"Silent" Dissemination of Klebsiella pneumoniae Isolates Bearing K-pneumoniae Carbapenemase in a Long-term Care Facility for Children and Young Adults in Northeast Ohio

Abstract

Klebsiella pneumoniae isolates harboring the K. pneumoniae carbapenemase gene (bla(KPC)) are creating a significant healthcare threat in both acute and long-term care facilities (LTCFs). As part of a study conducted in 2004 to determine the risk of stool colonization with extended-spectrum cephalosporin-resistant gram-negative bacteria, 12 isolates of K. pneumoniae that exhibited nonsusceptibility to extended-spectrum cephalosporins were detected. All were gastrointestinal carriage isolates that were not associated with infection.
Reassessment of the carbapenem minimum inhibitory concentrations using revised 2011 Clinical Laboratory Standards Institute breakpoints uncovered carbapenem resistance. To further investigate, a DNA microarray assay, PCR-sequencing of bla genes, immunoblotting, repetitive-sequence-based PCR (rep-PCR) and multilocus sequence typing (MLST) were performed.
The DNA microarray detected bla(KPC) in all 12 isolates, and bla(KPC-3) was identified by PCR amplification and sequencing of the amplicon. In addition, a bla(SHV-11) gene was detected in all isolates. Immunoblotting revealed "low-level" production of the K. pneumoniae carbapenemase, and rep-PCR indicated that all bla(KPC-3)-positive K. pneumoniae strains were genetically related (≥98% similar). According to MLST, all isolates belonged to sequence type 36. This sequence type has not been previously linked with bla(KPC) carriage. Plasmids from 3 representative isolates readily transferred the bla(KPC-3) to Escherichia coli J-53 recipients.
Our findings reveal the "silent" dissemination of bla(KPC-3) as part of Tn4401b on a mobile plasmid in Northeast Ohio nearly a decade ago and establish the first report, to our knowledge, of K. pneumoniae containing bla(KPC-3) in an LTCF caring for neurologically impaired children and young adults.

Full text

MAJOR ARTICLE
‘‘Silent’’ Dissemination of Klebsiella pneumoniae
Isolates Bearing K. pneumoniae Carbapenemase in
a Long-term Care Facility for Children and Young
Adults in Northeast Ohio
Roberto A. Viau,
1,a
Andrea M. Hujer,
2,3,a
Steven H. Marshall,
2
Federico Perez,
2,3
Kristine M. Hujer,
2,3
David F. Bricen
˜o,
9
Michael Dul,
7
Michael R. Jacobs,
4
Richard Grossberg,
8
Philip Toltzis,
7
and Robert A. Bonomo
2,3,5,6
1
Department of Medicine, Jacobi Medical Center, Albert Einstein College of Medicine, Bronx, New York;
2
Research Service, Louis Stokes Cleveland
Department of Veterans Affairs Medical Center,
3
Department of Medicine,
4
Department of Pathology,
5
Department of Pharmacology, and
6
Department
of Molecular Biology and Microbiology, Case Western Reserve School of Medicine,
7
Division of Pharmacology and Critical Care, Rainbow Babies and
Children's Hospital, Cleveland, and
8
Hattie Larlham Center for Children with Disabilities, Mantua, Ohio; and
9
Centro Internacional de Entrenamiento e
Investigaciones Medicas, Cali, Colombia
Background. Klebsiella pneumoniae isolates harboring the K. pneumoniae carbapenemase gene (bla
KPC
)are
creating a significant healthcare threat in both acute and long-term care facilities (LTCFs). As part of a study
conducted in 2004 to determine the risk of stool colonization with extended-spectrum cephalosporin-resistant
gram-negative bacteria, 12 isolates of K. pneumoniae that exhibited nonsusceptibility to extended-spectrum
cephalosporins were detected. All were gastrointestinal carriage isolates that were not associated with infection.
