Abstract
Thirty-eight AmpC-producing Klebsiella pneumoniae isolates identified from January to October 2006 in a large teaching hospital in the Czech Republic were analyzed. The AmpC cephalosporinase was identified as DHA-1, encoded by a plasmid-located complex class 1 integron, previously observed in a K. pneumoniae isolate from the Parisian region. The DHA-1 expression was inducible, and although in two isolates with higher resistance, the induction effect was masked at the phenotypic level. All of the isolates belonged to the international K. pneumoniae clone sequence type 11, split into two disseminated pulsed-field gel electrophoresis types. This is the first report on enterobacteriaceae with acquired AmpCs in the Czech Republic and possibly the first description of organisms with DHA-1 in the Central and Eastern Europe.
Introduction
In the Czech Republic, resistance of K. pneumoniae to newer generation cephalosporins has been frequent, with nearly 50% of invasive isolates collected within the European Antibiotic Resistance Surveillance System (EARSS) in 2006–2008 demonstrating resistance (www.rivm.nl/earss). By phenotypic observations, this resistance has been mediated by extended-spectrum β-lactamases (ESBLs) or by AmpC cephalosporinases (P. Urbá šková, unpublished data). The aim of this study was to analyze in detail the first K. pneumoniae isolates classified by susceptibility testing as probable AmpC producers in the Czech Republic.
Materials and Methods
Clinical isolates
The University Hospital in Plzeň is one of the largest Czech hospitals having ∼1,800 beds. In January 2006, the hospital laboratory recorded the first K. pneumoniae isolate resistant to cefoxitin and with reduced susceptibility to ceftazidime and cefotaxime showing the induction-like effect in the ESBL double-disk test 8 (blunted zones around the cefotaxime and ceftazidime disks from the side of the amoxicillin with clavulanate disk). By the end of October 2006, 36 nonduplicate isolates with such phenotype were retained for the further analysis, including all isolates of that kind identified in a survey from the mid-September to the end of October. Two other isolates (isolates 5 and 21) were resistant to cefoxitin, cefotaxime, and ceftazidime without any distortion of the zones around the cephalosporins' disks. Susceptibility of the all isolates to cefotaxime and ceftazidime was restored in the presence of 250 μg/ml cloxacillin (Polfa Tarchomin, Warsaw, Poland), which inhibits AmpC type β-lactamases. 8 Species was identified by the ENTEROtest 12 (Pliva Lachema Diagnostika, Brno, Czech Republic). Basic clinical information concerning the isolates is shown in Table 1.
HICU, hematooncological ICU; IMICU, internal medicine ICU; NICU, neurosurgical ICU; SICU, surgical ICU; BAL, bronchoalveolar lavage; AMP, ampicillin; AMS, ampicillin with sulbactam; ATM, aztreonam; CAZ, ceftazidime; CIP, ciprofloxacin; CTX, cefotaxime; FEP, cefepime; GEN, gentamicin; MEM, meropenem; PIP, piperacillin; TET, tetracycline; TZP, piperacillin with tazobactam; PFGE, pulsed-field gel electrophoresis; MIC, minimal inhibitory concentrations.
Susceptibility testing
Minimal inhibitory concentrations (MICs) of antimicrobials were determined by broth dilution as proposed by the European Committee for Antimicrobial Susceptibility Testing (EUCAST) and interpreted according to its guidelines 9 (www.eucast.org).
Plasmid transfer and replicon typing
Conjugation assays were performed as described previously, 3 with Escherichia coli A15 resistant to rifampin as a recipient. Selection was conducted on plates containing 64 μg/ml cefoxitin and 128 μg/ml rifampin (Sigma, St. Louis, MO). Plasmid DNA was purified with a NucleoBond® Xtra Midi kit (Machery-Nagel, Duren, Germany) and electroporated into E. coli DH5α cells. Transformants were selected with 64 μg/ml cefoxitin. The polymerase chain reaction–based replicon typing (PBRT) of plasmids was carried out according to Carattoli et al. 4
β-lactamase identification
β-lactamases were profiled by isoelectric focusing as described previously, 3 and observed with 0.5 mM nitrocefin (Oxoid, Basingstoke, United Kingdom), and with 0.5 mM nitrocefin with 0.3 mM cloxacillin. The multiplex PCR assay for ampC-like genes was performed according to Pérez-Pérez and Hanson. 17 The entire blaDHA-type genes were amplified and sequenced as described by Verdet et al. 26 Nucleotide sequences were determined using an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA), and analyzed by Lasergene Version 7.1.0 software (DNASTAR, Madison, WI) and the BLASTn option at the NCBI Web site (www.ncbi.nlm.nih.gov).
