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69―141（ 41 ）Surveillance of antimicrobial susceptibility ofEnterobacteriaceae pathogens isolated fromintensive care units and surgical units in Russia
IRINA PARTINA1, OLGA KALINOGORSKAYA1, SATOSHI KOJIMA2,VLADIMIR GOSTEV1, MARINA VOLKOVA1, VLADIMIR AGEEVETS1,YURI LOBZIN1 and SERGEY SIDORENKO1
Scientiﬁc Research Institute of Childrenʼs Infections,
Professor Popov Str. 9, Saint Petersburg 197022, Russia
2Global Business Division, Shionogi & CO., LTD.,
1–8 Doshomachi, 3-chome, Chuo-ku, Osaka 541–0045, Japan1(Received for publication August 6, 2015)

A total of 473 strains of Enterobacteriaceae, including Escherichia coli, Klebsiellaspp., Proteus spp., Citrobacter spp., Enterobacter spp., Serratia spp. andProvidencia spp., were isolated from patients admitted to intensive care units andsurgical units in Russia. About 90% of the isolates carried factors resistant to betalactams. The isolation rates of the extended-spectrum beta-lactamase (ESBL)
producer deﬁned in this study among E. coli, Klebsiella spp. and Proteus spp. were45%, 48% and 17%, respectively. In the settings with high prevalence of the ESBL
producer, ﬂomoxef, which belongs to the oxacephem subgroup, and carbapenems
retain their activity. The MIC50 of ﬂomoxef, meropenem and imipenem against total
isolates were 1 μg/mL, ≤0.063 μg/mL and 0.25 μg/mL, respectively. Fifty-ﬁve
carbapenem-resistant strains were isolated in this study. The carbapenem resistantrates of E. coli, Klebsiella spp. and Proteus spp. were 3%, 16% and 29%,respectively.

Introduction
Global spreading of antimicrobial resistance is one of the most serious threats for the healthcare system. The Russian Federation is one of the countries with an extremely high prevalence ofmulti-resistant bacteria in hospital settings. One surveillance study for selected European nations
including Russia reported that beta-lactamase–mediated mechanisms have spread widely among
Gram-negative bacilli, especially across the Eastern and Southern European nations1). This is
likely due to expanded use of beta-lactams without the activity against Gram-negative bacilli in42（ 42 ）THE JAPANESE JOURNAL OF ANTIBIOTICS

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daily therapy.
One way of controlling the spread of antimicrobial resistance is by introducing known antibiotics, which are not widely used but have important properties, into clinical practice. One suchantibiotic is ﬂomoxef, a cephem antibiotic of the oxacephem subgroup. Flomoxef has not beenmarketed in European countries and Russia.
To investigate the latest in vitro susceptibility to beta-lactams widely used in clinical practice, we conducted an epidemiological study of clinically signiﬁcant isolates of Enterobacteriaceae in Russia.

