Transcript based methods in the environment and hospital patients

Rapid and sensitive detection
of MRSA by PCR-based methods in
the environment and hospital patients.
by
Dr. Corinne Whitby
Department of Biological Sciences, Wivenhoe Park, University of Essex,
Colchester, Essex, CO4 3SQ.
Tel: +44 (0) 1206 872062, Fax: +44 (0) 1206 872592.
Email: [email protected].
MRSA ‘the super bug’
Methicillin-resistant Staphylococcus aureus (MRSA) was
first discovered in the UK in 1961.
It is a major pathogen associated with nosocomial disease
both in the community and in hospitals.
The number of MRSA infections is increasing with >1,600
deaths reported in 2006 in the UK alone.
SEM of MRSA
SEM of MRSA
Screening, treatments and prevention
Screening- by taking nasal swabs upon arrival at hospital.
Treatments- Vancomycin & teicoplanin antibiotics- but
resistant strains have been discovered. Additional antibiotics
are used to treat these severe infections.
Maggot therapy- to treat necrotic tissue from MRSA
infections.
Phage therapy- 95% efficacy against Staphylococcus isolates.
J. Infect. Dis. (2003). 187 (4): 613–24.
New antibiotics are currently in clinical
trials. e.g. Ceftaroline.
Prevention- handwashing, essential oils
e.g. Tea tree oil
Ruptured MRSA abscess
Genetics of MRSA
Methicillin resistance is mediated by the mecA gene encoding
for penicillin-binding protein 2a (PBP-2a) & allows S. aureus
to be oxacillin resistant.
Mueller Hinton agar showing
MRSA resistant to oxacillin
disk.
mecA gene is located on a mobile genetic element
called the staphylococcal cassette chromosome (SCC).
Expression of PBP-2a is controlled by mecR1 & mecI
regulator genes located upstream of mecA gene.
Isolates with mutations in the mec regulators may
phenotypically be highly resistant to methicillin & so
are also clinically relevant.
SCCmecII is most abundant in hospitals,
whereas SCCmecIV is most abundant
community-acquired MRSA.
Nature Reviews (2009) 7: 629
Diagnostic identification of MRSA
Traditional methods require culturing, phenotypic and
biochemical characterisation which is time-consuming.
Molecular methods e.g. Gene probing, DNA sequencing of
rRNA genes, FISH and PCR have all been used- but may
not differentiate between MSSA and MRSA.
Other methods include Real-time Q-PCR and MLST.
A PCR assay which detects polymorphisms in the 16S-23S
rRNA internal transcribed spacer region (ITS) may
provide a rapid alternative for identifying S. aureus spp.
Our Hypothesis:
In hospitals MRSA spreads between
the patient and the environment and vice versa and it is likely
that identical MRSA genotypes colonise both patient and
environment.
Our Aims:
• To identify MRSA in a hospital ITU, directly from the
environment without culturing & determine how the
environment may act as a reservoir for MRSA.
• To apply ITS-PCR to differentiate between MRSA and
MSSA isolates from both patient and the environment.
• To analyse the genetic diversity of mecA, mec regulator
genes in MRSA isolates cultured from both clinical and
environmental samples.
Methods/ Approaches
Sampling hospital
environment
Direct DNA
extraction
Clinical samples
Isolation & Screening
of MRSA isolates
Antimicrobial
susceptibility testing
PCR amplification
of ITS region
PAGE analysis
of ITS region
DNA extraction
PCR amplification
of mecA and mec
regulator genes
mecA gene sequencing,
RFLP analysis of mec
regulator genes
Results
110 environmental samples were taken from hospital
surfaces e.g. bed mattresses, floors, walls, desktops, beds,
computer keyboards, ventilator equipment and trolleys.
• Only 7 isolates were obtained from the environment and
all were MRSA (determined by PCR amplification of mecA
gene and culture).
• 60 isolates were obtained from clinical samples from
patients e.g. throat swab, sputum, blood culture, nose swab,
wound swab, abscess, trachaeal, hip fluid.
• 45% of the clinical isolates were MRSA (determined by
PCR amplification of mecA gene and culture).
PAGE analysis of ITS-PCR.
PAGE is a high resolution
method for fractionating DNA.
It allows rapid, high throughput
screening of samples.
PAGE analysis of ITS-PCR from 60 clinical isolates.
