Dissemination of Carbapenemase-Producing Enterobacteriaceae and Pseudomonas aeruginosa in Romania Laurent Dortet,a,b,c Mirela Flonta,d Yves-Marie Boudehen,b Elodie Creton,b Sandrine Bernabeu,a,b,c Anaïs Vogel,b Thierry Naasa,b,c Bacteriology-Hygiene Unit, Assistance Publique/Hôpitaux de Paris, Bicêtre Hospital, Le Kremlin-Bicêtre, Francea; Paris-Sud University, LabEx Lermit, Faculty of Medecine, Le Kremlin-Bicêtre, Franceb; Associated French National Reference Center for Antibiotic Resistance, Le Kremlin-Bicêtre, Francec; Clinical Hospital of Infectious Diseases, Microbiology Laboratory, Cluj-Napoca, Romaniad
Fifteen carbapenemase-producing Enterobacteriaceae isolates and 12 carbapenemase-producing Pseudomonas aeruginosa isolates were recovered from patients hospitalized between August 2011 and March 2013 at the Hospital of Infectious Disease, ClujNapoca, Romania. One KPC-, nine NDM-1-, four OXA-48-, and one VIM-4-producing Enterobacteriaceae isolates along with 11 VIM-2-producing and one IMP-13-producing P. aeruginosa isolates were recovered from clinical samples. All carbapenemase genes were located on self-conjugative plasmids and were associated with other resistance determinants, including extendedspectrum -lactamases and RmtC methylases.
arbapenem-hydrolyzing -lactamases have been increasingly reported worldwide among Enterobacteriaceae (1) and Pseudomonas spp. (2). The most clinically relevant carbapenemases encountered in Enterobacteriaceae belong to Ambler class A (KPC type), Ambler class B, metallo--lactamases (MBLs; e.g., IMP, VIM, and NDM types), or Ambler class D (OXA-48). In Pseudomonas aeruginosa, MBLs of VIM and IMP types are predominantly identified worldwide, whereas KPC-producing isolates were mostly described in South America (2). The aim of this study was to survey the occurrence and characterization of carbapenem-resistant Enterobacteriaceae and P. aeruginosa recovered at the Hospital for Infectious Diseases of Cluj-Napoca, Romania. From August 2011 to November 2013, carbapenem susceptibility was investigated for Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Serratia marcescens, and P. aeruginosa isolates that were recovered from clinical samples obtained from patients who had been hospitalized in three units of the hospital (Table 1). According to the EUCAST breakpoints (http://www.eucast.org), carbapenem-resistant isolates were recovered for 5.8% (36 of 625 isolates) of K. pneumoniae, 0.002% (2 of 1,114 isolates) of E. coli, 11.7% (10 of 85 isolates) of E. cloacae, 20.3% (16 of 79 isolates) of S. marcescens, and 55.9% (222 of 397 isolates) of the P. aeruginosa isolates. According to EUCAST guidelines for the detection of carbapenemase-producing Enterobacteriaceae (3), carbapenemase detection was performed using the Carba NP test in all Enterobacteriaceae with decreased susceptibility to carbapenems (inhibition zones of ⬍25 mm, ⬍23 mm, and ⬍25 mm to meropenem, imipenem, and ertapenem, respectively) and in 27 P. aeruginosa isolates that were resistant to carbapenems and ceftazidime (4, 5). Carbapenemase activity was detected in 5 K. pneumoniae, 1 E. coli, 5 E. cloacae, 4 S. marcescens, and 12 P. aeruginosa isolates (Table 1). All of those isolates were recovered from patients who had no history of travel outside Romania. Carbapenemase genes (blaKPC, blaVIM, blaIMP, blaNDM, and blaOXA-48) were sought by PCR (Table 1) and sequencing, as previously described (6). One E. coli, 4 E. cloacae, and the 4 S. marcescens isolates were positive for blaNDM-1. One KPCproducing and 4 OXA-48-producing K. pneumoniae isolates were identified. One E. cloacae isolate and 11 P. aeruginosa isolates were
