Isolation of NDM-producing Providencia rettgeri in Brazil

Sir, New Delhi metallo-β-lactamase (NDM) was first identified in 2009 in a Klebsiella pneumoniae isolated from a patient in a Swedish hospital who had been previously hospitalized in India in 2008.1 Since then, different bacterial species carrying the blaNDM gene have been isolated, mostly in the Indian subcontinent, but also in several other countries around the world. Here, we report the first description (to our knowledge) of NDM-1 in Brazil. In early 2013, a diabetic patient with peripheral vascular disease was admitted to a public hospital in southern Brazil (Rio Grande do Sul state) with a diabetic foot infection. Three weeks later, he was discharged from the hospital for ambulatory treatment with 500 mg of ciprofloxacin orally every 12 h. However, a month later he returned to the hospital with worsening of clinical signs and underwent toe amputation. Amoxicillin/clavulanate (1000/200 mg) intravenously every 8 h was initiated prior to surgery and maintained until patient discharge. A fragment of soft tissue from the toe sent for culture yielded growth of a carbapenem-resistant Providencia rettgeri, in which blaNDM-1 was detected. The patient recovered well and was discharged. Previous travel history to other countries was not established. The identification of the isolate was confirmed by the API 20E system (bioMérieux, Marcy l'Étoile, France) and 16S rRNA gene sequencing. It was resistant to imipenem (MIC 4 mg/L) and susceptible to ertapenem (MIC 0.5 mg/L) and meropenem (MIC 0.75 mg/L) using Etest (AB bioMérieux, Solna, Sweden) according to CLSI 2013 breakpoints.2 This resistance profile is very unusual in carbapenemase-producing bacteria. However, ertapenem susceptibility was also described in an NDM-producing P. rettgeri isolated from Israel.3 Screening for carbapenemase/metallo-β-lactamase production yielded positive results with imipenem/EDTA,4 but yielded negative results when using the modified Hodge test. PCR screening for β-lactamase genes (blaNDM, blaKPC, blaOXA-48, blaVIM, blaIMP, blaCTX-M, blaTEM, blaSHV and blaGES) followed by DNA sequencing identified only the presence of blaNDM-1. Antimicrobial susceptibility profiling analysis performed with Etest indicated that the isolate displayed a multidrug resistance profile. According to CLSI 2013,2 it was resistant to ceftazidime, cefotaxime and ciprofloxacin (MICs >256, 16 and >32 mg/L, respectively) and showed intermediate susceptibility to gentamicin (MIC 8 mg/L) and susceptibility to amikacin (MIC 3 mg/L). According to EUCAST breakpoints,5 it was resistant to tigecycline (MIC 2 mg/L). Infections due to multidrug-resistant carbapenemase-producing strains are commonly treated with polymyxin B and/or tigecycline. However, this isolate was intrinsically resistant to polymyxin B and presented intermediate susceptibility to tigecycline, creating a serious problem for the choice of therapy. The presence of visible plasmid DNA in the P. rettgeri isolate was not observed using alkaline lysis methodology.6 We also performed hybridization experiments to determine whether the blaNDM-1 gene was located in a low-copy plasmid (not detectable by ethidium bromide staining), but no hybridization was observed. These observations suggest that the blaNDM-1 gene in this strain is most likely chromosomally integrated. To characterize the genetic structure surrounding the blaNDM-1 gene, a tagmentation library from genomic DNA was made with the Nextera XT DNA Sample Preparation Kit (Illumina) and pair-end sequenced on an Illumina Miseq system. Contigs obtained after de novo assembling the reads with Velvet algorithms7 were used for Blast searches against GenBank. A contig that carried the NDM gene was then used as a reference to map the reads and check contig assembly with Geneious 6.1.5 (Biomatters, New Zealand). By this approach, we observed that blaNDM-1 was located inside a composite transposon, named Tn125 (10 092 bp), the same as that described in NDM-1-producing Acinetobacter baumannii isolated from Germany.8 Tn125 is bracketed by two copies of the insertion sequence ISAba125. In the A. baumannii isolate, Tn125 was found on the chromosome, corroborating our results. blaNDM-1 has been associated with different mobile elements; however, a complete form or variations of ISAba125 have been found upstream of the blaNDM-1 gene, suggesting that ISAba125 is responsible for mobilization of the gene. This insertion sequence has primarily been found in A. baumannii.8 In South America, the first description of NDM was in Uruguay in 2012, detected in the same species identified in our study (P. rettgeri).9 The Rio Grande do Sul state (Brazil) and Uruguay border one another. However, to assert that Uruguay was the source of NDM, it would be necessary to perform molecular typing of the isolates to assess their clonality. The other reports of NDM in South America were of a K. pneumoniae outbreak in Colombia10 and of A. baumannii in Paraguay,9 other countries that border Brazil. This first known detection of NDM in Brazil brings attention to the need for adopting effective measures to control the spread of this important carbapenemase in this country. This work was funded by research grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq, Fundação Carlos Chagas de Amparo a Pesquisa – FAPERJ and Instituto Oswaldo Cruz – IOC – FIOCRUZ. None to declare. We thank the Genomic Platform for DNA Sequencing PDTIS (Instituto Oswaldo Cruz).