research article detection of carbapenemase...

Research ArticleDetection of Carbapenemase-Producing Enterobacteriaceae inthe Baltic Countries and St Petersburg Area

Anastasia Pavelkovich12 Arta Balode3 Petra Edquist4 Svetlana Egorova5

Marina Ivanova2 Lidia Kaftyreva5 Irina Konovalenko6 Siiri Kotildeljalg1 Jana Lillo1

Lidia Lipskaya7 Jolanta Miciuleviciene8 Kristiine Pai1 Kristel Parv1 Katri Paumlrna1

Tiiu Roumloumlp1 Epp Sepp1 Jelena Štšepetova1 and Paul Naaber19

1 Department of Microbiology University of Tartu Ravila 19 50411 Tartu Estonia2 East-Tallinn Central Hospital Ravi 18 10138 Tallinn Estonia3 Rıga Stradins University 16 Dzirciema Street Rıga LV-1007 Latvia4 Smittskyddsinstitutet Folkhalsomyndigheten 171 82 Solna Sweden5 Institut Pasteur in Saint Petersburg Ul Mira 14 Saint Petersburg 197101 Russia6 St Petersburg Hospital No 31 Pr Dinamo 3 Saint Petersburg 197110 Russia7 St Petersburg Hospital No 40 Ul Borisova 9 Sestroretsk Saint Petersburg 197706 Russia8Vilnius City Clinical Hospital Antakalnio Street 57 LT-10007 Vilnius Lithuania9Quattromed HTI Laboratories Vaike-Paala 1 11415 Tallinn Estonia

Correspondence should be addressed to Paul Naaber paulnaaberquattromedee

Received 4 December 2013 Revised 18 January 2014 Accepted 21 January 2014 Published 4 March 2014

Academic Editor Karmen Torkar

Copyright copy 2014 Anastasia Pavelkovich et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The spread of carbapenemase-producing Enterobacteriaceae is a global problem however no exact data on the epidemiology ofcarbapenemase in theBaltic countries and St Petersburg area is availableWe aimed to evaluate the epidemiology of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in the Baltic States and St Petersburg Russia and to compare the differentmethods for carbapenemase detection From January to May 2012 all K pneumoniae (119899 = 1983) and E coli (119899 = 7774) clinicalisolates from 20 institutions in Estonia Latvia Lithuania and St Petersburg Russia were screened for carbapenem susceptibilityThe IMP VIM GIM NDM KPC and OXA-48 genes were detected using real-time PCR and the ability to hydrolyze ertapenemwas determined using MALDI-TOF MS Seventy-seven strains were found to be carbapenem nonsusceptible From these 15 Kpneumoniae strains hydrolyzed ertapenem and carried the 119887119897119886NDM gene All of these strains carried integron 1 and most carriedintegron 3 as well as genes of the CTX-M-1 group No carbapenemase-producing E coli or K pneumoniae strains were foundin Estonia Latvia or Lithuania however NDM-positive K pneumoniae was present in the hospital in St Petersburg Russia AMALDI-TOF MS-based assay is a suitable and cost-effective method for the initial confirmation of carbapenemase production

1 Introduction

Antibiotic resistance remains a major global public healthproblem that leads to increasing healthcare costs extralength of hospital stay and treatment failures [1] The recentEuropean Antimicrobial Resistance Surveillance Network(EARS-Net) report indicates that although the occurrence ofantibiotic resistance in gram-positive pathogens appears to

be stabilizing or even decreasing in some countries Europe-wide increase in antimicrobial resistance in gram-negativepathogens such asEscherichia coli andKlebsiella pneumoniaeis occurring [1]

An emerging problem is the spread and increasing preva-lence of carbapenemase-producing gram-negative bacteriaFor example outbreaks of Klebsiella pneumoniae carbapen-emase (KPC)-positive K pneumoniae occurred in the USA

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 548960 7 pageshttpdxdoiorg1011552014548960

2 BioMed Research International

in 2001 and subsequently spread throughout the world NewDelhi metallo-120573-lactamase (NDM-1)-positiveK pneumoniaewas imported from India and spread to the United Kingdomin 2010 [2] As high as a 50 prevalence of metallo-120573-lacta-mases were reported in K pneumoniae blood isolates inGreece [3]

In a recent European study most participants declaredsporadic cases or outbreaks of carbapenemase-producingEnterobacteriaceae [4] Although carbapenem nonsuscepti-ble E coli and K pneumoniae cases have also been reportedin the Baltic states no exact epidemiological data are avail-able and these cases were not confirmed using molecularmethods

The detection of carbapenemases in clinicalmicrobiologylabs is challenging [5] because phenotypic tests are time con-suming and difficult to interpret and the molecular methodsare not available in routine diagnostic labs in our regionAdditionally the increasing number of new carbapenemasesmakes molecular tests unsuitable for the initial detectionof carbapenemase production Recently a MALDI-TOF MS(matrix-assisted laser desorption ionization-time of flightmass spectrometry) assay has been described as a potentiallyuseful tool for the detection of120573-lactamase activity [6 7]Thistechnique is based on the detection of 120573-lactams degradationproducts However this method has not yet been introducedin routine diagnostics or validated in different geographicalregions

The aim of our study was to evaluate the epidemiology ofcarbapenemases in the Baltic countries and St Petersburgarea by applying different mass-spectrometry andmolecular-based detection methods