Materials and MethodsBacterial isolates and identiﬁcation
Isolates were collected in 2012 from local laboratories of 17 hospitals in the western part ofRussia. Thirteen institutes were in Saint Petersburg and four were in Moscow, Republic of Karelia, Stavropol Krai and Kurgan. Isolates were obtained from patients with hospital infections(pneumonia, intraabdominal infections, skin and soft tissue infections, infections of urinary tract,and sepsis) admitted to intensive care units and surgical units specializing in various areas of surgery. Isolation and identiﬁcation of bacteria were done in laboratories of healthcare institutions
by means of widely accepted methods. The collected isolates were submitted to the laboratory of
the Scientiﬁc Research Institute of Childrenʼs Infections. Matrix-assisted laser desorption ionization–time of ﬂight mass spectrometry (MALDI-TOF MS) has been used for the conﬁrmation ofidentiﬁcation of the submitted strains. Bacteria from isolated colonies were applied, using sterileloops, as thin ﬁlms on polished stainless steel target plates (Bruker Daltonik GmbH, Leipzig,Germany) in three replicates. The bacteria were then left to dry at room temperature for 1 min.
Subsequently 1.5 μL of the freshly prepared matrix solution, comprising a saturated α-cyano-4hydroxy-cinnamic acid in 50% acetonitrile with 2.5% triﬂuoroacetic acid, was applied to the samples and cocrystallized with them at room temperature for 10 min. The samples were applied to a
MicroFlex LT mass spectrometer (Bruker Daltonik GmbH, Leipzig, Germany), and the results
were analyzed by MALDI Biotyper 2.0 software package (Bruker Daltonik GmbH, Leipzig,Germany) according to the manufacturerʼs instructions.
Antimicrobial susceptibility testing
The susceptibility of Enterobacteriaceae to antimicrobial agents was assessed by serial microdilution in Mueller-Hinton broth (Becton, Dickinson & Co., Maryland, USA) in accordancewith Clinical and Laboratory Standards Institute (CLSI) recommendations and reading criteria in
96-well plates for immunological studies2). Substances of antibacterial products with known activity were used to prepare working antibiotic solutions. The following antibiotics were tested:Feb. 2016

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43（ 43 ）ampicillin, cefotaxime, ceftazidime, cefepime, cefoperazone, cefoperazone/sulbactam (ﬁxed ratioof 2 : 1), ﬂomoxef, imipenem, meropenem, amikacin, gentamicin, and ciproﬂoxacin. Flomoxef
was gifted from Shionogi & Co., Ltd. (Osaka, Japan) and other antibiotics were obtained from
Molekula GmbH (Munich, Germany). The following ranges of ﬁnal antibiotic concentrations in
the wells were used to determine the MIC: 0.063 to 64 μg/mL for ampicillin, cefotaxime, ceftazidime, cefepime, cefoperazone, ﬂomoxef, amikacin, gentamicin and ciproﬂoxacin; 0.063/0.032 to64/32 μg/mL for cefoperazone/sulbactam; 0.008 to 64 μg/mL for imipenem and meropenem. Each
new series of plates underwent three types of testing: testing of the suitability of the antibacterial
agents used with Escherichia coli strain ATCC 25922, monitoring of the growth of the strains
under the test conditions, and monitoring of the sterility of the substrate applied (Mueller-Hintonbroth).
Detection of ESBL-producing strain
ESBL production among E. coli, Klebsiella spp. and Proteus mirabilis was detected by themethod recommended by the CLSI3). In screening for ESBL production, if the MIC of cefotaxime
or ceftazidime was ≥2 μg/mL, the tested strain was classiﬁed as Screening (＋). Otherwise it wasclassiﬁed as Screening (−). For conﬁrmation of ESBL production, the ceftazidime MIC against
Screening (＋) strains was compared with the MIC of ceftazidime in combination with clavulanate (ﬁxed clavulanate concentration of 4 μg/mL). A ≥8-fold (≥3 twofold dilution) decrease in
the MIC of ceftazidime in combination with clavulanate versus the MIC of ceftazidime wasdeﬁned as ESBL (＋).

ResultsEscherichia coli
The results of testing E. coli susceptibility to antibiotics are presented in Table 1. Most of theisolates were non-susceptible to ampicillin, over 70% of the isolates showed MIC ＞64 μg/mL.
MIC50 values of the cephalosporins cefotaxime, ceftazidime, cefepime and cefoperazone were
64 μg/mL, 16 μg/mL, 8 μg/mL and 64 μg/mL, respectively. The MIC50 of cefoperazone/sulbactam
was 8 μg/mL, which was 8 times lower than that of cefoperazone alone. A synergistic effect of cefoperazone with a beta-lactamase inhibitor (sulbactam) was observed. The MIC50 of ﬂomoxef
was 1 μg/mL and its mode of isolates in the susceptibility distribution was at 0.125 μg/mL.
The isolates were most susceptible to carbapenems with MIC50 of 0.25 μg/mL for imipenem
and ≤0.063 μg/mL for meropenem.
Klebsiella spp.
The susceptibility of Klebsiella spp. is presented in Table 2. MIC50 values of cephalosporins,

Table 2. Susceptibility distribution of Klebsiella spp.