Genetic context of the blaDHA-1 gene
The genetic context of blaDHA-1 was analyzed by PCR mapping as described by Verdet et al. 26 Amplicons corresponding to the ampR gene and integronic gene cassettes aac(6′)-Ib-cr, blaOXA-1, catB3, and arr3 were sequenced using consecutive primers.
Typing
The isolates were typed by pulsed-field gel electrophoresis (PFGE) 23 using the restriction enzyme XbaI (Fermentas, Vilnius, Lithuania); the results were interpreted according to Tenover et al. 24 Multilocus sequence typing (MLST) was performed as described by Diancourt et al. 6 ; the database at www.pasteur.fr was used for assigning sequence types (STs).
β-lactamase-specific activity
The β-lactamase-specific activity (μmol of cephalothin hydrolyzed per 1 min by 1 mg of total protein) was measured in crude cell extracts by UV spectrophotometry. 13 The extracts were obtained from bacterial cultures grown without and with 64 μg/ml cefoxitin (AmpC inducer), and the assay was repeated with extracts preincubated for 5 min with 250 μmol/ml cloxacillin.
Results
Antimicrobial susceptibility
The results are shown in Table 1. For the 36 isolates with the induction phenotype, the cefotaxime, ceftazidime, and aztreonam MICs were 0.5–2, 2–16, and 1–2 μg/ml, respectively, whereas for the two highly resistant isolates 5 and 21, these values exceeded 16 μg/ml. On the other hand, there was no significant difference in cefepime MICs between the two types of isolates. All of the isolates were susceptible to meropenem and gentamicin but resistant to ciprofloxacin.
Plasmid transfer and replicon typing
The mating experiment did not yield transconjugants for any of the isolates. Plasmid DNA electroporation, performed for randomly chosen isolates 6, 11, and 15, produced E. coli transformants with increased MICs of cefotaxime and ceftazidime (data not shown). DNA of the transformants was subjected to PBRT; however, despite repeated attempts none of the replicon types included in the scheme were identified.
β-lactamase identification
The isoelectric focusing analysis revealed β-lactamases with isoelectric points (pIs) of 7.9, 7.6, and 7.4 in all of the isolates, whereas the transformants produced only the 7.9 and 7.4 enzymes. The pI 7.9 β-lactamases were not stained upon cloxacillin, indicating their AmpC type, and the multiplex PCR detected the blaDHA-type gene in all of the isolates. Sequencing of the entire gene from nine random isolates identified the DHA-1 enzyme. 2 Subsequently, PCR and sequencing revealed the pI 7.4 and 7.6 β-lactamases to be OXA-1 and SHV-1, respectively.
Genetic context of the blaDHA-1 gene
The PCR mapping of the region surrounding blaDHA-1 was performed for isolates 17 and 38 representing two different PFGE types (see below). The M. morganii ampR gene was identified next to blaDHA-1, with sequence identical to the one by Muratani et al. 15 and differing by one nucleotide from that reported originally 2 (the Glu90Gly amino acid change). The M. morganii DNA resided in a complex class 1 integron highly resembling that in the K. pneumoniae strain RBDHA from 2002 from the Paris region. 26 A large insertion (∼10 kb) with parts of operons sap and psp was present between ISCR1 and the M. morganii fragment. In the gene cassette array, the first cassette aac(6′)-Ib-cr differed by a single mutation from aac(6′)-Ib in RBDHA, and it was followed by identical cassettes blaOXA-1, catB3, and arr3. The region downstream the second copy of the sul1 gene had the RBDHA structure up to the IS6100 element. 26
Typing
Two PFGE types, A and B, with a high degree of similarity (83.9%) were found (Table 1). Type A grouped 31 isolates, including the 2 highly resistant isolates, and was split into 15 subtypes with subtype A6 prevailing (13 isolates); type B had 7 isolates in 4 subtypes. The MLST analysis was carried out for 11 isolates of both PFGE types. All these isolates were classified to ST11.
