International Journal of Antimicrobial Agents 44 (2014) 152–155

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International Journal of Antimicrobial Agents

journal homepage: http://www.elsevier.com/locate/ijantimicag

Short Communication

Emergence of carbapenemase-producing Gram-negative bacteria

in Saint Petersburg, Russia夽
Vladimir A. Ageevets a,b , Irina V. Partina a , Eugenia S. Lisitsyna c , Elena N. Ilina d ,
Yuri V. Lobzin a,e , Sergei A. Shlyapnikov e,f , Sergei V. Sidorenko a,e,∗
a
Research Institute of Children’s Infections, Professor Popov Str. 9, Saint Petersburg 197022, Russia
b
Saint Petersburg State University, Department of Microbiology, Universitetskaya Naberezhnaya Str. 7–9, Saint Petersburg 199034, Russia
c
Lytech Ltd., Malaya Semenovskaya Str. 3a, Moscow 107023, Russia
d
Research Institute for Physical-Chemical Medicine, Malaya Pirogovskaya Str. 1a, Moscow 119992, Russia
e
North-West State Medical University, Kirochnaya Str. 41, Saint Petersburg 191015, Russia
f
I.I. Dzhanelidze Institute of Emergency Medicine, Budapeshtskaya Str. 3, Saint Petersburg 192242, Russia

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

Article history: The emergence and spread of carbapenemase-producing Gram-negative bacteria represents a serious
Received 7 February 2014 public health concern. Here we show that of 477 Gram-negative isolates collected from 18 hospitals
Accepted 9 May 2014 between November 2011 and February 2013 in Saint Petersburg (Russia), minimum inhibitory concentra-
tions (MICs) were greater than the European Committee on Antimicrobial Susceptibility Testing (EUCAST)
Keywords: epidemiological cut-off value of at least one carbapenem antibiotic in 101 isolates (21.2%). The blaNDM-1
␤-Lactamase
gene was detected by PCR in 17 Klebsiella pneumoniae and 1 Acinetobacter nosocomialis isolate. Multilocus
Carbapenemase
sequence typing (MLST) revealed that all NDM-1-producing K. pneumoniae isolates belonged to sequence
Klebsiella pneumoniae
Acinetobacter nosocomialis
type 340 (ST340) and harboured genes encoding additional ␤-lactamases; presence of the blaCTX-M-1-like
gene correlated with aztreonam resistance, whilst its absence correlated with susceptibility. The epidemi-
ological situation in Saint Petersburg can be assessed as regional spread of NDM-1-producers. The blaKPC-2
gene was detected in two K. pneumoniae isolates (ST258 and ST273) and one Enterobacter cloacae isolate.
Two E. cloacae isolates harboured the blaVIM-4 gene, and one K. pneumoniae (ST395) isolate harboured the
blaOXA-48 gene. In NDM-1-producers, MICs of biapenem were the lowest compared with those of other
carbapenems. Most isolates were susceptible to tigecycline and polymyxin, except for one K. pneumoniae
isolate that was found to be polymyxin-resistant and one E. cloacae isolate that was tigecycline-resistant.
Only one patient with a urinary tract infection caused by KPC-2-producing K. pneumoniae had a history
of travel abroad (Southeast Asia). Thus, there is an actual threat of the emergence of an alarming endemic
situation with NDM-1-producers in Saint Petersburg.
© 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

