1. KPC-2 Amino Acid Sequence and KPC-3 Substitution
MSLYRRLVLLSCLSWPLAGFSATALTNLVAEPFAKLEQDFGGSIGVYAMDT
GSGATVSYRAEERFPLCSSFKGFLAAAVLARSQQQAGLLDTPIRYGKNALV
PWSPISEKYLTTGMTVAELSAAAVQYSDNAAANLLLKELGGPAGLTAFMR
SIGDTTFRLDRWELELNSAIPGDARDTSSPRAVTESLQKLTLGSALAAPQR
QQFVDWLKGNTTGNHRIRAAVPADWAVGDKTGTCGVYGTANDYAVVW
PTGRAPIVLAVYTRAPNKDDKHSEAVIAAAARLALEGLGVNGQ(GSEN)
Abstract Results
References
Summary
1. Centers for Disease Control and Prevention, Antibiotic
resistance threats in the United States, 2013.
2. Hayden M and Lin MY and Lolans K. et al. “Prevention of
Colonization and Infection by Klebsiella pneumoniae
Carbapenemase–Producing Enterobacteriaceae in Long-
term Acute-Care Hospitals.” Clinical Infectious Diseases
2015 Apr 15;60(8):1153-61
3. Marchaim D and Chopra T and Bogan C, et al. “The
burden of multidrug resistant organisms on tertiary
hospitals posed by patients with recent stays in long-term
acute care facilities.” American Journal of Infection
Control 2012 Oct;40(8):760-5
4. Kashikar A and Zhang Y and Tulpule A and Dunn M and
Herman K and Denys G and Bush K. ICAAC, Abstract C-
1095. (2015)
5. Bush K and Jacoby GA. “Updated Functional Classification
of Β-Lactamases .” Antimicrobial Agents and
Chemotherapy 54.3 (2010): 969–976.
KPC is a β-lactamase family that was first discovered on a
plasmid in K. pneumoniae, but since has been shown that
other Enterobacteriaceae can produce it. KPCs are causing
problems for healthcare due to the high resistance to current
antibiotics that is seen in pathogens carrying these enzymes.
KPC-producing organisms are part of a much larger group of
bacteria known as Carbapenem Resistant Enterobacteriaceae
(CRE). CRE, according to the CDC in a 2013 report, represent an
“immediate public health threat that requires urgent and
aggressive action.”[1] KPC-producing organisms are among the
most common CRE worldwide.[2] Carbapenemases are
organized into two groups: serine carbapenemases that have a
serine residue at the active site and metallo-β-lactamases
(MBLs) that have at least one zinc ion at its active site.[5]
Recommendations to reduce the occurrence of resistance
included patient screening and increased infection control.
Health centers and hospitals are at high risk spreading KPC
producing organisms. [3]
Indiana University Health began screening for CRE in 2009 with
former students of the biotech program creating susceptibility
profiles and screening for β-lactamases.[4] This study
compares the incidence of KPC from 2009-2013 [4] and 2014.
Materials and Methods
Acknowledgments
We thank previous Biotechnology students for their
contributions to this project, especially Yunliang
Zhang, Ankita Kashikar and Thomas Amick. We also
thank Dr. Rasheda Sultana for her advice and Dr.
Gerald Denys for providing the isolates.
• This study looked at isolates from a 2014 surveillance collection of
CRE in patient isolates that were located in two large hospitals
and fourteen care centers within the Indiana Health system.
• This study looked at 56 organism including K. pneumoniae (38), E.
cloacae (10), E. coli (4), E. aerogenes (2), C. freundii (1) and K.
oxytoca (1).
• At IUB, isolates were grown on using LB agar and LB medium
containing ampicillin
• Plasmid DNA isolated from CRE strains using a plasmid miniprep
kit. The purified plasmids were screened for the presence of the
gene blaKPC, using PCR and gel electrophoresis. The following
primers were used:
• Full gene sequencing of blaKPC was performed using Big Dye 3.1
kit. Alignments of gene sequences were analyzed using ExPASy,
BLAST and Clustal W.