2 Materials and Methods

21 Collection of Strains A total of 20 institutions from thefollowingBaltic countries and St Petersburg area participatedin our study Estonia (119899 = 5) Lithuania (119899 = 3) Latvia(119899 = 4) and the Saint-Petersburg region in Russia (119899 = 8)From January 1 to May 31 2012 all isolated E coli andK pneumoniae nonduplicated strains were screened forcarbapenem nonsusceptibility according to European Com-mittee of Antimicrobial Susceptibility Testing (EUCAST 20applied in Baltic states) or Clinical and Laboratory StandardInstitute (CLSI 2011 applied in Russia) standards in the par-ticipating labs Carbapenem nonsusceptibility to ertapenemandor meropenem was detected using disc-diffusion orbroth dilution methods Strains from all clinically relevantmaterials that were routinely sent to these microbiology labswere included Environmental and surveillance samples werenot included in our study In total 9757 strains were isolatedand screened for carbapenem nonsusceptibility and includedE coli from hospitalized patients (119899 = 4799) and outpatients(119899 = 2975) and K pneumoniae from hospitalized patients(119899 = 1631) and outpatients (119899 = 352) All screening positivestrains were sent to and further investigated in the Estoniancentral lab

22 Strains Data Institution (numbers of beds admissionsand patient days) strain-related (sampling date origin of

isolationmaterial and resistance pattern detected in thelocal labs) and patient-related data (patient age outpatientor hospitalized patient and department type) were alsocollected for the carbapenem nonsusceptible strains

23 Phenotypic Description of Strains Species identificationof these strains was confirmed in the central lab usingMALDI-TOF MS (Maldi Biotyper Bruker Daltonics GmbHGermany) Carbapenem nonsusceptibility was confirmedusing the agar-gradient method with ertapenem mero-penem and imipenem strips (Liofilchem Italy) on Muller-Hinton agar (Oxoid Limited UK) according to the manu-facturerrsquos recommendations andwas interpreted according toEUCAST standards (httpwwweucastorg)

24 Mass-Spectrometry-Based Confirmation of Carbapene-mase Production Carbapenem nonsusceptible strains wereanalyzed using a mass-spectrometry-based semi quantitativeresistance assay method that was described previously byBurckhardt and Sparbier [8 9] and modified according toDr Katrin Sparbierrsquos recommendations (personal communi-cation)

Bacterial strains were grown for 18 to 24 h on 5 sheepblood agar plates (Microlabor Estonia) at 36∘CThe cells werethen resuspended in thirtymicroliters of 045NaCl solutionwith or without 1 glitre ertapenem (MSD USA) in 15-mLEppendorf tubes and the amount of bacteria filled a 1-120583L-inoculation loop (corresponding to 3ndash6 times 108 cells 1mL of aMcFarland 1-2)The suspension was incubated for 2 h at 37∘Cunder agitation and then centrifuged for 2min at 13000 rpmat 20∘C One microliter of the cleared supernatant was addedto each target spot and dried at room temperature and1 120583L of matrix (HCCA [120572-cyano-4-hydroxycinnamic acid]high-pressure liquid chromatography [HPLC] grade FlukaGermany) was added to each target spot

MALDI-TOF MS analysis was performed using aMicroflex LT instrument (Bruker Daltonics GmbH Ger-many) and 96-spot polished-steel targets The specificallymodified protocol was used (0 to 1 000Da 12 to 17 laserintensity) For one spectrum approximately 200 to 300 shotswere summed and the HCCA peak at 379Da was used forcalibration The mass spectrum between 438 and 530Da wasanalyzed using 30 software flexAnalysis (Bruker DaltonicsGmbH Germany) Carbapenem nonsusceptible strains wereanalyzed in six reaction sets and each set contained positive(carbapenemase-producing strain) and negative controls(strain incapable of carbapenemase production) and duringeach reaction set the characteristic mass spectrum of pureertapenem was measured

An automated evaluation algorithm for the MALDI-TOF MS-based functional 120573-lactamase assay (MSBL assay)was implemented using Bruker Daltonics software prototype(which is underdevelopment and not yet commercially avail-able) that was employed in cooperation with Sparbier et al[10] This software automatically calculates the logarithm ofthe sum ratio of the intensity of all peaks that correspondedto the hydrolyzed form and the sum of the intensities ofall peaks that corresponded to the non-hydrolyzed form of

BioMed Research International 3

the antibiotic (logRQ) The results were displayed in a boxplot diagram [10] This automated evaluation was used as acontrol method for the visual analysis of the spectra

25 Molecular Characterization of Strains Total bacteriaDNA from the clinical samples was extracted using theQIAamp DNA Mini Kit (Qiagen Germany) Plasmid DNAwas extracted using the NucleoSpin Plasmid Quick Pure kit(Macherey-Nagel France)

Genes encoding IMP VIM GIM NDM OXA-48 andKPC-group carbapenemases were detected using a multiplexreal-time PCR reaction that was based on the protocolspublished previously by Mendes et al [11] Poirel et al [12]Samuelsen et al [13] and Chen et al [14] Amplification wasperformed in a 20-120583Lmixture that contained 10120583L of a PowerSYBR Green PCRMaster Mix (Applied Biosystems UK) andsix pairs of primers (concentration rate is 10120583M) in 3 differentPCRs (Table 1(a)) using Applied Biosystems StepOnePlusReal-Time PCR system (Life Technologies USA) The PCRconditions were as follows 1 cycle at 50∘C for 2min initialdenaturation at 94∘C for 5min 35 cycles of 94∘C for 20 s 53∘Cfor 45 s for the blaIMP blaVIM and blaGIM genes or 60∘C for45 s for the blaOXA-48 blaNDM and blaKPC genes and 72∘C for30 s and amelt curve step at 95∘C for 15 s 60∘C for 1min and95∘C for 15 s

The presence of different blaCTX-M subgroups was deter-mined using a multiplex real-time PCR protocol [15] thatdisplayed the group designation (CTX-M-1-2-9 and 825) ofthe blaCTX-M-positive isolates The method used one primerpair that was specific for all four groups Four TaqMan probeswere included in the reactions and bound to PCR productsaccording to the CTX-M subgroup