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positive for blaVIM. Finally, 1 P. aeruginosa isolate produced an IMP-type carbapenemase. To evaluate their clonal relationship, 14 carbapenemase-producing enterobacterial isolates (n ⫽ 5, K. pneumoniae; n ⫽ 5, E. cloacae; and n ⫽ 4, S. marcescens) (see Fig. S1 in the supplemental material) and the 12 P. aeruginosa isolates (see Fig. S2) were subjected to Diversilab, a semiautomated repetitive sequence-based PCR (rep-PCR) (bioMérieux, La Balmes-Les-Grottes, France). A cutoff value of 95% similarity was used to discriminate independent clones, as recommended by the manufacturer. We identified 2 clones of K. pneumoniae, 3 clones of E. cloacae, 2 clones of S. marcescens, and 2 clones of P. aeruginosa, of which the VIM-2producing clone was predominant (Table 2). Multilocus sequence typing analysis was performed on carbapenemase-producing K. pneumoniae, E. coli, E. cloacae, and P. aeruginosa isolates, as previously described (7, 8) (Table 2). The KPC-2-producing K. pneumoniae isolates belonged to the pandemic ST258 clone (9). The OXA-48-producing K. pneumoniae isolates belonged to ST101 that had been prevalent among OXA48-producing K. pneumoniae isolated in Europe (10), including Romania (11). The NDM-1-producing E. coli isolates belonged to the successful and virulent clone ST131, as previously observed with a strain that originated from India (12, 13), Thailand (14), and Târgu-Mures¸, a town located in the central part of Romania (11). The NDM-1-producing E. cloacae isolates were of three different sequence types (STs) (ST93, ST171, ST255). Of note, the VIM-4-producing E. cloacae and the NDM-1-producing E. cloa-
Received 29 June 2015 Returned for modification 3 August 2015 Accepted 17 August 2015 Accepted manuscript posted online 24 August 2015 Citation Dortet L, Flonta M, Boudehen Y-M, Creton E, Bernabeu S, Vogel A, Naas T. 2015. Dissemination of carbapenemase-producing Enterobacteriaceae and Pseudomonas aeruginosa in Romania. Antimicrob Agents Chemother 59:7100 –7103. doi:10.1128/AAC.01512-15. Address correspondence to Laurent Dortet, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.01512-15. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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TABLE 1 Clinical features of the carbapenemase-producing isolates and patient characteristics Patient
Sexa
Age, yr
Hospitalization department
Species
Date of isolation (mo/day/yr)
Carbapenemase
Site of isolation
Antimicrobial treatmentb
Outcome
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
M M F M M M M F M M M F M M M M F F M M M M F M M M M
73 59 16 82 53 62 70 80 66 77 73 42 83 81 62 45 71 65 64 64 90 56 76 73 31 31 63
ICUc Ambulatory Surgery ICU Surgery Surgery Surgery Ambulatory Surgery ICU Infectious diseases Infectious diseases Surgery Infectious diseases Surgery ICU ICU Infectious diseases ICU ICU Infectious diseases Surgery Infectious diseases Infectious diseases Infectious diseases Infectious diseases Surgery
P. aeruginosa E. cloacae K. pneumoniae P. aeruginosa E. cloacae E. cloacae S. marcescens S. marcescens P. aeruginosa E. cloacae P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa K. pneumoniae P. aeruginosa P. aeruginosa S. marcescens P. aeruginosa K. pneumoniae K. pneumoniae K. pneumoniae S. marcescens P. aeruginosa P. aeruginosa E. coli E. cloacae
8/22/2011 9/26/2011 9/30/2011 10/6/2011 3/19/2012 8/14/2012 3/3/2013 4/2/2013 4/10/2013 4/24/2013 5/14/2013 6/25/2013 8/13/2013 8/20/2013 8/19/2013 8/19/2013 8/20/2013 9/11/2013 9/18/2013 9/19/2013 4/5/2013 11/7/2012 3/19/2013 10/21/2013 11/11/2013 11/11/2013 11/20/2013
