Journal of Medical Microbiology (2016), 65, 547–553 DOI 10.1099/jmm.0.

000254

Risk factors for KPC-producing Klebsiella

pneumoniae: watch out for surgery
Kesia Esther da Silva,1 Wirlaine Glauce Maciel,1
avia Patussi Correia Sacchi,1,2 Cecilia Godoy Carvalhaes,3
Fl
Fernanda Rodrigues-Costa,3 Ana Carolina Ramos da Silva,3
Mariana Garcia Croda,2 Fabio Juliano Negr~ ao,1 Julio Croda,1,3,4
Ana Cristina Gales and Simone Simionatto1
2

1
Correspondence Laboratório de Pesquisa em Ci^
encias da Saúde, Universidade Federal da Grande Dourados -
Simone Simionatto UFGD, Dourados, Mato Grasso do Sul, Brazil
simonesimionatto@ufgd.edu.br 2
Hospital Universit
ario de Dourados, Universidade Federal da Grande Dourados - UFGD,
Dourados, Mato Grasso do Sul, Brazil
3
Laboratório ALERTA, Disciplina de Infectologia, Departamento de Medicina, Universidade Federal
de S~ao Paulo - UNIFESP, S~
ao Paulo, Brazil
4
Fundaç~ao Osvaldo Cruz, Campo Grande, Mato Grasso do Sul, Brazil

Received 11 December 2015
Accepted 21 March 2016
This study describes the molecular characteristics and risk factors associated with carbapenem-
resistant Klebsiella pneumoniae strains. Risk factors associated with KPC-producing K.
pneumoniae strains were investigated in this case-control study from May 2011 to May 2013.
Bacterial identification was performed by matrix-assisted laser desorption/ionization-time-of-flight
mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility was determined by broth
microdilution. Carbapenemase production was assessed by both modified Hodge test (MHT)
and ertapenem hydrolysis using MALDI-TOF MS. The presence of b-lactamase-encoding genes
was evaluated by PCR and DNA sequencing. Alterations in genes encoding K. pneumoniae
outer membrane proteins were analysed by PCR and DNA sequencing as well as SDS-PAGE.
Genetic relatedness among strains was determined by pulsed-field gel electrophoresis. This
study included 94 patients. Longer hospitalisation, mechanical ventilation, catheters, and previous
surgery were associated with KPC-producing K. pneumoniae. Sixty-eight strains showed
resistance to carbapenems. Carbapenemase production was detected by MHT in 67 K.
pneumoniae strains and by MALDI-TOF MS in 57. The presence of the blaKPC-2 gene was
identified in 57 strains. The blaKPC-2 gene was not found in 11 carbapenem-resistant K.
pneumoniae; instead, the blaCTX–M-1-like, blaCTX–M-2-like, blaCTX-M-8 like, blaCTX-M-14-like and
blaSHV-like genes associated with OmpK35 and OmpK36 alterations were observed. Thirty-three
KPC-producing K. pneumoniae strains were clonally related, and patients infected with these
strains had a higher mortality rate (78.78 %). Our results show that KPC-producing K.
pneumoniae was associated with several healthcare-related risk factors, including recent surgery.

INTRODUCTION matter of great concern (Vatopoulos, 2008). They cause

The emergence of carbapenem-resistant Klebsiella pneumo-
niae has been reported in many countries and has become a
Abbreviations: ICU, intensive care unit; MALDI-TOF MS, matrix-assisted
laser desorption/ionization-time-of-flight mass spectrometry; KPC, Kleb-
siella pneumonia carbapenemase; MHT, modified Hodge test; CLSI,
Clinical and Laboratory Standards Institute; PFGE, pulsed field gel elec-
trophoresis; OMP, outer membrane protein; ESBL, extended-spectrum
beta-lactamase.
numerous diseases, are hard to treat and have the potential
to spread within healthcare facilities. Infections with these
organisms are associated with high rates of morbidity and
mortality (Souli et al., 2012).
The emergence and dissemination of K. pneumoniae that
harbour carbapenemase-encoding genes poses a significant
threat to the control and treatment management of noso-
comial infections and have been associated with hospital
outbreaks in various geographical regions (Gupta et al.,