To detect integrons real-time PCRwas performed as pre-viously described [16] with a reaction mixture of 25 120583L thatcontained 5120583L ofDNA template 125 120583L of TaqMan universalmaster mix (Applied Biosystem USA) 6mMMgCl

2 04 120583M

of each primer (int1-(LC1-LC5) int2-(LC2-LC3) and int3-(LC1-LC2)) and 02 120583M of each probe (int1 int2 and int3Table 1(b)) The programme was as follows 10 min initialstep at 95∘C followed by 45 cycles at 95∘C for 30 sec and60∘C for 1min and it was performed using a 7500 real-timePCR detection system (Applied-Biosystem USA) and opticalgrade 96-well plates Assayswere performed in triplicate anda negative control was included on each plate

26Quality Control Strains In the carbapenemases detectionstudies (MALDI-TOF based assay and real-time PCR) thefollowing control strains were used Swedish Institute forCommunicable Disease Control (Sweden) carbapenemasesdetection control set including IMP-positive Pseudomonasaeruginosa Ps540 VIM-positiveK pneumoniaeCCUG58547GIM-positive P aeruginosa NDM-1-positive K pneumo-niae K275 OXA-48-positive K pneumoniae Oxa241 KPC-positive K pneumoniae K271 and MBL-positive Klebsiellapneumoniae KSKS2823 and EuSCAPE (European surveyon carbapenemase-producing Enterobacteriaceae) projectquality control set (provided by NEQAS) including KPC-positive K pneumoniae 1940 KPC-2-positive K pneumoniae

0

5

10

15

20

25

30

35

LT EERULV

Carb

apen

em n

onsu

scep

tible

stra

ins (

)

Figure 1 Percentages of carbapenem nonsusceptible Klebsiellapneumoniae strains in particular hospitals (dots) and the countryaverages (lines) LT Lithuania LV Latvia RU Russia St Petersburgand EE Estonia

1944 KPC-3-positive K pneumoniae 1942 OXA-48-positiveK pneumoniae 1943 VIM-1-positive K pneumoniae 1945NDM-1-positiveK pneumoniae strains 1946 and 1948 IMP-1-positiveK pneumoniae 1947 and NDM-1-positive Enterobac-ter aerogenes 1941

In CTX-M detection studies we used E coli CCUG55971(CTX-M-1 group) E coli CCUG55972 (CTX-M-2) E coliCCUG55970 (CTX-M-9) and E aerogenes isolate Rio3(CTX-M-825) as positive controls These strains were pro-vided from Swedish Institute for Communicable DiseaseControl

For integron detections the following strains were usedas positive templates E colipBadint1 E colipGEMTeasyint2 and E colipBadint3 provided from the Department ofBacteriology of University of Limoges (France)

For negative controls wild-type E coli andK pneumoniaeclinical strains were used

3 Results

According to initial screening and identification confirma-tion 80 isolates appeared to be nonsusceptible to carbape-nem After minimal inhibitory concentration (MIC) confir-mation using the agar-gradient method 77 strains had MICvalues above the epidemiological cut-off according to theEUCAST standard for any tested carbapenem (nonwild typestrains) including 73 K pneumoniae and 4 E coli strainsThe prevalence of the carbapenem nonsusceptible strains inthe institutions of the participating countries is shown inFigure 1

31 Carbapenemase ProductionConfirmation byMALDI-TOFMS Of the 77 phenotypically carbapenem nonsusceptiblestrains 15 were able to hydrolyze ertapenem as detected bythe MSBL assayThe spectra of all carbapenemase-producingstrains showed the hydrolyzed decarboxylated product ofertapenem (4505Da) which corresponded to high-strengthcarbapenemase (Figure 2) The visual- and software-basedevaluations of the results gave the same results An example of


Table 1 (a) Primers for the detection of carbapenemase-encoding genes (b) Primers and probes used for multiplex real-time PCR detectionof classes 1 2 and 3 integrons

(a)

Reaction number Primers Oligonucleotide sequence (51015840-31015840) Volume for reaction

PCRMix 1

IMP (forward) GAATAGRRTGGCTTAAYTCTC 25 120583LIMP (reverse) CCAAACYACTASGTTATC 25 120583LVIM (forward) GTTTGGTCGCATATCGCAAC 025 120583LVIM (reverse) AATGCGCAGCACCAGGATAG 025 120583LGIM (forward) TCAATTAGCTCTTGGGCTGAC 025 120583LGIM (reverse) CGGAACGACCATTTGAATGG 025 120583L

PCRMix 2

NDM (forward) CAATATTATGCACCCGGTCG 05 120583LNDM (reverse) ATCATGCTGGCCTTGGGGAA 05 120583L

OXA 48 (forward) GCGTGGTTAAGGATGAACAC 05 120583LOXA 48 (reverse) CATCAAGTTCAACCCAACCG 05 120583L

PCRMix 3

KPC (forward) GGCAGTCGGAGACAAAACC 05 120583LKPC (reverse) CCCTCGAGCGCGAGTCTA 05 120583L

(b)

Primersprobes Oligonucleotide sequence (51015840-31015840) LocationintI1-LC1 GCCTTGATGTTACCCGAGAG intI1intI1-LC5 GATCGGTCGAATGCGTGTintI2-LC2 TGCTTTTCCCACCCTTACC intI2intI2-LC3 GACGGCTACCCTCTGTTATCTCintI3-LC1 GCCACCACTTGTTTGAGGA intI3intI3-LC2 GGATGTCTGTGCCTGCTTGintI1-probe ATTCCTGGCCGTGGTTCTGGGTTTT intI1intI2-probe TGGATACTCGCAAACAAGTTATTTTTACGCTG intI2intI3-probe CGCCACTCATTCGCCACCCA intI3

the ertapenem hydrolyzation spectra visualised with softwareprototype is presented in Figure 3

