl positive - spa type t448 and agr type III.
Isolates of this clone was found to cause community associated ear, skin and
soft tissue infections.
Along with the egc gene cluster they carried plasmid pIB485 - sed and sej. 84
MRSA of this clone -Reported from Australia, Ireland and Germany.

OTHER CLONAL COMPLEXES (CCS)

CC30; ST30-MRSA-IV
spa type t021/ Multi-drug resistant CA MRSA
Single pvl positive CA-MRSA isolate of this group was isolated from breast
abscess.
Previously reported from New Zealand, South Pacific, USA, Australia,
Germany, Switzerland, the UK and Hong Kong.

CC9; ST109

13 MSSA isolates under this clone

spa type t209.
Isolated from carriers
All carried exfoliative toxin gene etb.
Previously reported among veterinary isolates

ST-672

MRSA and MSSA isolates

spa type t3841.
Five of these isolates were found to be MRSA, which carried SCCmec I
4 HA infections/ 1 from carrier
First reported from South India in 2010

85

SUMMARY

S. aureus isolates from four different populations, viz.,

hospital-associated
infections,
community-associated
infections, HIV-infected patients and healthy carriers from
various communities at risk, collected from Chennai, South
India were characterized for virulence and resistance
determinants and epidemiological types by molecular
methods and compared.

A total of 769 S. aureus

251 from hospitalized patients,

225 from outpatients,
70 from HIV positive patients
223 carrier isolates from healthy individuals in closed communities.

The overall nasal carriage rate of S. aureus among healthy

communities - 26%.

86

The overall prevalence of MRSA in this study was

found to be 48%.

MRSA

hospital-associated infections - 81.7%

community associated infections -58.3%
HIV-infected patients - 22.8%
healthy carriers - 7.1%

Our study reports the highest prevalence of MRSA of

82% among hospital-associated infections from the
Indian subcontinent.
87

RESISTANCE

All the tested isolates were sensitive to vancomycin and linezolid.

Highest resistance was observed for erythromycin (58.5%).

About 10 (2.7%) MRSA isolates showed constitutive clindamycin

resistance.

About 19% of MRSA and 12% MSSA showed inducible

clindamycin resistance (D-test positive, ermA gene +).

Mupirocin resistance (mupA) was detected in 21% of MRSA from

HAI and 8% of MSSA from community (carrier and clinical).

About 1% of MSSA isolates from community and hospitalassociated infections showed fusidic acid resistance (fusC gene).

Multi-drug resistance was found to be significantly higher among

MRSA than MSSA.

88

VIRULENCE

All virulence factors except lukM and ETD were detected in this study.

The overall prevalence of pvl was found to be 36.3%.

PVL was detected both in MRSA and MSSA in all groups of the study.

Among superantigen enterotoxins, sea (39.5%) was the predominant.

No significant difference in the virulence profile was obtained between

MRSAs causing community and hospital associated infections.

Superantigen-enterotoxins, chp and MSCRAMMs (clfB, ebp and bbp)

were high among clinical isolates.
89

MSCRAMMs- fib and cna were found to be high among carrier

isolates.

MOLECULAR EPIDEMIOLOGY

SCCmec type I, III, IV & V were detected among MRSA.

SCCmec type V was the predominant. None of the MRSA carried SCCmec type
II.

The major agr subtype in this study was found to be agr II followed by agr I.

agr-IV was detected in about 8% of MSSA and carried either eta and etb or etb
alone.

A total of 78 different spa types were obtained, of which spa type t657, t852
were predominant among MRSA.

spa type t4615 and t3841 were predominant among MSSA.

The major sequence type was found to be ST772 (Bengal Bay clone) a single
locus variant of ST1 and was found to be CA-MRSA.

90

ST772-MRSA-V - multidrug- resistant CA-MRSA causing both

community-associated (20.88%) and hospital associated (41.83%)
infections. Circulating in the community asymptomatically.

ST22-MRSA-IV epidemic (UK) clone of CA-MRSA was detected in

4.40% and 13.33% of hospital- and community-associated infections
respectively.