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K. E. Silva and others
2011; Hoenigl et al., 2012). Carbapenemases, including
enzymes of Ambler classes A Klebsiella pneumonia carbape-
nemase (KPC), Guiana extended espectrum (GES) and
Serratia marescens enzyme types (SME), B (metallo-b-lacta-
mases) Imipenemase (IMP), Verona imipenemase (VIM)
and New Delhi metallo-b-lactamase types (NDM), and D
Oxacilinase-48 (OXA-48) can hydrolyse almost all
b-lactams. In addition, the rates of carbapenem resistance
among K. pneumoniae have dramatically increased
(Queenan & Bush, 2007). The most frequently encountered
carbapenemase is KPC (Leung et al., 2012). KPC occurs
most commonly in K. pneumoniae, but its production has
also been reported in other Enterobacteriaceae species
(Casadevall & Pirofisk, 2000; Silva et al., 2015). Several out-
breaks of KPC-producing bacteria have occurred. These
outbreaks can be explained by the fact that the KPC-encod-
ing gene generally resides on transposons, which are carried
by conjugative plasmids, increasing its potential to spread
(Gupta et al., 2011) and making this enzyme an interna-
tional clinical and public health concern (Leung et al.,
2012).
Although many studies have reported on the drug resis-
tance profile of K. pneumoniae worldwide (Gupta et al.,
2011; Souli et al., 2012; Carvalhaes et al., 2013), there is
limited information regarding the epidemiology of this
species in Mid-Eastern Brazil. A case-control and molec-
ular study was performed in a public hospital to identify
the risk factors associated with carbapenem-resistant
K. pneumoniae strains. Understanding the risk factors
associated with these strains in healthcare facilities may
be important for targeting interventions and reducing
hospital transmission.
METHODS
Case-control study. To identify risk factors, a case-control study
was conducted. Patients hospitalized between May 2011 and May
2013 at a tertiary teaching hospital located in the city of Dourados,
in Mato Grosso do Sul (a central-western Brazilian state), were
included in this study. A case was defined as a patient who pre-
sented KPC-producing K. pneumoniae strains isolated from clinical
cultures from any source during the study period. Controls were
patients presenting non-carbapenemase-producing K. pneumoniae.
For each case one control was selected from patients admitted
within the study period matched by age, clinical manifestation and
hospital ward. All medical, nursing and microbiological records of
patients hospitalized during the study period were reviewed. Clinical
records from in-patients were reviewed, and the following data were
recorded: demographics; medical history and co-morbid conditions;
residence in a healthcare institution prior to hospital admission;
location prior to admission; ward of admission; hospital course;
invasive procedures; mechanical ventilation use; treatment with
immunosuppressors; antibiotic exposure history; source of infection;
and outcome.
Recorded co-morbidities included diabetes mellitus, cardiovascular dis-
ease, renal failure, chronic obstructive pulmonary disease, alcoholism,
substance misuse, HIV infection, decubitus ulcers, active cancer and
hypertension. All antibiotics that were administered for 24 h during
the current hospitalization were recorded. Both individual and cumula-
tive antibiotic exposures were evaluated.
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Bacterial strains. The carbapenem-resistant K. pneumoniae isolates
during the study period were obtained from 47 patients. Patients’
identification and demographic data were recorded. Colonization
was defined as the isolation of strains without clinical manifestation