32 Molecular Detection of Carbapenemase-Encoding GenesWhen analysing these 77 carbapenem nonsusceptible isolatesfor carbapenemase-encoding genes we found 15 isolatesthat were positive for the blaNDM gene All of the other 62isolateswere negative for the blaIMP blaVIM blaGIM blaOXA-48blaNDM or blaKPC genes The 15 isolates with the NDM genewere also found to hydrolyze ertapenem by the MSBL assay

33 Characterization of Strains All carbapenemase produc-ers were identified as K pneumoniae and were isolated fromone St Petersburg (Russia) hospital (Table 2) Most of thesestrains were isolated from intensive care patients and wereisolated from different clinical materials All of these strainscarried integron 1 and most carried integron 3 and the CTX-M-1 group of genes All strains were resistant to all of thetested carbapenems fluoroquinolones and aminoglycosidesWhen comparing the minimal inhibitory concentrationranges of the carbapenemase producers and nonproducerswe found that the producers had imipenem MICs ge8 120583gmLand nonproducers had MICs le4 120583gmL For the other car-bapenems the ranges overlapped

4 Discussion

This was the first study to investigate carbapenem resistancein the Baltic countries and St Petersburg area that includedpatients and sample types from the multiple largest medicalinstitutions in Estonia Latvia Lithuania and St Petersburgand to apply molecular methods for characterization ofstrains We found high variation in carbapenem nonsus-ceptibility (MICs above the epidemiological cut-off values)between different institutions within particular countries(highest in Russia from 0 to 30) However variationsbetween the country averages were not so remarkableAlthough we found carbapenem nonsusceptible strains inthe Baltic states (Estonia Latvia and Lithuania) as wasdescribed in previous EARS-Net reports none of these wereactually carbapenemase producersTherefore after screeningnearly 10000 E coli and K pneumoniae strains we conclu-de that carbapenemases are not yet a problem in Balticcountries In these carbapenem nonsusceptible strains someother mechanisms for example production of CTX-M aswell as acquired cephalosporinases along with porin loss orchanges in efflux are suspected In these cases carbapenemresistance was usually low (relatively low MICs) and thusinfections could be treatedwith higher doses of carbapenemsThese strains appear to exhibit low spreading potential


449995times10

4

1005

400 420 440 460 480 500 520 540

Ert inc pos contr 0 A3 MS raw

Inte

nse (

au)

mz

(a)

450377476251

498338

times104

10

05

00

Ert inc II 0 D2 MS raw

400 420 440 460 480 500 520 540Inte

nse (

au)

mz

(b)

450080

476032

497980

519930

600040002000

0

Ert 0 A1 MS raw

400 420 440 460 480 500 520 540Inte

nse (

au)

mz

(c)

450250

0

8000600040002000

Ert inc rus280 0 D7 MS raw

400 420 440 460 480 500 520 540Inte

nse (

au)

mz

(d)

450165476076

498025

times104

151005

Ert inc neg contr 0 A4 MS raw

400 420 440 460 480 500 520 540

Inte

nse (

au)

mz

(e)

Figure 2 MALDI-TOF MS spectrum showing ertapenem and itsdegradation products Spectrum related to (a) positive control(Klebsiella pneumoniae metallo-120573-lactamase positive) hydrolyzeddecarboxylated product of ertapenem (4505Da) (b) incubatedertapenem solution hydrolyzed decarboxylated product ofertapenem (4505Da) ertapenem molecule (4765Da) andertapenem sodium adduct (4985Da) (c) pure ertapenem solutionhydrolyzed decarboxylated product of ertapenem (4505Da)ertapenem molecule (4765Da) ertapenem sodium adduct(4985Da) and ertapenem disodium adduct (5205Da) (d) NewDelhi Metallo-120573-lactamase-positive strain (Klebsiella pneumoniae)hydrolyzed decarboxylated product of ertapenem (4505Da)(e) negative control (noncarbapenemase-producing Klebsiellapneumoniae) hydrolyzed decarboxylated product of ertapenem(4505Da) ertapenemmolecule (4765Da) and ertapenem sodiumadduct (4985Da)

On the contrary carbapenemase producers are associatedwith therapy failures outbreaks and increased mortality[17] However our study revealed high prevalence of NDM-type carbapenemase-producing K pneumoniae from oneSt Petersburg hospital Infection control and antimicrobial

Log R

Q

Erti

ncco

ntro

lEr

tinc

eeitk

b297

Erti

ncee

itkb 298

Erti

ncru

s 132

Erti

ncru

s134

Erti

ncru

s 173

Erti

ncru

s218

Erti

ncru

s260

Erti

ncru

s 265

Erti

ncru

s266

Erti

ncru

s 269

Erti

ncru

s 279

Erti

ncru

s 301

Erti

ncru

s 73

Erti

ncru

s 78

Erti

ncru

s84

Ertapenem

10

05

00

Figure 3 Evaluation of the MALDI-TOFMS spectra of 16 differentKlebsiella pneumoniae strains using the new software prototypeDifferences in the hydrolysis rates of the different strains can easilybe detected green area no hydrolysis yellow area intermediatehydrolysis red area hydrolysis

resistance surveillance institutions of neighboring BalticStates should be alerted when spreading of this strain occurs

In our study we also evaluated different methods suchas MALDI-TOF MICs of different carbapenems and real-time PCR for the detection of carbapenemases in E coli andK pneumoniae isolates In this set of strains we found goodcorrelation between functional assay such as the MALDI-TOF assay and molecular detections of specific carbapene-mase genes Thus from a sensitivity and specificity point ofview we found these methods to be equal However all ofthese methods have some advantages and disadvantages asdiscussed below The limitation of our study for evaluatingthe different methods was the epidemiological situation inthe studied region where only one type (NDM) of carbapen-emase was present

