Enterobacter hormaechei replaces virulence with carbapenem resistance via porin loss

Abstract

Pathogenic Enterobacter species are of increasing clinical concern due to the multidrug-resistant nature of these bacteria, including resistance to carbapenem antibiotics. Our understanding of Enterobacter virulence is limited, hindering the development of new prophylactics and therapeutics targeting infections caused by Enterobacter species. In this study, we assessed the virulence of contemporary clinical Enterobacter hormaechei isolates in a mouse model of intraperitoneal infection and used comparative genomics to identify genes promoting virulence. Through mutagenesis and complementation studies, we found two porin-encoding genes, ompC and ompD, to be required for E. hormaechei virulence. These porins imported clinically relevant carbapenems into the bacteria, and thus loss of OmpC and OmpD desensitized E. hormaechei to the antibiotics. Our genomic analyses suggest porin-related genes are frequently mutated in E. hormaechei, perhaps due to the selective pressure of antibiotic therapy during infection. Despite the importance of OmpC and OmpD during infection of immunocompetent hosts, we found the two porins to be dispensable for virulence in a neutropenic mouse model. Moreover, porin loss provided a fitness advantage during carbapenem treatment in an ex vivo human whole blood model of bacteremia. Our data provide experimental evidence of pathogenic Enterobacter species gaining antibiotic resistance via loss of porins and argue antibiotic therapy during infection of immunocompromised patients is a conducive environment for the selection of porin mutations enhancing the multidrug-resistant profile of these pathogens.

Keywords: Enterobacter cloacae complex; antimicrobial resistance; infection and pathogenesis; porins; virulence.

Fig. 1.

E. hormaechei ST78 is virulent in a mouse model of peritonitis. (A) Survival curves of mice infected i.p. with 10⁸ colony-forming units (CFU) of indicated E. hormaechei ST171 and ST78 isolates. Number of isolates tested per ST indicated in parentheses. n = 5 to 10 mice per isolate, and 5 mice for PBS control. P < 0.0001, log-rank (Mantel-Cox) test. (B and C) ST78 survival curve data shown in (A), separated by ST78 clade (B) and isolates belonging to ST78 clade D (C). In (B), number of isolates tested per clade indicated in parentheses. In (C), n = 10 mice per isolate, and 5 mice for PBS control. (D) Bacterial burdens of NR3072 and NR3033 in the peritoneal lavage fluid, blood, spleen, and lungs of mice, 16 h after i.p. injection with 10⁸ CFU. The horizontal dotted line indicates the limit of detection. n = 5 mice per isolate. Error bars indicate geometric SD. **P < 0.01, *P < 0.05, Mann–Whitney test.

Fig. 2.

Comparative genomics of E. hormaechei isolates. (A) Maximum-likelihood phylogeny of ST78 isolates. Clade designations have been previously described (14). (B) The genomes of six ST78 clade D isolates were compared via maximum-likelihood phylogeny, core-genome single-nucleotide substitution (SNV) counts, and clustering of orthologous genes. The first heatmap to the right of the phylogeny shows counts of isolate-pair SNVs, while the second heatmap shows the number of genes from ortholog clustering that are not shared between a pair of isolates. Through these comparisons, we uncovered that NR3033 features a 2,384-nucleotide deletion that disrupts yddG and ompD and a deletion of a guanine in nucleotide position 410 of ompC, resulting in a frameshift. (C) Neighbor-joining phylogeny of 6,833 E. hormaechei assemblies from NCBI. Pie charts display the proportions of porin genes ompD and ompC in an intact state (present) or with disruptive mutations: presumed gene deletion (not found), disruption by insertion sequence (IS), or frameshift/truncation. The large pie charts on the left depict porin status proportions for all isolates in the phylogeny. Smaller pie charts show porin gene status of subsets with at least 50 isolates that descend from an at least 500-SNVs-long phylogeny branch (highlighted in yellow). Additionally, porin gene status was tabulated for the 12 most frequent sequence types (ST), indicated with a red branch coloring and a label. ompD pie charts have a thicker border than ompC pie charts. *ompC was duplicated in two isolates.

Fig. 3.

Porins are required for the virulence of E. hormaechei ST78 clade D isolates. (A) Survival curves of mice i.p. infected with 10⁸ CFU of NR3072, NR3072 ∆ompC, NR3072 ∆ompD, and NR3072 ∆ompC ∆ompD. n = 10 mice per strain. ***P < 0.001, log-rank (Mantel-Cox) test. (B) Survival curves of mice i.p. infected with 10⁸ CFU of NR3072 ∆ompC ∆ompD attTn7::Cm and NR3072 ∆ompC ∆ompD attTn7::ompC+ompD. n = 10 mice per strain. **P < 0.01, log-rank (Mantel-Cox) test. (C) Survival curves of mice i.p. infected with 10⁸ CFU of NR2339, NR2339 ∆ompC ∆ompD, and NR2339 ∆ompC ∆ompD attTn7::ompC+ompD. n = 10 mice per strain. **P < 0.01, log-rank (Mantel-Cox) test. (D) Bacterial burdens of NR3072, NR3072 ∆ompC ∆ompD, and NR3072 ∆ompC ∆ompD attTn7::ompC+ompD in peritoneal lavage fluid, blood, spleen, and lungs 16 hours after i.p. infection. The horizontal dotted line indicates the limit of detection. n = 15 mice per strain. Error bars indicate geometric SD. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, or not significant (ns), Mann–Whitney test.

Fig. 4.

Both OmpC and OmpD contribute to carbapenem sensitivities of E. hormaechei ST78 clade D isolates. (A) Growth curves of NR3033, NR3072, NR2339, and their porin gene-manipulated derivatives in 0.5 µg/mL meropenem. n = 6 biological replicates per strain. Error bars indicate SE of the mean. (B–D) Minimum inhibitory concentrations (MICs) of meropenem (B), ertapenem (C), and imipenem (D) against NR3033, NR3072, NR2339, and their porin gene-manipulated derivatives. n = 3 to 6 biological replicates per strain for each antibiotic. Error bars indicate SE of the mean. ****P < 0.0001, ***P < 0.001, or not significant (ns), one-way ANOVA with Sidák’s multiple comparison test.

Fig. 5.

Porins are dispensable for virulence in immunocompromised hosts. (A) Schematic of E. hormaechei infection in the cyclophosphamide model of immunosuppression. (B) Survival curves of cyclophosphamide-treated mice i.p. infected with 10⁸ CFU of E. coli DH5α λpir or E. hormaechei ST78 strains NR3033, NR3072, NR3072 ∆ompC ∆ompD, or NR2339 ∆ompC ∆ompD. n = 10 mice per E. hormaechei strain and PBS control, n = 5 mice for DH5α λpir. (C) Schematic of meropenem-treated ex vivo human whole blood bacteremia model. (D) Burdens over time of NR3072 attTn7::Hyg (“WT”) and NR3072 ∆ompC ∆ompD attTn7::Cm (“∆ompC ∆ompD”) during coinfection of human whole blood, with or without meropenem treatment. The horizontal dotted line indicates the limit of detection. n = 4 biological replicates (4 different blood donors). Error bars indicate geometric SD. ****P < 0.0001, one-way ANOVA with Sidák’s multiple comparison test.