Presented at 2016 Research Symposium & Poster Fair April 2, 2016 Bloomington, IN
Introduction
KPC
Forward 5’ ATGTCACTGTATCGCCGTCT 3’
Reverse 5’ TTTTCAGAGCCTTACTGCCC 3’
In this study, two types of KPCs were observed, KPC-2 and KPC-3.
For Central Indiana, KPC-2 and KPC-3 are the only KPC variants that
have been observed.
Below is the amino acid sequence for KPC-2. Brackets are added to
denote the position of the primers used and the highlighted amino
acid indicates the position and substitution of KPC-3.
Forward
Primer
Reverse
Primer
KPC-3 Y
1.5kpb
1.0kpb
0.5kpb
184182179177176173 185+Con 186 SM8
ECL
19 EA3 -Con 175 187
ECL
20 183
Image of PCR product gels. Three-digits number refer to Klebsiella
pneumoniae isolates and ECL is Enterobacter cloacae. The KPC
gene is 893 base pairs long.
When a positive band was observed the PCR product was then
sequenced.
KPC genetic screening results from 2014 isolates. In total
there were 41 KPC producing organisms observed.
Antibiotic resistance is on the rise and poses a grave threat to
the public. A common resistance mechanism is through the
production of enzymes called β-lactamases that render β-
lactam antibiotics inert and unable to kill the bacteria. The goal
of my lab is to study the bacteria that produce these enzymes
in the Carbapenem-Resistant Enterobacteriaceae or CRE
Project. The purpose of my specific project is to identify various
β-lactamases in clinical isolates from Indianapolis area hospitals
and healthcare centers. β-Lactamase genes I screened for
include those that code for TEM, CTX-M and Klebsiella
pneumoniae carbapenemase (KPC), enzymes frequently
identified in historical CRE from Indiana. This poster will only
focus on KPC findings.
The screening process for these isolates included growing the
cells in a LB broth containing ampicillin. The cells then
underwent Qiagen plasmid miniprep to isolate the plasmids.
PCR was performed on the plasmids to identify a known β-
lactamase gene blaKPC. If a positive signal was observed on a
Southern blot, the sample was submitted for sequencing for
confirmation of the presence of the gene.
Isolates collected from 2014 (n=56) have been screened for
genes producing KPC β-lactamases. Among these isolates, at
least 41 carry KPC genes. Of the 45 KPC-producing isolates, 3
produce KPC-2 and 28 produce KPC-3 enzymes. Additional
isolates from 2015 are being screened for comparative
purposes.
The majority of CRE from Indianapolis produce KPC
carbapenemases, with a predominance of KPC-3 to KPC-2
enzymes. Additional monitoring will be necessary to determine
whether other mechanisms of resistance may be operative and
identify more β-lactamases.
The number of CRE Isolates decreased from 2012 to 2013
then plateaued. Data from 2009-2013 from Kashikar et al.
[4]
Screening Indianapolis Antibiotic-Resistant Clinical
• The predominant carbapenemase in
Indiana CRE was KPC-3 compared to
KPC-2.
• KPC-2-like and KPC-3-like enzymes
differed by one or two amino acid
substitutions from themselves, and
from their parent enzymes.
• The majority of the isolates that
produced KPC carbapenemases were K.
pneumoniae.
• Out of the 56 isolates screened, only 41
isolates produced KPC variants.
• For 2014, the number of CRE isolates
has decreased compared to the upward
trend seen from 2009-2013.
• At this time it is unclear why the
prevalence of KPC producing organisms
is decreasing, but it may be due to
improved infection control or changes
in antibiotic usage patterns.
Future Directions
Isolates from 2014 will continue to be screened for
other β-lactamases including TEM, SHV, CTX-M,
SME, NDM and VIM.
Isolates from 2015 have been received and will be
monitored for KPCs and other β-lactamases.
0 5 10 15 20 25 30
KPC-3-like
KPC-2-like
KPC3
KPC2
Number Isolates
KPCVariants
Number of KPC variations in 2014 Isolates
P
E
R
C
E
N
T
A
G
E
C
R
E
Decreased Identification of CRE in Central
Indiana
from 2009 - 2014
1.1
2.3
3.0
1.7 1.6
0
1
2
3
4
Isolates for KPC β-Lactamases from 2014
Kevin Knight and Karen Bush | Indiana University, Bloomington, IN