Whereas the molecular methods and the usage of car-bapenems and the combinations of carbapenems and car-bapenemase inhibitors have been available for years forcarbapenemase detection the MALDI-TOF assay is a novelmethod that is not fully standardized and has not beentested for different strains sets and epidemiological sit-uations However carbapenem resistance detection usingMALDI-TOF MS has many advantages when comparedto synergyinhibition-based phenotypic methods or PCRFirst depending on the type of carbapenemase the resultscan be available as soon as 2-3 h after the start of incuba-tion This is especially useful in an outbreak situation whenthe carbapenemase has already been identified The princi-ple of degradation-product monitoring is universal for thedetection of other enzymatic resistance mechanisms suchas extended spectrum 120573-lactamases (ESBL) Second thismethod is comparatively easy to perform [8] Third thecost per determination is relatively low and is less than oneEUR per reaction [8] The only problem may be the costof the equipment However MALDI-TOF MS has becomeincreasingly used in routine diagnostic labs over recent yearsfor the identification of pathogens


Table 2 Comparison of carbapenem nonwild type Klebsiella pneumoniae strains with and without carbapenemase production

Carbapenemaseproducers(119899 = 15)

Carbapenemasenonproducers

(119899 = 58)

Country

Russia 15 7Estonia 0 37Latvia 0 8

Lithuania 0 6

Department

Intensive care 14 8Surgical 0 16Medical 1 31

Outpatients 0 3

Material

Blood 1 1Puswound 2 11

Urine 5 39Lower respiratory 7 6Upper respiratory 0 1

Genes detected (number ofpositive strains)

NDM 15 0IMP VIM GIM KPC or OXA48 0 0

CTX-M-1 group 13 50CTX-M-2 group 0 2CTX-M-8 or 9 0 0Integron 1 15 52Integron 2 0 1Integron 3 111 251

MIC values mgL(rangemedian)

Ertapenem ge322 0125ndashge3262

Meropenem 4ndashge32123 0032ndash4053

Imipenem 8ndashge32324 0094ndash4054

Resistance (numbers119877 + 119868tested strains)

Ciprofloxacin 1515 4951Gentamycin 15155 34495

1

119875 = 004 234119875 = 0001 5119875 = 001

Unfortunately a semiquantitative assay does not allowfor specification of the enzyme Currently a software toolsupporting an automated evaluation is under developmentwhich will be able to calculate the hydrolysis capacity (BrukerDaltonicsGmbHGermany) [18] Aprototype of this softwarewas also used in our study to compare it with the manualmethod and it was easy to use and gave reliable results withour strain set

PCR is the fastest way to determine which family of120573-lactamase is present However PCR techniques are notused in the majority of routine laboratories because of highcosts absence of real-time equipment and qualified per-sonnel In addition this method can detect only previouslydescribed enzymes Mutations in a carbapenemase-encodinggene could lead to negative results

We found that of the carbapenem nonsusceptible strainsmost carbapenemase producers and nonproducers carriedthe CTX-M 1 group of ESBL genes and class 1 integronsAlthough the class 3 integron was also found in carbapene-mase nonproducers it was more common in NDM-positiveK pneumoniae strains In previous studies a high prevalence

of class 1 integrons in K pneumoniae and its relation withNDM genes have been reported [19ndash21] However rela-tions between carbapenem nonsusceptibility (carbapene-mase related or nonrelated) and the presence of class 3integrons have not yet been described and require furtherinvestigation

In conclusion carbapenemase-producing E coli or Kpneumoniae were not found in Baltic countries but NDM-positive K pneumoniae was present in St Petersburg Inour current epidemiological and economic situation theMALDI-TOF assay seems to be suitable and cost-effectivemethod for the initial confirmation of carbapenemase pro-duction

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper


Acknowledgments

The authors are grateful to Ruta Ambrazaitiene GintarasMakstutis Dace Rudzite Tatjana Djundika Nataliya Ved-ernikova Olga Morozova Tatyana Kurchikova Maria Pay-setchkaya Marina Smirnova Svetlana Rudenko Irina Zolo-tuhhina Kaisa Kirs Krista Loivukene Natalja Kamoninaand Anna Tisler for their help in strain collection Theywould also like to thank Dr Katrin Sparbier and ChristophLange (Bruker Daltonics GmbH Germany) for assistance inthe performance and interpretation of the MALDI-TOF MSassay Dr O Barraud (Department of Bacteriology MedicalFaculty University of Limoges France) who kindly providedintegron-positive control strains the Swedish Institute forCommunicable Disease Control for the set of carbapene-mase and ESBL-positive control strains and methodologyThis study was supported by grants of the Baltic Antibi-otic Resistance collaborative Network (BARN) EuropeanUnion through the European Regional Development Fund(ARMMD Project no 32070111-0013) Estonian Ministry ofEducation and Research (target financing no SF0180132s08)and Estonian Science Foundation (Grant no 9059)

References

[1] ldquoMultidrug antibiotic resistance increasing in Europerdquo ECDC2012 httpwwwecdceuropaeuenpressnews layoutsformsNews DispFormaspxID=563ampList=8db7286c-fe2d-476c-9133-18ff4cb1b568

[2] A Marra ldquoNDM-1 a local clone emerges with worldwideaspirationsrdquo Future Microbiology vol 6 no 2 pp 137ndash141 2011

[3] EARS-Net Annual Report 2011 httpwwwecdceuropaeuenpublicationspublicationsantimicrobial-resistance-surveil-lance-europe-2011pdf

