Surveillance and outbreak reports Carbapenemase-producing Klebsiella pneumoniae in the Czech Republic in 2011

J Hrabák (Jaroslav.Hrabak@lfp.cuni.cz)1, C C Papagiannitsis1, V Študentová1, V Jakubu2, M Fridrichová2, H Zemlickova2, Czech Participants of European Antimicrobial Resistance Surveillance Network3 1. Department of Microbiology, Faculty of Medicine and University Hospital in Plzeň, Charles University in Prague, Plzeň, Czech Republic 2. National Reference Laboratory for Antibiotics, National Institute for Health, Prague, Czech Republic 3. The members of the network are listed at the end of the article


Spread of carbapenemase-producing
Enterobacteriaceae and Pseudomonas spp.has been observed in many countries across the world [1][2][3].Carbapenemase producers are usually resistant to almost all of the effective antibiotics (such as betalactams, aminoglycosides, fluoroquinolones).Therapy of infections caused by such bacteria is limited to few choices (such as colistin and/or a combination therapy) with unpredictable effect [4].Therefore, prevention of their spread in healthcare settings and in the community is a big challenge for medicine today.

Beta-lactamase identification, bla gene environment mapping
Detection of bla genes, encoding important carbapenemase types, was performed by PCR using specific primers for bla OXA-48 , bla IMP , bla NDM , bla VIM and bla KPC [2,[16][17][18].The gene environment of bla KPC was determined by PCR mapping as proposed by Naas et al. [17].Mapping of the VIM-encoding integrons was performed by PCR [16].For detection of bla CMY -type genes, a PCR assay was employed [19].PCR products were sequenced on both strands.

Conjugation and transformation
To check transferability of the resistance genes on a conjugative plasmid, conjugal transfer was carried out by broth mating, using rifampin-resistant Escherichia coli A15 as previously described [20].Transconjugants were selected with 50 mg/L ampicillin and 60 mg/L rifampin.Transformation experiments were performed with plasmid extracts, purified using a Qiagen Plasmid Maxi Kit (Qiagen GmbH, Hilden, Germany), and E. coli DH5alpha chemically competent cells as a recipient.Transformants were selected with 50 mg/L ampicillin.

Plasmid analysis
Plasmid content was visualised after S1 linearisation followed by PFGE separation [21].Localisation of bla VIM , bla KPC and bla CMY genes was analysed by hybridisation.The bla-specific probes were prepared from PCR amplicons using a BrightStar Psoralen-Biotin kit (Applied Biosystems, Prague, Czech Republic).DNA after S1 linearisation and PFGE separation was transferred on BrightStar-Plus Positively Charged Nylon Membrane (Applied Biosystems, Prague, Czech Republic) according to manufacturer recommendations, and hybridised for 24 h at 42 °C.Detection of membranes was performed by BrightStar BioDetect Kit (Applied Biosystems, Prague, Czech Republic).PCR-based replicon typing (PBRT) of plasmids was performed as proposed by Carattoli et al. [22], using total DNA from transconjugants/transformants or from clinical isolates that were non-successful in conjugation and transformation experiments.IncF plasmids were further characterised by replicon sequence typing (RST) [23].Plasmids carrying bla KPC were identified by PCR mapping as proposed by Baraniak et al. [24].

Results
MALDI-TOF MS meropenem hydrolysis assay confirmed carbapenemase activity in 15 of the 102 isolates analysed.Ethylene-diamine tetra-acetic acid (EDTA)-meropenem combined disk test confirmed metallo-beta-lactamase production in six of the isolates.The respective aminophenylboronic acid-meropenem test was positive for KPC production in the remaining nine isolates.All of the suspected isolates based on the phenotypic tests were positive in MALDI-TOF MS meropenem hydrolysis assay.