1. Introduction resistance has been reported worldwide. Carbapenem resistance

For more than 30 years, use of carbapenems has been crucial
for the therapy of life-threatening nosocomial infections caused
by Gram-negative bacteria resistant to other groups of antibi-
otics. However, in recent years the development of carbapenem
夽 The results of this study were presented in part at the 23rd European Congress
of Clinical Microbiology and Infectious Diseases (ECCMID), 27–30 April 2013, Berlin,
Germany [P1287].
∗ Corresponding author. Present address: Research Institute of Children’s Infec-
tions, Professor Popov Str. 9, Saint Petersburg 197022, Russia. Tel.: +7 963 316 0808;
fax: +7 812 234 9691.
E-mail address: sidorserg@gmail.com (S.V. Sidorenko).
arises from several mechanisms and their combinations (deficiency
of porin expression, efflux, AmpC hyperproduction, and alter-
ations in penicillin-binding proteins). However, carbapenemases
(enzymes that hydrolyse carbapenems), the genes encoding which
are mapped to different mobile genetic elements, pose the main
threat. Carbapenemases were first found in environmental species
of low clinical significance; however, since the 1990s an increasing
number of reports have dealt with the detection of chromosome-
and plasmid-borne carbapenemases in Gram-negative bacteria.
Acquisition of Ambler class A (KPC), class B (IMP, VIM, NDM), also
known as metallo-␤-lactamases (MBLs), and class D (OXA) car-
bapenemases is the most important aspect in Europe [1].
In Russia, the first MBLs were detected in hospital-acquired
Pseudomonas aeruginosa isolates and, at present, sequence type