of infection. Clinical infection was defined by medical diagnosis
according to clinical criteria (sepsis, fever, changes in frequency or
colour of secretions, or new radiological findings) associated with
the decision to initiate antibiotic therapy, as well as isolation of
one strain of KPC-producing K. pneumoniae (Casadevall & Pirofisk,
2000). This study was conducted with the approval of the Research
Ethics Committee from the Universidade Federal da Grande Doura-
dos (no. 039439/2012).
Bacterial identification, susceptibility testing and phenotypic
assays. Bacterial species were identified using the VITEK 2 automated
system (bioMerieux), and confirmed by matrix-assisted laser desorp-
tion/ionization-time-of-flight mass spectrometry (MALDI-TOF MS)
using a Microflex LT spectrometer (Bruker Daltonics), as previously
described (Fehlberg et al., 2014). The MICs of antimicrobials were deter-
mined by broth microdilution according to guidelines from the Clinical
and Laboratory Standards Institute (CLSI) (CLSI, 2013). Preliminary
screening for the presence of carbapenemases was performed by the
modified Hodge test (MHT) according to CLSI guidelines (CLSI, 2014).
Positive results obtained with the MHT were confirmed by ertapenem
hydrolysis using MALDI-TOF MS, as previously described (Carvalhaes
et al., 2013).
PCR amplification and sequencing of b-lactamase encoding
genes. The presence of b-lactamase genes (blaTEM-like, blaSHV-like,
blaCTX-M-1-like, blaCTX–M-2-like, blaCTX-M-8-like, blaCTX-M-14-like, blaGES-
like, blaKPC-like, blaSME-like, blaNDM-like, blaIMP-like, blaSPM-like, blaVIM-
like, blaSIM-like, blaGIM-like, blaNDM-like and blaOXA-48-like) was evaluated
by PCR followed by sequencing using specific primers, as previously
described (Fehlberg et al., 2014). The DNA sequences and their
derived protein sequences were analysed using the Lasergene Soft-
ware Package (DNASTAR) and compared with the sequences
deposited in GenBank.
Molecular typing by pulsed-field gel electrophoresis The genetic
relationship among the KPC-2-producing K. pneumoniae strains was
determined by pulsed-field gel electrophoresis (PFGE) using the restric-
tion enzyme SpeI (New England BioLabs) (Silva et al., 2011). The
restriction patterns were analysed using the BioNumerics software v.3.0
(Applied Maths). Percentage similarity between fingerprints was scored
by the Dice coefficient (Dice, 1945). The unweighted pair group method
with arithmetic mean and a 1.00 % tolerance limit was used to recon-
struct a dendrogram. Dendrogram and cluster analyses were performed
using the algorithms available within the BioNumerics software package
v.6.0 (Applied Maths).
Outer membrane protein analysis. The outer membrane proteins
(OMPs) of K. pneumoniae strains were analysed by SDS-PAGE using
membrane extracts from bacteria grown overnight in nutrient broth and
gels stained with Coomassie blue (Carvalhaes et al., 2010). Alterations of
OmpK35- and OmpK36-encoding genes were also investigated by PCR
and DNA sequencing (Correa et al., 2013).
Statistical analysis. All clinical data were entered into a Research
Electronic Data Capture (Redcap) database and SAS v.9.2 (SAS Insti-
tute), and these were used to analyse the univariate and multivariate
models. Dichotomized and categorical data were analysed with the chi-
squared test or Fisher’s exact test. For continuous variables, the t-test or
ANOVA was used. Bivariate analyses were performed to verify the asso-
ciations between the dependent and independent variables, and those
achieving a pre-specified level of significance (P<0.20) were included in
Journal of Medical Microbiology 65