VIM-2 NDM-1 KPC-2 VIM-2 VIM-4 NDM-1 NDM-1 NDM-1 VIM-2 NDM-1 VIM-2 VIM-2 IMP-13 VIM-2 OXA-48 VIM-2 VIM-2 NDM-1 VIM-2 OXA-48 OXA-48 OXA-48 NDM-1 VIM-2 VIM-2 NDM-1 NDM-1
Blood Urine Urine Blood Urine Urine Urine Urine Urine Urine Urine Urine Urine Urine Drain Urine Urine Urine Urine Wound Urine Urine Urine Urine Ear Ear Urine
Not available Not available Not available Not available Not available Not available Not available Not available Not available Not available GEN, COL IMP Not available IMP, GEN, COL Not available IMP, COL, CIP GEN Not available CRO, CIP CRO, CIP CRO, IMP, GEN, CIP Not available CRO, CIP, GEN MEM, GEN Not available Not available Not available
Improved Improved Improved Improved Improved Improved Improved Improved Improved Expired Improved Improved Improved Expired Improved Improved Improved Improved Improved Improved Improved Improved Improved Improved Improved Improved Improved
a
M, male; F, female. CIP, ciprofloxacin; COL, colistin; CRO; ceftriaxone; IMP, imipenem; GEN, gentamicin; MEM, meropenem. c ICU, intensive care unit. b
cae isolates were of the same clone, ST93, indicating that this clone might be prevalent in this hospital. The IMP-13-producing P. aeruginosa isolate belonged to a new ST, ST1982. This ST is closely related to the ST357 clone that was previously described to be associated with the regional dissemination of IMP-7 in central Europe (15), but it is not related to ST621, an ST currently spreading worldwide (16). The VIM-2-producing P. aeruginosa isolates belonged to a new ST, ST2026. This ST is close to the ST233 that was recently described to be associated with the dissemination of VIM-2 in Bucharest (17). Mating-out assays were performed using enterobacterial clinical strains as donors and the azide-resistant E. coli J53 as the recipient strain. E. coli transconjugants were obtained for all enterobacterial donor isolates (Table 2). Plasmid DNA of transconjugants was extracted by using the Kieser method, analyzed by agarose gel electrophoresis, and typed by using PCR-based replicon typing (18). The blaKPC-2 plasmids belonged to the IncFIIk group. All blaNDM-1-positive plasmids belonged to untypeable incompatibility groups. In addition, specific PCRs for the detection of IncN2 (IncN2-for: 5=-GTCTAACGAGCTTACCGA AG-3=, IncN2-rev: 5=-GTTTCAACTCTGCCAAGTTC-3=) and IncHI1B (IncHI1B-for: 5=-GGAGCGATGGATTACTTCAGTA C-3=, IncHI1B-rev: 5=-TGCCGTTTCACCTCGTGAGTA-3=) that were previously described to be prevalent in blaNDM-1-carrying plasmids also remained negative (14, 19). As previously observed, the rmtC gene, encoding a 16S RNA methylase responsible for resistance to all aminoglycosides, was located on the blaNDM-1-
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carrying plasmid (11). The IncL/M-type specific to the archetypal 61.8-kb self-conjugative plasmid harboring the blaOXA-48 gene was detected in all OXA-48 transconjugants by using primers previously described (20). The blaVIM-4 plasmids belonged to the IncFII group. Whole DNA extracted from P. aeruginosa clinical isolates was electroporated into an electrocompetent P. aeruginosa CIP104116 recipient strain. Transformants were obtained for all of the P. aeruginosa isolates, and the plasmids for both, blaVIM-2harboring and blaIMP-13-harboring plasmids, belonged to the IncFIC group (Table 2). PCR mapping showed that the blaKPC-2 gene was part of Tn4401, as previously described (1). The blaNDM-1 gene was preceded by a truncated copy of the