Diagnostic Microbiology and Infectious Disease 82 (2015) 326–330

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Diagnostic Microbiology and Infectious Disease

journal homepage: www.elsevier.com/locate/diagmicrobio

Molecular epidemiology of KPC-2–producing Enterobacteriaceae

(non–Klebsiella pneumoniae) isolated from Brazil☆
Carolina Padilha Tavares a, Polyana Silva Pereira a, Elizabeth de Andrade Marques b, Celio Faria Jr. c,
Maria da Penha Araújo Herkenhoff de Souza d, Robmary de Almeida e, Carlene de Fátima Morais Alves f,
Marise Dutra Asensi a, Ana Paula D’Alincourt Carvalho-Assef a,⁎
a
Laboratório de Pesquisa em Infecção Hospitalar, Oswaldo Cruz Institute, RJ, Brazil
b
Departamento de Microbiologia e Imunologia, Faculdade de Ciências Médicas, Universidade do Estado do Rio de Janeiro, RJ, Brazil
c
Laboratório Central de Saúde Pública do Distrito Federal, DF, Brazil
d
Laboratório Central de Saúde Pública do Espírito Santo, ES, Brazil
e
Laboratório Central de Saúde Pública de Goiás, GO, Brazil
f
Laboratório Central de Saúde Pública de Minas Gerais, MG, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: In Brazil, since 2009, there has been an ever increasing widespread of the blaKPC-2 gene, mainly in Klebsiella
Received 20 October 2014 pneumoniae. This study aims to assess the molecular epidemiology and genetic background of this gene in
Received in revised form 31 March 2015 Enterobacteriaceae (non–K. pneumoniae) species from 9 Brazilian states between 2009 and 2011. Three hundred
Accepted 13 April 2015
eighty-seven isolates were analyzed exhibiting nonsusceptibility to carbapenems, in which the blaKPC-2 gene was
Available online 16 April 2015
detected in 21.4%. By disk diffusion and E-test, these isolates exhibited high rates of resistance to most of the
Keywords:
antimicrobials tested, including tigecycline (45.6% nonsusceptible) and polymyxin B (16.5%), the most resistant
KPC species being Enterobacter aerogenes and Enterobacter cloacae. We found great clonal diversity and a variety of
Enterobacteriaceae blaKPC-2–carrying plasmids, all of them exhibiting a partial Tn4401 structure. Therefore, this study demonstrates
Carbapenem resistance the dissemination of KPC-2 in 9 Enterobacteriaceae species, including species that were not previously described
Tn4401 such as Pantoea agglomerans and Providencia stuartii.
© 2015 Elsevier Inc. All rights reserved.