the multivariable analysis. Logistic regression analysis was used to esti-
mate the crude and adjusted odds ratios.
RESULTS
Patients of the study and outbreak description
The patients were hospitalized in different hospital wards,
and their ages ranged from 2 to 80 years. Prior to isola-
tion of carbapenem-resistant K. pneumoniae, all patients
had received antimicrobial regimens, which included
penicillins, third- or fourth-generation cephalosporins,
quinolones, aminoglycosides, carbapenems and polymyx-
ins. A total of 68 carbapenem-resistant K. pneumoniae
strains were recovered from 47 patients between 2 and
40 days following admission, and of these, 36 had a his-
tory of previous hospitalization in our facilities or in
other hospitals. The remaining patients had no history of
previous hospitalization and were admitted to the inten-
sive care unit (ICU) directly from the emergency room.
Of the strains that were identified, 65 % (n = 44) of
these were isolated from the ICU and 35 % (n = 24)
were isolated from other hospital wards. Furthermore,
51 % (n = 35) of the identified strains were obtained
from urine culture, 19 % (n = 13) from swabs, 15 % (n
= 10) from tracheal aspirates, 9 % (n = 6) from blood
culture and 6 % (n = 4) from surgical wounds.
The case control study was performed with 94 patients (47
cases and 47 controls) and there were no significant differ-
ences (P>0.05) among cases and controls with regard to
baseline demographics. In the multivariable analysis, KPC-
producing K. pneumoniae strains were associated with long-
term hospitalization, use of mechanical ventilation, central
venous and urinary catheters, and previous surgery
(Table 1). The analysis of data on patient outcomes revealed
that KPC-producing K. pneumoniae patients had a higher
mortality rate than patients infected with carbapenem-sus-
ceptible K. pneumoniae (47.2 and 19.3 %, respectively; P =
0.01).
Susceptibility testing
Carbapenem-resistant K. pneumoniae strains showed
resistance to the antibiotics tested by broth microdilu-
tion as follows: meropenem (MIC50, >8 mg l–1), imipe-
nem (MIC50, >16 mg l–1) and ertapenem (MIC50, >32
mg l–1). Carbapenemase production was detected by
MHT in 67 strains; however, carbapenem hydrolysis was
detected by MALDI-TOF MS in only 57 of the 67
strains (85.0 %).
Molecular testing and PFGE
PCR amplification and sequencing showed that the bla
KPC-2 gene was present in 57 carbapenem-resistant strains.
The blaKPC-2 gene was not detected in 11strains. However,
the presence of other genes was observed, including bla
CTX–M-1-like, blaCTX–M-2-like, blaCTX-M-8 like, blaCTX-M-14-like
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Risk factors for KPC: watch out for surgery
and blaSHV-like. Eight blaCTX-M-2-positive strains also car-
ried blaCTX-M-8-like, blaCTX-M-14-like and blaSHV-like genes.
The presence of blaGES-like, blaSME-like, blaNDM-like, blaVIM-
like, blaSIM-1, blaGIM-1 and blaOXA-48-like genes was not
detected.
Carbapenem hydrolysis and genes that encode carbapene-
mases were not detected in 11 K. pneumoniae strains. In
these strains, blaCTX and blaSHV genes were identified, and
alterations in the OmpK35 and OmpK36 proteins were
investigated. According to the SDS–PAGE results, 11 K.
pneumoniae strains presented two bands, probably corre-
sponding to OmpA and one of the main porins (either
OmpK35 or OmpK36), suggesting that they have lost at
least one of the main porins. PCR analysis of OMP-encod-
ing genes showed altered amplicons of at least one main
OMP-encoding gene, including a lack of amplification
(ten isolates) or enhanced amplicon size (one isolate),
which suggests the presence of an insertion element. PCR
amplification of the blaCTX, blaSHV, ompk35 and ompk36
genes is shown in Table 2.
For PFGE analysis, 10 out of 57 KPC-producing
K. pneumoniae strains collected from the same patients on
the same days and anatomical sites were excluded. PFGE
analysis of the remaining 47 KPC-producing K. pneumoniae
strains identified 33 (70.21 %) with more than 88.80 % sim-
ilarity, as shown in the dendrogram (Fig. 1, cluster A). Of
these 33 isolates, 24 (72.72 %) were obtained from urine
cultures, and 29 (82.35 %) were isolated from ICU patients.
Analysis of the data revealed that patients infected or colo-
nized with this predominant clonal strain had a higher mor-
tality rate (78.78 %; P0.01) than patients infected with
other strains. Ten KPC-producing K. pneumoniae strains,
which were isolated from the same patients and collected
on different days and from different sites of infection, were
not included in PFGE analysis.
DISCUSSION
Due to the wide use of antibiotics, the number of carbape-
nem-resistant K. pneumoniae strains is increasing. The
global emergence and spread of carbapenemase genes
among K. pneumoniae strains is a severe challenge for public
health (Queenan & Bush, 2007; Monteiro et al., 2009; Souli
et al., 2012). During the 24-month study period, 47 patients
were infected or colonized by carbapenem-resistant K.
pneumoniae strains in our hospital. The proportion of KPC-
positive cases with carbapenem-resistant K. pneumoniae
strains was very high (83.82 %). Furthermore, the presence
of KPC was associated with higher morbidity and mortality
rates.
In our study, most KPC-2 positive isolates (57 %) were
recovered from patients hospitalised in the ICU. Those
patients had several co-morbidities and were subjected to
aggressive medical interventions, including broad-spec-
trum antibiotics. These results are in agreement with pre-
viously reported findings that showed that prior
549
K. E. Silva and others