The major HA-MRSA clone in this study was found to be ST239

MRSAIII, which was found to be resistant to mupirocin and also
exhibited inducible clindamycin resistance.

Other MLST clones circulating in Chennai ST368, ST1208, ST678,

ST45, ST30
91

HIGHLIGHTS OF THE STUDY

We report two novel spa types; t9036 & t9037.

First report - MRSA isolates with mupirocin and inducible

clindamycin resistance.

First study to compare clinical isolates from hospital settings,

HIV patients with the carrier isolates from healthy community.

MRSA among HIV patients is very low compared to community

and hospitalized patients.

First to report the virulence factors of S. aureus infections

among HIV patients.

mecA negative SCCmec elements from highly susceptible S.

aureus was reported for the first time.

92

CONCLUSION

93

Antibiotic resistance was significantly high among hospital associated isolates compared to
other groups.

No significant difference in the prevalence of virulence genes between clinical isolates of

community- and hospital associated infection, but low in HIV associated infection.

Antibiotic resistance was significantly high among HA-MRSA compared to MRSA from
community-associated infections and HIV-infected patients.

Prevalence of MRSA (2%) among healthy individuals from various high risk communities
indicates that CAMRSA isolates are circulating in the community asymptomatically.

Genotyping showed that the carrier S. aureus isolates from community were highly diverse
compared to clinical isolates.

The emergence of MDR-CA-MRSA clones among hospitalized patients

shows

that

CA-MRSA

has

acquired

additional

drug

resistance

determinant on entering the hospital settings. This study has shown

that in the Indian scenario, CA-MRSA isolates have infiltrated into
the

hospital

settings,

acquired

multiple

antibiotic

determinants and have become endemic in hospitals.

94
resistance

PUBLICATIONS
1.

Nagarajan A, Arunkumar K, Saravanan M, et al. Use of triplex PCR for rapid detection of PVL and differentiation of MRSA from
methicillin resistant coagulase negative Staphylococci. J of Clin Diagn Res 2012. Accepted. Impact factor 0.113. Indexed in
Pubmed, Medline, Scopus.

2.

Nagarajan A, Saravanan M, Padma K. Emergence of Methicillin-Resistant Staphylococcus aureus ST239 with High-Level Mupirocin
and Inducible Clindamycin Resistance in a Tertiary Care Center in Chennai, South India. J Clin Microbiol 2012, 50:3412-3413.
Impact factor 4.153. Pubmed ID: 22855516
3.

Nagarajan A, Arunkumar K, Saravanan M, et al. Detection of fusidic acid resistance determinants among Staphylococcus aureus
isolates causing skin and soft tissue infections from a tertiary care centre in Chennai, South India. BMC Infectious Diseases 2012,
12 (Suppl 1):P45 doi: 10.1186/1471-2334-12-S1-P45. Impact factor 3.253. Pubmed ID: 3344753

4.

Nagarajan A, Arunkumar K, Saravanan M, et al. PVL positive methicillin resistant Staphylococcus aureus breast abscess infection
among post-partum women in Chennai, South India. BMC Infectious Diseases 2012, 12 (Suppl 1):O13 doi: 10.1186/1471- 2334-12S1-O13. Impact factor 3.253. Cited by [2]. Pubmed ID: 3344695

5.

Nagarajan A, Saravanan M, Betsy SDB, et al. Mupirocin resistant HA-MRSA with inducible clindamycin resistance causing skin
infections in a tertiary care centre from Chennai, South India. International Conference on Emerging Infectious Diseases 2012 poster
and oral presentation abstracts. Emerg Infect Dis [serial on the Internet]. 2012. Impact factor 6.53 (Official Journal of CDC,
USA)

6.

Nagarajan A, Ananthi M, Krishnan P, et al. Emergence of Panton- Valentine leucocidin among community- and hospital-associated
meticillin-resistant Staphylococcus aureus in Chennai, South India. J Hosp Infect. 2010 Nov; 76(3): 269-71. Impact factor 3.11.
Cited by [2]. Pubmed ID: 20621389

7.