http://dx.doi.org/10.1016/j.ijantimicag.2014.05.004
0924-8579/© 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
 V.A. Ageevets et al. / International Journal of Antimicrobial Agents 44 (2014) 152–155
235 (ST235) VIM-2-producing P. aeruginosa isolates are widely
spread not only in Russia but also in Belarus and Kazakhstan
[2]. Carbapenemase detection in Escherichia coli (VIM-4) was first
reported in 2012 from Moscow (Russia) [3]. In addition, a prelimi-
nary report on the outbreak of hospital-acquired infections (HAIs)
due to NDM-type-producing Klebsiella pneumoniae was published
in Saint Petersburg (Russia) [4]. In 2013, NDM-1-producing K. pneu-
moniae ST340 was isolated from a patient with a urinary tract
infection in Saint Petersburg [5].
This study describes carbapenemase-producing Gram-negative
bacteria causing outbreaks and sporadic cases of HAIs in Saint
Petersburg.
2. Methods
2.1. Bacterial isolates and identification
Consecutive, non-duplicate, Gram-negative isolates were
prospectively collected between November 2011 and February
2013 from the local laboratories of 18 hospitals in Saint Petersburg.
These isolates were obtained from unique patients with HAIs of
different localisations (respiratory, urinary, intra-abdominal, skin
and soft-tissue). Bacterial isolates and anonymous case report
forms were sent to the referral laboratory (Research Institute
of Children’s Infections, Saint Petersburg, Russia). Species iden-
tification was performed using a microflexTM MALDI Biotyper
2.0 (software version 3.1.1.0) (Bruker Daltonics, GmbH, Bremen,
Germany) according to the manufacturer’s instructions.
2.2. Antimicrobial susceptibility testing
The following antibiotics were tested: ampicillin; cefotaxime;
ceftazidime; cefepime; cefepime/clavulanic acid; aztreonam;
imipenem; ertapenem; meropenem; biapenem; gentamicin;
amikacin; tigecycline; polymyxin B; ciprofloxacin; and fosfomycin.
Biapenem and fosfomycin were obtained from Meiji Seika Pharma
Co., Ltd. (Tokyo, Japan) and the other antibiotics were obtained
from Molekula GmbH (Munich, Germany) as laboratory-grade
powders. Minimum inhibitory concentrations (MICs) for all antibi-
otics, except fosfomycin, were determined using the microdilution
method in Mueller–Hinton broth (Becton, Dickinson & Co., Sparks,
MD) and were interpreted according to Clinical and Laboratory
Standards Institute (CLSI) criteria [6]. The MIC of fosfomycin was
determined using the agar dilution method in Mueller–Hinton agar
(bioMérieux, Marcy-l’Étoile, France) with the addition of 25 mg/L
glucoso-6-phosphate (Molekula GmbH).
2.3. Detection of carbapenemase activity
Carbapenemase activity was detected using two phenotypic
tests: (i) the modified Hodge test (MHT) performed according
to CLSI recommendations [6]; and (ii) mass spectrometric mea-
surement of meropenem hydrolysis in a solution performed as
described previously [7] with modifications as follows. Cultures
of the tested strains were incubated overnight on Mueller–Hinton
agar (bioMérieux). A heavy inoculum (3.8–4.2 on the McFarland
scale) was prepared in 1 mL of 0.9% NaCl (pH 6.8). The resulting
suspension was centrifuged and the supernatant was discarded.
To each Eppendorf tube with pelleted cells, 15 ␮L of meropenem
solution (200 mg/L) in 0.9% NaCl was added. Following this, cells
were re-suspended, incubated for 3 h at 37 ◦ C and pelleted by
centrifugation. The mass spectra of the resulting supernatant
(1 ␮L) were obtained using a microflexTM matrix-assisted laser
desorption/ionisation time-of-flight (MALDI-TOF) mass spectrom-
eter (Bruker Daltonics) equipped with flexAnalysis 3.3 software. A
153
positive result was defined as the absence of detectable peaks cor-
responding to the native form of meropenem (384 kDa). To avoid
false-positive results due to spontaneous hydrolysis of meropenem
as well as false-negative results, the following control samples were
tested in each experiment: pure solution of 0.9% NaCl (pH 6.8);
200 mg/L meropenem solution in 0.9% NaCl; meropenem solution
with carbapenemase-producing K. pneumoniae; and meropenem
solution with carbapenemase-negative K. pneumoniae. However,
we could not exclude false-negative results with isolates producing
enzymes with low carbapenemase activity.
2.4. PCR and sequencing
Bacterial DNA was extracted using a DNA-express Kit (Lytech
Ltd., Moscow, Russia) according to the manufacturer’s instructions.
Genes of 11 clinically relevant carbapenemases (blaIMP , blaVIM ,
blaNDM , blaSPM , blaAIM , blaDIM , blaGIM , blaSIM , blaKPC , blaBIC and
blaOXA-48 ) were detected by multiplex PCR using primers and condi-
tions described previously [8]. Genes encoding extended-spectrum
␤-lactamases (ESBLs) (blaTEM-like , blaSHV-like , blaCTX-M-1-like ,
blaCTX-M-2-like , blaCTX-M-9-like , blaCTX-M-25-like , blaPER-1-like ,
blaPER-2-like , blaVEB-like , blaGES-like , blaOXA-2-like and blaOXA-10-like )
were analysed as described previously [9]. In addition, genes
encoding AmpC ␤-lactamases (blaMOX-1 , blaMOX-2 , blaCMY-1–11 ,
blaLAT-1–4 , blaBIL-1 , blaDHA-1 , blaDHA-2 , blaACC , blaMIR-1T , blaACT-1 and
blaFOX-1–5b ) were analysed as described previously [10].
The entire coding regions of blaNDM , blaKPC , blaOXA and blaVIM
were additionally amplified and sequenced using the following
self-designed primers: NDMfor, 5 -agccgctgcattgatgctga-3 ; NDM-
rev, 5 -cggaatggctcatcacgat-3 ; VIMfor, 5 -tcgcaagtccgttagccca
ttc-3 ; VIMrev, 5 -cccgggaatgacgacctct-3 ; KPCfor, 5 -tcgaaca
ggactttggcggct-3 ; KPCrev, 5 -cctcgctgtgcttgtcatcctt-3 ; OXAfor, 5 -
gggcgaaccaagcatttttac-3 ; and OXArev, 5 -ggcgcagccctaaaccatc-3 .
The amplicons were then sequenced using an ABI Prism® BigDye®
Terminator Cycle Sequencing Ready Reaction Kit and ABI 3730
Genetic Analyzer (Applied Biosystems, Foster City, CA).
2.5. Multilocus sequence typing (MLST)
MLST of K. pneumoniae was performed using the proto-
col available at http://www.pasteur.fr/recherche/genopole/PF8/
mlst/Kpneumoniae.html.
3. Results and discussion
A total of 477 isolates of Gram-negative bacteria were collected
from the participating centres. The MIC of at least one carbapenem
antibiotic was found to be above the European Committee on
Antimicrobial Susceptibility Testing (EUCAST) epidemiological cut-
off value (http://www.eucast.org) in 101 isolates (21.2%). These
isolates were tested for possible carbapenemase production by
MHT and MALDI-TOF mass spectrometry (MALDI-TOF/MS) assays,
and the presence of bla genes was detected by PCR. Sequencing was
used for precise identification of the carbapenemase genes.
Of 101 isolates with reduced carbapenem susceptibility,
24 (23.8%) displayed carbapenemase activity according to the
MALDI-TOF/MS assay and harboured genes encoding differ-
ent carbapenemases (Table 1). Two of these were negative by
the MHT. Notably, none of the isolates was positive by MHT
but negative by the MALDI-TOF/MS assay. All carbapenemase-
producers were resistant to ampicillin and first- to third-generation
cephalosporins. In the remaining 77 isolates (76.2%) lacking
carbapenemase activity, MICs of carbapenems were <4.0 mg/L.
Reduced outer-membrane permeability and/or AmpC ␤-lactamase
hyperproduction is considered to be the most probable mechanism
of resistance.
 154 V.A. Ageevets et al. / International Journal of Antimicrobial Agents 44 (2014) 152–155