1. Introduction repeat sequences. There are 3 more frequently described isoforms of

The emergence of Gram-negative pathogens resistant to many anti-
microbial classes has become a serious threat in the last decades
(Nordmann et al., 2011). KPC-type carbapenemases have been a major
determinant of carbapenem resistance in Klebsiella pneumoniae
(Nordmann et al., 2011) as well as in many other Enterobacteriaceae
(Tzouvelekis et al., 2012). This enzyme was initially described during a
surveillance study in the United States in 2001, quickly spreading
throughout the world (Yigit et al., 2001).
KPC affords resistance to all beta-lactam antibiotics available, and its
gene has been described in mobile elements, such as plasmids and a
Tn3-based transposon (Tn4401) associated with insertion sequence
(IS) structures (Naas et al., 2008). This transposon consists of a
transposase (tnpA), a resolvase (tnpR), the blaKPC gene, and 2 insertion
sequences (ISKpn6 and ISKpn7). It is flanked by two 39-bp inverted
☆ This study was performed in Laboratório de Pesquisa em Infecção Hospitalar, Oswaldo
Cruz Institute, Brazil.
⁎ Corresponding author. Tel.: +55-212-5621-636.
E-mail address: anapdca@ioc.fiocruz.br (A.P.D.’A. Carvalho-Assef).
this transposon (isoforms “a”, “b”, and “c”) (Cuzon et al., 2011). This
structure, associated to the blaKPC gene, has also been described inserted
within plasmids of different sizes and incompatibility groups (Andrade
et al., 2011; Cuzon et al., 2010; Pereira et al., 2013).
KPC-producing K. pneumoniae was first described in Brazil in 2006
(Monteiro et al., 2009), and its incidence has increased significantly. In
2010, a great dispersion of this gene was observed in several hospitals
in different Brazilian cities and states (Pereira et al., 2013). Pereira
et al. (2013) observed that the spread of the blaKPC-2 gene in
K. pneumoniae is both due to the dispersion of Tn4401 in different clones
and the dissemination of a clonal complex (CC11), in which the se-
quence types (STs) 437 and 11 have played an important role.
Regarding other Enterobacteriaceae species, there are few reports in
Brazil, such as Escherichia coli (D'Alincourt Carvalho-Assef et al., 2010),
Enterobacter cloacae (Zavaski et al., 2009), Salmonella spp. (Miriagou
et al., 2003), Serratia marcescens (Del Peloso et al., 2010), and Klebsiella
oxytoca (Almeida et al., 2013). However, the dissemination mechanisms
of the blaKPC gene in these species are still unclear. Therefore, the aim of
this study is to assess the molecular epidemiology and genetic back-
ground of the blaKPC gene in isolates belonging to 9 different species of
Enterobacteriaceae recovered from 9 Brazilian states between 2009
and 2011.

