Short Communication
OpenAccess
Molecular Epidemiology of KPC-2 Producing
Klebsiella pneumoniae
Cristina Motta Ferreira1*, William Antunes Ferreira2, Lucyane Mendes Silva3, Thiago Costa
Barbosa4, Victor Costa De Souza3 and Felipe Gomes Naveca3
1PhD in Tropical and Infectious Disease. Fundação de Hematologia e Hemoterapia do Amazonas –HEMOAM, Brasil
2PhD in Tropical and Infectious Disease. Fundação de Dermatologia Tropical e Venereologia Alfredo da Matta- FUAM, Brasil
3Fellowship of FAPEAM. Fundação de Hematologia e Hemoterapia do Amazonas –HEMOAM, Brasil
4Fellowship of Master Science in Science Applied in Haematology. Fundação de Hematologia e Hemoterapia do Amazonas –HEMOAM, Brasi
5Master in Tropical Patology. Instituto Leônidas e maria Deane-FIOCRUZ-Amazonia, Brasil
6PhD in Microbiology. Instituto Leônidas e Maria Deane-FIOCRUZ-Amazonia, Brasil
2PhD in Tropical and Infectious Disease. Fundação de Dermatologia Tropical e Venereologia Alfredo da Matta- FUAM, Brasil
3Fellowship of FAPEAM. Fundação de Hematologia e Hemoterapia do Amazonas –HEMOAM, Brasil
4Fellowship of Master Science in Science Applied in Haematology. Fundação de Hematologia e Hemoterapia do Amazonas –HEMOAM, Brasi
5Master in Tropical Patology. Instituto Leônidas e maria Deane-FIOCRUZ-Amazonia, Brasil
6PhD in Microbiology. Instituto Leônidas e Maria Deane-FIOCRUZ-Amazonia, Brasil
*Corresponding author: Cristina Motta Ferreira, PhD in Tropical and Infectious Disease, Fundação de Hematologia e Hemoterapia do Amazonas-
HEMOAM. Av. Constantino Nery, 4327. Chapada. CEP: 69055-002. Manaus- Amazonas, Brasil. Tel: 55-92-36550139; E-mail:
@
Received: 01 June, 2017; Accepted: 03 July, 2017; Published: 14 July, 2017
Citation: Cristina Motta Ferreira et al. (2017) Molecular Epidemiology of KPC-2 Producing Klebsiella pneumoniae. SOJ Microbiol
Infect Dis 5(3):1-3. DOI: 10.15226/sojmid/5/3/00176
Abstract Top
We describe the molecular epidemiology of the KPC-2 K.
pneumoniae among public hospitals in the Amazon State, Brazil. A
total of 4.4% (5/113) of the K. pneumoniae isolates were identified as
KPC-producing and CTX-M-109 beta-lactamase. Further, MultiLocus
Sequence Typing identified three clones STs11, 40 and 2230. To the
best of our knowledge, the present study demonstrates for the very
first time, detection of the multidrug resistant K. pneumoniae (KPC-
2), ST2230 clone, in Brazil.
Keywords: ST2230; CTX-M-109; Carbapenem Resistance; Antimicrobial; Multi Resistant
Keywords: ST2230; CTX-M-109; Carbapenem Resistance; Antimicrobial; Multi Resistant
Text
Nosocomial infections caused by carbapenemase–
producing bacteria has increased worldwide and have become
a serious public health threat [1]. Infections resulting from
this pathogen may lead to a serious life-threatening illness
[2] and are characterized by a resistant phenotypic profile to
monobactams, carbapenems, broad spectrum cephalosporins,
fluoroquinolones and aminoglycosides [2-4]. These bacteria
pose a significant risk for hospitalized patients, particularly for
neonates, immune-compromised, diabetics or patients with
alcohol-associated disorders or bloodstream infections, as well
as those receiving advanced medical care [2,3]. The antibiotic
therapies available to treat these infections are restricted to
tigecycline, polymyxins and occasionally aminoglycosides
[3,5]. A number of KPC variants have been described among
which the KPC-2 and -3 are the most prevalent [3,5]. In this
study, we described the molecular epidemiology of five KPC-producing
K. pneumoniae among public hospitals in the Amazon
region. In the year 2015, a total of 133 clinical samples of K.
pneumoniae were obtained from inpatients from two public
hospitals in Manaus-Brazil. The biochemical identification and
susceptibility test of these isolates were performed by VITEK-2
automated system protocols (bioMérieux, France). The screening
detection for Extended-Spectrum Β-Lactamase (ESBL) and for
carbapenemase production were performed according to CLSI
(2015) recommendations [6]. Plasmid DNA was extracted with
the use of PureLink™ Quick Plasmid Miniprep Kit (INVITROGEN,
CARLSBAD, CA, USA). Further, molecular analysis was performed
by Polymerase Chain Reactions (PCRs). PCR amplifications
for blaCTX-M gene were performed according to Villegas, et al.