MLST, multilocus sequence typing; MIC, minimum inhibitory concentration; FEP, cefepime; FEP/CLA, cefepime/clavulanic acid; AZT, aztreonam; ERT, ertapenem; IPM, imipenem; MER, meropenem; BIA, biapenem; GEN, gentamicin;
According to PCR and sequencing analysis, presence of the

16–256

4–128
blaNDM-1 gene was confirmed in 17 K. pneumoniae isolates and
FOS

128

128

256
64

32
1 Acinetobacter nosocomialis isolate obtained from four different

Total no. of isolates, number of isolates positive by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS), and number of isolates positive by the modified Hodge test (MHT).
0.015–0.06
hospitals. Eleven NDM-1-producing K. pneumoniae isolates were

0.03–0.06

0.03–0.06
obtained during the entire study period (15 months) from hospi-
tal A, and we were unable to detect any trends in incidence. Five
PMB

0.25

0.06

0.06

0.06
16
isolates were recovered during the last 3 months of the study from
hospital D. One isolate each of K. pneumoniae and A. nosocomialis
0.12–0.25

0.025–0.5
was detected in hospitals B and C, respectively. Carbapenemase
activity was detected by MALDI-TOF/MS assay in all of the iso-
0.25

0.12

0.25

0.25

0.06
TIG

4
lates; however, the MHT failed to detect carbapenemase activity
in two K. pneumoniae isolates. These results support previous find-
>256

>256

>256

>256

>256

>256

>256
CIP

ings regarding the poor performance of the MHT in the detection of

2

MBL-producers [11]. All NDM-1-producing K. pneumoniae isolates

>256

>256

>256
AMK

belonged to the widely spread ST340; Sweden is the closest country

16

16

2
to Saint Petersburg, where ST340 was also detected [12].
>128

>128

>128
GEN

128

Isolates in the present study were subdivided into two clus-

0.5

0.5
64
1

ters on the basis of minor differences in resistance patterns and

0.12–2.0

the presence of genes encoding ␤-lactamases. Thirteen isolates

0.12–32

2.0–8.0

from hospitals A, B and D harboured blaSHV-like , blaCTX-M-1-like and

>64
BIA

32

64

blaCTX-M-2-like genes and were resistant to aztreonam. However,

four isolates from hospital A were susceptible to aztreonam and
8–64
MER

1–4
>64

>64
64

64

16

16

lacked blaCTX-M-1-like genes. We can explain this either by several

episodes of introduction of NDM-1-producing K. pneumoniae ST340
32–64

0.5–1
4–64

from abroad or by microevolution within a single genetic lineage

IPM

>64

>64

>64

16

(e.g. acquisition or loss of a mobile element carrying a blaCTX-M-1-like

32–64

gene). An A. nosocomialis isolate from hospital C was highly

ERT

2–8
>64

>64

>64

>64

resistant to cefepime and carbapenems but was susceptible to

16

16

aminoglycosides and ciprofloxacin.