http://dx.doi.org/10.1016/j.diagmicrobio.2015.04.002
0732-8893/© 2015 Elsevier Inc. All rights reserved.
 C.P. Tavares et al. / Diagnostic Microbiology and Infectious Disease 82 (2015) 326–330
2. Materials and methods
2.1. Clinical strains
From January 2009 to December 2011, the Laboratório de Pesquisa
em Infecção Hospitalar in Instituto Oswaldo Cruz (Rio de Janeiro),
which contributes to the Nosocomial Infections Monitoring Network as-
sociated to the Health Ministry of Brazil, received a total of 387 clinical
non–K. pneumoniae Enterobacteriaceae (NKPE) isolates exhibiting
nonsusceptibility to at least 1 of the carbapenems (imipenem,
meropenem, or ertapenem), recovered from 9 Brazilian states: Espírito
Santo (ES), Minas Gerais (MG), Rio de Janeiro (RJ), the Federal District
(DF), Goiás (GO), Pernambuco (PE), Alagoas (AL), Ceará (CE), and
Bahia (BA). The isolates were identified by conventional techniques.
The blaKPC gene and its allelic variant were detected and identified by
PCR and sequencing, respectively (Peirano et al., 2009).
2.2. Antimicrobial susceptibility testing
Antimicrobial susceptibility testing of all KPC-producing isolates was
performed and interpreted based on CLSI recommendations (CLSI,
2014) by the disk diffusion method with the following antimicrobials
(Oxoid, Hampshire, UK): ceftazidime (30 μg), cefotaxime (30 μg), cefe-
pime (30 μg), aztreonam (30 μg), sulfamethoxazole/trimethoprim
(25 μg), ciprofloxacin (5 μg), gentamicin (10 μg), and amikacin
(30 μg). The MIC values were determined by Etest® (AB Biodisk,
Solna, Sweden) for imipenem, meropenem, ertapenem, tigecycline,
and polymyxin B according to the manufacturer's instructions. Broth
microdilution was performed to confirm resistance to polymyxin B
(CLSI, 2014). The tests were interpreted based on CLSI 2014 breakpoints
(CLSI, 2014), except for tigecycline and polymyxin B for which EUCAST
2014 breakpoints were adopted (EUCAST, 2014).
2.3. Analysis of genetic diversity
For molecular typing, pulsed-field gel electrophoresis (PFGE) and
multilocus sequence typing (MLST) (in the case of E. coli) were
employed. On PFGE, XbaI or SmaI (for Providencia stuartii) digestion
was used to analyze multiple isolates harboring blaKPC belonging to
the same species (Ribot et al., 2006). Banding patterns were visually
inspected and subsequently analyzed with BioNumerics 6.6 software
(Applied Maths, Sint-Martens-Latem, Belgium) with Dice's coefficient
analysis and bandwidth tolerance set at 1.5%. Isolates with at least 80%
of similarity were considered as the same clonal group. MLST of E. coli
isolates was performed according to MLST database from Pasteur Insti-
tute (http://www.pasteur.fr/recherche/genopole/PF8/mlst/EColi.html).
2.4. Flanking regions of blaKPC
The genetic environment of the blaKPC gene was analyzed by multi-
plex PCR according to the primers described by Naas et al. (2008) except
for the amplification of the ISKpn7 region, in which the primers
described by Kitchel et. al. (2009) were adopted. The Tn4401 isoform
was determined by ISKpn7 amplicon sequencing. The positive and
negative controls for KPC-2 were K. pneumoniae ATCC BAA 1705 and
ATCC BAA 1706, respectively.
2.5. Extended spectrum beta-lactamase (ESBL) gene detection
PCR and sequencing were performed to amplify and identify other
beta-lactamase–coding genes (CTX-M, SHV, and TEM) (Cuzon et al.,
2010; Mendonça et al., 2007).
327
2.6. Genetic environment of the blaKPC gene
Plasmid analyses were performed by restriction digestion with S1
nuclease (Barton et al., 1995). The plasmids were transferred to a
nylon membrane and hybridized with digoxigenin-labeled blaKPC-2
probe generated by the PCR DIG detection system (Roche Diagnostics,
Mannheim, Germany) (Sambrok and Russel, 2001).
3. Results
Of all NKPE nonsusceptible to carbapenems isolates studied, blaKPC was
detected in 21.4% (83/387), representing 9 different Enterobacteriaceae
species. All of the isolates had the KPC-2 variant.
The distribution of blaKPC-2 among species was Enterobacter
aerogenes (38 out of 121 isolates, 31.4%), E. cloacae (17 out of 100 iso-
lates, 17%), E. coli (13 out of 104 isolates, 12.5%), Pantoea agglomerans
(4 out of 10 isolates, 40%), P. stuartii (4 out of 11 isolates, 36.4%),
Citrobacter freundii (3 out of 8 isolates, 37.5%), K. oxytoca (2 out of 9 iso-
lates, 22.2%), S. marcescens (1 out of 19 isolates, 5.3%), and Morganella
morganii (1 out of 5 isolates, 20%). They were recovered from surveil-
lance swabs (n = 26, 31.3%), urine (n = 13, 15.7%), respiratory tract
samples (n = 12, 14.5%), skin and soft tissue (n = 10, 12%), blood
(n = 5, 6%), and other sites (n = 10, 12%). In 8.4% (n = 7) of the isolates,
the anatomical site of isolation was not reported. Most of the KPC-2–
producing isolates were recovered from the DF (39.8% n = 33) and RJ
state (15.7% n = 13) (Table 1).
Through PFGE analysis, there was a great clonal diversity among
E. coli and E. cloacae isolates, 12 and 16 clonal groups, respectively. In
E. aerogenes, 9 clonal groups were evident, with a predominant group
(A, 65.8%) detected in 8 different hospitals from the DF and 3 different
hospitals from GO, over an 11-month period. All P. stuartii isolates
belonged to the same clonal group, detected in the same hospital in
MG. The C. freundii isolates were pertinent to 3 different clonal groups.
Regarding P. agglomerans and K. oxytoca, the isolates were designated
to 2 clonal groups (Table 1).
Through E. coli MLST analysis, the isolates belonged to 9 STs, 4 new
STs that presented 2 new alleles of dinB and icdA genes (ST629 and
ST630) and 2 new profiles (ST631 and ST632). Three different clonal
groups by PFGE (EcE, EcK, and EcL) belonged to ST2, and 2 others
(EcG and EcH) were identified as ST43.
According to CLSI (2014) breakpoints, there were high rates of resis-
tance to most of the antimicrobials tested. Gentamicin and amikacin
were the antimicrobials with the lowest resistance rates (39.8% and
34.9%, respectively). Through E-test, most of the isolates were resistant
to carbapenems, as was expected, with MIC50 ≥32 mg/L for all
carbapenems tested. Individually, several species displayed lower
rates of MIC50, E. coli and C. freundii exhibiting the lowest MIC50
(Table 2).
Approximately 47% of the isolates were nonsusceptible to tigecyc-
line (MIC50 = 1 mg/L and MIC range 0.125–16 mg/L) (Table 2). These
isolates were species of E. aerogenes (60.5%; 23/38), E. cloacae (52.9%;
9/17), P. stuartii (75%; 3/4), P. agglomerans (25% 1/4), C. freundii
(33.3%; 1/3), K. oxytoca (50%; 1/2), and S. marcescens (100%; 1/1).
Regarding polymyxin B, 16.5% of resistance was apparent, excluding
species intrinsically resistant (MIC50 = 0.75 mg/L and MIC range
0.19–32 mg/L) (Table 2). To confirm the polymyxin B resistance, broth
microdilution (the gold standard method) was used to test these iso-
lates, and all of the isolates were still considered resistant with the
same MIC values. These isolates were species of E. aerogenes (26.3%;
10/38), C. freundii (33.3%; 1/3), and K. oxytoca (50% 1/2). Of the 10
E. aerogenes isolates resistant to polymyxin B, 9 belonged to clonal
group A, representing 90% of the polymyxin-resistant E. aerogenes
strains (Table 2).
In our study, the association of KPC-2 with other beta-lactamases
was evident in 44 isolates (53%). The blaTEM gene was detected in
45.8% of the isolates. Through sequencing, 2 different allelic variants
328 C.P. Tavares et al. / Diagnostic Microbiology and Infectious Disease 82 (2015) 326–330