insertion sequence ISAba125 and followed by bleMBL, a bleomycin resistance gene, as observed for most NDM-1-positive enterobacterial isolates (6, 21). The blaOXA-48 gene was bracketed by two IS1999 elements, forming a functional composite transposon, Tn1999.2, as previously described (10). Concerning P. aeruginosa, the carbapenemase-encoding genes were found to be part of the class 1 integrons. The blaVIM-2 gene was found alone in In56 (GenBank accession number AF191564) or associated with an upstream-located blaOXA-1 gene. The blaIMP-13 gene was found to be part of In320 (GenBank accession number AJ628135), with aacA4 gene encoding the AAC-6=-1b-N-acetyltransferase that confers high-level resistance to tobramycin and, to a lesser extent, amikacin but not to gentamicin. In conclusion, this study highlights the dissemination in Ro-
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7, 8, 23 18
1, 4, 9, 14, 16, 19, 24, 25 VIM-2 11, 12, 17 VIM-2 13 IMP-13
S. marcescens A B
P. aeruginosa A A B
NDM-1 NDM-1
Q, Ami Q, Ami Q, Ami
SXT, Q, Ami, TET SXT, Q, Ami, TET
SXT, Q, Ami, TET, Cm SXT, Q, Ami, TET, Cm SXT, Q, Ami SXT, Q, Ami, TET, Cm
Q, Ami
b
Size, kb Associated resistance determinant(s)d
Genetic environment
Untypeable Untypeable
Untypeable Untypeable Untypeable IncFII
Untypeable
CTX-M-15, TEM-1, OXA-1, OXA-10, RmtC TEM-1, OXA-1, RmtC CTX-M-15, TEM-1, OXA-1, RmtC CTX-M-15, TEM-1, OXA-1, AAC-6=-1b
ca. 100 None ca. 100 OXA-1 ca. 120 AAC-6=-1b
ca. 100 CTX-M-15, TEM-1, OXA-1, RmtC ca. 100 CTX-M-15, TEM-1, OXA-1, OXA-10, RmtC
ca. 120 ca. 60 ca. 100 ca. 100
ca. 150 CTX-M-15, TEM-1, OXA-1, RmtC
Class 1 integron Class 1 integron Class 1 integron
Truncated ISAba125, bleMBL Truncated ISAba125, bleMBL
Truncated ISAba125, bleMBL Truncated ISAba125, bleMBL Truncated ISAba125, bleMBL Class 1 integron
Truncated ISAba125, bleMBL
IncFIIK ca. 140 CTX-M-15, TEM-1, OXA-1, OXA-9, AAC-6=-1b Tn4401 IncL/M OXA-48 ca. 62 CTX-M-15, TEM-1, OXA-9, AAC-6=-1b-cr Tn1999.2 IncL/M OXA-48 ca. 62 CTX-M-15, TEM-1, OXA-9 Tn1999.2
ST2026 IncFIC ST2026 IncFIC ST1982 IncFIC
NA NA
ST255 ST171 ST93 ST93
ST131
ST258 ST101 ST101
Incompatibility
Plasmid carrying the carbapenemase
Isolate numbers referred to those of patients recapitulated in Table 1. Ami, aminoglycosides; Cm, chloramphenicol; Q, fluoroquinolones; SXT, sulfamethoxazole-trimethoprim; TET, tetracycline. c NA, not available. d Resistance markers coharbored by the carbapenemase gene-carrying plasmid are underlined.
a
6, 10 2 27 5
E. cloacae A B C C
NDM-1 NDM-1 NDM-1 VIM-4
NDM-1
26
E. coli A
SXT, Q, Ami, Cm SXT, Q, Ami, TET Q
Non--lactam-associated Carbapenemase resistanceb STc
KPC-2 OXA-48 OXA-48
No. of isolatesa
K. pneumoniae A 3 B 15, 20, 21 B 22
Species and clones
TABLE 2 Genetic features associated with carbapenemase producers
Dortet et al.
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mania of a wide variety of carbapenemases, including KPC-2, NDM-1, VIM-4, and OXA-48, in Enterobaceriaceae and VIM-2 and IMP-13 in P. aeruginosa. It also highlights the high occurrence of RmtC 16S RNA methylase among NDM-1-producing Enterobacteriaceae.
10.
11.
ACKNOWLEDGMENTS We thank Platform Genotyping of Pathogens and Public Health (Institut Pasteur, Paris, France) for coding multilocus sequence type (MLST) alleles and profiles and making them available at www.pasteur.fr/mlst. This work was partially funded by the University Paris-Sud, France. L.D. and T.N. are members of the Laboratory of Excellence in Research on Medication and Innovative Therapeutics (LERMIT) supported by grant ANR-10-LABX-33 from the French National Research Agency.
