Enhanced killing of antibiotic-resistant bacteria enabled by massively parallel combinatorial genetics

Abstract

New therapeutic strategies are needed to treat infections caused by drug-resistant bacteria, which constitute a major growing threat to human health. Here, we use a high-throughput technology to identify combinatorial genetic perturbations that can enhance the killing of drug-resistant bacteria with antibiotic treatment. This strategy, Combinatorial Genetics En Masse (CombiGEM), enables the rapid generation of high-order barcoded combinations of genetic elements for high-throughput multiplexed characterization based on next-generation sequencing. We created ∼ 34,000 pairwise combinations of Escherichia coli transcription factor (TF) overexpression constructs. Using Illumina sequencing, we identified diverse perturbations in antibiotic-resistance phenotypes against carbapenem-resistant Enterobacteriaceae. Specifically, we found multiple TF combinations that potentiated antibiotic killing by up to 10(6)-fold and delivered these combinations via phagemids to increase the killing of highly drug-resistant E. coli harboring New Delhi metallo-beta-lactamase-1. Moreover, we constructed libraries of three-wise combinations of transcription factors with >4 million unique members and demonstrated that these could be tracked via next-generation sequencing. We envision that CombiGEM could be extended to other model organisms, disease models, and phenotypes, where it could accelerate massively parallel combinatorial genetics studies for a broad range of biomedical and biotechnology applications, including the treatment of antibiotic-resistant infections.

Keywords: combination therapy; drug resistance; multifactorial genetics; synthetic biology; systems biology.

Fig. 1.

Overview, validation, and application of the CombiGEM technology to identify genetic combinations that combat antibiotic resistance. (A) Outline of the CombiGEM assembly strategy. Transcription factor (TF) expression constructs are barcoded (BC), and four restriction sites (1A, 1B, 2A, 2B) are positioned as shown. The pairs 1A/1B and 2A/2B consist of unique restriction sites that generate compatible overhangs within the pair but are incompatible with the other pair. The barcoded vectors are pooled and digested with enzymes 1B + 2A. Inserts are generated from vectors by PCR and digested with 1A + 2B. A one-pot ligation reaction produces a pairwise combinatorial library, which can be further digested and ligated with the same insert pool to produce higher-order combinations. In this work, barcodes are separated by 6 base pairs. (B) Distribution of high-throughput sequencing reads among combinations in the pairwise library. A total of 1.9 million reads were distributed among 34,554 combinations, recovering 98% of the possible 35,343 pairwise combinations. (C) Experimental outline. E. coli NDM-1 cells containing a CombiGEM library were diluted into cultures with and without anhydrotetracycline (aTc) and grown to middle logarithmic phase. Each culture was then further diluted into cultures with and without antibiotic (Abx), and with and without aTc. DNA from each condition was harvested at early and late logarithmic-growth stages and processed for high-throughput sequencing (HT-seq) with Illumina HiSeq. Comparisons of abundances of barcoded gene combinations among different conditions revealed genotypes that conferred desired phenotypes. (D) Heatmap of S scores for specific hits across experiments. Combinations marked as potentiating combinations represent genetic combinations that drop out upon addition of both ceftriaxone and aTc. Neutral combinations showed neutral S scores in the presence of both ceftriaxone and aTc. Ceftriaxone was applied in two different concentrations: 64 µg/mL (low) and 256 µg/mL (high). Gene pairs correspond to numbers 1, qseB + bolA; 2, lrp + xylR; 3, eCFP + norR; and 4, pdhR + yohL.

Fig. 2.

Antibiotic time-kill curves for NDM-1 E. coli with genetic combinations identified through CombiGEM. (A) Interacting gene pairs. Potentiating combinations identified through S-score analysis, qseB + bolA and lrp + xylR, exhibited enhancement of ceftriaxone activity upon induction with aTc, with up to 3–5 orders of magnitude greater killing compared with ceftriaxone alone or aTc alone. (B) Control genetic combinations with neutral S scores showed only minor potentiation of ceftriaxone. E. coli MG1655 cells with no genetic combinations (labeled MG1655) were effectively killed by ceftriaxone down to the limit of detection (100 CFU/mL). NDM-1 E. coli with no genetic combinations (labeled NDM-1) exhibited significantly less killing by ceftriaxone compared with E. coli MG1655. (C) Individual constituent genes of potentiating combinations revealed less effective potentiation of ceftriaxone than the corresponding combinations. In all experiments in this figure, ceftriaxone was administered at a concentration of 192 µg/mL and aTc at 100 ng/mL. Error bars represent SEM (small error bars are obscured by symbols).

Fig. 3.

Lethal genetic combinations potentiate antibiotic activity against NDM-1 bacteria. (A) Interacting gene pairs. When used in combination with ceftriaxone, lethal genetic combinations identified via log-ratio analysis achieved reductions of viable cell counts of up to 6 orders of magnitude and suppressed growth for over 10 h in time-kill assays. (B) Control gene pairs. Random combinations with lethal genes, such as rstA + oxyR and cadC + allR, showed significantly less potentiation of antibiotic activity of ceftriaxone and less sustained growth suppression over time compared with lethal combinations identified through CombiGEM. (C) Individual genes. The single gene constituents of the torR + metR combination had weaker effects on bacterial killing compared with the torR + metR combination, when applied together with ceftriaxone. In all experiments in this figure, ceftriaxone was administered at 192 µg/mL and aTc at 100 ng/mL. Error bars represent SEM (small error bars are obscured by symbols).

Fig. 4.

Phagemid-based delivery of genetic combinations enhance killing of NDM-1 E. coli with ceftriaxone. (A) NDM-1 E. coli were infected with M13 phagemids containing combination constructs and treated with antibiotic and aTc where appropriate, which is indicated as time t = 0 on the following graphs. (B) Phagemids containing torR + metR and nhaR + melR, when coadministered with ceftriaxone and induced with aTc, achieved amplified killing of NDM-1 E. coli bacteria. (C) In contrast to the results in B, phagemid-based delivery of a control genetic combination did not result in enhanced killing of NDM-1 E. coli. In all experiments in this figure, ceftriaxone was administered at 192 µg/mL and aTc at 100 ng/mL. Error bars represent SEM (small error bars are obscured by symbols).