Table 1. Summary of risk factors associated with KPC-producing K. pneumoniae

CI, Confidence interval; OR, odds ratio.

Risk factor Case patients (n = 47) Control patients (n = 47) Univariable analysis Multivariable analysis

OR (95% CI) P OR (95% CI) P

Mean age (years) 58.7 46.9

Comorbidities
Diabetes mellitus 10 (21.2) 6 (12.7) 0.76 (0.29–2.02) 0.59
Chronic heart failure 12 (21.0) 8 (13.7) 0.60 (0.22–1.60) 0.30
Chronic renal failure 1 (1.7) 5 (8.6) 5.28 (0.59–46.21) 0.09
Alcoholism 7 (12.2) 4 (6.9) 0.52 (0.14–1.91) 0.32
Substance misuse 9 (15.7) 3 (5.1) 0.29 (0.07–1.13) 0.06
Pulmonary disease 25 (43.8) 9 (15.5) 0.23 (0.09–0.56) <0.01
HIV infection 1 (1.7) 2 (3.45) 2.00 (0.17–22.69) 0.56
Ulcers 7 (12.2) 5 (8.6) 0.67 (0.20–2.26) 0.52
Cancer 8 (14.0) 0 (0.0) 0.04 (0.01–0.88) <0.01
Hypertension 11 (19.3) 14 (24.1) 1.33 (0.54–3.24) 0.52
Hospitalization
ICU stay 38 (80.8) 23 (48.9) 0.22 (0.09–0.57) <0.01
Length of hospital stay 37 (67.9) 10 (17.2) <0.01 2.23 (1.05–4.72) 0.03
Previous hospital 36 (63.1) 23 (39.6) 0.38 (0.18–0.81) 0.01
admission
Previous surgery 22 (38.6.) 2 (3.4) 0.05 (0.01–0.25) <0.01 35.98 (9.42– 137.48) <0.01
Use of 0 (0.0) 1 (1.7) 3.00 (0.11–75.19) 0.31
immunosuppressive
Presence of device
Urinary catheter 16 (28.0) 3 (5.1) 0.13 (0.03–0.51) <0.01 37.55 (9.61–146.69) <0.01
Mechanical ventilation 30 (52.6) 3 (5.1) 0.04 (0.01–0.17) <0.01 17.80 (5.64–56.13) <0.01
Central venous catheter 20 (35.0) 0 (0.0) 0.01 (0.01–0.26) <0.01 15.48 (3.83–62.56) <0.01

antimicrobial exposure in hospitalised patients, especially
severely ill patients, is the main force driving the spread of
carbapenem-resistant organisms (Hernandez-Alles et al.,
1999; Hoenigl et al., 2012; Patel et al., 2008; Souli et al.,
2012).
Assessment of the factors that predict carbapenem resis-
tance by multivariable analysis demonstrated that long-
term hospitalization, mechanical ventilation, central venous
and urinary catheters, and previous surgery were associated
with KPC-producing K. pneumoniae. These risk factors

Table 2. Phenotypic screening and mechanisms of resistance detected in K. pneumoniae strains

P, Positive; N, negative.

