Enterobacter bugandensis: a novel enterobacterial species associated with severe clinical infection

Abstract

Nosocomial pathogens can cause life-threatening infections in neonates and immunocompromised patients. E. bugandensis (EB-247) is a recently described species of Enterobacter, associated with neonatal sepsis. Here we demonstrate that the extended spectrum ß-lactam (ESBL) producing isolate EB-247 is highly virulent in both Galleria mellonella and mouse models of infection. Infection studies in a streptomycin-treated mouse model showed that EB-247 is as efficient as Salmonella Typhimurium in inducing systemic infection and release of proinflammatory cytokines. Sequencing and analysis of the complete genome and plasmid revealed that virulence properties are associated with the chromosome, while antibiotic-resistance genes are exclusively present on a 299 kb IncHI plasmid. EB-247 grew in high concentrations of human serum indicating septicemic potential. Using whole genome-based transcriptome analysis we found 7% of the genome was mobilized for growth in serum. Upregulated genes include those involved in the iron uptake and storage as well as metabolism. The lasso peptide microcin J25 (MccJ25), an inhibitor of iron-uptake and RNA polymerase activity, inhibited EB-247 growth. Our studies indicate that Enterobacter bugandensis is a highly pathogenic species of the genus Enterobacter. Further studies on the colonization and virulence potential of E. bugandensis and its association with septicemic infection is now warranted.

Figure 1

Virulence assessment of EB-247 in insect and mouse model. (A) Survival rates of G. mellonella after the infection of different bacterial pathogens. G. mellonella were infected and observed until 7 days post infection. For each group 10 larvae were infected with ~10²bacterial cfu and incubated at 37 °C. The data shown are obtained from the three independent experiments. (B) G. mellonella response to high dose infection. To observe the dose dependent virulence of EB-247, ~10⁴ cfu were infected to G. mellonella (n = 10) and monitored for several hours post infection. (C) Intra-gastric (IG) infection of BALB/c mice (n = 4) with EB-247 and SB 300. (D) Intra-peritoneal (IP) infection of BALB/c (n = 4) with EB-247 and SB 300. For both the IG and IP route of infection, the mice were infected with 10⁵ and 10² cfu of EB-247 and SB 300 respectively. For both routes of infection, the mice were sacrificed on day 2 post infection and the bacterial colonization was assessed in different organs. (E) Cytokines measurement of mice following intra-peritoneal infection. The blood samples from individual mice infected with IP were collected by retro orbital bleeding and cytokines were estimated using a bead-based immunoassay technique. The dotted lines in the graphs indicate minimum detection limits. ns, not significant; *statistically significant (p < 0.05, t test).

Figure 2

Growth of EB-247 in human serum and physical characterization of EB-247 O-antigens and their role in cytokine induction in vitro. (A) The growth pattern of EB-247 in 50% human serum. Five µL from overnight cultures of individual strains were suspended in human serum and incubated at 37 °C for 22 h in a 96-well microtiter plate. The OD₆₀₀ was measured in every hour by the incubator cum microtiter plate reader. Data represented means ± SD of measurements performed in triplicate. (B) Characterization of EB-247 lipopolysaccharide (LPS). The LPS of individual strains were extracted by using the hot phenol water method and electrophoresed in a Tris-Glycine polyacrylamide gel. Further, the gel was stained by using a conventional silver staining protocol. (C) Estimation of fold change in the mRNA expression of IL-1, IL-6 and TNF-α upon LPS stimulation in BMDM cells. The cells were stimulated with 0.5 µg/mL of LPS isolated from EB-247 and SB 300. The BMDM cells were harvested 4 h post stimulation and the cDNA was prepared from the isolated total RNA. In addition, quantitative gene expression was measured using qRT PCR. The data represents the mean with standard deviation of three individual experiments with three replicates of each.

Figure 3

Whole genome transcriptome analysis using an RNA-Seq approach. (A) Experimental design of the RNA-seq approach. Schematic illustration of the steps that are employed during the sequencing procedure. (B) Differential expression analysis of normalized reads. After normalization, the sequence reads were analyzed by using the DESeq2, baySeq and edgeR packages. Individual packages were run independently and genes that were common according to all three different analyses were considered as differentially regulated. (C) Correlation between the average read counts of individual genes regulated in both LB and serum. Red and green dots represent the individual genes that are up and down regulated respectively, whereas the black dots represent the genes that are not differentially regulated. Each dot represents the paired coordinate value between the mean numbers of reads of individual genes obtained from experiments from three biological repeats. The correlation coefficient value was determined by using the approach ‘Goodness of Fit’ of GraphPad Prism V5.

Figure 4

Transcriptional profile of differentially regulated genes grouped according to their most likely functions in EB-247 adaptation to and survival in human serum. (A) Iron uptake, (B) regulatory function, (C) chaperons, (D) others, (E) transport and binding, (F) energy metabolism, and (G) membrane proteins. The circles represent the number of reads mapped for a single gene. Green color represents the reads obtained from the sample grown in serum, while red represents reads obtained from EB-247 grown in LB. The data shown is the result of three independent biological experiments.

Figure 5

Schematic representation of mechanisms associated with EB-247 iron acquisition and differential expression analysis of genes. (A) Enterobactin mediated iron uptake. (B) Hemin mediated iron uptake. (C) Ferrichrome mediated iron uptake. (D) Aerobactin mediated iron uptake. (E) EfeUOB mediated iron transport. (F) FeoABC mediated iron transport.

Figure 6

Differential expression of the pga operon in response to human serum and growth inhibition of EB-247 by MccJ25. (A) Relative fold-change in the mRNA expression level of the pga operon. EB-247 was treated with human serum and the total RNA was isolated. The differential expression was examined using qRT PCR. Data is presented as mean ± standard deviation of three individual experiments with three replicates each. (B) Growth curve of EB-247 in presence of MccJ25. EB-247 was incubated in 25% serum and LB with a concentration of 0.6 µg/mL of MccJ25. The growth was monitored over 600 min. The data for the growth curves are the representatives of three individual experiments.