[4] C Glasner B Albiger G Buist et al ldquoCarbapenemase-prod-ucing enterobacteriaceae in Europe a survey among nationalexperts from 39 countries February 2013rdquo Eurosurveillance vol18 no 28 article 3 2013

[5] P Nordmann M Gniadkowski C G Giske et al ldquoIdentifica-tion and screening of carbapenemase-producing Enterobacte-riaceaerdquo Clinical Microbiology and Infection vol 18 no 5 pp432ndash438 2012

[6] J Hrabak V Studentova RWalkova et al ldquoDetection of NDM-1 VIM-1 KPC OXA-48 and OXA-162 carbapenemases byMALDI-TOF mass spectrometryrdquo Journal of Clinical Microbi-ology vol 50 no 7 pp 2441ndash2443 2012

[7] LWangCHanW SuiMWang andX Lu ldquoMALDI-TOFMSapplied to indirect carbapenemase detection a validated pro-cedure to clearly distinguish between carbapenemase-positiveand carbapenemase-negative bacterial strainsrdquo Analytical andBioanalytical Chemistry vol 405 no 15 pp 5259ndash5266 2013

[8] I Burckhardt and S Zimmermann ldquoUsing matrix-assistedlaser desorption ionization-time of flight mass spectrometry todetect carbapenem resistance within 1 to 25 hoursrdquo Journal ofClinical Microbiology vol 49 no 9 pp 3321ndash3324 2011

[9] K Sparbier S Schubert U Weller C Boogen and MKostrzewa ldquoMatrix-assisted laser desorption ionization-timeof flight mass spectrometry-based functional assay for rapiddetection of resistance against 120573-lactam antibioticsrdquo Journal ofClinical Microbiology vol 50 no 3 pp 927ndash937 2012

[10] K Sparbier K Lange J Jung S Schubert and M KostrzewaldquoAn automated evaluation algorithm for then MALDI-TOFMS based functional 120573-lactamase assayrdquo ASMS A101 2013httpwwwbrukercomruproductsmass-spectrometry-and-separationsliteratureliterature-roomhtmleID=dam frontendpushampstream=1ampdocID=55242

[11] R E Mendes K A Kiyota J Monteiro et al ldquoRapid detectionand identification of metallo-120573-lactamase-encoding genes bymultiplex real-time PCR assay and melt curve analysisrdquo Journalof Clinical Microbiology vol 45 no 2 pp 544ndash547 2007

[12] L Poirel C Heritier V Tolun and P Nordmann ldquoEmergenceof oxacillinase-mediated resistance to imipenem in Klebsiellapneumoniaerdquo Antimicrobial Agents and Chemotherapy vol 48no 1 pp 15ndash22 2004

[13] Oslash Samuelsen C M Thilesen L Heggelund A N Vada AKummel and A Sundsfjord ldquoIdentification of NDM-1-prod-ucing Enterobacteriaceae in Norwayrdquo Journal of AntimicrobialChemotherapy vol 66 no 3 pp 670ndash672 2011

[14] L Chen K D Chavda J R Mediavilla et al ldquoMultiplex real-time PCR for detection of an epidemic KPC-producing Kleb-siella pneumoniae ST258 clonerdquo Antimicrobial Agents andChemotherapy vol 56 no 6 pp 3444ndash3447 2012

[15] C I Birkett H A Ludlam N Woodford et al ldquoReal-time Taq-Man PCR for rapid detection and typing of genes encodingCTX-M extended-spectrum 120573-lactamasesrdquo Journal of MedicalMicrobiology vol 56 part 1 pp 52ndash55 2007

[16] O BarraudM C Baclet F Denis andM C Ploy ldquoQuantitativemultiplex real-time PCR for detecting class 1 2 and 3 integronsrdquoJournal of Antimicrobial Chemotherapy vol 65 no 8 Article IDdkq167 pp 1642ndash1645 2010

[17] P Nordmann T Naas and L Poirel ldquoGlobal spread of car-bapenemase producing Enterobacteriaceaerdquo Emerging Infec-tious Diseases vol 17 no 10 pp 1791ndash1798 2011

[18] M Kostrzewa and D Orth ldquoBruker Daltonik GmbH MALDITOF based microbial identification for the 21st century andbeyondrdquo inProceedings of the 23rd EuropeanCongress of ClinicalMicrobiology and Infectious Diseases April 2013

[19] R Farzana S Shamsuzzaman and K ZMamun ldquoIsolation andmolecular characterization of New Delhi metallo-120573-lactamase-1 producing superbug in Bangladeshrdquo Journal of Infection inDeveloping Countries vol 7 no 3 pp 161ndash168 2013

[20] D Yong M A Toleman C G Giske et al ldquoCharacterizationof a new metallo-120573-lactamase gene bla NDM-1 and a novelerythromycin esterase gene carried on a unique genetic struc-ture in Klebsiella pneumoniae sequence type 14 from IndiardquoAntimicrobial Agents and Chemotherapy vol 53 no 12 pp5046ndash5054 2009

[21] S Derakhshan S N Peerayeh F Fallah B Bakhshi M Rahbarand A Ashrafi ldquoDetection of class 1 2 and 3 integrons amongKlebsiella pneumoniae isolated from children in Tehran hospi-talsrdquo Pediatric Infectious Diseases vol 1 no 4 pp 164ndash168 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


in 2001 and subsequently spread throughout the world NewDelhi metallo-120573-lactamase (NDM-1)-positiveK pneumoniaewas imported from India and spread to the United Kingdomin 2010 [2] As high as a 50 prevalence of metallo-120573-lacta-mases were reported in K pneumoniae blood isolates inGreece [3]