Table 1
Clonal groups and distribution of KPC-positive Enterobacteriaceae isolates from 9 different geographic regions of Brazil.

Species Pulsotypes and MLSTa of Enterobacteriaceae (no. of isolates per Brazilian state) Total (%)

Northeast Southeast Midwest

CE PE BA AL RJ ES MG DF GO

E. aerogenes EaH (1) EaF (6) EaA (23)b EaA (2)b 38 (45.8)
EaG (2) EaB (1)
EaC (1)
EaD (1)
EaI (1)
E. cloacae EclC (2) EclG (1) EclH (1) EclM (1) EclB (1) EclA (1) 17 (20.5)
EclJ (1) EclM (1) EclK (1) EclF (1) EclD (1)
EclL (1) EclI (1) EclE (1)
EclN (1)
EclO (1)
E. coli EcI/ST39 (1) EcJ/ST632 (1) EcA/ST629 (1) EcE/ST2 (1) EcG/ST43 (1) EcF/ST305 (2) 13 (15.7)
EcK/ST2 (1) EcB/ST630 (1) EcH/ST43 (1)
EcC/ST631 (1)
EcD/ST479 (1)
EcL/ST2 (1)
P. stuartii PsA (4) 4 (4.8)
P. agglomerans PaA (3) 4 (4.8)
PaB (1)
C. freundii CfA (1) CfB (1) CfC (1) 3 (3.6)
K. oxytoca KoA (1) KoB (1) 2 (2.4)
M. morganii 1 1 (1.2)
S. marcescens 1 1 (1.2)
Total (%) 4 (4.8) 10 (12) 2 (2.4) 1 (1.2) 13 (15.7) 5 (6) 9 (10.8) 33 (39.8) 6 (7.3) 83 (100)
a
MLST was performed only for E. coli isolates.
b
The isolates were retrieved from 8 hospitals from DF and 3 hospitals from GO between March 2010 and February 2011.