0.12–0.25

In the case of NDM-1-producers, MICs of biapenem were the

>128

lowest compared with those of other carbapenems (Table 2). MICs

AZT

>64

>64

>64

>64

>64

>64

of biapenem, meropenem and imipenem were ≤8.0 mg/L in 16

MIC range (mg/L)

(88.9%), 1 (5.6%) and 6 (33.3%) of 18 NDM-1-producers, whilst

FEP/CLA

MIC of ertapenem was >8.0 mg/L in all isolates. These data may
>256

>256

>128

>128

>128

>128

>128

AMK, amikacin; CIP, ciprofloxacin; TIG, tigecycline; PMB, polymyxin B; FOS, fosfomycin; N/D, not determined.

be of clinical significance because it was shown that imipenem

8

and meropenem could provide some therapeutic benefit in treat-

>256

>256

>128

>128

>128

>128

>128

>128
FEP

ing infections caused by carbapenemase-producing K. pneumoniae

strains with MICs ≤ 8.0 mg/L [13]. The high activity of biapenem
SHV-like, TEM-like,

TEM-like, SHV-like,

against NDM-1-producing strains was also demonstrated by Liv-

ermore et al. [14]. All 18 NDM-1-producers were susceptible to
CTX-M-1-like,

CTX-M-1-like,

CTX-M-1-like,

CTX-M-1-like,
CTX-M-2-like

CTX-M-2-like

CTX-M-2-like

CTX-M-2-like

CTX-M-2-like

CTX-M-2-like
␤-lactamases

tigecycline and polymyxin B, and 14 (77.8%) were susceptible to

Additional

TEM-like,

TEM-like,
SHV-like,

SHV-like,

fosfomycin according to the EUCAST cut-off values.

Two E. cloacae isolates from hospital B harboured the blaVIM-4
–

gene and were positive by the MALDI-TOF/MS assay and MHT. MICs
of imipenem for both isolates and those of meropenem for one iso-
ST258

ST273

ST395
ST340

ST340
MLST

N/D

N/D

ND

late were in the susceptible category according to CLSI criteria but

were above the EUCAST epidemiological cut-off value. Isolates were
A (7); B (1);

resistant to cefepime and aztreonam, demonstrated synergism

Hospital

isolates)

between cefepime and clavulanic acid, and harboured blaTEM-like ,

(no. of

D (5)

G (1)
A (4)

B (2)
C (1)

E (1)
F (1)

F (1)
Genotypes and phenotypes of carbapenemase-producers.

blaCTX-M-1-like and blaCTX-M-2-like genes. All VIM-4-producers were

sensitive to polymyxin B and tigecycline.
The blaKPC-2 gene was detected by PCR and sequencing of three
17/17/15
MALDI+/
isolates/

isolates that were positive by the MALDI-TOF/MS assay and MHT.

MHT+a
No. of

1/1/1

2/2/2

2/2/2

1/1/1

1/1/1

A KPC-2-producing K. pneumoniae isolate from hospital G belonged

to the main globally spread lineage ST258 [1]. A KPC-2-positive K.
pneumoniae ST273 isolate was obtained from hospital F from the
K. pneumoniae

K. pneumoniae
Acinetobacter

urine sample of a patient with recent history of hospitalisation in

nosocomialis
Enterobacter
pneumoniae

E. cloacae
Klebsiella

Vietnam. This isolate was resistant to polymyxin B. We found no

Species

cloacae

records of the detection of KPC-2-producing K. pneumoniae ST273;

however, colistin-resistant but carbapenem-susceptible isolates
belonging to ST273 have been reported from Italy [15]. The patient
Carbapenemase

was successfully treated with a combination of tigecycline and

meropenem; however, 2 weeks later, E. cloacae harbouring KPC-
OXA-48
NDM-1

2 carbapenemase resistant to tigecycline was isolated from urine.