Methods. Reassessment of the carbapenem minimum inhibitory concentrations using revised 2011 Clinical
Laboratory Standards Institute breakpoints uncovered carbapenem resistance. To further investigate, a DNA
microarray assay, PCR-sequencing of bla genes, immunoblotting, repetitive-sequence-based PCR (rep-PCR) and
multilocus sequence typing (MLST) were performed.
Results. The DNA microarray detected bla
KPC
in all 12 isolates, and bla
KPC-3
was identified by PCR
amplification and sequencing of the amplicon. In addition, a bla
SHV-11
gene was detected in all isolates.
Immunoblotting revealed ‘‘low-level’’ production of the K. pneumoniae carbapenemase, and rep-PCR indicated that
all bla
KPC-3
-positive K. pneumoniae strains were genetically related ($98% similar). According to MLST, all isolates
belonged to sequence type 36. This sequence type has not been previously linked with bla
KPC
carriage. Plasmids from
3 representative isolates readily transferred the bla
KPC-3
to Escherichia coli J-53 recipients.
Conclusions. Our findings reveal the ‘‘silent’’ dissemination of bla
KPC-3
as part of Tn4401b on a mobile plasmid
in Northeast Ohio nearly a decade ago and establish the first report, to our knowledge, of K. pneumoniae containing
bla
KPC-3
in an LTCF caring for neurologically impaired children and young adults.
Long-term care facilities (LTCFs) are essential com-
ponents of healthcare delivery to many patients.
Unfortunately, LTCFs are also recognized as ‘‘reser-
voirs of antibiotic resistance’’ [1]. In the past 3
decades numerous outbreaks of multidrug-resistant
gram-negative and gram-positive organisms have been
described in LTCFs [2,3]. The spread of antibiotic-
resistant pathogens transmitted from LTCFs to wider
healthcare delivery systems that serve a large region is
now appreciated as a major challenge in the design of
effective infection control and antibiotic utilization
strategies in the care of the elderly [4]. Among gram-
negative bacteria, Escherichia coli–andKlebsiella
pneumoniae–producing extended-spectrum b-lactamases
(ESBLs), as well as carbapenem-resistant Acinetobacter
baumannii and Pseudomonas aeruginosa,arethemost
significantthreatsinthissetting[5–10]. Especially
Received 7 October 2011; accepted 9 December 2011.
a
R. A. V. and A. M. H. contributed equally to this work.
Correspondence: Robert A. Bonomo, MD, Infectious Diseases Section, Louis
Stokes Cleveland Veterans Affairs Medical Center, 10701 E Blvd, Cleveland, OH,
44106 (robert.bonomo@va.gov).
Clinical Infectious Diseases 2012;54(9):1314–21
Published by Oxford University Press on behalf of the Infectious Diseases Society of
America 2012.
DOI: 10.1093/cid/cis036
1314 dCID 2012:54 (1 May) dViau et al

concerning has been the national and global spread of carba-
penem-resistant K. pneumoniae harboring bla
KPC
,belonging
to sequence type (ST) 258 [7,11,12].
Although not as well documented as in adult patients,
antibiotic-resistant gram-negative organisms are present in
healthcare settings serving children, including pediatric
LTCFs [13–16]. In a surveillance study examining the anti-
biotic susceptibility of normal flora of children residing in an
LTCF in Cleveland, Ohio, nearly 40% of subjects were col-
onized with resistant bacteria, and .60% of organisms were
resistant to .2 antibiotics tested [14]. Invasive devices were
found to b e a significant risk factor for colonization by resistant
gram-negative bacteria [14].
Little is known about the spread of bla
KPC
harboring strains
or whether the same risk factors are present in children and
adults. Unfortunately, the clinical detection of bla
KPC
is under-
mined by heterogeneous expression of carbapenem resistance.
Ertapenem minimum inhibitory concentrations (MICs) are the
most sensitive for detection of K. pneumoniae carbapenemase
(KPC) but may lack specificity, and therefore additional phe-
notypic tests (ie, modified Hodge test and boronic acid disk)
have been devised [17–20]. MICs of carbapenems are dependent
not only on the presence and the level of expression of bla
KPC
but also on changes in outer membrane proteins [7,21,22].
