The most important resistance mechanism against β-lactam drugs is the production of carbapenemases. In this study, we report the first identification of Klebsiella pneumoniae carbapenemase (KPC)-2 and New Delhi metallo-β-lactamase (NDM)-1 in Enterobacter hormaechei subps. oharae from Brazil. The detection of carbapenemases was done by phenotypic assays, PCR, and DNA sequencing, whereas the identification was performed by conventional techniques, sequencing of the 16S rDNA gene, and hsp60-genotyping. Molecular typing was performed using pulsed-field gel electrophoresis, and antimicrobial susceptibility was surrogated by the Etest methodology. Using the whole genome sequencing approach, we searched for resistance genes, plasmid incompatibility group genes, and the genetic environment of blaNDM and blaKPC. The plasmid identification was done by restriction digests with the S1 nuclease followed by hybridization using digoxigenin labeled specific probes. The isolate was considered multiresistant, being susceptible to amikacin and polymyxin B. We observed the following resistance genes: blaCTX-M-15, blaACT-7, blaTEM-1, blaOXA-1, aadA1, aadA2, strA, strB, aac(3)-II, qnrB1, and aac(6')-Ib-cr and incompatibility group plasmid genes IncA/C, IncHI2, and IncN. The blaKPC gene was found associated to the transposon Tn4401 isoform b in plasmid with 50 kb (IncN) and blaNDM-1 was flanked by a truncated ISAba125 and bleMBL in plasmid with 160 kb (IncA/C). This study showed the coproduction of two important carbapenemases (KPC-2 and NDM-1) associated with mobile genetic elements of worldwide epidemiological importance (Tn4401 and ISAba125, respectively), reinforcing the idea that urgent measures are necessary to reduce and prevent the spreading of these carbapenemases primarily in the hospital settings.

Small RNAs influence the gene expression at the post-transcriptional level by guiding messenger RNA (mRNA) cleavage, translational repression, and chromatin modifications. In addition to model plants, the microRNAs (miRNAs) have been identified in different crop species. In this work, we developed a specific pipeline to search for coffee miRNA homologs on expressed sequence tags (ESTs) and genome survey sequences (GSS) databases. As a result, 36 microRNAs were identified and a total of 616 and 362 potential targets for Coffea arabica and Coffea canephora, respectively. The evolutionary analyses of these molecules were performed by comparing the primary and secondary structures of precursors and mature miRNAs with their orthologs. Moreover, using a stem-loop RT-PCR assay, we evaluated the accumulation of mature miRNAs in genomes with different ploidy levels, detecting an increase in the miRNAs accumulation according to the ploidy raising. Finally, a 5' RACE (Rapid Amplification of cDNA Ends) assay was performed to verify the regulation of auxin responsive factor 8 (ARF8) by MIR167 in coffee plants. The great variety of target genes indicates the functional plasticity of these molecules and reinforces the importance of understanding the RNAi-dependent regulatory mechanisms. Our results expand the study of miRNAs and their target genes in this crop, providing new challenges to understand the biology of these species.