Table 1. Susceptibility distribution of E. coli44（ 44 ）THE JAPANESE JOURNAL OF ANTIBIOTICS
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69―145（ 45 ）cefotaxime, ceftazidime, cefepime and cefoperazone were ＞64 μg/mL, 64 μg/mL, 32 μg/mL and＞64 μg/mL, respectively. The MIC50 of cefoperazone/sulbactam was 32 μg/mL, which was lower
than that of cefoperazone alone. The MIC50 of ﬂomoxef was 2 μg/mL and its mode of isolates inthe susceptibility distribution was at 0.125 μg/mL.
The isolates were most susceptible to carbapenem antibiotics with MIC50 of 0.5 μg/mL toimipenem and 0.125 μg/mL to meropenem. The susceptibility distribution of Klebsiella spp.
showed some carbapenem-resistant isolates (MIC ≥4 μg/mL). The resistance rate was 14%(27/187) to imipenem and 16% (30/188) to meropenem.
Proteus spp.
The results of Proteus spp. were separately described by indole production in Table 3. One
was an indole-negative strain, P. mirabilis, and the other was an indole-positive group. Of the 52
isolates, 43 were P. mirabilis.
Most of the P. mirabilis isolates were non-susceptible to ampicillin, over 70% were showing
MIC ≥64 μg/mL. MIC50 values of cephalosporins, cefotaxime, ceftazidime, cefepime and cefoperazone were 32 μg/mL, 2 μg/mL, 4 μg/mL and 32 μg/mL, respectively. Addition of sulbactam tocefoperazone resulted in a MIC50 shift to a lower value. The MIC50 of cefoperazone alone, 32 μg/mL, was shifted to 1 μg/mL by addition of sulbactam. The MIC50 of ﬂomoxef was 0.5 μg/mL andits susceptibility distribution pattern was similar to cefepime; both modes of isolates in the susceptibility distribution were at 0.125 μg/mL.
The indole-positive group was less susceptible to antibiotics than P. mirabilis. MIC50 ofcefotaxime, ceftazidime, cefepime, cefoperazone and ﬂomoxef were ＞64 μg/mL, 8 μg/mL, 8 μg/mL, 64 μg/mL and 32 μg/mL, respectively.
Among the Proteus spp., only one isolate (P. mirabilis) was resistant to meropenem (MIC ≥4 μg/mL) but 15 isolates including 3 indole-positive isolates were resistant to imipenem (MIC ≥4 μg/mL). Lower susceptibility to imipenem, which is a natural feature of Proteus species, wasobserved.
Enterobacteriaceae encoding chromosomal AmpC beta-lactamases
Table 4 presents results for Citrobacter spp., Enterobacter spp., Serratia spp. and Providencia
spp. Bacteria of these species were classiﬁed into one group based on the mechanism of resistance to beta-lactam antibiotics. Bacteria in this group have inducible gene expression of chromosomal AmpC beta-lactamases4). MIC50 of cephalosporins, cefotaxime, ceftazidime, cefepime andcefoperazone were ＞64 μg/mL, 8 μg/mL, 8 μg/mL and ＞64 μg/mL, respectively. The MIC50 ofcefoperazone with sulbactam was 8 μg/mL, which was 16 times lower than that of cefoperazonealone. The MIC50 of ﬂomoxef against these species was 4 μg/mL and the susceptibility distribution was as broad as that of the cepharosporins.