In this study we describe the ‘‘silent dissemination’’ and
earliest report of KPC-producing K. pneumoniae in an LTCF
caring for children and young adults with neurodevelopmental
impairments. As part of a study conducted in 2004 to de-
termine the risk of stool colonization by extended-spectrum
cephalosporin-resistant gram-negative bacteria, we identified
12 strains of K. pneumoniae that exhibited nonsusceptibility
to extended-spectrum cephalosporins. Reassessment of car-
bapenem MICs using recently revised breakpoints uncovered
carbapenem resistance. Genetic analysis revealed that a sin-
glesequencetypenotpreviously reported to contain bla
KPC
had disseminated as early as 2004 in Northeast Ohio in this
LTCF. Introduction of bla
KPC
into our region occurred be-
fore the description of the spread of ST 258, recognition of
Tn4401, the KQ element, or the mobile genetic elements
harboring this carbapenemase gene [23,24].
MATERIALS AND METHODS
Setting and Selection of Strains
The study facility cares for 130 nonambulatory children and
young adults with severe neurodevelopmental abnormalities.
During the course of the study, the ages of the residents
ranged between 5 and 47 years, with 81% distributed between
ages 11 and 29 years.
The current investigation was derived from a larger study
in which patient stool samples were collected between 1 July
and 30 December 2004. The goal of the study was to evaluate
the changes in gastrointestinal flora as a result of residence in
the facility. Approximately 10-g portions of whole stool were
collected from participating residents every 2 weeks by care-
givers. Samples were placed in anaerobic containers (Anaerobic
Systems) on site and then transported at ambient temperature
to a research microbiology laboratory within 24 hours. The
specimens were transferred to cryovials containing cooked
meat medium (BBL) and glycerol, and stored at 270°C.
After an analysis of the epidemiology of antibiotic-resistant
gram-negative organisms in a nearby pediatric intensive care
unit revealed that colonization by resistant organisms was
frequent in patients transferred from an LTCF [14], archived
frozen specimens were assessed for the presence of bacteria
expressing resistance to extended-spectrum cephalosporins.
Samples from the first 50 subjects enrolled in the larger study
were selected. Subjects were excluded if .2 successive sam-
ples were missing or the subject was hospitalized in an acute-
care facility during the period of sample collection. The
study was approved by the Institutional Review Board of
University Hospitals Case Medical Center, and parents or
legal guardians provided written informed consent.
Antimicrobial Susceptibility Testing
Single colonies of K. pneumoniae were recovered from frozen
stocks, and MICs of 17 agents were determined using Sen-
sititre GNXF trays (Trek Diagnostic Systems) as described
elsewhere [18]. American Type Culture Collection control
strains P. aeruginosa 27853 and E. coli 25922 were used as
quality control strains for susceptibility testing. Twelve iso-
lates of K. pneumoniae obtained were identified as non-
susceptible to third-generation cephalosporins using the
then current National Committee for Clinical Laboratory
Standards (NCCLS) criteria and were selected for study [25].
Carbapenem MIC results were reinterpreted according to
criteria issued by the Clinical Laboratory Standards Institute
(CLSI) in 2011 [26]. Breakpoints established by the US Food
and Drug Administration were used to interpret MIC results
for tigecycline (susceptible was defined as MIC #2 mg/L; resistant,
as MIC $8 mg/L). In addition, modified Hodge tests (MHTs)
with imipenem and meropenem were performed [27].
b-Lactamase Gene Screening Using a Microarray Detection
System
The Check-Points ESBL/AmpC/KPC/NDM-1 assay (Check-
MDR CT101; Check-Points) is a DNA low-density microarray
testing method for detecting bla
TEM
,bla
SHV
,bla
CTX-M
,bla
KPC
,
6 additional groups of AmpCs (bla
CMY-2-like
,bla
DHA
,bla
FOX
,
bla
CMY-1-like/MOX
,bla
ACC
,andbla
MIR
/
ACT
), and the bla
NDM-1
metallo-b-lactamase genes [28–30]. Briefly, genomic DNA was
extracted from colonies of bacteria grown overnight using the
DNeasy blood and tissue kit (Qiagen Sciences). Microarray
KPC-Bearing Klebsiella in Pediatric LTCF dCID 2012:54 (1 May) d1315

assays were performed according to instructions of the
manufacturer, generating templates of the target bla DNA
sequence that are amplified, hybridized, and then detected by
the instrument.
