Abstract
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, Cluj-Napoca, 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 extended-spectrum β-lactamases and RmtC methylases.
TEXT
Carbapenem-hydrolyzing β-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-β-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 KPC-producing and 4 OXA-48-producing K. pneumoniae isolates were identified. One E. cloacae isolate and 11 P. aeruginosa isolates were positive for blaVIM. Finally, 1 P. aeruginosa isolate produced an IMP-type carbapenemase.
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 | M | 73 | ICUc | P. aeruginosa | 8/22/2011 | VIM-2 | Blood | Not available | Improved |
| 2 | M | 59 | Ambulatory | E. cloacae | 9/26/2011 | NDM-1 | Urine | Not available | Improved |
| 3 | F | 16 | Surgery | K. pneumoniae | 9/30/2011 | KPC-2 | Urine | Not available | Improved |
| 4 | M | 82 | ICU | P. aeruginosa | 10/6/2011 | VIM-2 | Blood | Not available | Improved |
| 5 | M | 53 | Surgery | E. cloacae | 3/19/2012 | VIM-4 | Urine | Not available | Improved |
| 6 | M | 62 | Surgery | E. cloacae | 8/14/2012 | NDM-1 | Urine | Not available | Improved |
| 7 | M | 70 | Surgery | S. marcescens | 3/3/2013 | NDM-1 | Urine | Not available | Improved |
| 8 | F | 80 | Ambulatory | S. marcescens | 4/2/2013 | NDM-1 | Urine | Not available | Improved |
| 9 | M | 66 | Surgery | P. aeruginosa | 4/10/2013 | VIM-2 | Urine | Not available | Improved |
| 10 | M | 77 | ICU | E. cloacae | 4/24/2013 | NDM-1 | Urine | Not available | Expired |
| 11 | M | 73 | Infectious diseases | P. aeruginosa | 5/14/2013 | VIM-2 | Urine | GEN, COL | Improved |
| 12 | F | 42 | Infectious diseases | P. aeruginosa | 6/25/2013 | VIM-2 | Urine | IMP | Improved |
| 13 | M | 83 | Surgery | P. aeruginosa | 8/13/2013 | IMP-13 | Urine | Not available | Improved |
| 14 | M | 81 | Infectious diseases | P. aeruginosa | 8/20/2013 | VIM-2 | Urine | IMP, GEN, COL | Expired |
| 15 | M | 62 | Surgery | K. pneumoniae | 8/19/2013 | OXA-48 | Drain | Not available | Improved |
| 16 | M | 45 | ICU | P. aeruginosa | 8/19/2013 | VIM-2 | Urine | IMP, COL, CIP | Improved |
| 17 | F | 71 | ICU | P. aeruginosa | 8/20/2013 | VIM-2 | Urine | GEN | Improved |
| 18 | F | 65 | Infectious diseases | S. marcescens | 9/11/2013 | NDM-1 | Urine | Not available | Improved |
| 19 | M | 64 | ICU | P. aeruginosa | 9/18/2013 | VIM-2 | Urine | CRO, CIP | Improved |
| 20 | M | 64 | ICU | K. pneumoniae | 9/19/2013 | OXA-48 | Wound | CRO, CIP | Improved |
| 21 | M | 90 | Infectious diseases | K. pneumoniae | 4/5/2013 | OXA-48 | Urine | CRO, IMP, GEN, CIP | Improved |
| 22 | M | 56 | Surgery | K. pneumoniae | 11/7/2012 | OXA-48 | Urine | Not available | Improved |
| 23 | F | 76 | Infectious diseases | S. marcescens | 3/19/2013 | NDM-1 | Urine | CRO, CIP, GEN | Improved |
| 24 | M | 73 | Infectious diseases | P. aeruginosa | 10/21/2013 | VIM-2 | Urine | MEM, GEN | Improved |
| 25 | M | 31 | Infectious diseases | P. aeruginosa | 11/11/2013 | VIM-2 | Ear | Not available | Improved |
| 26 | M | 31 | Infectious diseases | E. coli | 11/11/2013 | NDM-1 | Ear | Not available | Improved |
| 27 | M | 63 | Surgery | E. cloacae | 11/20/2013 | NDM-1 | Urine | Not available | Improved |
M, male; F, female.