were defined, blaTEM-141 (36.8%) and blaTEM-141-like (63.2%), a novel alle-
lic variant differing from the blaTEM-141 in 3 nucleotides (GeneBank ac-
cession no. KR024889). The frequency of blaSHV detection was 6%, and
through sequencing, 4 different allelic variants were found: blaSHV-2a
(16.7%), blaSHV-63(16.7%), blaSHV-120 (16.7%), and blaSHV-165 (33.3%).
The blaCTX-M genes were detected in 22.9% of the isolates, and the allelic
variants were blaCTX-M-8 (10.5%), blaCTX-M-9 (15.8%), blaCTX-M-15 (36.8%),
blaCTX-M-59 (31.6%), and blaCTX-M-97 (5.3%) (Table 2).
By multiplex PCR, a partial Tn4401 structure was present in all iso-
lates. The inverted repeats and their transposition-generated target
site duplications were absent in all isolates. The tnpA region was detect-
ed in 48.2% of the isolates; ISKpn6, in 97.8%; and ISKpn7, in 57.8%. The 3
regions together (tnpA, ISKpn6 and ISKpn7) were detected in 31.3% of
the isolates (n = 26). In 91.7% (44/48), there were no deletions in the
ISKpn7 region, suggesting isoform “b”, and in 8.3% (4/44), a deletion of
69 bp was found, suggesting isoform “d” (Table 2).
Restriction with S1 nuclease and hybridization analysis revealed that
blaKPC was found in plasmids in all isolates. The KPC-carrying plasmids
varied from ~14 to 120 kb, and the most prevalent plasmids present
~14 kb (n = 35) and ~42 kb (n = 24) (Table 2). In 15.7% (13/83) of
the isolates, more than 1 plasmid carrying the blaKPC gene was detected.
4. Discussion
This study describes the resistance profiles, genetic diversity, and gene-
tic environment of the blaKPC-2 gene among 9 different Enterobacteriaceae
species isolated from Brazil, during a 2-year study. Since 2009, there has
been an ever widespread of KPC-producing K. pneumoniae in several
Brazilian states (Pereira et al., 2013; Seki et al., 2011). Beyond the dis-
semination of K. pneumoniae, KPC in other Enterobacteriaceae species
has also been recorded (Petrella et al., 2008). In our study, there was a
KPC production rate of 21.4% in carbapenem-nonsusceptible isolates.
All of the isolates produced KPC-2, the most reported variant in Brazil
and throughout the world (Walsh, 2010).
All of the isolates were nonsusceptible to at least 1 of the carbapenems,
and 2 isolates were resistant to all of the antimicrobials tested except to
tigecycline (1 of the isolates was nonsusceptible) and polymyxin B.
A high rate of resistance to tigecycline (45.6%) among E. aerogenes,
E. cloacae, P. agglomerans, C. freundii, K. oxytoca, and S. marcescens isolates
was observed. E. aerogenes, K. oxytoca, and C. freundii isolates resistant to
polymyxin B (16.5%) were also found.
Resistance to tigecycline and mainly to polymyxin is very worrisome
because these drugs are one of the few options left to treat infections
caused by multiresistant KPC-producing bacteria. Polymyxin resistance
has been witnessed in KPC-producing K. pneumoniae isolates (Lee et al.,
2009), including those from Brazil (Pereira et al., 2013). To our knowledge,
this is the first report of E. aerogenes strains resistant to polymyxin B in
Brazil. Most of polymyxin-resistant E. aerogenes isolates belonged to
clone A. This is a worrisome fact (it may indicate the adaptation of this
clone and possible spread) and mainly warrants a more judicious applica-
tion of this antimicrobial agent.
The transposon Tn4401 is described to be the origin of blaKPC gene
acquisition and dissemination. It consists of a transposase (tnpA), a
resolvase (tnpR), the blaKPC gene, and 2 putative IS elements, ISKpn6
and ISKpn7 (Naas et al., 2008). In Brazil, the blaKPC gene has been asso-
ciated with Tn4401 variant “b” in both K. pneumoniae as well as other
Enterobacteriaceae species (Andrade et al., 2011; Pereira et al., 2013).
Although isoform “b” was also the most prevalent in this study, the
complete transposon (tnpA, ISKpn6, and ISKpn7) was only amplified in
31.3% of the isolates (n = 26). Thus, these findings suggest the diversity
of the genetic environment surrounding blaKPC in the Enterobacteriaceae
isolates from Brazil.
Besides the dissemination of blaKPC-2 through Tn4401, the participa-
tion of epidemic clones is being described in the dispersion of this gene.
There is an epidemic clone of K. pneumoniae delineated worldwide,
identified as ST258 by MLST. In Brazil, the dissemination of 2 major
clones in K. pneumoniae (ST437 and ST11) related to ST258 was also
identified (Pereira et al., 2013).
In this study, through PFGE analysis, we observed clonal spread of
some species in some Brazilian states. Despite this, for now, no preva-
lent clone was considered responsible for the spread of KPC in NKPE
species in Brazil.
Regarding E. coli isolate MLST analysis, 2 isolates that belonged to ST43
were found, which is equivalent to ST131 on the UoW (MLST scheme of
University of Warwick, Warwick Medical School, Coventry, UK)
(Matsumura et al., 2012). This ST has been described as highly virulent
Table 2
Susceptibility profile and molecular characteristics of all KPC-2–producing isolates studied.