VIM-producing isolates
Five of the six VIM-1-producing K. pneumoniae were isolated from one hospital (A5) in Prague (Table 1).In all five isolates, MICs of meropenem were in the susceptible category according to the EUCAST criteria, ranging from 1 to 2 mg/L.The five isolates were resistant to all antibiotics tested, except colistin.The variable region of their class 1 integron containing bla VIM-1 gene is described in Table 1.Neither transconjugants nor transformants were obtained from any of the five isolates detected in hospital A5.A bla VIM -specific probe hybridised strongly with a band corresponding to the chromosomal material, which confirmed the chromosomal location of the bla VIM-1 -containing integron.All isolates belonged to ST11, which is a common clone of K. pneumoniae that possesses extended spectrum (ESBL)-and AmpC-beta-lactamases [25,26].The sixth VIM-4-producing strain was detected in October 2011 in a patient admitted to the hospital in the Czech Republic after the medically assisted repatriation from a hospital in Northern Greece.Carbapenemresistant K. pneumoniae (isolate no.V624; Table 1) was isolated from blood immediately after the admission to the hospital.The isolate belonged to ST1029, a novel sequence type, which is a single locus variant (SLV) of ST383 and was first reported in Greece in 2009 [19].The strain produced VIM-4 and CMY-4 beta-lactamases as described in ST383 by Papagiannitsis et al. [19].However, no production of KPC enzyme was identified in our strain, contrary to the Greek strain.The class 1 integron consisting of a sole bla VIM-4 gene cassette was harboured by a conjugative plasmid of A/C replicon type.A similar plasmid harbouring bla VIM-1 was described by Samuelsen et al. in a patient repatriated from Greece in 2005 [27].Immediately after the isolation of the carbapenem-resistant K. pneumoniae isolate, recommended isolation precautions were set up in the hospital and no transfer of the strain to another patient was found.

KPC-producing isolates
In the Czech Republic, the first KPC-producing K. pneumoniae isolate was obtained from a patient repatriated from a hospital in Italy to hospital A41 in Prague in July 2011.A carbapenem-resistant isolate producing KPC-3 was cultivated from a urine sample (isolate no.V514; Table 2).From August till December, five more KPC-3producing K. pneumoniae strains were identified in different patients.Their molecular and epidemiological characteristics are summarised in Table 2. Three of these patients were hospitalised on the same ward as, but without direct contact to, the index case, while the remaining two patients were hospitalised in the same time period but in different hospital wards (Figure 1).
Another KPC-producing isolate was recovered from a patient repatriated from a hospital on Greece to hospital A6 in Prague (isolate no.V597; Table 2).The strain was recovered from a blood sample.A second patient (isolate no.V640; Table 2) hospitalised in the same room as the previous one, was colonised with a strain of the same PFGE pattern and ST.The last case was detected in hospital A51 in Prague.This strain (isolate no.V601; Table 2) was obtained from the respiratory tract of a patient repatriated from a hospital on Crete (Greece).No spread to other patients was detected.No difference was detected in the PFGE patterns of ST258 and ST512 isolates.
According to the EUCAST criteria, the detected KPCproducing isolates were susceptible only to gentamicin (Table 3).MICs of colistin, which is sometimes the drug of the last choice in carbapenemase-producing Enterobacteriaceae infections, were in the resistant category (8-16 mg/L).Plasmid profiling with S1 linearisation of all clinical isolates showed a common profile with plasmids approximately 40, 110 and 200 kb in size [24].All KPC-producing isolates harboured bla KPC -positive plasmids of similar size (approximately 110 kb).Those bla KPC -encoded plasmids were negative for all replicon sequences included in the PBRT panel.However, by the RST method, the KPC-encoding plasmids were positive for the FII k replicon.Using PCRbased mapping, the plasmids were identified as the pKpQIL type [24].Both bla KPC-2 and bla KPC-3 were part of the transposon Tn4401, isoform a.No transconjugants were obtained from KPC producers.KPC-encoding plasmids were only transferred by transformation of plasmid DNA obtained from isolate V597.MICs of the transformant are shown in Table 3.All of the patients repatriated to the Czech Republic had been hospitalised in intensive care units in the countries they were repatriated from.In two hospitals in the Czech Republic (A6 and A51), isolation precautions were set up immediately after the identification of carbapenem-resistant K. pneumoniae isolate.