Polymerase Chain Reaction Confirmation of b-Lactamase Genes
Polymerase chain reaction(PCR)amplificationofbla
TEM
,
bla
SHV
,andbla
KPC
genes was achieved using established
primers and amplified with a MJ Research Gradient Cycler
Model PTC 225 (MJ Research) using thermocycling con-
ditions adjusted to the primer melting temperatures [21,22,
31]. In addition to bla genes, we tested for the presence of
qnrA,qnrB,qnrC,aac(6#), rmtA,rmtB,rmtC,andrmtD genes
by PCR using the primers and thermocycling conditions val-
idated in other studies [23,32]. Positive controls included well-
characterized isolates in our collection [31,32].
DNA Sequencing
Amplicons obtained using primers for bla
KPC,
bla
SHV
,and
bla
TEM
were sequenced at a commercial sequencing facility
(MCLAB). Additionally, the upstream sequence of bla
KPC
positive strains was also obtained by PCR amplification us-
ing primers described elsewhere [22,24]. Sequence data were
analyzed using Lasergene 7.2 software (DNAstar), and se-
quences were compared with BLAST online software (http://
blast.ncbi.nlm.nih.gov), using the megablast algorithm.
Repetitive-Sequence-Based PCR
Genetic similarity among strains was investigated by
repetitive-sequence-based PCR (rep-PCR) using the Diversi-
Lab strain typing system (Bacterial BarCodes; bioMe
´rieux), as
described elsewhere [21]. DNA was isolated from strains using
the MoBio UltraClean Microbial DNA Isolation Kit (MoBio),
following the manufacturer’s instructions for gram-negative
bacteria. Isolated DNA was amplified using the DiversiLab
Klebsiella kit, and amplicons were analyzed on an Agilent 2100
Bioanalyzer (Agilent). Dendrograms and comparisons were
generated by the DiversiLab Software (bioMe
´rieux). Isolates
with .95% similarity were considered of the same clone type.
Strains from our collection were used as comparators [21].
Multilocus Sequence Typing
Multilocus sequence typing (MLST) was performed on all
K. pneumoniae strains as described by Diancourt [33]. Se-
quences of 7 housekeeping genes (rpoB,gapA,mdh,pgi,
phoE,infB, and tonB) were compared with the MLST data-
base (http://www.pasteur.fr/recherche/genopole/PF8/mlst/).
Immunoblotting
Immunoblotting for assessment of KPC production was per-
formed according to established methods [22].
Mating Experiments
To investigate whether there was transfer of bla genes, 3 repre-
sentative bla
KPC-3
producing strains susceptible to rifampin
were selected for overnight culture in lysogeny broth (LB)
with rifampin-resistant E. coli J-53. After overnight growth in
LB, cells were plated on Mueller Hinton (MH) agar contain-
ing ampicillin (100 mg/L) and rifampin (100 mg/L). Colonies
were replated on the same selective MH agar. These strains
were screened by PCR for bla
KPC
, as described elsewhere [34].
RESULTS
The first 50 subjects were enrolled in the study between 1 July
and 30 December 2004 and were considered for inclusion in
the current analysis. Sixteen subjects were excluded owing
to inadequate longitudinal sampling of specimens as defined
in the Methods section, and 8 additional subjects were ex-
cluded owing to transfer to an acute care hospital during the
period of observation. Among the remaining 26 residents,
specimens were collected for a median of 11.5 weeks (range,
10–26 weeks). Stool specimens from 12 of the 26 subjects were
positive for K. pneumoniae expressing resistance to extended-
spectrum cephalosporins. In 4 of the 12 positive subjects the
organism was cultivated from only 1 specimen, but in the
remainder, excretion occurred over a median of 9 weeks
(range, 4–26 weeks).