In a recent European study most participants declaredsporadic cases or outbreaks of carbapenemase-producingEnterobacteriaceae [4] Although carbapenem nonsuscepti-ble E coli and K pneumoniae cases have also been reportedin the Baltic states no exact epidemiological data are avail-able and these cases were not confirmed using molecularmethods

The detection of carbapenemases in clinicalmicrobiologylabs is challenging [5] because phenotypic tests are time con-suming and difficult to interpret and the molecular methodsare not available in routine diagnostic labs in our regionAdditionally the increasing number of new carbapenemasesmakes molecular tests unsuitable for the initial detectionof carbapenemase production Recently a MALDI-TOF MS(matrix-assisted laser desorption ionization-time of flightmass spectrometry) assay has been described as a potentiallyuseful tool for the detection of120573-lactamase activity [6 7]Thistechnique is based on the detection of 120573-lactams degradationproducts However this method has not yet been introducedin routine diagnostics or validated in different geographicalregions

The aim of our study was to evaluate the epidemiology ofcarbapenemases in the Baltic countries and St Petersburgarea by applying different mass-spectrometry andmolecular-based detection methods

2 Materials and Methods

21 Collection of Strains A total of 20 institutions from thefollowingBaltic countries and St Petersburg area participatedin our study Estonia (119899 = 5) Lithuania (119899 = 3) Latvia(119899 = 4) and the Saint-Petersburg region in Russia (119899 = 8)From January 1 to May 31 2012 all isolated E coli andK pneumoniae nonduplicated strains were screened forcarbapenem nonsusceptibility according to European Com-mittee of Antimicrobial Susceptibility Testing (EUCAST 20applied in Baltic states) or Clinical and Laboratory StandardInstitute (CLSI 2011 applied in Russia) standards in the par-ticipating labs Carbapenem nonsusceptibility to ertapenemandor meropenem was detected using disc-diffusion orbroth dilution methods Strains from all clinically relevantmaterials that were routinely sent to these microbiology labswere included Environmental and surveillance samples werenot included in our study In total 9757 strains were isolatedand screened for carbapenem nonsusceptibility and includedE coli from hospitalized patients (119899 = 4799) and outpatients(119899 = 2975) and K pneumoniae from hospitalized patients(119899 = 1631) and outpatients (119899 = 352) All screening positivestrains were sent to and further investigated in the Estoniancentral lab

22 Strains Data Institution (numbers of beds admissionsand patient days) strain-related (sampling date origin of

isolationmaterial and resistance pattern detected in thelocal labs) and patient-related data (patient age outpatientor hospitalized patient and department type) were alsocollected for the carbapenem nonsusceptible strains

23 Phenotypic Description of Strains Species identificationof these strains was confirmed in the central lab usingMALDI-TOF MS (Maldi Biotyper Bruker Daltonics GmbHGermany) Carbapenem nonsusceptibility was confirmedusing the agar-gradient method with ertapenem mero-penem and imipenem strips (Liofilchem Italy) on Muller-Hinton agar (Oxoid Limited UK) according to the manu-facturerrsquos recommendations andwas interpreted according toEUCAST standards (httpwwweucastorg)

24 Mass-Spectrometry-Based Confirmation of Carbapene-mase Production Carbapenem nonsusceptible strains wereanalyzed using a mass-spectrometry-based semi quantitativeresistance assay method that was described previously byBurckhardt and Sparbier [8 9] and modified according toDr Katrin Sparbierrsquos recommendations (personal communi-cation)

Bacterial strains were grown for 18 to 24 h on 5 sheepblood agar plates (Microlabor Estonia) at 36∘CThe cells werethen resuspended in thirtymicroliters of 045NaCl solutionwith or without 1 glitre ertapenem (MSD USA) in 15-mLEppendorf tubes and the amount of bacteria filled a 1-120583L-inoculation loop (corresponding to 3ndash6 times 108 cells 1mL of aMcFarland 1-2)The suspension was incubated for 2 h at 37∘Cunder agitation and then centrifuged for 2min at 13000 rpmat 20∘C One microliter of the cleared supernatant was addedto each target spot and dried at room temperature and1 120583L of matrix (HCCA [120572-cyano-4-hydroxycinnamic acid]high-pressure liquid chromatography [HPLC] grade FlukaGermany) was added to each target spot

MALDI-TOF MS analysis was performed using aMicroflex LT instrument (Bruker Daltonics GmbH Ger-many) and 96-spot polished-steel targets The specificallymodified protocol was used (0 to 1 000Da 12 to 17 laserintensity) For one spectrum approximately 200 to 300 shotswere summed and the HCCA peak at 379Da was used forcalibration The mass spectrum between 438 and 530Da wasanalyzed using 30 software flexAnalysis (Bruker DaltonicsGmbH Germany) Carbapenem nonsusceptible strains wereanalyzed in six reaction sets and each set contained positive(carbapenemase-producing strain) and negative controls(strain incapable of carbapenemase production) and duringeach reaction set the characteristic mass spectrum of pureertapenem was measured

An automated evaluation algorithm for the MALDI-TOF MS-based functional 120573-lactamase assay (MSBL assay)was implemented using Bruker Daltonics software prototype(which is underdevelopment and not yet commercially avail-able) that was employed in cooperation with Sparbier et al[10] This software automatically calculates the logarithm ofthe sum ratio of the intensity of all peaks that correspondedto the hydrolyzed form and the sum of the intensities ofall peaks that corresponded to the non-hydrolyzed form of







2 04 120583M



0

5

10

15

20

25

30

35

LT EERULV

Carb

apen

em n

onsu

scep

tible

stra

ins (

)






3 Results





(a)


PCRMix 1


PCRMix 2



PCRMix 3


(b)





4 Discussion



449995times10

4

1005

400 420 440 460 480 500 520 540


Inte

nse (

au)

mz

(a)

