The document discusses horizontal gene transfer of antibiotic resistance genes within Enterobacteriaceae bacteria. It summarizes key species such as E. coli, K. pneumoniae, and E. cloacae that can transfer genes horizontally. It also discusses various antibiotic resistance mechanisms like beta-lactamases and provides classifications. The document analyzes spread of multidrug resistance in different regions of Asia and provides case studies showing evidence of horizontal gene transfer between bacteria. It analyzes how antibiotic pressure can promote horizontal gene transfer at the proteomic level by overexpressing protective proteins in bacteria.

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Enterobacteriaceae: intestinal
bacteria and horizontal gene
transfer
Hanna E. Sidjabat, PhD
Overview
 Key species within Enterobacteriaceae –
horizontal gene transfer – E. cloacae, K.
pneumoniae and E. coli.
 Antimicrobial resistance mechanisms –
horizontal expansion of antibiotic resistance
genes and case studies.
 What changes during antibiotic pressure –
promote horizontal gene transfer at the
proteomic level.
Why emerging antibiotic
resistance is important?
 Evolution to multidrug resistance and pan-drug
resistance.
 Clear relationship between antibiotic resistance
and mortality.
 Increase in infected population (2010 – 2030):
elderly, immunocompromised, ICU patients,
transplant patients and patients undergo surgery.
World Economic Forum 2013

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Antibiotic resistance mechanisms
Antibiotic resistance mechanisms
 Enzymatic
 mutation of the target binding site
 efflux pumps
 modification of the outer membrane proteins
 Beta-lactam – most commonly used antibiotics.
 Resistance by beta-lactamases – most
commonly reported.
Classification of β-lactamases
Ambler molecular
classification
Description Examples of enzymes
Class A
Serine β-lactamase
- Cephalosporinases (ESBLs)
- Usually clavulanic acid susceptible
except KPC
TEM, SHV, CTX-M
KPC
Class B
Metallo β-lactamase
(MBL)
- Metal ion (Zn++)
- Carbapenemases
- Not inhibited by clavulanic acid
- Inhibited by aztreonam
IMP, VIM, NDM
Class C
AmpC β-lactamase
- Clavulanic acid resistant
- Chromosomally encoded in
certain Gram negatives
- Plasmid-encoded
CMY, DHA,ACT, BIL,
FOX
Class D
Serine β-lactamase
- Oxacillinases
- Clavulanic acid susceptible
- Carbapanemases
OXA
Blue = cephalosporinase,red = carbapanemase

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East Asia:
• CTX-M, CTX-M-14 or CTX-M-9 group (all parts in East Asia)
• CTX-M-15 ST131 (nearly all parts in East Asia)
• CMY-2 and/or DHA (China, Taiwan, Japan, Korea)
• KPC ST131 (China, Taiwan)
• IMP ST131 (Taiwan)
• NDM (China, Hong Kong, Taiwan, Japan, Korea)
South Asia :
• CTX-M-15, CMY-2 (Indian subcontinent)
• CTX-M-15 ST131 (India, Pakistan)
• ST131 (Bangladesh)
• NDM (Indian subcontinent)
• OXA-48 co-producing NDM (Central India)
• KPC (importation from India to France)
West Asia:
• CTX-M-15
• CTX-M-1 group
• CTX-M-15 ST131
• NDM
• OXA-48
South East Asia:
• CTX-M (all region in South East Asia)
• CTX-M-15 ST131 (Indonesia)
• CMY-2 (Singapore, Thailand, Malaysia)
• DHA (Singapore, Thailand, Philippines)
• NDM (Singapore, Thailand, Malaysia)
• NDM ST131 (Thailand)
• IMP (Singapore)
Red = East Asia
Purple = Southeast Asia
Green = South Asia
Blue = Central Asia
Yellow = Western Asia
Key multidrug resistant E. coli four regions in Asia
Sidjabat HE & Paterson DL. 2015.Multidrug-resistant Escherichia coli in Asia:
epidemiology and management.Expert Review of Anti-infectiveTherapy.13:575-91.
Cases with evidence of horizontal gene
transfer in Enterobacteriaceae
Vertical spread
or clonal spread
Outbreak like situation.
Spread of antibiotic resistance
Plasmid
Chromosome
Conjugation
Hanna Sidjabat – Superbugs 2013