There were no characteristics distinguishing between res-
idents whose specimens were K. pneumoniae positive and
residents whose specimens were not; they were of similar age
(median, 16.2 vs 19.9 years for those with positive vs those
with negative specimens); similar proportions had been ex-
posed to oral (64% vs 80%) or intravenous (28% vs 26%)
antibiotics during the previous 12 months; and similar
proportions had been hospitalized in the previous 12 months
(35% vs 27%) (all P..10). The residents carrying K. pneu-
moniae isolates were housed in all 5 of the facility’s pods, and all
had been born in, and referred from, cities in Ohio.
The antimicrobial susceptibility testing results of the
extended-spectrum cephalosporin-resistant K. pneumoniae
isolates are summarized in Table 1. All 12 isolates were re-
sistant to ceftazidime, cefotaxime, and aztreonam based on
2011 CLSI breakpoints. Reassessment of the carbapenem
MICs using 2011 CLSI breakpoints revealed that all iso-
lates were resistant to ertapenem and imipenem, 1 isolate
was susceptible to meropenem, and 2 isolates were suscep-
tible to doripenem [26]. The MHT was positive using imi-
penem and meropenem for all isolates, including the
meropenem-susceptible strain (Kp-11). All strains were
susceptible to cefepime, amikacin, colistin, and polymyxin B,
and 9 were susceptible to gentamicin and tobramycin. Consis-
tent with quinolone-prescribing practices in children, all isolates
1316 dCID 2012:54 (1 May) dViau et al

Table 1. Antimicrobial Susceptibility Testing of Klebsiella pneumoniae Isolates Collected From a Long-term Care Facility
MIC (mg/L) and Interpretation
Antibiotic Kp-4 Kp-5 Kp-7 Kp-8 Kp-9 Kp-10 Kp-11 Kp-12 Kp-14 Kp-15 Kp-16 Kp-17
Ciprofloxacin #0.25 S #0.25 S #0.25 S #0.25 S #0.25 S #0.25 S #0.25 S #0.25 S #0.25 S #0.25 S #0.25 S #0.25 S
Gentamicin #1S #1S 8I #1S #1S .8R #1S #1S #1S #1S 8I #1S
Amikacin #4S #4S #4S #4S #4S #4S #4S #4S #4S #4S #4S #4S
Tobramycin #1S #1S 8I #1S #1S 8I #1S #1S #1S #1S 8I #1S
Trimethoprim/sulfamethoxazole #0.5/9 S #0.5/9 S #0.5/9 S #0.5/9 S #0.5/9 S #0.5/9 S #0.5/9 S #0.5/9 S #0.5/9 S #0.5/9 S #0.5/9 S #0.5/9 S
Colistin
a
0.5 S 0.5 S 0.5 S 0.5 S 1 S 0.5 S 0.5 S 0.5 S 0.5 S 0.5 S 0.5 S 1 S
Polymyxin B
a
0.5S0.5S2S1S1S0.5S1S1S1S1S1S1S
Tigecycline
b
0.5 S 0.5 S 0.5 S 0.5 S 1 S 0.5 S #0.25 S 4 I 0.5 S 0.5 S 1 S 4 I
Piperacillin/tazobactam .64/4 R .64/4 R .64/4 R .64/4 R .64/4 R .64/4 R .64/4 R .64/4 R .64/4 R .64/4 R .64/4 R .64/4 R
Ceftazidime 16 R 16 R 16 R 16 R 16 R 16 R 16 R .16 R 16 R 16 R 16 R .16 R
Cefotaxime 8 R 4 R 4 R 8 R 8 R 8 R 8 R 16 R 4 R 4 R 8 R 16 R
Cefepime 4 S 4 S 4 S 4 S 4 S 4 S 8 S 8 S 4 S 8 S 8 S 4 S
Aztreonam .16 R .16 R .16 R .16 R .16 R .16 R .16 R .16 R .16 R .16 R .16 R .16 R
Meropenem 4 R 2 I 4 R 8 R 8 R 8 R #1S 4R 4R 4R 4R 4R
Imipenem 4 R 4 R 4 R 4 R 4 R 4 R 4 R 4 R 4 R 4 R 4 R 4 R
Doripenem 2 I 2 I .2R 1S .2R 2I 1S .2R 2I 2I 2I 2I
Ertapenem 4 R 4 R 4 R 4 R .4R 4R 4R 4R 2R .4R 4R .4R
Modified Hodge test
c
111111111111
Unless otherwise specified, susceptibility tests were interpreted according to 2011 Clinical Laboratory Standards Institute (CLSI) criteria. For aztreonam and ceftazidime, isolates were considered susceptible at minimum