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1. Nordmann P, Dortet L, Poirel L. 2012. Carbapenem resistance in Enterobacteriaceae: here is the storm! Trends Mol Med 18:263–272. http://dx .doi.org/10.1016/j.molmed.2012.03.003. 2. Potron A, Poirel L, Nordmann P. 2015. Emerging broad-spectrum resistance in Pseudomonas aeruginosa and Acinetobacter baumannii: mechanisms and epidemiology. Int J Antimicrob Agents 45:568 –585. http://dx .doi.org/10.1016/j.ijantimicag.2015.03.001. 3. EUCAST. 2013. EUCAST guidelines for detection of resistance mechanisms and specific resistances of clinical and/or epidemiological importance. http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files /Resistance_mechanisms/EUCAST_detection_of_resistance_mechanisms _v1.0_20131211.pdf. 4. Dortet L, Brechard L, Poirel L, Nordmann P. 2014. Impact of the isolation medium for detection of carbapenemase-producing Enterobacteriaceae using an updated version of the Carba NP test. J Med Microbiol 63:772–776. http://dx.doi.org/10.1099/jmm.0.071340-0. 5. Dortet L, Poirel L, Nordmann P. 2012. Rapid detection of carbapenemase-producing Pseudomonas spp. J Clin Microbiol 50:3773–3776. http: //dx.doi.org/10.1128/JCM.01597-12. 6. Poirel L, Dortet L, Bernabeu S, Nordmann P. 2011. Genetic features of blaNDM-1-positive Enterobacteriaceae. Antimicrob Agents Chemother 55: 5403–5407. http://dx.doi.org/10.1128/AAC.00585-11. 7. Dortet L, Poirel L, Al Yaqoubi F, Nordmann P. 2012. NDM-1, OXA-48, and OXA-181 carbapenemase-producing Enterobacteriaceae in Sultanate of Oman. Clin Microbiol Infect 18:E144 –E148. http://dx.doi.org/10.1111 /j.1469-0691.2012.03796.x. 8. Girlich D, Poirel L, Nordmann P. 2015. Clonal distribution of multidrug-resistant Enterobacter cloacae. Diagn Microbiol Infect Dis 81:264 – 268. http://dx.doi.org/10.1016/j.diagmicrobio.2015.01.003. 9. Munoz-Price LS, Poirel L, Bonomo RA, Schwaber MJ, Daikos GL, Cormican M, Cornaglia G, Garau J, Gniadkowski M, Hayden MK, Kumarasamy K, Livermore DM, Maya JJ, Nordmann P, Patel JB, Paterson DL, Pitout J, Villegas MV, Wang H, Woodford N, Quinn JP. 2013. Clinical epidemiology of the global expansion of Klebsiella pneu-
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Fifteen carbapenemase-producing Enterobacteriaceae isolates and 12 carbapenemase-producing Pseudomonas aeruginosa isolates were recovered from patients hospitalized between August 2011 and March 2013 at the Hospital of Infectious Disease, ClujNapoca, Romania. One KPC-, nine NDM-1-, four OXA-48-, and one VIM-4-producing Enterobacteriaceae isolates along with 11 VIM-2-producing and one IMP-13-producing P. aeruginosa isolates were recovered from clinical samples. All carbapenemase genes were located on self-conjugative plasmids and were associated with other resistance determinants, including extendedspectrum -lactamases and RmtC methylases.
arbapenem-hydrolyzing -lactamases have been increasingly reported worldwide among Enterobacteriaceae (1) and Pseudomonas spp. (2). The most clinically relevant carbapenemases encountered in Enterobacteriaceae belong to Ambler class A (KPC type), Ambler class B, metallo--lactamases (MBLs; e.g., IMP, VIM, and NDM types), or Ambler class D (OXA-48). In Pseudomonas aeruginosa, MBLs of VIM and IMP types are predominantly identified worldwide, whereas KPC-producing isolates were mostly described in South America (2). The aim of this study was to survey the occurrence and characterization of carbapenem-resistant Enterobacteriaceae and P. aeruginosa recovered at the Hospital for Infectious Diseases of Cluj-Napoca, Romania. From August 2011 to November 2013, carbapenem susceptibility was investigated for Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Serratia marcescens, and P. aeruginosa isolates that were recovered from clinical samples obtained from patients who had been hospitalized in three units of the hospital (Table 1). According to the EUCAST breakpoints (http://www.eucast.org), carbapenem-resistant isolates were recovered for 5.8% (36 of 