Discussion
Carbapenemase-producing enterobacteria seem to be uncommon in the Czech Republic with only three reported cases in the period of 2009 and 2010 and six cases in 2012 [1,5,7].In 2011, two outbreaks and a few cases of VIM-and KPC-producing K. pneumoniae were reported.The K. pneumoniae species was the only member of the Enterobacteriaceae family found to produce carbapenemases in that year in the Czech Republic.We believe that the situation is not underestimated because, since the mandatory official guideline was issued by the Ministry of Health in 2012, all carbapenem-resistant enterobacteria have been sent to the NRL for Antibiotics for confirmation of carbapenemase production and epidemiological typing.
The situation of VIM-1-producing K. pneumoniae in the hospital A5 seems to have been endemic.Even if no epidemiological connection among the isolates could be found (such as hospitalisation on the same ward, use of the same medical procedure or the same medical personnel), most of them were recovered the same time period between May and October 2011 (Table 1, Figure 2).Therefore, the occurrence of these isolates could be considered as an outbreak, but we were not able to identify an index case nor reservoir of the strains.Therefore, our hypothesis was based on molecular typing of the isolates only.
The increasing incidence of KPC-producing K. pneumoniae observed in the Czech Republic in 2011 was initially caused by the repatriation of infected patients from Italy (KPC-3, ST512) and Greece (KPC-2, ST258), followed by an outbreak with an ST512 strain in Hospital A41.All isolates showed identical PFGE patterns and belonged to ST512, supporting the theory of an outbreak.
This ST is a single locus variant of a widely spread KPC-2-producing ST258 clone.ST512 was first reported from Israel among the isolates producing KPC-3 carbapenemase [28].All KPC-producing isolates detected in this study were resistant to colistin.Resistance to this drug in KPC-producing K. pneumoniae isolates is being described more and more frequently [29,30].Treatment options for infections caused by carbapenemase-producing Enterobacteriaceae are seriously limited until new classes of antibiotics are found; therefore it is necessary to understand the epidemiological principles of the spread of such bacteria and to set up efficient infection control measurements.
It can be assumed that the repatriated patients acquired the carbapenemase-producing Enterobacteriaceae in the foreign countries, since their transport was organised through specialised medical assistance and they were admitted to a Czech hospital without delay.Molecular typing data also confirm this theory.In all of the described patients, screening (such as rectal swab, sputum, urine, wound swab etc.) for identification of carbapenemase-producing Enterobacteriaceae was performed in the intensive care units abroad in a way corresponding to what is recommended in the national guidelines issued by the NRL for Antibiotics [31].
Until mid-2012, there was no official document in the Czech Republic on isolation precautions for patients colonised or infected by carbapenemase-producing Enterobacteriaceae.However, recommendations regarding diagnostic procedures, screening, and specific hygienic measurements were available from the NRL for Antibiotics [31].Recently, an official guideline for the management of imported cases of carbapenemase-producing Enterobacteriaceae including infection control procedures has been approved and published through a bulletin of the Ministry of Health of the Czech Republic [32].In this document, screening procedures on medical wards with confirmed occurrence of carbapenemase-producing Enterobacteriaceae are described in detail.The recommended screening is based on rectal swabs collected from patients hospitalised on the same ward or in possible contact with an infected or colonised patient.Other tissues sampled for standard screening in intensive care units (such as sputum, urine, different swabs) should also be tested for carbapenemase-producing Enterobacteriaceae.For patients with suspected or proven carbapenemaseproducing Enterobacteriaceae, strict isolation procedures have to be set up.
In 2012 and 2013, there has not been a further increase in the occurrence of carbapenemase-producing Enterobacteriaceae in the Czech Republic, and only one outbreak (five patients) and four sporadic cases have been noted until mid-2013 (data not shown).An almost similar number of carbapenem-non-susceptible isolates has been sent for confirmation of carbapenemase production from routine laboratories in 2012 as in 2011.Only two imported cases of VIM-1-producing K. pneumoniae and NDM-4-producing Enterobacter cloacae have been detected [33].This situation signalises that the proposed preventive recommendations have been able to stabilise or even decrease the incidence of carbapenemase-producing Enterobacteriaceae in our country.

Table 2
Characterisation of KPC-producing Klebsiella pneumoniae isolates recovered from Czech hospitals in 2011 (n=9)

Table 3
Susceptibility of carbapenemase-producing Klebsiella pneumoniae and their transconjugants/transformants, Czech Republic, 2011 (n=7) TRAN V597: transformant of V597.As the MICs of isolates of the same clone were similar, we show in the Table only representative isolates of each clone and their transformant/ transconjugant. b