inhibitory concentrations (MICs) of #4 mg/L; intermediate at MICs of 8 mg/L; and resistant at $16 mg/L. For cefotaxime, meropenem, imipenem, and doripenem, #1 mg/L was considered susceptible; 2 mg/L,
intermediate; and $4 mg/L, resistant. For ertapenem, #0.25 mg/L was considered susceptible; 0.5 mg/L, intermediate; and $1 mg/L, resistant.
Abbreviations: I, intermediate; MIC, minimum inhibitory concentration; R, resistant; S, susceptible.
a
CLSI breakpoints for Pseudomonas aeruginosa were applied.
b
Susceptibility tests were interpreted according to Food and Drug Administration criteria.
c
Performed with imipenem and meropenem, following CLSI recommendations. Plus signs indicate positive results.
KPC-Bearing Klebsiella in Pediatric LTCF dCID 2012:54 (1 May) d1317

were susceptible to ciprofloxacin, whereas 2 strains were non-
susceptible to tigecycline (Kp-12 and Kp-17).
PCR and Microarray Analysis
Using the Check-Points ESBL/AmpC/KPC/NDM-1 micro-
array and PCR/DNA sequencing, all the K. pneumoniae
strains were found to contain bla
KPC-3
and bla
SHV-11
genes;
bla
TEM-1
was found in 3 isolates (Table 2). PCR amplification
did not reveal qnrA,qnrB,qnrC,rmtA,rmtB,rmtC,rmtD,or
aac(6#). These findings were consistent with the suscepti-
bility results reported in Table 1.
Rep-PCR and MLST
All strains of K. pneumoniae were found to be related (.98%
similarity) by rep-PCR (Figure 1). Comparison with our
nationwide collection of KPC strains showed that these
strains were significantly different from any others we had
previously examined (data not shown). As a result, we were
prompted to perform MLST; all strains were identified as ST
36, a sequence type not previously associated with bla
KPC
.
Immunoblotting
We confirmed the PCR results for KPC by performing an
immunoblot with a highly specific antibody that recognizes
KPC-2/3. Low-level expression was observed (data not shown).
Conjugation Experiments
Conjugation demonstrated transfer of bla
KPC-3
on a mobile
element from 3 isolates to E. coli J-53 recipients. The transfer
of bla
KPC-3
was verified by PCR and sequencing of the
amplicon.
DISCUSSION
In this study we characterized isolates possessing bla
KPC-3
dating back to 2004, which to the best of our knowledge
represents the earliest detection of such a resistance de-
terminant in an LTCF caring for children and young adults.
This singular observation places the global epidemic of
bla
KPC
spreading in healthcare facilities in an entirely new
light. First, the patient population of LTCF’s caring for
children typically is very different from residents in adult
facilities, with the former predominantly composed of pa-
tients with significant neurological, muscular, and de-
velopmental disabilities but with few other comorbidities.
Therefore, the epidemiologic characteristics of resistant
microorganisms in pediatric facilities may differ significantly
from those in institutions caring primarily for elderly adults.
Few studies have been conducted in facilities caring for
children, and unlike in older adults, we know little regarding
the significance of bla
KPC
isolates in children in LTCFs and
the long-term consequences of this colonization. This study
draws attention to this concerning occurrence.