625 isolates) of K. pneumoniae, 0.002% (2 of 1,114 isolates) of E. coli, 11.7% (10 of 85 isolates) of E. cloacae, 20.3% (16 of 79 isolates) of S. marcescens, and 55.9% (222 of 397 isolates) of the P. aeruginosa isolates. According to EUCAST guidelines for the detection of carbapenemase-producing Enterobacteriaceae (3), carbapenemase detection was performed using the Carba NP test in all Enterobacteriaceae with decreased susceptibility to carbapenems (inhibition zones of ⬍25 mm, ⬍23 mm, and ⬍25 mm to meropenem, imipenem, and ertapenem, respectively) and in 27 P. aeruginosa isolates that were resistant to carbapenems and ceftazidime (4, 5). Carbapenemase activity was detected in 5 K. pneumoniae, 1 E. coli, 5 E. cloacae, 4 S. marcescens, and 12 P. aeruginosa isolates (Table 1). All of those isolates were recovered from patients who had no history of travel outside Romania. Carbapenemase genes (blaKPC, blaVIM, blaIMP, blaNDM, and blaOXA-48) were sought by PCR (Table 1) and sequencing, as previously described (6). One E. coli, 4 E. cloacae, and the 4 S. marcescens isolates were positive for blaNDM-1. One KPCproducing and 4 OXA-48-producing K. pneumoniae isolates were identified. One E. cloacae isolate and 11 P. aeruginosa isolates were
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positive for blaVIM. Finally, 1 P. aeruginosa isolate produced an IMP-type carbapenemase. To evaluate their clonal relationship, 14 carbapenemase-producing enterobacterial isolates (n ⫽ 5, K. pneumoniae; n ⫽ 5, E. cloacae; and n ⫽ 4, S. marcescens) (see Fig. S1 in the supplemental material) and the 12 P. aeruginosa isolates (see Fig. S2) were subjected to Diversilab, a semiautomated repetitive sequence-based PCR (rep-PCR) (bioMérieux, La Balmes-Les-Grottes, France). A cutoff value of 95% similarity was used to discriminate independent clones, as recommended by the manufacturer. We identified 2 clones of K. pneumoniae, 3 clones of E. cloacae, 2 clones of S. marcescens, and 2 clones of P. aeruginosa, of which the VIM-2producing clone was predominant (Table 2). Multilocus sequence typing analysis was performed on carbapenemase-producing K. pneumoniae, E. coli, E. cloacae, and P. aeruginosa isolates, as previously described (7, 8) (Table 2). The KPC-2-producing K. pneumoniae isolates belonged to the pandemic ST258 clone (9). The OXA-48-producing K. pneumoniae isolates belonged to ST101 that had been prevalent among OXA48-producing K. pneumoniae isolated in Europe (10), including Romania (11). The NDM-1-producing E. coli isolates belonged to the successful and virulent clone ST131, as previously observed with a strain that originated from India (12, 13), Thailand (14), and Târgu-Mures¸, a town located in the central part of Romania (11). The NDM-1-producing E. cloacae isolates were of three different sequence types (STs) (ST93, ST171, ST255). Of note, the VIM-4-producing E. cloacae and the NDM-1-producing E. cloa-
Received 29 June 2015 Returned for modification 3 August 2015 Accepted 17 August 2015 Accepted manuscript posted online 24 August 2015 Citation Dortet L, Flonta M, Boudehen Y-M, Creton E, Bernabeu S, Vogel A, Naas T. 2015. Dissemination of carbapenemase-producing Enterobacteriaceae and Pseudomonas aeruginosa in Romania. Antimicrob Agents Chemother 59:7100 –7103. doi:10.1128/AAC.01512-15. Address correspondence to Laurent Dortet, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.01512-15. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Carbapenemase Producers from Romania
TABLE 1 Clinical features of the carbapenemase-producing isolates and patient characteristics Patient
Sexa
Age, yr
Hospitalization department
Species
Date of isolation (mo/day/yr)
Carbapenemase
Site of isolation
Antimicrobial treatmentb
Outcome
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
M M F M M M M F M M M F M M M M F F M M M M F M M M M
73 59 16 82 53 62 70 80 66 77 73 42 83 81 62 45 71 65 64 64 90 56 76 73 31 31 63