Nagarajan A, Saravanan M, Padma K. High prevalence of exfoliative toxins among carrier isolates of Staphylococcus aureus from
healthy individuals from various communities in Chennai, South India. Indian J Microbiology 2012. Impact factor 0.5

95

8.

Betsy SDB, Nagarajan A, Padma K, et al. Clindamycin resistance among Staphylococcus aureus causing skin and ear infections
from Chennai, South India. BMC Infectious Diseases 2012, 12 (Suppl 1):P70 doi: 10.1186/1471-2334-12-S1-P70. Impact factor
3.253. Pubmed ID: 3344777

SELECTED PRESENTATION AT INTERNATIONAL CONFERENCES

1.

Nagarajan A, Saravanan M, Betsy SDB, Sivakumar G, Sinha B and Padma K.

Mupirocin resistant HA-MRSA with inducible clindamycin resistance causing skin
infections in a tertiary care centre from Chennai, South India. International Conference
on Emerging Infectious Diseases 2012, March 11 -14, 2012, Atlanta, Georgia, USA.

2.

Nagarajan. A, K. Arunkumar, M. Saravanan, G. Sivakumar, Padma K. Detection of

fusidic acid resistance determinants among Staphylococcus aureus isolates causing skin
and soft tissue infections from a tertiary care center in Chennai, South India.
International Science Symposium on HIV and Infectious diseases 2012. Jan 20 22,
2012, Chennai, India.

3.

Nagarajan A, Ananthi M, Prabha C, Hans JL, Padma K. Emergence of PVL positive

methicillin resistant S. aureus in Chennai, South India. International Conference on
Understanding and Managing Pathogenic Microbes (UMPM 2010), IMTECH,
Chandigarh, India. January 22 24, Abstract No. P36.

4.

Nagarajan A, Saravanan M and Padma Krishnan. Methicillin Sensitive

Staphylocococcus aureus carrying mecA negative SCCmec elements. ISSSI 2012,
Lyon, France.

5.

ISSSI 2010 University of Bath, England

96

ACKNOWLEDGEMENTS

Dr. Padma Krishnan

Dr. Thangam Menon

Dr. Sujatha Narayanan

Dr. Banu Sinha, Germany

97

ICMR BMBF (Germany) for funding the research and research training

ACKNOWLEDGEMENTS
Collaborators
Dr. Soumya Swaminathan NIRT
Dr. Sumathi G MMC
Dr. G. Sivakumar MMC
Dr. G.Narendran NIRT
Dr. Chandrasekar, GHTM
Dr. J. Suria Kumar, GHTM
Dr. C. Prabha, NIAIDS, USA
Teaching Faculty
Prof. Dr. Elancheziyan Manickan
Dr. Srivani Ramesh
Dr. K. Veeramani, ASO
Former Directors
Dr. Balasubramanian
Dr. Srinivasan

Juniors
Mr. Saravanan
Mr. Kaushik
Ms. Betsy Soundarya
Dr. Thangalaxmi
Dr. Ramasamy
Mr. P. Nagaraj

UICIC trainees
Ms. E. Padmasini
Ms. R. Gayathri
Ms. R. Praveena
Ms. M. Akila
Mr. S. Sugumar
Ms. S. Subbulakshmi

Seniors
Dr. Ananthi
Dr. Mahalaksmi
Dr. Padmavathy
Dr. C. Anitha
Mr. Karthikeyan
Ms. Jasmine Shahina
Ms. Anuswedha
Dr. D. Prabhu

Dr. K. Arun Kumar

Ms. Jayanthi Marie

Lab staffs
Dr. Kownhar
Ms. Ramani

Friends
Dr. S. Senthil Kumar
Ms. Divya Bajoria
Dr.Bharathi
Ms. Kousalya
Ms. Karthiga
Mr. Mohinder, Germany
Ms. Nathiya, Germany
Dr. Viswanathan, Germany
98
Dr. Osthuysen, Germany
Mr. Eddy, Germany

Thank you

99