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Vertical spread
or clonal spread
Outbreak like situation.
Horizontal spread
Horizontal gene transfer.
Plasmid
Chromosome
Conjugation
Hanna Sidjabat – Superbugs 2013
Spread of antibiotic resistance
 44-yr old woman in USA.
 Small bowel transplantation in
2005.
 In June 2008 was presented
with bacteremia with E. coli and
E. cloacae.
 Piperacillin-tazobactam ->
amikacin -> meropenem.
 In Jul 2008, E. coli and P.
mirabilis (urine).
 In Aug 2008, E. coli and K.
pneumoniae.
 In Nov 2008, P. aeruginosa, S.
marcescens and K.
pneumoniae.
• Burn patient was colonised with carbapenem resistant
Enterobacteriaceae.
• The patient received various antibiotics over prolonged period.
• The colonisation occurred for 8 months.
• The plasmids harbouring resistance genes were transferred from
E. cloacae to E. coli.
• The resistant organisms could not be isolated – no antibiotic.
Sidjabat HE et al. 2014. J Clin Microbiol. 52:3816-8.

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A. IMP-4-producing E. coli. B. IMP-4-producing E. cloacae.
Clonal spread and horizontal
spread in larger scale
CTX-M-15 producing isolates and plasmids
Sample
ID
Ec103
Ec73
Ec90
Ec81
Ec88
Ec97
Ec98
Ec73
Ec133
Ec100
Ec112
Ec69
Ec68
Ec83
CTX-M type
CTX-M-15
CTX-M-15
CTX-M-9
CTX-M-9
CTX-M-15
CTX-M-9
CTX-M-15
CTX-M-15
CTX-M-15
CTX-M-15
CTX-M-15
CTX-M-15
CTX-M-9
CTX-M-9
O25b-ST131 – pandemic E. coli
ISOLATES CTX-M-15 PLASMIDS

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Sample
ID
Ec173
Ec29
Ec108
Ec156
Ec202
Ec127
Ec206
Ec96
Ec116
Ec107
Ec112
Ec125
Ec113
Ec86
Ec191
Ec124
Ec122
Ec123
Ec88
Ec129
CMY-2 producing isolates and plasmids
Enzyme
CMY-2
CMY-2
CMY-2
CMY-2
CMY-2
CMY-2
CMY-2
CMY-2
CMY-2
CMY-2
CMY-2
CMY-2
CMY-2
CMY-2
CMY-2
CMY-2
CMY-2
CMY-2
CMY-2
Inc I1
ISOLATES CMY-2 PLASMIDS
Sidjabat, et al,IJAA 2014
Genetic surroundings of blaNDM from Australia
Complete plasmid sequence of NDM
plasmids from Australia and New Zealand

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Changes at the bacterial proteome -
antibiotic pressure
 IMP-producing E. coli (n=4)
 NDM-producing E. coli (n=3)
 KPC-producing E. coli (n=3)
o E. coli ATCC 25922 –
susceptible E. coli.
All the above E. coli are carbapenemase
Producers – resistant to the last resort
of antibiotics.
Cultured at sub-MICs.
Analysed using liquid-chromatography mass-
Spectrometry.
Clinical isolates
Principal Component Analysis
No antibiotic and
grown in ciprofloxacin
Meropenem
Principal Component Analysis
Meropenem
NDM
IMP
KPC
No antibiotic and
grown in ciprofloxacin

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PCA: variables - sample clustering
Variables that led to
sample clustering
Outer membrane protein A
No Antibiotic
Ciprofloxacin
Meropenem
HU DNA-binding proteins
MEM
MEM
CIPCIP No AbNo Ab
Role:
Stabilize DNA and prevent its denaturation under extreme
environmental conditions

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GroEL and GroES chaperonin complex
To protect newly synthesized or stress-denatured polypeptides from misfolding and
aggregating. Help to buffer the effects of environmental changes.
MEM
MEM
Enterobacterial complement
resistance protein (TraT)
Role:
Regulates conjugation to prevent unproductive conjugation.
TraT encodes an outer membrane protein.
MEM
Summary
 Clonal analysis of bacteria and plasmids can help
the understanding of the mechanisms of spread
of antibiotic resistance genes.
 Bacteria are capable in surviving in the harsh
environment due antibiotic pressure by over-
expressing protective proteins. Antibiotic
pressure can promote the horizontal gene
transfer.

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the University of Queensland, St. Lucia Campus
Thank you:
Prof David Paterson
Prof Anders Cervin
AlexanderWailan,BSc Hons
Dr.Witchuda Kamolvit
Jolene Gien,BSc Hons
Contact:
h.sidjabat@uq.edu.au