Species Antimicrobials Allelic variants of ESBL genes Size of KPC- Tn4401

plasmid (n) (% isolates positive)
No. of isolates resistant or intermediary MIC50 (range)
(disk diffusion)

C.P. Tavares et al. / Diagnostic Microbiology and Infectious Disease 82 (2015) 326–330
CAZ CTX FEP ATM SXT CIP AMI GEN IPM MER ERT TGC PB blaSHV (n) blaTEM (n) blaCTX-M (n) Neg Most tnpA ISKpn6 ISKpn7
frequent isoform
(range)

E.ae 38 38 38 38 24 37 27 11 ≥32 ≥32 ≥32 1.5 0.75 SHV-120 (1) TEM-141 (3) CTX-M-59 (6) 18 24 kb (29) 28.9 97.4 “b” – 57.9
(n = 38) (0.38 to ≥32) (0.38 to ≥32) (0.38 to ≥32) (0.125–16) (0.38–32) TEM-141-like (10) CTX-M-8 (1) (14–88 kb) “d” – 2.6
CTX-M-97 (1) Neg – 39.5
CTX-M-15 (1)
E.cl 16 17 17 17 12 11 8 11 ≥32 12 ≥32 1 0.75 SHV-2a (1) TEM-141 (7) CTX-M-8 (1) 2 42 kb (7) 56.3 100 “b” – 64.7
(n = 17) (0.5 to ≥32) (0.047 to ≥32) (0.047 to ≥32) (0.5–4) (0.19–2) TEM-141-like (8) CTX-M-9 (2) (14–120 kb) Neg – 35.3
CTX-M-15 (6)
CTX-M-9 (1)
E.co 8 12 11 12 6 10 3 5 12 4 4 0.19 0.75 TEM-141 (4) 8 42 kb (8) 78.6 100 “b” – 61.5
(n = 13) (3 to ≥32) (0.38 to ≥32) (0.75 to ≥32) (0.19–1) (0.5–2) TEM-141-like (1) (14–60 kb) Neg – 38.5
P.st 0 4 4 4 3 4 4 3 ≥32 4 6 1.5 NE 4 14 kb (4) 100 100 Neg – 100
(n = 4) (4 to ≥32) (0.75 to ≥32) (1.5 to ≥32) (1–2)
P.ag 4 4 4 4 1 4 2 3 ≥32 ≥32 ≥32 1 0.32 SHV-63 (1) TEM-141-like (4) 42 kb (3) 75 100 “b” – 25
(n = 4) (24 to ≥32) (≥32) (≥32) (0.75–3) (0.25–1.5) SHV-165 (2) (24–42 kb) “d” – 75
C.fr 3 3 3 3 1 2 0 0 0.75 0.25 6 1 1 3 42 kb (2) 66.7 66.7 “b” – 33.3
(n = 3) (0.75) (0.25) (4 to ≥32) (0.5–6) (0.5–12) (18–42 kb) Neg – 66.7
K.ox 2 2 2 2 2 2 1 0 0.25 to ≥32 0.19 to ≥32 6 to ≥32 0.5–2 0.38–12 TEM-141-like (1) 1 24–42 kb 0 100 “b” – 50
(n = 2) Neg – 50
c
M.mo 0 1 1 1 1 1 1 1 ≥32 2 1.5 0.75 NE 1 14–24 kb 100 100 Neg – 100
(n = 1)
S.ma 1 1 1 1 1 0 1 1 ≥32 ≥32 ≥32 2 NEc 1 24–88 kb 100 100 Neg – 100
(n = 1)
All (%) 86.7 100 83.1 97.6 61.4 85.5 57.8 42.2 ≥32 ≥32 ≥32 1 0.75 5 38 19 37 14 kb (35) 48.2 97.8 “b” – 53
(0.25 to ≥32) (0.047 to ≥32) (0.047 to ≥32) (0.125–16) (0.19–32) (6.0%) (45.8%) (22.9%) (44.6) 42 kb (24) “d” – 4.8
(14–120 kb) Neg– 42.2