CIP, ciprofloxacin; COL, colistin; CRO; ceftriaxone; IMP, imipenem; GEN, gentamicin; MEM, meropenem.
ICU, intensive care unit.
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-2-producing clone was predominant (Table 2).
TABLE 2.
Genetic features associated with carbapenemase producers
| Species and clones | No. of isolatesa | Carbapenemase | Non-β-lactam-associated resistanceb | STc | Plasmid carrying the carbapenemase |
Associated resistance determinant(s)d | Genetic environment | |
|---|---|---|---|---|---|---|---|---|
| Incompatibility | Size, kb | |||||||
| K. pneumoniae | ||||||||
| A | 3 | KPC-2 | SXT, Q, Ami, Cm | ST258 | IncFIIK | ca. 140 | CTX-M-15, TEM-1, OXA-1, OXA-9, AAC-6′-1b | Tn4401 |
| B | 15, 20, 21 | OXA-48 | SXT, Q, Ami, TET | ST101 | IncL/M OXA-48 | ca. 62 | CTX-M-15, TEM-1, OXA-9, AAC-6′-1b-cr | Tn1999.2 |
| B | 22 | OXA-48 | Q | ST101 | IncL/M OXA-48 | ca. 62 | CTX-M-15, TEM-1, OXA-9 | Tn1999.2 |
| E. coli | ||||||||
| A | 26 | NDM-1 | Q, Ami | ST131 | Untypeable | ca. 150 | CTX-M-15, TEM-1, OXA-1, RmtC | Truncated ISAba125, bleMBL |
| E. cloacae | ||||||||
| A | 6, 10 | NDM-1 | SXT, Q, Ami, TET, Cm | ST255 | Untypeable | ca. 120 | CTX-M-15, TEM-1, OXA-1, OXA-10, RmtC | Truncated ISAba125, bleMBL |
| B | 2 | NDM-1 | SXT, Q, Ami, TET, Cm | ST171 | Untypeable | ca. 60 | TEM-1, OXA-1, RmtC | Truncated ISAba125, bleMBL |
| C | 27 | NDM-1 | SXT, Q, Ami | ST93 | Untypeable | ca. 100 | CTX-M-15, TEM-1, OXA-1, RmtC | Truncated ISAba125, bleMBL |
| C | 5 | VIM-4 | SXT, Q, Ami, TET, Cm | ST93 | IncFII | ca. 100 | CTX-M-15, TEM-1, OXA-1, AAC-6′-1b | Class 1 integron |
| S. marcescens | ||||||||
| A | 7, 8, 23 | NDM-1 | SXT, Q, Ami, TET | NA | Untypeable | ca. 100 | CTX-M-15, TEM-1, OXA-1, RmtC | Truncated ISAba125, bleMBL |
| B | 18 | NDM-1 | SXT, Q, Ami, TET | NA | Untypeable | ca. 100 | CTX-M-15, TEM-1, OXA-1, OXA-10, RmtC | Truncated ISAba125, bleMBL |
| P. aeruginosa | ||||||||
| A | 1, 4, 9, 14, 16, 19, 24, 25 | VIM-2 | Q, Ami | ST2026 | IncFIC | ca. 100 | None | Class 1 integron |
| A | 11, 12, 17 | VIM-2 | Q, Ami | ST2026 | IncFIC | ca. 100 | OXA-1 | Class 1 integron |
| B | 13 | IMP-13 | Q, Ami | ST1982 | IncFIC | ca. 120 | AAC-6′-1b | Class 1 integron |
Isolate numbers referred to those of patients recapitulated in Table 1.
Ami, aminoglycosides; Cm, chloramphenicol; Q, fluoroquinolones; SXT, sulfamethoxazole-trimethoprim; TET, tetracycline.
NA, not available.
Resistance markers coharbored by the carbapenemase gene-carrying plasmid are underlined.
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 OXA-48-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-Mureş, 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. cloacae 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′-GTCTAACGAGCTTACCGAAG-3′, IncN2-rev: 5′-GTTTCAACTCTGCCAAGTTC-3′) and IncHI1B (IncHI1B-for: 5′-GGAGCGATGGATTACTTCAGTAC-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-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-2-harboring 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 Romania 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.
Supplementary Material
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.
Footnotes
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.01512-15.
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