Second, all isolates were resistant to ceftazidime and cefo-
taxime, the basis for inclusion in the study. Phenotypic char-
acterization showed all isolates were susceptible to cefepime,
which initially led us to suspect that they might contain an
AmpC b-lactamase. However, evidence of an AmpC was not
detected. Therefore, this study not only confirms the difficulty in
detecting KPC strains using previous CLSI break points [18,25]
Figure 1. Dendrogram depicting .98% similarity and band patterns of
Klebsiella pneumoniae carbapenemase–producing K. pneumoniae strains
typed by repetitive-sequence-based polymerase chain reaction.
Table 2. Molecular Analysis of Klebsiella pneumoniae Collected
From a Long-term Care Facility for Children and Young Adults
Strain MLST bla
KPCa
bla
NDM-1
bla
AmpC
bla
CTX-M
bla
SHVb
bla
TEMb
Kp-4 36 12 2 2WT 2
Kp-5 36 12 2 2WT 2
Kp-7 36 12 2 2WT WT
Kp-8 36 12 2 2WT 2
Kp-9 36 12 2 2WT 2
Kp-10 36 12 2 2WT WT
Kp-11 36 12 2 2WT 2
Kp-12 36 12 2 2WT 2
Kp-14 36 12 2 2WT 2
Kp-15 36 12 2 2WT 2
Kp-16 36 12 2 2WT WT
Kp-17 36 12 2 2WT 2
Plus signs indicate present. Minus signs indicate absent. Abbreviations: MLST,
multilocus sequence typing: WT, wild type.
a
All bla
KPC-3,
as determined by sequencing.
b
WT indicates non-extended-spectrum b-lactamase bla
SHV
and bla
TEM
genes,
which were confirmed by sequence analysis (ie, bla
SHV-11
and bla
TEM-1
,
respectively).
1318 dCID 2012:54 (1 May) dViau et al

but also indicates that the major pathway of dissemination of
bla
KPC
in the time near its first detection may have been largely
‘‘silent.’’
Theimplicationsofthispatternofspreadforbla
KPC
genes
are very important. The case of bla
KPC
dissemination in
a manner that did not initially trigger detection by routine
phenotypic testing may have been an important early selec-
tive advantage mechanism in which the bla gene sampled
a variety of genetic backgrounds and range of sequence types
that eventually led to a more permanent incorporation into
the most advantageous genetic background. Moreover, ST 36
may have been a predecessor strain; a group of bacteria in
which bla
KPC
resided for a while before eventually finding ST
258. As a point of reference, ST 36 and ST 258 are not closely
related. They differ in 5 of the 7 housekeeping genes used to
determinesequencetype(http://www.pasteur.fr/recherche/
genopole/PF8/mlst/).
Carbapenem breakpoint guidelines in 2004 did not iden-
tify these isolates as carbapenem resistant, even when ex-
amining the ertapenem MICs [25]. For instance, strain Kp-
11 showed positive MHT, whereas MICs against ertapenem
and meropenem were 4 and #1 mg/L, respectively. The re-
vised 2011 CLSI criteria with lower breakpoints favor de-
tection of carbapenem resistance [26].
Although we expected that susceptibility to quinolones
would be preserved because children are not treated with
these antimicrobials, bla
KPC
often coexists with quinolone-
resistance determinants among K. pneumoniae isolated from
adults [21]. Among these isolates, PCR did not reveal the
presence of plasmid-mediated quinolone-resistance genes,
or aminoglycoside-modifying enzyme genes. The presence of
nonsusceptibility to tigecycline in 2 of the isolates was not
anticipated, because tigecycline was approved for release by
theUSFoodandDrugAdministrationinJuly2005afterthe
collection of these isolates.