Strain MHT MALDI- blaCTX–M-1 blaCTX–M-8 blaCTX–M-14 blaSHV ompk35 (expected size/ ompk36 (expected size/
TOF MS size found; bp) size found; bp)

1 N N P N P N 1148/1148 1148/2000
2 P N N P P N 1148/1148 1148/N
3 P N P N N P 1148/N 1148/1148
4 P N P P N N 1148/1148 1148/N
5 P N P N P N 1148/1148 1148/N
6 P N P N P P 1148/1148 1148/N
7 P N P N N N 1148/N 1148/1148
8 P N P N P N 1148/1148 1148/N
9 P N P N P N 1148/1148 1148/N
10 P N P N N N 1148/N 1148/1148
11 P N P N N N 1148/N 1148/1148

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 Risk factors for KPC: watch out for surgery

Strains Ward Site of infection Outcome

60 65 70 75 80 85 90 95 100
84.6 21673 * P4 RECTAL SWAB RECOVERY
52203 IUC A TRACHEAL ASPIRATES DEATH

47309 P3 URINE RECOVERY

9030 P3 URINE RECOVERY
53962 * IUC A NASAL SWAB DEATH

66650 IUC B URINE DEATH

34099 IUC B URINE DEATH

34575 IUC A URINE DEATH

39559 IUC A URINE RECOVERY

99.1
4749 IUC A URINE DEATH
80.0 5255 IUC A URINE DEATH

59130 IUC A URINE DEATH

59799 IUC A URINE DEATH

92.3 6197 IUC A TRACHEAL ASPIRATES DEATH

62538 IUC A URINE DEATH

63433 IUC A URINE DEATH
48496 P3 URINE RECOVERY
57528 A IUC B BLOOD CULTURE DEATH
53957 IUC B BLOOD CULTURE DEATH
42318 IUC B URINE RECOVERY
88.8
33156 IUC B BLOOD CULTURE DEATH

55118 P4 BLOOD CULTURE DEATH

26748 * IUC A RECTAL SWAB DEATH
77.6
26748 * IUC A RECTAL SWAB RECOVERY

979 IUC B URINE RECOVERY

47309 IUC B URINE DEATH

77.6 44839 IUC B URINE DEATH

54676 IUC A TRACHEAL ASPIRATES DEATH

17991 IUC B URINE DEATH

23816 IUC B URINE DEATH

IUC A URINE DEATH
46793
70.7
46422 IUC A URINE DEATH
50366 IUC B URINE DEATH

40154 IUC B URINE DEATH

34521 IUC A URINE DEATH

84.6 50366 P3 TRACHEAL ASPIRATES RECOVERY

67.5
55570 P4 BLOOD CULTURE RECOVERY

14981 IUC B URINE RECOVERY

92.0 2012.1 IUC B URINE RECOVERY

88.3 1568 IUC A URINE RECOVERY
66.4
2780 P4 BLOOD CULTURE RECOVERY
3686 * P3 NASAL SWAB RECOVERY

90.9 4126 * P3 RECTAL SWAB RECOVERY

84.0 5539 P4 TRACHEAL ASPIRATES RECOVERY
72.5
5708 P4 TRACHEAL ASPIRATES RECOVERY
2012.2 * P4 NASAL SWAB RECOVERY
22340 P3 SCAR RECOVERY

Fig. 1. Dendrogram displaying the genetic relatedness of 47 KPC-producing K. pneumoniae strains recovered in a Brazilian
teaching hospital, based on PFGE data and carbapenemase content. The 33 strains showing 88.80 % similarity are grouped
above (cluster A). Asterisks indicate the colonizing strains.