ICUc Ambulatory Surgery ICU Surgery Surgery Surgery Ambulatory Surgery ICU Infectious diseases Infectious diseases Surgery Infectious diseases Surgery ICU ICU Infectious diseases ICU ICU Infectious diseases Surgery Infectious diseases Infectious diseases Infectious diseases Infectious diseases Surgery
P. aeruginosa E. cloacae K. pneumoniae P. aeruginosa E. cloacae E. cloacae S. marcescens S. marcescens P. aeruginosa E. cloacae P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa K. pneumoniae P. aeruginosa P. aeruginosa S. marcescens P. aeruginosa K. pneumoniae K. pneumoniae K. pneumoniae S. marcescens P. aeruginosa P. aeruginosa E. coli E. cloacae
8/22/2011 9/26/2011 9/30/2011 10/6/2011 3/19/2012 8/14/2012 3/3/2013 4/2/2013 4/10/2013 4/24/2013 5/14/2013 6/25/2013 8/13/2013 8/20/2013 8/19/2013 8/19/2013 8/20/2013 9/11/2013 9/18/2013 9/19/2013 4/5/2013 11/7/2012 3/19/2013 10/21/2013 11/11/2013 11/11/2013 11/20/2013
VIM-2 NDM-1 KPC-2 VIM-2 VIM-4 NDM-1 NDM-1 NDM-1 VIM-2 NDM-1 VIM-2 VIM-2 IMP-13 VIM-2 OXA-48 VIM-2 VIM-2 NDM-1 VIM-2 OXA-48 OXA-48 OXA-48 NDM-1 VIM-2 VIM-2 NDM-1 NDM-1
Blood Urine Urine Blood Urine Urine Urine Urine Urine Urine Urine Urine Urine Urine Drain Urine Urine Urine Urine Wound Urine Urine Urine Urine Ear Ear Urine
Not available Not available Not available Not available Not available Not available Not available Not available Not available Not available GEN, COL IMP Not available IMP, GEN, COL Not available IMP, COL, CIP GEN Not available CRO, CIP CRO, CIP CRO, IMP, GEN, CIP Not available CRO, CIP, GEN MEM, GEN Not available Not available Not available
Improved Improved Improved Improved Improved Improved Improved Improved Improved Expired Improved Improved Improved Expired Improved Improved Improved Improved Improved Improved Improved Improved Improved Improved Improved Improved Improved
a
M, male; F, female. CIP, ciprofloxacin; COL, colistin; CRO; ceftriaxone; IMP, imipenem; GEN, gentamicin; MEM, meropenem. c ICU, intensive care unit. b
cae isolates were of the same clone, ST93, indicating that this clone might be prevalent in this hospital. The IMP-13-producing P. aeruginosa isolate belonged to a new ST, ST1982. This ST is closely related to the ST357 clone that was previously described to be associated with the regional dissemination of IMP-7 in central Europe (15), but it is not related to ST621, an ST currently spreading worldwide (16). The VIM-2-producing P. aeruginosa isolates belonged to a new ST, ST2026. This ST is close to the ST233 that was recently described to be associated with the dissemination of VIM-2 in Bucharest (17). Mating-out assays were performed using enterobacterial clinical strains as donors and the azide-resistant E. coli J53 as the recipient strain. E. coli transconjugants were obtained for all enterobacterial donor isolates (Table 2). Plasmid DNA of transconjugants was extracted by using the Kieser method, analyzed by agarose gel electrophoresis, and typed by using PCR-based replicon typing (18). The blaKPC-2 plasmids belonged to the IncFIIk group. All blaNDM-1-positive plasmids belonged to untypeable incompatibility groups. In addition, specific PCRs for the detection of IncN2 (IncN2-for: 5=-GTCTAACGAGCTTACCGA AG-3=, IncN2-rev: 5=-GTTTCAACTCTGCCAAGTTC-3=) and IncHI1B (IncHI1B-for: 5=-GGAGCGATGGATTACTTCAGTA C-3=, IncHI1B-rev: 5=-TGCCGTTTCACCTCGTGAGTA-3=) that were previously described to be prevalent in blaNDM-1-carrying plasmids also remained negative (14, 19). As previously observed, the rmtC gene, encoding a 16S RNA methylase responsible for resistance to all aminoglycosides, was located on the blaNDM-1-