Upstream sequencing of bla
KPC-3
revealed that this bla gene
was located in Tn4401b in all strains. This variant does not
contain the 100–base pair deletion described in Tn4401a. The
level of KPC expression was examined by detecting the
b-lactamase via immunoblotting. High-level KPC production
was not observed, supporting the lower carbapenem MICs
among these strains (data not shown) [22]. Our mating ex-
periments suggested that transmission of a plasmid-bearing
bla
KPC-3
among different K. pneumoniae may also have oc-
curred. Analysis of the genetic context of the plasmids in these
strains is underway. The Check-Points microarray also con-
firmed the transfer of the bla
KPC-3
gene into the J-53 E. coli
recipients; however, the bla
SHV-11
from isolates Kp-4, Kp-5,
and Kp-11 did not transfer.
All KPC-producing strains were found to be closely related
using molecular typing techniques that have been previously
validated for K. pneumoniae, this suggested clonal relatedness.
We have not yet determined whether this resistance gene or
plasmid was introduced in the facility from a single source and
disseminated or was introduced by multiple patients coming
from referring hospitals.
It was also intriguing that MLST revealed the introduction
of a new sequence type not known before to harbor bla
KPC
.
Other studies have found bla
KPC
in STs 11, 14, 258, 277, 337,
338, 339, 340, and 588 [35]. To our knowledge, this is the
first report of bla
KPC-3
in a K. pneumoniae isolate of ST 36.
This sequence type has been reported in Spain, Northern
Europe, and Korea [35–38], and it is linked to bla
CTX-M-15
,
bla
SHV-11
,qnrS1, bla
OXA-1
,andaac(6#)-Ib-cr genes. Our
strains harbored a bla
SHV-11
gene. In addition, 3 were posi-
tive for bla
TEM-1
(Table 2). Because these bla genes are often
found in mobile elements, the results are not unexpected.
Finally, the commercial microarray system used to analyze
the isolates demonstrated 100% sensitivity and specificity for
the detection of bla
KPC
,bla
SHV
,andbla
TEM
in this study. The
analysis of the bla genes using this method was far more
efficient than by conventional PCR. This assay gave us
a ‘‘snapshot’’ of the b-lactamase background of antibiotic
resistance in these gram-negative species. Evidence is accu-
mulating that this microarray can offer real-time in-
formation potentially leading to the choice of appropriate
antibiotic therapy.
In summary, this study reveals the silent dissemination of
bla
KPC-3
as part of Tn4401b in Northeast Ohio in 2004 and
establishes the first report to our knowledge of K. pneumoniae
producing KPC in pediatric LTCFs. An examination of the
literature reveals that only 1 bla
KPC
containing E. coli isolate
in Ohio was reported as part of the MYSTIC program’s
1999–2005 isolate survey and the SENTRY Antimicrobial
Surveillance program from 2000 to 2004 did not report any
bla
KPC
containing isolates from Ohio [39,40]. Our findings
are all the more striking because the organisms were discov-
ered in relatively young patients, a population not previously
identified as being at risk for harboring KPC-expressing or-
ganisms. The molecular background of these strains (all ST
36) suggests that a unique sequence type not previously re-
ported to carry bla
KPC
was involved in the clonal spread. This
report also highlights the importance of the revised 2011 CLSI
guidelines for detecting carbapenem resistance mediated by
bla
KPC
. We plan to perform a detailed molecular analysis of
other enteric isolates and the mobile plasmids recovered from
this survey to gain deeper insight into the transmission dy-
namics and genetic background. Most importantly, the find-
ings of bla
KPC
on a mobile genetic element in an LTCF
providing care to children and young adults with significant
neurodevelopmental abnormalities points to an important
area for enhanced infection control efforts.
KPC-Bearing Klebsiella in Pediatric LTCF dCID 2012:54 (1 May) d1319

Notes
Acknowledgment. The authors would like to especially acknowledge
the early contributions of Dr Michael Dul to this project.
Financial support. This work was supported by the Veterans Affairs
Merit Review Program, VISN 10 Geriatric Research Education and Clinical
Center, Elan Pharmaceuticals, the National Institutes of Health (grant RO1
AI063517-07), and Steris Corporation (institutional grant to F. P.).
Potential conflicts of interest. All authors: No reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
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