450377476251

498338

times104

10

05

00


400 420 440 460 480 500 520 540Inte

nse (

au)

mz

(b)

450080

476032

497980

519930

600040002000

0

Ert 0 A1 MS raw

400 420 440 460 480 500 520 540Inte

nse (

au)

mz

(c)

450250

0

8000600040002000


400 420 440 460 480 500 520 540Inte

nse (

au)

mz

(d)

450165476076

498025

times104

151005


400 420 440 460 480 500 520 540

Inte

nse (

au)

mz

(e)



Log R

Q

Erti

ncco

ntro

lEr

tinc

eeitk

b297

Erti

ncee

itkb 298

Erti

ncru

s 132

Erti

ncru

s134

Erti

ncru

s 173

Erti

ncru

s218

Erti

ncru

s260

Erti

ncru

s 265

Erti

ncru

s266

Erti

ncru

s 269

Erti

ncru

s 279

Erti

ncru

s 301

Erti

ncru

s 73

Erti

ncru

s 78

Erti

ncru

s84

Ertapenem

10

05

00









(119899 = 58)

Country


Lithuania 0 6

Department


Outpatients 0 3

Material












1

119875 = 004 234119875 = 0001 5119875 = 001









Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







2 04 120583M



0

5

10

15

20

25

30

35

LT EERULV

Carb

apen

em n

onsu

scep

tible

stra

ins (

)






3 Results





(a)


PCRMix 1


PCRMix 2



PCRMix 3


(b)





4 Discussion



449995times10

4

1005

400 420 440 460 480 500 520 540


Inte

nse (

au)

mz

(a)

450377476251

498338

times104

10

05

00


400 420 440 460 480 500 520 540Inte

nse (

au)

mz

(b)

450080

476032

497980

519930

600040002000

0

Ert 0 A1 MS raw

400 420 440 460 480 500 520 540Inte

nse (

au)

mz

(c)

450250

0

8000600040002000


400 420 440 460 480 500 520 540Inte

nse (

au)

mz

(d)

450165476076

498025

times104

151005


400 420 440 460 480 500 520 540

Inte

nse (

au)

mz

(e)



Log R

Q

Erti

ncco

ntro

lEr

tinc

eeitk

b297

Erti

ncee

itkb 298

Erti

ncru

s 132

Erti

ncru

s134

Erti

ncru

s 173

Erti

ncru

s218

Erti

ncru

s260

Erti

ncru

s 265

Erti

ncru

s266

Erti

ncru

s 269

Erti

ncru

s 279

Erti

ncru

s 301

Erti

ncru

s 73

Erti

ncru

s 78

Erti

ncru

s84

Ertapenem

10

05

00









(119899 = 58)

Country


Lithuania 0 6

Department


Outpatients 0 3

Material












1

119875 = 004 234119875 = 0001 5119875 = 001









Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



(a)


PCRMix 1


PCRMix 2



PCRMix 3


(b)





4 Discussion



449995times10

4

1005

400 420 440 460 480 500 520 540


Inte

nse (

au)

mz

(a)

450377476251

498338

times104

10

05

00


400 420 440 460 480 500 520 540Inte

nse (

au)

mz

(b)

450080

476032

497980

519930

600040002000

0

Ert 0 A1 MS raw

400 420 440 460 480 500 520 540Inte

nse (

au)

mz

(c)

450250

0

8000600040002000


400 420 440 460 480 500 520 540Inte

nse (

au)

mz

(d)

450165476076

498025

times104

151005


400 420 440 460 480 500 520 540

Inte

nse (

au)

mz

(e)



Log R

Q

Erti

ncco

ntro

lEr

tinc

eeitk

b297

Erti

ncee

itkb 298

Erti

ncru

s 132

Erti

ncru

s134

Erti

ncru

s 173

Erti

ncru

s218

Erti

ncru

s260

Erti

ncru

s 265

Erti

ncru

s266

Erti

ncru

s 269

Erti

ncru

s 279

Erti

ncru

s 301

Erti

ncru

s 73

Erti

ncru

s 78

Erti

ncru

s84

Ertapenem

10

05

00









(119899 = 58)

Country


Lithuania 0 6

Department


Outpatients 0 3

Material












1

119875 = 004 234119875 = 0001 5119875 = 001









Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


449995times10

4

1005

400 420 440 460 480 500 520 540


Inte

nse (

au)

mz

(a)

450377476251

498338

times104

10

05

00


400 420 440 460 480 500 520 540Inte

nse (

au)

mz

(b)

450080

476032

497980

519930

600040002000

0

Ert 0 A1 MS raw

400 420 440 460 480 500 520 540Inte

nse (

au)

mz

(c)

450250

0

8000600040002000


400 420 440 460 480 500 520 540Inte

nse (

au)

mz

(d)

450165476076

498025

times104

151005


400 420 440 460 480 500 520 540

Inte

nse (

au)

mz

(e)



Log R

Q

Erti

ncco

ntro

lEr

tinc

eeitk

b297

Erti

ncee

itkb 298

Erti

ncru

s 132

Erti

ncru

s134

Erti

ncru

s 173

Erti

ncru

s218

Erti

ncru

s260

Erti

ncru

s 265

Erti

ncru

s266

Erti

ncru

s 269

Erti

ncru

s 279

Erti

ncru

s 301

Erti

ncru

s 73

Erti

ncru

s 78

Erti

ncru

s84

Ertapenem

10

05

00









(119899 = 58)

Country


Lithuania 0 6

Department


Outpatients 0 3

Material












1

119875 = 004 234119875 = 0001 5119875 = 001









Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





(119899 = 58)

Country


Lithuania 0 6

Department


Outpatients 0 3

Material












1

119875 = 004 234119875 = 0001 5119875 = 001









Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article detection of carbapenemase...

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