Isolates 5 and 21
Isolates 5 and 21 showed a higher level of resistance to β-lactams and lack of the phenotypic induction effect. The β-lactamase-specific activity was measured in extracts of these two isolates and two selected inducible isolates. For the inducible isolates 17 and 38 the activity increased by 8.54 and 3.59 upon cefoxitin, respectively, whereas for the highly resistant isolates 5 and 21 these ratios were 2.18 and 6.47, respectively. Preincubation of the extracts with cloxacillin resulted in >95% inhibition of the activity, confirming that most of that was produced by DHA-1. These results indicated that in the highly resistant isolates DHA-1 expression was inducible. Moreover, the expression levels in the isolates 5 and 21 were comparable to those in the isolates 17 and 38 (data not shown).
Discussion
This work documents further the geographic spread of K. pneumoniae producing plasmid-mediated DHA-1. Organisms with DHA-type β-lactamases have been identified in several survey or population studies, often in Asia and North America,1,7,12,15,16,21,28 and different kinds of analyses in European or nearby countries.11,14,19,26 To our knowledge, this is the first report of DHA-producing isolates of K. pneumoniae in Central and Eastern Europe, and one of the few descriptions of nosocomial outbreaks due to these organisms.20,22,25 The study demonstrated a high dissemination potential of K. pneumoniae DHA-1 in the Czech hospital; the first isolate was recorded in January 2006, and between the mid-September and the end of October 2006 as many as 25 patients were infected or colonized with that. Among all K. pneumoniae blood isolates recovered in the center over the whole 2006 (n = 72), such isolates were more frequent (n = 25) than ESBL producers (n = 17) (T. Bergerová, J. Hrabák, unpublished data).
The predominance of the PFGE type A isolates indicated the clonal character of the spread, while the remarkable diversity of PFGE patterns in both types suggests rapid diversification. Located on a nonconjugative and PBRT-non-typeable plasmid(s), 4 the blaDHA-1-carrying class 1 integron had a complex structure identified 4 years earlier in a K. pneumoniae strain in France, originating from one of several blaDHA-1 escapes from the M. morganii chromosome. 26 Interestingly, the outbreak strain was able to segregate variants with increased resistance and the induction effect masked; mechanism might rely on permeability alterations and/or efflux. All of the isolates were of the ST11 clone, which underlines the role of K. pneumoniae international clones in the spread of resistance. ST11 forms with ST258 and ST270 the clonal complex CC11 of intercontinental distribution [www.pasteur.fr]. It has been one of the main CTX-M-15-producing epidemic K. pneumoniae clones in Hungary, 5 and in 2006 it was identified in the same hospital in Plzeň with the SHV-5 ESBL. 10 The lack of the MLST data on the previously reported K. pneumoniae with DHA-1 makes it difficult to suggest any possible source of its emergence in the Czech Republic. The more recent observations indicate the continuing spread of this or similar organisms in the country. The EARSS-CZ data for 2008 reported 47.6% of all invasive K. pneumoniae isolates to be resistant to expanded-spectrum cephalosporins [www.rivm.nl/earss], 16.9% of which (n = 120) had the AmpC-mediated phenotype, being present in almost each hospital participating in the program (P. Urbášková, unpublished data).
Footnotes
Acknowledgments
The study reported here was partially financed by the research project grants MSMT 2E08003 and MSM 0021620819 from the Ministry of Education, Czech Republic, and by the grant PBZ-MNiSW-04/I/2007 from the Ministry of Science and Higher Education, Poland.
Disclosure Statement
No competing financial interests exist.