Table 3-B. Susceptibility distribution of Proteus spp. (indole-positive group)

Table 3-A. Susceptibility distribution of Proteus mirabilis46（ 46 ）THE JAPANESE JOURNAL OF ANTIBIOTICS
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Screening (＋): MIC of ceftazidime ≥2 μg/mL or cefotaxime ≥2 μg/mL.
ESBL (＋): ≥8-fold decrease in MIC of ceftazidime in combination with clavulanate versus their MICof ceftazidime alone among Screening (＋) strains.
Carbapenem resistant contains strains of MIC of meropenem ≥4 μg/mL or imipenem ≥4 μg/mL.

Table 5-A. Detection of ESBL-producing strain

Table 4. Susceptibility distribution of Enterobacteriaceae encoding chromosomal AmpC beta-lactamases*
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Table 5-C. Susceptibility distribution of ESBL (＋) strains

Table 5-B. Activity of ﬂomoxef and reference antibiotics for Screening (＋) and Screening (−)
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69―149（ 49 ）The MIC50 of carbapenems was 0.5 μg/mL for imipenem and ≤0.063 μg/mL for meropenem.
Detection and susceptibility of ESBL-producing strain
Comparison of the susceptibility distribution between cefoperazone and that of cefoperazone
with sulbactam indicated the presence of ESBL-producing strains in E. coli, Klebsiella spp. andProteus spp. To further understand the results, we classiﬁed possible ESBL producers and carbapenem-resistant strain producers (Table 5-A).
For ESBL screening, cephalosporin non-susceptible isolates were classiﬁed as Screening(＋). For ESBL conﬁrmation, isolates showing synergy between ceftazidime and clavulanate in
Screening (＋) were deﬁned as ESBL (＋). The ESBL (＋) detection rate was 45% in E. coli,
48% in Klebsiella spp. and 17% in Proteus species.
The carbapenem-resistant rate was 3% for E. coli, 16% for Klebsiella spp. and 29% for Proteus spp. Most of them (39/49) were included in the ESBL (−) subgroup, which did not show
synergy between ceftazidime and clavulanate (data not shown).
To clarify the unique activity of ﬂomoxef among cephems, we compared activities against
Screening (＋) and Screening (−) subgroups (Table 5-B). The MIC50 of cephems, except for ﬂomoxef, against the Screening (＋) subgroup was 32 μg/mL or higher. Intriguingly, the MIC50 ofﬂomoxef was 2 μg/mL. The activities of ﬂomoxef and major beta-lactams against ESBL (＋) arepresented in Table 5-C. Meropenem was the most potent with an MIC50 ≤0.063 μg/mL and themode of the isolates in the susceptibility distribution was at ≤0.063 μg/mL. The MIC50 of ﬂomoxef was 1 μg/mL and the mode of the MIC distribution was 0.125 μg/mL.

Discussion
This study showed that beta-lactam non-susceptible Enterobacteriaceae had spread widely inintensive care units and surgical units in Russia. The obtained results generally correspond to
those published earlier concerning the spread of ESBL in Russia. In 1999, it was reported that theprevalence of ESBL among Klebsiella spp. was more than 40%5), and in 2009, this parameterreached 67%6).
In the settings with high prevalence of ESBL-producer in Russia, ﬂomoxef retained activity
against cephalosporin-resistant isolates of Enterobacteriaceae. The advantages of ﬂomoxef overother cephalosporins can be explained by its resistance to ESBL hydrolysis7). In Eastern Asia,
ﬂomoxef is recently being re-evaluated in the face of globally spreading ESBL-producing bacteria8〜11). Of course, it should be noted that ﬂomoxef is not a universal antibiotic. The range ofﬂomoxef MIC50 against Enterobacteriaceae, except for the indole-positive Proteus group, is0.5–4 μg/mL, which is lower than those of cepharosporins. However, the MIC90 values ofﬂomoxef in this study against E. coli, Klebsiella spp. and Enterobacteriaceae encoding chromo-50（ 50 ）THE JAPANESE JOURNAL OF ANTIBIOTICS