VIM-4

KPC-2
Table 1

In this case, in vivo transmission of the blaKPC gene was sus-

a

pected. KPC-2-producers carried different additional ESBL genes

 V.A. Ageevets et al. / International Journal of Antimicrobial Agents 44 (2014) 152–155
Table 2
Carbapenem minimum inhibitory concentration (MIC) distribution for NDM-1-producers.
Antibiotic n (%) at each MIC (in mg/L)
0.12 0.25 0.5 1 2
Biapenem 1 (5.6) 3 (16.7) 2 (11.1) 0 1 (5.6)
Meropenem 0 0 0 0
Imipenem 0 0 0 0
Ertapenem 0 0 0 0
and retained susceptibility to amikacin. To our knowledge, this is
the first report of KPC-2 in Russia.
The blaOXA-48 gene was detected in one K. pneumoniae ST395
isolate that was positive by the MALDI-TOF/MS assay and MHT,
did not harbour genes encoding additional ESBLs, and was resis-
tant to ␤-lactams and ciprofloxacin but retained susceptibility to
aminoglycosides, tigecycline and polymyxin B.
Outbreaks of healthcare-associated infections due to NDM-
1-producing K. pneumoniae ST340 were detected in at least
three hospitals in Saint Petersburg. Thus, according to the epi-
demiological scale and stages of the nationwide expansion of
carbapenem-resistant Enterobacteriaceae proposed by a group
of European experts [16], regional spread of NDM-1-producing
K. pneumoniae ST340 is observed in Saint Petersburg (stage 3,
more than one epidemiologically related outbreak confined to
hospitals, which are part of a regional referral network, sug-
gesting a regional autochthonous inter-institutional transmission).
According to self-assessment of the epidemiological stage of
carbapenemase-producing Enterobacteriaceae by national experts,
the occurrence of NDM-1-producers is significantly lower in
countries near Saint Petersburg. In Finland, Norway and Sweden,
either sporadic or single outbreaks caused by NDM-1-producers
were reported, and data from Estonia are not available [17]. Thus,
there is an actual threat of the emergence of an alarming endemic
situation in a Baltic region city with 5 million residents. The
epidemiological situation with other carbapenemases is not so
alarming in Saint Petersburg. KPC-2-producers caused sporadic
hospital outbreaks (stage 2b), VIM-4-producers caused a single
outbreak (stage 2a), and there was a single case of infection by
OXA-48-producers (stage 1).
Screening for Enterobacteriaceae isolates with carbapenem
MICs above EUCAST epidemiological cut-off values (or corre-
sponding zone diameters) in hospital laboratories and subsequent
detection of carbapenemase activity by the MALDI-TOF/MS assay
and molecular typing of selected isolates in a referral laboratory
should be proposed for regional surveillance and early detection of
carbapenemase-producing Enterobacteriaceae.
Funding: This study was partially supported by The Rus-
sian Foundation for Basic Research (RFBR) [research project no.
14-04-00563] and by a grant from the Russian Government
[12197р/23100].
Competing interests: YVL has served as a board member of Merck
Sharp & Dohme, Pfizer, Novartis and Astellas; SAS has served as a
board member of Merck Sharp & Dohme, Pfizer and AstraZeneca
and has served on the speakers’ bureaus of Merck Sharp & Dohme,
Pfizer and AstraZeneca; SVS has received funding from R-Pharm,
Pfizer and AstraZeneca, has served as a board member of Merck
Sharp & Dohme, Pfizer, AstraZeneca and Astellas, and has served on
the speakers’ bureaus of Merck Sharp & Dohme, Pfizer, AstraZeneca
and Astellas. All other authors declare no competing interests.
Ethical approval: Not required.
155
4 8 16 32 64 >64
7 (38.9) 2 (11.1) 0 2 (11.1) 0 0
0 0 1 (5.6) 5 (27.8) 0 9 (50.0) 3 (16.7)
0 5 (27.8) 1 (5.6) 8 (44.4) 2 (11.1) 1 (5.6) 1 (5.6)
0 0 0 1 (5.6) 5 (27.8) 2 (11.1) 10 (55.6)
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