E.ae = E. aerogenes; E.cl = E. cloacae; E.co = E. coli; P.st = P. stuartii; P.ag = P. agglomerans; C.fr = C. freundii; K.ox = K. oxytoca; M.mo = M. morganii; S.ma = S. marcescens; CAZ = ceftazidime; CTX = cefotaxime; FEP = cefepime;
ATM = aztreonam; SXT = trimethoprim/sulfamethoxazole; CIP = ciprofloxacin; AMI = amikacin; GEN = gentamycin; IPM = imipenem; MER = meropenem; ERT = ertapenem; TGC = tigecycline; PB = polymyxin B; NE = not evaluated.

329
330 C.P. Tavares et al. / Diagnostic Microbiology and Infectious Disease 82 (2015) 326–330
and is associated with the global dissemination of the CTX-M-15 enzyme
(Coque et al., 2008). However, none of these ST43 isolates produced CTX-
M–type enzymes, but they both produced KPC-2 and TEM. The associa-
tion of ESBLs with KPC has usually been described in K. pneumoniae
(Bradford et al., 2004; Kassis-Chikhani et al., 2010). Nevertheless, it has
also been reported among other Enterobacteriaceae (Petrella et al., 2008).
These results disclose the variety of blaKPC-2–carrying plasmids
among Enterobacteriaceae isolates. The same has been shown for
K. pneumoniae in Brazil (Pereira et al., 2013). However, plasmids with
about 14 kb and about 42 kb were the most frequently found among
the isolates. Other studies are peremptory to confirm whether the plas-
mids presenting the same size are really the same.
5. Conclusions
This study clearly demonstrates the dissemination of KPC-2 to NKPE
species in many Brazilian states, including species that were not previously
described as harboring blaKPC, such as P. agglomerans and P. stuartii.
Therefore, the importance of prompt recognition of these isolates
and the establishment of proper therapeutic and infection control
measures to reduce their spread among patients and within hospitals
are clearly emphasized.
A part of this study was presented at the 51st Annual Interscience
Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL,
USA, 2011, and at the XXVI Latin American Microbiology Conference,
Santos, SP, Brazil, 2012.
Author disclosure statement
All authors report no conflicts of interest relevant to this article.
Funding
This work was funded by research grants from Conselho Nacional de
Desenvolvimento Científico e Tecnológico, Fundação Carlos Chagas de
Amparo à Pesquisa, and Instituto Oswaldo Cruz.
Ethical approval
The study was approved by Fiocruz Ethical Committee and Brazilian
National Committee for Research Ethics under the reference number
CAAE-000012/011-02.
Acknowledgments
We thank the following microbiologists who provided some of the
strains: Ivoneide Barroso, Maria Iracema Patricio, Valdelúcia Cavalcanti,
Letiano da Silva, and Maria Pia from the Public Health Laboratories of
Alagoas, Ceará, Pernambuco, Piauí, and Rio de Janeiro, respectively.
We also thank the Genomic Platform for DNA Sequencing PDTIS
(Instituto Oswaldo Cruz) and the Institute Pasteur MLST database cura-
tors for coding the MLST alleles and profiles for E. coli.
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