400 bp
300 bp
ITS-type 6 is
predominant.
200 bp
1 6 6 7 7 1516 6 6 8 9 10 6 11 9 9 12 6 7 6 8 6 6 6 13 13 6 2
ITS-type
100 bp
400 bp
300 bp
200 bp
100 bp
ITS type 1:
EMRSA-16 NCTC13143.
ITS type 2:
MSSA NCTC6571
PAGE analysis of ITS-PCR from 7 environmental
MRSA isolates.
ITS-type 6 also detected in 1
environmental isolate.
ITS-type 3,4 and 5 were unique to
environmental isolates i.e. not found in
the clinical isolates suggesting different
genotypes were colonizing the
environment compared to the patient.
ITS type 2: MSSA NCTC6571
400 bp
300 bp
200 bp
3 3 3 4 5 6 2 2
100 bp
Example PAGE of ITS-types directly from 110 hospital
environmental samples without culturing.
400 bp
300 bp
ITS-type 6 also found in
several environmental
samples.
200 bp
100 bp
400 bp
300 bp
200 bp
100 bp
ITS type 1: EMRSA16 NCTC13143.
ITS type 2: MSSA
NCTC6571
Distribution of ITS-types from S. aureus isolates
(both environmental and clinical) that were mecA+
or mecA-.
Total number of isolates
35
30
25
20
Number of mecA-
15
Number of mecA+
10
5
0
1
2 3
4
5 6
7 8
9 10 11 12 13 14 15 16 17
ITS subtype
17 different ITS profiles were identified: 6 patterns for MRSA, 7
patterns for MSSA, and 4 patterns for both MSSA and MRSA.
22 isolates that were mecA+ were from ITS type 6.
Analysis of mecA and mec regulatory genes (mecI, mecRI).
mecA gene PCR amplification: No mecA PCR products were obtained from the
environment but mecA PCR products obtained for all 7 environmental & 27 clinical
isolates.
mecI gene PCR amplification:
No environmental isolates generated mecI
PCR products.
M
C206
MseI
C224 M
MseI
Uncut Uncut M
C206 C224
2 clinical isolates (C206, C224) generated
mecI PCR products and were MRSA.
Restriction digestion with MseI generated
bands of 350, 130 bp, and <30bpconsistent with other workers.
mecRI PCR amplification: 2 environmental
& 17 clinical isolates generated mecRI PCR
products. All were MRSA except 1 clinical
isolate which was MSSA.
600 bp
400 bp
200 bp
PCR amplification and restriction
digest of mecI genes from 2 clinical
S. aureus isolates.
Conclusions I
• ITS-PCR was successfully used to directly detect S. aureus spp. from hospital
environments without culture and may be applied for rapid screening.
• 56% of isolates were MRSA & represented ITS- types 1,3,4,5,16 & 17. Screen
for these?
• ITS-type 6 predominated clinical samples with MRSA representatives found in
both environmental isolates and environmental samples. Screen for these also?
• Different ITS-types were colonizing the environment compared to the patient
and were MRSA. Thus despite stringent disinfection surfaces are a reservoir
for MRSA which have the potential to transfer to the patient.
• No isolate had ITS-profiles identical to the control strain EMRSA-16, a
common organism recovered from hospitals in the UK- these S. aureus spp. may
have arisen from multiple external sources.
Conclusions II
• No mecA PCR products were obtained directly from the environment- MRSA
was below detection limits. A ‘nested’ PCR amplification approach may improve
sensitivity?
• mecA PCR products were obtained for all 7 environmental & 27 clinical isolates
and corroborated the antibiotic susceptibility testing.
• BLASTN analysis of the mecA sequences from clinical and environmental
isolates had 98-100% identity to mecA genes from S. aureus spp.
• Only two clinical isolates (C206, C224) generated mecI PCR products. Both
were MRSA. No environmental isolates generated mecI PCR products.
• mecRI gene was detected in 19 isolates (18 of which were MRSA).
• It is possible that MRSA strains only have mecA and acquire the mec regulator
genes later.
• Large genetic diversity obtained with all S. aureus isolates suggest there is a
broad-range of MRSA clones in the hospital and gene transfer may occur
whereby mecA homologues may be acquired from the environmental reservoir.
Acknowledgements
We thank:
Hospital Infection Society,
Santander
University of Essex
for help with funding this work.