[7]. For carbapenemase production (blaKPC), we designed one
pair of primers (blaKPCFW-5´-ATGTCACTGTATCGCCGTC-3´)
and (blaKPCRV-5´-TTACTGCCCGTTGACGCC-3´). For blaNDM
production gene, we designed another pair of primers
(blaNDMFW-5´-GCCCAATATTATGCACCCGG-3´);(blaNDMRV-5´-
CGCAGCTTGTCGGCCAT-3´). The sequencing of the blaKPC and
blaNDM amplicons were performed at ABI 3130 sequencer,
(Applied Biosystems, Foster City, CA). These sequences were
analyzed with a software geneious V.10.0.10. Multilocus Sequence
Typing was performed according to Institut Pasteur´s MLST
scheme (http://bigsdb.web.pasteur.fr. ) and for blaCTX-M gene, the
blastn was made (http://blast.ncbi.nlm.nih.gov/Blast.cg ). The
total of the 113 isolates of the K. pneumoniae were obtained from
different biological sites such as urine 44.2% (50/113), blood
30% (34/113), tracheal aspirate 20.3% (23/113), wound 1.76%
(2/113) and fecal 0.88% (1/113). The susceptibility test of them
showed resistance of 100% to ceftriaxone and cefepime; 69.9%
(79/113) to gentamicin; 64.6% (73/113) to ciprofloxacin; 60.1%
(68/113) to ceftazidime; 54.8% (62/113) to cefoxitin, and 4.4%
(5/113) to tigecycline. Five of the 113 K. pneumoniae isolates 4.4%
(5/113), showed MICs to ertapenem of ≥ 8μg/mL, meropenem ≥
8μg/mL and 16μg/mL, and to imipenem 4 μg/mL, 8 μg/mL and
16μg/mL. These five isolates presented also susceptibility to
amikacin, two of them to tigecycline, and three to colistin. They
were identified as ESBL and KPC-producing bacteria, and were
isolated from blood (2/5), urine (1/5) and tracheal aspirate
(2/5), from five patients admitted at the intensive care unit, three
of them were male (22, 42, 65 years-old), one female (30 yearsold),
and one newborn. The obtained sequence of the blaCTX-M gene
showed similarity with CTX-M-109, and the sequence of the blaKPC
plasmidial gene showed identity with the sequence of blaKPC-2.
We didn´t find the presence of blaNDM plasmidial gene. The MLST
analysis showed that these five KPC-producing isolates belonged
to the ST11(1/5), ST340 (3/5) and ST2230 (1/5). The ST2230
was identified as a new clone, in the Amazon Brazilian region
(GenBank accession: KY689923; KY689924; KY689925).Two
of the KPC-producing isolates not deposited at the GenBank, had
the identical sequence of blaKPC-2 gene and were the same clone
(ST340). Multidrug resistant bacteria are a substantial threat
to public health because their resistance to all the antibiotic
options available, and the most serious infections occurs in
health care settings caused by K. pneumoniae [8]. Concerned with
this problem, the World Health Organization (WHO) developed
a global priority pathogens list, in which K. pneumoniae is at
the priority one (critical) (http://www.who.int/medicines/
publications/global-priority-list-antibiotic-resistant-bacteria/
en/ ). The endemic dissemination of the carbapenemase variant
KPC-2 and KPC-3, has been reported in the USA (5.7%), Italy
(89.5%), Canada (89.3%), and China (63%) [9-12]. The ST258
clone, predominant in European countries, has contributed
significantly to the worldwide distribution of the carbapenem
resistance [4]. In Asia, there are different clones such as ST392
and ST395, which also harbor the blaKPC-2 gene, whereas in Brazil,
different STs such as ST437, ST340 and ST842, harboring the
KPC-2 variant, have been described and isolated from different
healthcare settings [12,13].The spread of the antibiotic resistance
genes maybe be associated with the dissemination of clones, and
other factors such as plasmids, and transposons – i.e. Horizontal
Gene Transfer (HGT) - between bacteria [4,14,15]. Furthermore,
the capacity of the K. pneumoniae to colonize medical equipment
and to survive for long periods of time, at extreme temperature
in the environment, explains why this pathogen can also be
isolated from hospital environment and play an important role in
the horizontal transfer of antibiotic resistance, acting also as an
efficient donor and receptor [4,13,16-19]. The events observed
in the present study, suggest that asymptomatic carriers, such as
health professionals and their flow between different hospitals;
patient or other people who move through hospitals, may be
contributing to the local dissemination of these clones, which
may affect vulnerable patients [13]. The ST11 and ST340 clones
were isolated from inpatients at the same general hospital. These
clones were observed in different states of Brazil such as Ceará,
Pernambuco, Rio de Janeiro, Piauí, Alagoas and Federal District
[13]. To date, only one clone (ST841) was described in Manaus,
Amazonas [13]. The ST2230 clone, that has been described
before in China, was isolated from a blood sample of a newborn
from a maternity hospital, being the first description in the city
and in Brazil, and possibly representing an important fact, once
it is associated with multidrug resistance. Pereira, et al. detected
resistant clones to carbapenems, amikacin, tigecycline and colistin
[13]. In the present study, the clones were sensitive to colistin and
to amikacin. Since the KPC-producing bacteria present a resistance
profile to different classes of antibiotics, such as monobactams,
carbapenems and broad spectrum cephalosporins, the last
therapeutic options that remain is polymyxin B, colistin and
fosfomycin [3,13,19]. A serious prophylactic action in the health
centers with focus on control measure of the environment, contact
precautions, hand hygiene, early identification of asymptomatic
carriers, antimicrobial stewardship and the implementation of
the infection control guideline, are obligatory to prevent, control
or at least to reduce the risks of dissemination of those virulent
clones [20]. Preventive measures are necessary to avoid the
dissemination of these highly pathogenic clones among hospitals
and KPC producing K. pneumoniae positive patients, should be
isolated and treated according standard guidelines.
Acknowledgments
We thank the team of the curators of the Institut Pasteur
MLST system (Paris, France) for importing novel alleles, profiles
and/or isolates at http://bigsdb.web.pasteur.fr
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