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somal AmpC beta-lactamases were ＞64 μg/mL. It has been reported from Taiwan that high rates
of resistance were observed for ﬂomoxef among Enterobacteriaceae carrying AmpC betalactamases and/or carbapenemase such as metallo-beta-lactamase or KPC (Klebsiella pneumoniae carbapenemase)12). The non-susceptible strains in our study could carry those beta lactamases.
Our ﬁndings show that ﬂomoxef may be considered as a treatment option of community- andhospital-acquired localized infections caused by Enterobacteriaceae in Russia. This should help
prevent the overuse of carbapenems.
Acknowledgements
We thank all investigators who participated in this study and all medical facilities that submitted bacterial strains for this study. We also thank T. YAMAGUCHI, Dr. T. SATO, and Dr. H. MAKIof Discovery Research Laboratory for Core Therapeutic Areas, Shionogi & Co., Ltd. for helpful
discussions and Dr. J. SHIMADA of Shionogi & Co., Ltd. Counselor for comments that greatly improved the manuscript.
Ethical approval: Not required.
Conﬂicts of interest
This study was conducted at Scientiﬁc Research Institute of Childrenʼs Infections under thecontract research for Nycomed Russia/CIS.
SATOSHI KOJIMA is an employee of Shionogi & Co., Ltd.

References
1) JONES, R. N.; M. FLONTA, N. GURLER, et al.: Resistance surveillance program report for selectedEuropean nations (2011). Diagn. Microbiol. Infect. Dis. 78: 429〜436, 2014
2) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically;
Approved standard—ninth edition M07-A9, CLSI, Wayne, PA. 20123) Clinical and Laboratory Standards Institute. Performance standard for antimicrobial susceptibility
testing. Nineteenth Informational Supplement Document M100-S22. CLSI, Wayne, PA. 20124) LIVERMORE, D. M.: beta-Lactamases in laboratory and clinical resistance. Clin. Microbiol. Rev. 8:557〜584, 1995
5) SIDORENKO, S. V.; L. S. STRACHUNSKII, L. I. AKHMEDOVA, et al.: The results of a multicenter study ofthe comparative activity of cefepime and other antibiotics against the causative agents of severe
hospital infections (the Micromax program). Antibiot. Khimioter. 44: 7〜16, 1999
6) KOZLOV, R. S. & A. A. NIKULIN: Ertapenem—a new carbapenem. Clin. Microbiol. Antimicrob.
Chemoth. 11: 40〜55, 2009
7) JACOBY, G. A. & I. CARRERAS: Activities of β-lactam antibiotics against Escherichia coli strains
producing extended spectrum β-lactamases. Antimicrob. Agents Chemother. 34: 858〜862, 1990
8) ITO, A.; Y. M. TATSUMI, T. WAJIMA, et al.: Evaluation of antibacterial activities of ﬂomoxef against

Feb. 2016

9)

10)

11)
12)

THE JAPANESE JOURNAL OF ANTIBIOTICS

69―151（ 51 ）ESBL producing Enterobacteriaceae analyzed by Monte Carlo Simulation. Jpn. J. Antibiotics 66:71〜86, 2013
YANG, Q-W.; H. ZHANG, J-W. CHENG, et al.: Flomoxef showed excellent in vitro activity against
clinically important Gram-positive and Gram-negative pathogens causing community- andhospital-associated infections. Diagn. Microbiol. Infect. Dis. 81: 269〜274, 2015
YANG, Q-W.; H. ZHANG, J-W. CHENG, et al.: In vitro activity of ﬂomoxef and comparators against
Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis producing extended-spectrum
β-lactamases in China. Int. J. Antimicrob. Agents 45: 485〜490, 2015
CUI, L-Q.; Y. LI, Y. Lv, et al.: Antimicrobial resistance surveillance of ﬂomoxef in China. J. Infect.
Chemother. 21: 402〜404, 2015
WANG, J-T.; U-I. WU, T-L.Y. LAUDERDALE, et al.: Carbapenem-nonsusceptible Enterobacteriaceae
in Taiwan. PLoS ONE 10: e0121668, 2015