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K. E. Silva and others
probably represent severe underlying illness and therefore
increased susceptibility to infection, leading to increased
risk of infection from multidrug-resistant organisms. Simi-
lar risk factors for KPC have been described (Souli et al.,
2012; Correa et al., 2013). Prior studies demonstrated asso-
ciations between KPC-producing K. pneumoniae infection
and length of hospitalization, use of central venous
catheters, ICU stay and exposure to specific broad-spectrum
antimicrobial agents, such as carbapenems, cephalosporins,
fluoroquinolones and penicillins (Papadimitriou-Olivgeris
et al., 2012; Tumbarello et al., 2014). Despite surgery
being reported as a risk factor for carbapenem-resistant K.
pneumoniae infection/colonization, molecular analysis
to detect carbapenemase genes was not performed in this
study (Kofteridis et al., 2014). Thus, to our knowledge, this
study is the first to show an association between surgical
procedures and KPC-producing K. pneumoniae. The KPC-2
strains associated with surgery were isolated over 6 months.
Although correlations among sex, operating room and sur-
gical procedures were not observed, patients who undergo
surgery are generally hospitalized long-term, which could
increase the risk of infection by KPC. Conversely, contact
between patients, hands or contaminated medical equip-
ment may have contributed to the dissemination of these
strains. The impossibility of identifying a common source
in an environmental reservoir is the major limitation of this
study.
To analyse the mechanisms of antimicrobial resistance,
phenotypic and molecular assays of carbapenem-resistant
K. pneumoniae isolates were performed. In vitro antimi-
crobial resistance to carbapenems was observed in all K.
pneumoniae strains. False positives for carbapenemase
production was found using MHT in 11 strains.
According to Wang et al. (2011), the false positive
results observed using MHT probably occur due to low-
level hydrolysis of ertapenem by extended-spectrum b-
lactamases (ESBLs), particularly those of the CTX-M
type. Although carbapenemase-encoding genes were not
identified in 11 K. pneumoniae strains, blaCTX-M and bla
SHV genes were detected. The SDS-PAGE results, in con-
junction with PCR analysis, showed that ompK35 and
ompK36 were modified by mutations or insertion
sequences in all ESBL strains. Therefore, these porin
alterations had contributed to the reduction of outer
membrane permeability and susceptibility to carbape-
nems. Increased levels of resistance to carbapenems
were probably caused by decreased expression of OMPs
and/or increased efflux, as described by Correa et al.
(2013). Our results are in agreement with those found
by Carvalhaes et al. (2010) who reported carbapenem
resistance due to ESBL production coupled with porin
loss.
The PFGE results were evaluated to explore the genetic
relationship among KPC-producing K. pneumoniae.
Analysis of 47 KPC-producing K. pneumoniae identified
a predominant clonal type (cluster A) that comprised
33 strains (70.21 %), which were associated with ICU
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patients (87.87 %), urine infections (72.72 %) and a high
mortality rate (78.78 %). Outbreaks of KPC-producing
K. pneumoniae isolated from ICU patients have been
described (Gupta et al., 2011; Tofteland et al., 2013;
Yang et al., 2013) with mortality rates ranging from
52.80 to 66.70 % (Zarcotou et al., 2011; Qureshi et al.,
2012). However, our results showed a higher mortality
rate (78.78 %) associated with the KPC outbreak. This
high mortality rate may be overestimated, as patients
infected with KPC-producing K. pneumoniae had several
co-morbidities and invasive devices, which may have
contributed to the worsening of clinical symptoms and
consequent progression to death. Nevertheless, these
results suggest that infection control measures should be
reinforced in our hospital to control the spread of KPC
enzymes and to reduce mortality rates.
Our findings showed that KPC-producing K. pneumoniae
strains were associated with several healthcare-related risk
factors. Furthermore, this is the first study to demonstrate
an association between surgical procedures and KPC. Early
and accurate detection, in conjunction with effective infec-
tion control measures, are of utmost importance to control
the spread of carbapenemase-producing organisms. There-
fore, highly intensive efforts are required to control the
spread of these organisms in our hospital.
ACKNOWLEDGEMENTS
This work was partially supported by the Brazilian National Research
Council (CNPq grants 480949/2013-1) and the Support Foundation
for the Development of Education, Science and Technology in the
State of Mato Grosso do Sul (FUNDECT grants 05/2011 and 04/
2012). K.E.S. and W.G.M received a scholarship from Coordenaç~ao
de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). A.C.G. is
a researcher from the National Council for Science and Technological
Development (CNPq), Ministry of Science and Technology, Brazil
(process number 307816/2009-5). A.C.G. has recently received
research funding and/or consultation fees from AstraZeneca, MSD,
Novartis, Thermo Fisher Scientific and bioMerieux. The other
authors have no conflict of interest to declare.
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