Phenotypic characteristics

The ISS strains showed aerobic, motile, rod shape, Gram stain negative characteristics; colonies were pale yellow in color, formed within 24–36 h at 35 °C on R2A, TSA, and blood agar. Growth was observed at 1–8% NaCl and in pH range 5–7. The Vitek and BioLog systems as well as MALDI-TOF profiles identified the ISS strains as E. ludwigii. The MALDI-TOF profile scores for the tested strains were 2.16 (E. ludwigii) and 2.10 (E. asburiae). In general, no noticeable phenotypic differences were observed among the Enterobacter species tested including E. bugandensis EB-247T, whose genome is closer to ISS strains. As reported earlier, all these five ISS Enterobacter isolates were resistant to cefazolin, cefoxitin, oxacillin, penicillin and rifampin, while for ciprofloxacin and erythromycin, strains were either resistant or intermediate resistant. For gentamycin and tobramycin some strains were resistant, some intermediate resistant, and some susceptible [9].

Molecular phylogeny

The 16S rRNA gene sequencing of all five isolates placed them within the Enterobacter group and showed maximum similarity (99.6%) with E. bugandensis EB-247T, E. cancerogenus LMG 2693, E. ludwigii EN-119, and E. mori R18–2 (99 to 100%). Since 16S rRNA gene sequencing analysis is insufficient to differentiate Enterobacter species, polygenic and whole genome-based analyses were further attempted. All ISS strains were phylogenetically characterized by the gyrB locus (~ 1.9 kb) and showed that the ISS isolates form a close group with E. bugandensis EB-247T and 153_ECLO strains (> 99%) while MBRL 1077 isolate was exhibiting 97% similarity with high bootstrap value.

MLST analysis

The genomic contigs of the ISS isolates were searched for gene sequences of dnaA, fusA, gyrB. leuS, pyrG, rplB, and rpoB, which are standardized for the use of MLST analysis and reported for E. cloacae species [29]. The good congruence between the single-gene reconstructions and the concatenate reinforced the stability of the genealogy were observed. The reconstruction was based on the RAxML algorithm [37] and the resulting MLST tree (Fig. 1) shows that the ISS isolates are phlylogenetically related to E. bugandensis clinical strains (EB-247, strain 153_ECLO, and isolate MBRL 1077).

Fig. 1 Multiple-locus sequence types (MLST) analysis of ISS strains and related species of the Enterobacter. The obtained genomic contigs of the ISS isolates (in bold) were searched for gene sequences of dnaA, fusA, gyrB, leuS, pyrG, rplB, and rpoB, which are standardized for the use in MLST analysis and reported for E. cloacae species [29]. The retrieved sequences were compared with the sequence types deposited at the Enterobacter MLST database, concatenated according to the MLST scheme. The reconstruction was based on the RAxML algorithm [4], and the bootstrap values were calculated using 1000 replicates. The bar indicates 2% sequence divergence Full size image

SNP analysis

Even though MLST analysis was clearly able to genomically resolve the ISS isolates to species level and distinguish them from other members of the genus Enterobacter, whole genome SNP analysis, SNP tree analysis excluding plasmid sequences, was carried out to validate these results. The snpTree does not ignore any nucleotide positions and is able to consider 100% of the chromosomal genome. All the available WGS of the Enterobacter genus reference genomes from GenBank were used for SNP analysis with snpTree. Of the 22 total nucleotide sequences; 58,121 positions were found in all analyzed genomes and 3832 positions in the dataset were used to confer the final tree (Fig. 2). The snpTree analyses confirmed and gave a strong validation to the MLST/gyrB data, confirming that all ISS isolates are E. bugandensis but strain MBRL 1077 grouped differently from the members of the E. bugandensis group.

