E. aerogenes isolates C10, D2, D3, and E9 were obtained between 2006 and 2012 from patients hospitalized in wards or intensive care units at the Hospital de Clínicas of the Universidade Federal do Paraná (Curitiba, Brazil). The main selection criterion for genome sequencing was the MDR phenotype, particularly in carbapenem resistant isolates. The negative laboratory tests for carbapenemases were also taken into account, as divergent enzymes or alternative resistance mechanisms could be relevant to the observed MDR phenotypes. C10 and D2 samples were isolated from different body sites of the same patient. Isolates were grown in selective medium with an ertapenem disk (10 ug) and stored at −80°C in trypticase soy broth containing glycerol 15%. Identification of isolates was performed using Vitek® 2 Compact (BioMérieux S.A., Marcy l'Etoile, France) and by mass spectrometry using Microflex LT instrument (Bruker Daltonics, Bremen, Germany). This study was carried out in accordance with the Brazilian legislation and was approved by the Institutional Ethics Review Board of the Hospital de Clínicas, Universidade Federal do Paraná (IRB#: 2656.263/2011-11). Our study involved only bacterial isolates and no human specimens were analyzed or stored. Further, we used no patient information other than the anatomical sites from where the isolates were collected. Therefore, the same Ethics Review Board exempted us from obtaining informed consent forms.

Genomic DNA was extracted using DNeasy 96 Blood & Tissue Kit (QIAGEN Silicon Valley, Redwood City, USA). DNA quality was assessed using a Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, USA). DNA quantification was performed using Qubit (Thermo Fisher Scientific Inc., Waltham, USA). Illumina sequencing libraries with an average fragment size of 550 bp were prepared using Illumina TruSeq DNA PCR-free LT Kit (Illumina Inc., San Diego, USA). Whole-genome sequencing of paired-end (PE) libraries was performed using a HiSeq 2500 instrument in RAPID run mode (Illumina Inc., San Diego, USA) at the Life Sciences Core Facilities of the State University of Campinas (São Paulo, Brazil). Quality-based trimming and filtering was performed using Trimmomatic version 0.32 (Bolger et al., 2014). PE reads were assembled de novo using Velvet version 1.2.10 (Zerbino and Birney, 2008) and contigs were scaffolded using SSPACE version 3.0 (Boetzer et al., 2011). Gene predictions and annotations were performed using NCBI Prokaryotic Genome Automatic Annotation Pipeline (PGAAP; Angiuoli et al., 2008).

Antibiotic resistance-related genes were predicted using the ResFinder database version 2.1 (Zankari et al., 2012) with the following parameters: “all databases” were used for antimicrobial configuration, type of reads as “assembled genomes/contigs” and thresholds of 98 identity and 80% coverage between sequences. This dataset of resistance genes was complemented with BLASTp searches against the ARDB (Antibiotic Resistance Genes Database) version 1.1 (Liu and Pop, 2009) using “resistance gene complete” database, 40% identity and e-value of 0.0001.

All isolates showed MDR profile and had increased MIC for at least one carbapenem. Information regarding collection date and site, clinical setting, PFGE profile and antimicrobial resistance profiles of each isolate are available in Table 1. Among the four analyzed samples, C10 and D2 were isolated from different body sites of the same patient within a short period of time (a month) and belong to the same PFGE profile. These genomes allow one to analyze the possible genome plasticity between the isolates. D3 and E9 samples were isolated from two patients with an interval of collection date greater than 5 years. D3 and E9 were also interesting because of their sensitivity to meropenem and resistance to ertapenem and imipenem. Surprisingly, E9 showed resistance to carbapenems but not to 3rd (ceftazidime and cefotaxime) and 4th generation (cefepime) cephalosporins (Table 1). All isolates possessed bla_AmpC and bla_TEM, as detected by PCR. The gene bla_CTX-M2 was found in all isolates except E9. Phenotypic tests (i.e., Modified Hodge test and double-disk synergy) to detect carbapenemases were positive for C10, D2, and E9. However, no class A, B, and D carbapenemase encoding genes were detected by PCR. All isolates tested negative in carbapenem hydrolysis assays.