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carrying plasmid (11). The IncL/M-type specific to the archetypal 61.8-kb self-conjugative plasmid harboring the blaOXA-48 gene was detected in all OXA-48 transconjugants by using primers previously described (20). The blaVIM-4 plasmids belonged to the IncFII group. Whole DNA extracted from P. aeruginosa clinical isolates was electroporated into an electrocompetent P. aeruginosa CIP104116 recipient strain. Transformants were obtained for all of the P. aeruginosa isolates, and the plasmids for both, blaVIM-2harboring and blaIMP-13-harboring plasmids, belonged to the IncFIC group (Table 2). PCR mapping showed that the blaKPC-2 gene was part of Tn4401, as previously described (1). The blaNDM-1 gene was preceded by a truncated copy of the insertion sequence ISAba125 and followed by bleMBL, a bleomycin resistance gene, as observed for most NDM-1-positive enterobacterial isolates (6, 21). The blaOXA-48 gene was bracketed by two IS1999 elements, forming a functional composite transposon, Tn1999.2, as previously described (10). Concerning P. aeruginosa, the carbapenemase-encoding genes were found to be part of the class 1 integrons. The blaVIM-2 gene was found alone in In56 (GenBank accession number AF191564) or associated with an upstream-located blaOXA-1 gene. The blaIMP-13 gene was found to be part of In320 (GenBank accession number AJ628135), with aacA4 gene encoding the AAC-6=-1b-N-acetyltransferase that confers high-level resistance to tobramycin and, to a lesser extent, amikacin but not to gentamicin. In conclusion, this study highlights the dissemination in Ro-
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7, 8, 23 18
1, 4, 9, 14, 16, 19, 24, 25 VIM-2 11, 12, 17 VIM-2 13 IMP-13
S. marcescens A B
P. aeruginosa A A B
NDM-1 NDM-1
Q, Ami Q, Ami Q, Ami
SXT, Q, Ami, TET SXT, Q, Ami, TET
SXT, Q, Ami, TET, Cm SXT, Q, Ami, TET, Cm SXT, Q, Ami SXT, Q, Ami, TET, Cm
Q, Ami
b
Size, kb Associated resistance determinant(s)d
Genetic environment
Untypeable Untypeable
Untypeable Untypeable Untypeable IncFII
Untypeable
CTX-M-15, TEM-1, OXA-1, OXA-10, RmtC TEM-1, OXA-1, RmtC CTX-M-15, TEM-1, OXA-1, RmtC CTX-M-15, TEM-1, OXA-1, AAC-6=-1b
ca. 100 None ca. 100 OXA-1 ca. 120 AAC-6=-1b
ca. 100 CTX-M-15, TEM-1, OXA-1, RmtC ca. 100 CTX-M-15, TEM-1, OXA-1, OXA-10, RmtC
ca. 120 ca. 60 ca. 100 ca. 100
ca. 150 CTX-M-15, TEM-1, OXA-1, RmtC
Class 1 integron Class 1 integron Class 1 integron
Truncated ISAba125, bleMBL Truncated ISAba125, bleMBL
Truncated ISAba125, bleMBL Truncated ISAba125, bleMBL Truncated ISAba125, bleMBL Class 1 integron
Truncated ISAba125, bleMBL
IncFIIK ca. 140 CTX-M-15, TEM-1, OXA-1, OXA-9, AAC-6=-1b Tn4401 IncL/M OXA-48 ca. 62 CTX-M-15, TEM-1, OXA-9, AAC-6=-1b-cr Tn1999.2 IncL/M OXA-48 ca. 62 CTX-M-15, TEM-1, OXA-9 Tn1999.2
ST2026 IncFIC ST2026 IncFIC ST1982 IncFIC
NA NA
ST255 ST171 ST93 ST93
ST131
ST258 ST101 ST101
Incompatibility
Plasmid carrying the carbapenemase
Isolate numbers referred to those of patients recapitulated in Table 1. Ami, aminoglycosides; Cm, chloramphenicol; Q, fluoroquinolones; SXT, sulfamethoxazole-trimethoprim; TET, tetracycline. c NA, not available. d Resistance markers coharbored by the carbapenemase gene-carrying plasmid are underlined.
a
6, 10 2 27 5
E. cloacae A B C C
NDM-1 NDM-1 NDM-1 VIM-4
NDM-1
26
E. coli A
SXT, Q, Ami, Cm SXT, Q, Ami, TET Q
Non--lactam-associated Carbapenemase resistanceb STc
KPC-2 OXA-48 OXA-48
No. of isolatesa
K. pneumoniae A 3 B 15, 20, 21 B 22
Species and clones
TABLE 2 Genetic features associated with carbapenemase producers
Dortet et al.
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Carbapenemase Producers from Romania
mania of a wide variety of carbapenemases, including KPC-2, NDM-1, VIM-4, and OXA-48, in Enterobaceriaceae and VIM-2 and IMP-13 in P. aeruginosa. It also highlights the high occurrence of RmtC 16S RNA methylase among NDM-1-producing Enterobacteriaceae.
10.
11.
ACKNOWLEDGMENTS We thank Platform Genotyping of Pathogens and Public Health (Institut Pasteur, Paris, France) for coding multilocus sequence type (MLST) alleles and profiles and making them available at www.pasteur.fr/mlst. This work was partially funded by the University Paris-Sud, France. L.D. and T.N. are members of the Laboratory of Excellence in Research on Medication and Innovative Therapeutics (LERMIT) supported by grant ANR-10-LABX-33 from the French National Research Agency.
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