Fig. 2 Single nucleotide polymorphism (SNP) based phylogenetic tree, showing the relationship between the ISS isolates (in bold) and members of the Enterobacter genus. The tree was generated using CSI Phylogeny [28] version 1.4 Full size image

SNP identification within ISS strains was carried out using GATK HaplotypeCaller. Filtered SNP calls and indels (after removal of false positives) are given in the Additional file 1: Table S1. Post-filtration analyses showed that there were 9, 12, 15, 13, and 0 SNPs seen in IF2SWB1, IF2SWB5, IF2SWP2, IS2WP3 and IS3SWP2, respectively. Further 6, 0, 4, 6, and 0 indels were seen in IF2SWB1, IF2SWB5, IF2SWP2, IS2WP3 and IS3SWP2, respectively (Additional file 1: Table S1). A maximum of 15 SNPs was observed among ISS isolates, probably being clonal in origin, with a very recent common ancestor. However, it should be noted that 4 strains were isolated from location #2 (space toilet) and one strain from the exercise platform (ARED).

ANI values and digital DNA-DNA hybridization

The ANI values for the ISS strains were maximum against E. bugandensis EB-247, 153_ECLO, and MBRL 1077 strains (> 95%) as were those of MLST analyses, and the ANI values of rest of the Enterobacter genomes tested were < 91% (Table 1). The digital DNA-DNA hybridization (dDDH) results of the ISS strain showed high similarity with E. bugandensis EB-247 (89.2%), 153_ECLO (89.4%), and MBRL 1077 (64%) strains whereas dDDH value was < 44.6% to all the other available Enterobacter reference genomes (Table 1). Based on various molecular analyses attempted during this study all five ISS Enterobacter strains were phenotypically and genotypically identified as E. bugandensis.

Table 1 Digital DDH and ANI values of ISS strains and comparison with various Enterobacter species Full size table

Functional characteristics

A detailed genome analysis of all five ISS strains and 3 clinical isolates were carried out to understand its genetic makeup. A total of 4733 genes were classified as carbohydrate metabolism (635 genes), amino acid and derivatives (496 genes), protein metabolism (291 genes), cofactors, vitamins, prosthetic groups, pigments (275 genes), membrane transport (247 genes), and RNA metabolism (239 genes) (Fig. 3). To test antimicrobial resistance at genomic level, the ISS strains were further compared with nosocomial isolates (1291 genomes) having more than 95% ANI identity with the ISS strains, which taxonomically identified them as same species. Genomes of the clinical strains of E. bugandensis 247, 153_ECLO, and MBRL-1077, whose ANI values were > 95%, were used for the genetic comparison to further broaden the picture.

Fig. 3 Metabolic functional profiles and subsystem categories distribution of strain IF3SW-P2. 4733 genes were identified that dominated by carbohydrate metabolism followed by amino acid and derivatives Full size image

Features playing a broad role and implemented by the same domain such as Spectinomycin 9-O-adenylyltransferase and Streptomycin 3-O-adenylyltransferase (EC 2.7.7.47) were only present in E. bugandensis 247 due to the probable lack of selective pressure that might have been encountered by the ISS isolates (Table 2). The predicted arsenic resistance (arsenic resistance protein, ArsH) noticed in E. bugandensis 247 but not in other strains should be phenotypically tested to confirm the resistance properties conferred in strain E. bugandensis 247 and cross checked with the ISS strains for their inability to degrade arsenic. Trace metals detected in ISS potable water samples, but typically below potability requirements, included arsenic, barium, chromium, copper, iron, manganese, molybdenum, nickel, lead, selenium, and zinc. No mercury or cadmium was detected and the arsenic levels varied from nondetectable in water samples to a maximum of 3.8 μg/L [38].

Table 2 Comparative analyses of antimicrobial gene profiles of E. bungandensis isolated from ISS and clinical sources Full size table

Global comparison of ISS genomes with other Enterobacter genomes

A visualization program was reported to be invaluable [36] in determining the genotypic differences between closely related prokaryotes. Visualizing a prokaryote genome as a circular image has become a powerful means of displaying informative comparisons of one genome to a number of others. Using BRIG, a global visual comparison of ISS isolates with other Enterobacter WGS from the GenBank Microbial Genomes Resource was carried out. The resulting output of the BRIG analysis [36], a visualization image, showed draft genome assembly information, read coverage, assembly breakpoints, and collapsed repeats. The mapping of unassembled sequencing reads of the ISS genomes against fully annotated E. cloacae central reference sequences is depicted in Fig. 4.