We obtained between 16,841,714 and 25,138,390 150 bp PE reads per library. After genome assembly, 5,833,521 bp were assembled in 58 scaffolds for C10, 5,821,782 bp were assembled in 57 scaffolds for D2, 5,584,745 bp were assembled in 55 scaffolds for D3 and 5,637,471 bp were assembled in 59 scaffolds for E9. By using the NCBI Prokaryotic Annotation Pipeline, we were able to predict 5,363, 5,380, 5,067, and 5,129 protein-coding sequences in each of the genomes listed above, respectively. Genomic features of the four sequenced genomes are summarized in Table 1.

A total of 18 enzymes related to antibiotic resistance were identified using ResFinder, ARDB and PGAAP (Table 2). All isolates harbor genes related to: (i) aminoglycoside resistance (genes aacA4 and aadA); (ii) beta-lactam resistance, including genes belonging to class A beta-lactamases (TEM family), class B beta-lactamases (Ribonuclease Z), class C beta-lactamases (CMY/LAT/MOX/ACT/MIR/FOX family) and class D beta-lactamases (OXA-9); (iii) bacitracin resistance (gene bacA), and (iv) sulphonamide resistance (gene sul1; Table 2). Genes sul2 and rmtD were only identified in E. aerogenes D3. The gene sul2 has been implicated on sulphonamide resistance for inducing high expression levels of the enzyme dihydropteroate synthase (Sköld, 2001), while rmtD has been related to aminoglycoside resistance and this variant was identified for the first time in South America in a P. aeruginosa isolate in 2005 (Doi et al., 2007). Interestingly, E. aerogenes D3 was isolated in 2006, indicating that this variant has spread amongst Enterobacteriaceae in Brazil since its first report (Doi et al., 2007).

Although the four isolates showed carbapenem-resistance, no carbapenemase gene was identified using molecular detection or in silico analysis. Hence, it is likely that these isolates employ alternative mechanisms to counter carbapenem effects. Various multidrug efflux transporters were found in the genomes described here (Table 2). They belong to four superfamilies: the major facilitator superfamily (MFS), multidrug and toxic compound extrusion (MATE), ATP-binding cassette (ABC) and resistance-nodulation-cell division (RND). RND type of transporters has been often associated with multidrug resistance of Gram-negative bacteria (Nikaido, 1998). In particular, the RND type genes forming the AcrA-AcrB-TolC efflux pump were found in multiple copies in our isolates (Table 2). Experimental evolution studies of E. aerogenes under successive imipenem exposure reported alterations in membrane permeability with complete loss of porins (e.g., Omp35 and Omp36) and overexpression of AcrAB-TolC efflux pumps (Bornet et al., 2003; Thiolas et al., 2005; Lavigne et al., 2012). As a result of efflux pump expression, the E. aerogenes isolates showed resistance to carbapenems and other antibiotics, especially fluoroquinolones (Bornet et al., 2003; Thiolas et al., 2005; Lavigne et al., 2012). Given the multiple copies of genes encoding efflux pumps in our isolates, it is possible that an increased expression of AcrAB-TolC efflux pumps could contribute to the observed carbapenem-resistant profiles.

E. aerogenes is an emergent nosocomial pathogen with a diversity of mechanisms to circumvent antimicrobial activity. Here we reported the phenotypic screens, genome sequencing, and prediction of putative resistance gene repertoires of four multidrug-resistant E. aerogenes isolated between 2006 and 2012. The data reported here may help understand the biochemistry, evolution, and epidemiology of this important pathogen. The material provided in this work may be used in future comparative genomics and molecular epidemiology studies aiming to clarify the resistance profiles and dynamics of multidrug-resistant Enterobacteriaceae species.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.