Emergence of NDM-1-Producing Enterobacteriaceae in Belgium^▿

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The New Delhi metallo-β-lactamase (NDM-1) is a class B β-lactamase that confers resistance to virtually all β-lactams, including carbapenems. Initially reported in Sweden (16) in Klebsiella pneumoniae and Escherichia coli isolates from a patient transferred from India, NDM-producing isolates have been reported mostly in the United Kingdom, India, and Pakistan (7) but are currently spreading worldwide (14), including in Europe (12). Here, we report on the first five isolates of NDM-1-producing Escherichia coli (n = 2), K. pneumoniae, Morganella morganii, and Enterobacter cloacae (n = 1 each) that were detected in three patients subsequent to their transfer to Belgium from Pakistan, Montenegro, and Kosovo/Serbia.

Case 1 (2) was a 52-year-old patient suffering from diabetes who had to be hospitalized in Pakistan in May 2010 for a necrotic wound infection following a traffic accident. He was transferred to a hospital in Brussels, Belgium, where he died after 2 weeks from uncontrolled severe sepsis.

Case 2 (2) was a mentally disabled, 44-year-old epileptic man who was admitted to a hospital in the area of Antwerp, Belgium. He developed seizures while swimming in a hotel's pool in Montenegro and was hospitalized in Podgorica, where he developed ventilator-associated pneumonia.

Case 3 was an immunosuppressed 46-year-old female treated for many years with prednisolone for systemic lupus erythematosus. She was consecutively hospitalized in Serbia and in Kosovo because of leg wound necrosis and went back in Namur, Belgium, to avoid amputation.

Screening for intestinal carriage of carbapenemase producers was carried out upon admission by using rectal swab, wound, and sputum specimens that were cultured by direct plating on ChromID extended-spectrum β-lactamase (ESBL) agar (bioMérieux). The recovered isolates were identified to the species level with MicroFlex LT (Bruker Daltonik). Microbiological analysis of clinical samples revealed extensively resistant E. coli in patient 1, K. pneumoniae and E. coli isolates in patient 2, and Morganella morganii and E. cloacae isolates in patient 3 (Table 1).

By disc diffusion, all isolates displayed reduced susceptibility or resistance to carbapenems (diameter of inhibition zone between 7 and 18 mm for imipenem and meropenem), and a synergy was evidenced by double-disc test between imipenem and EDTA (420 μg). Also, Vitek2 suggested the presence of a carbapenemase (data not shown).

MIC determination by the microdilution method of the Clinical and Laboratory Standards Institute (CLSI) (4) confirmed that four out of five isolates showed high-level resistance to all penicillins as well as to all expanded-spectrum cephalosporins and to aztreonam. Only the M. morganii isolate remained susceptible to cefepime (MIC = 8 μg/ml), to aztreonam (MIC = 2 μg/ml), and to all carbapenems but ertapenem (MIC = 4 μg/ml). Using the Etest, all isolates were resistant to all carbapenems tested, which had higher MICs, varying between 1.5 and >32 μg/ml, than those observed by microdilution. This could possibly be explained by the presence of isolated tiny colonies growing inside the inhibition zone, which must be taken into account when interpreting the Etest and which were not detected under the growth conditions used for microdilution. In any case, this underlines that all MIC techniques are not equivalent, which could have implications for patient management according to current CLSI interpretative guidelines. All the isolates but K. pneumoniae remained susceptible to tigecycline and to colistin (except M. morganii, which is intrinsically resistant to all the polymyxins).

Analysis of β-lactamase genes was performed by ESBL/KPC array (Check-Points, Wageningen, Netherlands) (5, 8) and by PCR sequencing targeting carbapenemases (1), bla_NDM-1 (for NDM-1, NDMFW [5′-ACTTGGCCTTGCTGTCCTT], NDMRV [5′-CATTAGCCGCTGCATTGAT], NDMseqFW [5′-CGCACCTCATGTTTGAATTCGCC], NDMseqRV [5′-GTCGCAAAGCCCAGCTTCGC]), plasmidic AmpC (10), and rRNA methylase coding genes (15). Plasmids were extracted with the Qiagen plasmid kit and were electroporated in E. coli Top10 using GenePulserII (Bio-Rad), and the incompatibility group of the plasmids was determined by PCR replicon typing (3).

Molecular features are presented in Table 1. bla_NDM-1 was detected in each strain. In addition, PCR sequencing detected various additional β-lactamases (bla_TEM-1, bla_OXA-1, bla_OXA-9, bla_CMY-16, bla_CMY-58, bla_SHV-12, and bla_CTX-M-15), rRNA methylases (rmtB, armA), and genes affecting the quinolones (qnrA6-B1 and -B2) cotransfered or not with bla_NDM-1 by electroporation. Multiple plasmids (48 kb to 190 kb) were recovered in the clinical isolates, but bla_NDM-1 was detected in transformants on plasmids of 130, 150, and 170 kb. For patients 2 and 3, the fact that bla_NDM-1 was detected in 2 different species on different genetic supports and associated to different comarkers is consistent with acquisition overseas of multiple NDM-1-expressing strains rather than transfer in vivo.

Ec10158, Ec10243, and Kp10197 isolates were typed by pulsed-field gel electrophoresis (PFGE) by using the XbaI restriction enzyme, and multilocus sequence typing (MLST) was performed according to http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html and http://mlst.ucc.ie/mlst/dbs/Ecoli/. The two E. coli isolates were not clonally related. Ec10158 originating from Pakistan belonged to ST101 and had a sequence type identical to that of the NDM-expressing E. coli 271 recovered from an Australian patient transferred from Bangladesh (11), and Ec10243 belonged to ST782, which is related to sequence type complex 69 found in uropathogenic E. coli (13). A Kp10197 isolate from Serbia/Kosovo belonged to ST15, a single-allele variant of the ST14, which was described for NDM-1-producing K. pneumoniae from India (16). However, ST14/15 is a globally widespread K. pneumoniae complex, so that the use of this criteria to infer relatedness between isolates recovered in different countries is probably not as relevant as comparison of their bla_NDM-1 genetic supports.

These five Enterobacteriaceae clinical isolates are the first characterized NDM-1-producing Enterobacteriaceae from Belgium. Interestingly, two NDM-1-producing K. pneumoniae strains were also recently reported in Austria from a patient transferred from Kosovo (17) and in Denmark from a women transferred from Bosnia and Herzegovina (6). Our observation also further highlights the frequent association of bla_NDM-1 with a high diversity of resistance genes affecting β-lactams, aminoglycosides, and quinolones. The detection of a prolonged asymptomatic carriage of NDM-positive Enterobacteriaceae in two patients (cases 2 and 3) suggests the real possibility of transmission of these organisms among hospitalized patients, though no secondary cases did occur locally in any of the three hospitals, probably thanks to early application of strictly enforced barrier precautions (http://www.nsih.be/surv_carba/carbapenemase_fr.asp). The rapid dissemination of NDM-1 enzymes worldwide and the dramatic consequences for treatment and for infection control measures warrant a high degree of awareness and of monitoring (9).