A Genome-Scale Antibiotic Screen in Serratia marcescens Identifies YdgH as a Conserved Modifier of Cephalosporin and Detergent Susceptibility

Abstract

Serratia marcescens, a member of the order Enterobacterales, is adept at colonizing health care environments and is an important cause of invasive infections. Antibiotic resistance is a daunting problem in S. marcescens because, in addition to plasmid-mediated mechanisms, most isolates have considerable intrinsic resistance to multiple antibiotic classes. To discover endogenous modifiers of antibiotic susceptibility in S. marcescens, a high-density transposon insertion library was subjected to sub-MICs of two cephalosporins, cefoxitin, and cefepime, as well as the fluoroquinolone ciprofloxacin. Comparisons of transposon insertion abundance before and after antibiotic exposure identified hundreds of potential modifiers of susceptibility to these agents. Using single-gene deletions, we validated several candidate modifiers of cefoxitin susceptibility and chose ydgH, a gene of unknown function, for further characterization. In addition to cefoxitin, deletion of ydgH in S. marcescens resulted in decreased susceptibility to multiple third-generation cephalosporins and, in contrast, to increased susceptibility to both cationic and anionic detergents. YdgH is highly conserved throughout the Enterobacterales, and we observed similar phenotypes in Escherichia coli O157:H7 and Enterobacter cloacae mutants. YdgH is predicted to localize to the periplasm, and we speculate that it may be involved there in cell envelope homeostasis. Collectively, our findings provide insight into chromosomal mediators of antibiotic resistance in S. marcescens and will serve as a resource for further investigations of this important pathogen.

Keywords: AmpC; Serratia marcescens; TIS; TnSeq; YdgH; cefepime; cefoxitin; cephalosporin; ciprofloxacin; fluoroquinolone.

FIG 1

Serratia marcescens ATCC 13880 transposon-insertion sequencing reveals genes important for in vitro growth. (A) S. marcescens genes binned by percentage of TA sites disrupted. Overlaid is the hidden Markov model-based analysis assignment (EL-ARTIST pipeline) of whether insertions within the gene are underrepresented, differ by domain within the gene (“domain”), or are found to be distributed neutrally within the gene. The left histogram peak contains predominantly underrepresented genes where insertions are tolerated in only a low percentage of sites. (B) Venn diagram illustrating overlap between S. marcescens ATCC 13880 (GenBank accession number CP072199) underrepresented genes (red) with E. coli K-12 underrepresented genes (yellow), with the K-12 KEIO collection as an additional comparator (blue). (C) Genes underrepresented in S. marcescens ATCC 13880 but not in E. coli K-12, either by EL-ARTIST analysis or by the analysis resulting from the KEIO collection. Clusters of orthologous genes (COG) categories of those genes are tabulated. (D) Lipid A 4-amino-4-deoxy-L-arabinose modification operon, demonstrating that transposon insertions are underrepresented (red) in arnD, arnE, and arnF. btuD and nlpC are genes flanking this operon.

FIG 2

Antibiotic screen revealing S. marcescens genes important for growth and survival. (A) Screen schematic. An input library (containing about 2 million insertion mutants) is grown to an optical density (OD) of 0.1 and divided between the following 4 conditions for an additional 6 h of growth: LB alone; LB plus cefoxitin 4 μg/ml; LB plus cefepime 0.025 μg/ml; and LB plus ciprofloxacin 0.05 μg/ml. (B) Under the screening conditions, the library without drug selection undergoes a more than 2-log expansion, while those with antibiotic decrease in CFU to a similar extent (about 0.5 log), with the library in cefoxitin undergoing expansion after 4 h, likely due to upregulation of AmpC beta-lactamase. Libraries were harvested for analysis at 6 h. (C) Number of genes with insertion mutants showing a ≥4-fold change at 6 h with a Mann-Whitney U P value of ≤ 0.01, compared to outgrowth in LB alone. All data are tabulated in Table S4 in the supplemental material. (D) Venn diagram illustrating genes showing coordinate enrichment or depletion. Individual genes comprising the depicted sets are tabulated in Table S5.

FIG 3

Whole-genome screen for modifiers of cefoxitin susceptibility. (A) Volcano plot illustrating candidate genes important for cefoxitin susceptibility. On the x axis is the log₂ fold change (log₂FC) in insertion mutant abundance in cefoxitin (FOX) compared to that in no drug. The y axis is the inverse Mann-Whitney U P value (1/MWU p-val), which roughly measures the concordance between mutants with insertions at individual TA sites across a gene. Genes were depleted (red) if the log₂FC was ≤2 and the 1/MWU p-val was ≥100. Genes were enriched (blue) if the log₂FC was ≥2 and the 1/MWU p-val was ≥100. (B) Selected genes enriched or depleted in cefoxitin were tabulated. Genes important for envelope integrity and peptidoglycan recycling were depleted. ydgH, an enriched, poorly characterized gene, is also highlighted. (C) Growth of various S. marcescens gene deletion mutants in cefoxitin 4 μg/ml. Growth is depicted as CFU_mutant/CFU_Wt (in cefoxitin) divided by CFU_mutant/CFU_Wt (in LB alone). Although no mutant had large defects in LB alone, due to stochastic errors in dilution to starting CFU, this improved the repeatability of the experiment. *, P ≤ 0.05; **, P ≤ 0.01; unpaired two-tailed t test, unadjusted.

FIG 4

YdgH contributes to basal S. marcescens cefoxitin susceptibility. (A) The YdgH locus on the y axis with transposon-insertion (Tn) reads on the y axis, demonstrating the large enrichment of YdgH insertion mutants in cefoxitin (below, compared to the input library; above, on the same scale). (B) The ΔydgH mutant had increased growth in cefoxitin compared to that of the wild type (Wt) at multiple drug concentrations and at multiple time points. (C) Schematic illustrating optical density at 600 nm (OD₆₀₀) ratio used in subsequent figures. The OD₆₀₀ of the ΔydgH mutant at 4 h is divided by that of the Wt. (D) Exogenous ydgH rescues the cefoxitin phenotype in the ΔydgH mutant. *, P ≤ 0.05; **, P ≤ 0.01; unpaired two-tailed t test, unadjusted.

FIG 5

YdgH deletion leads to decreased cephalosporin susceptibility and increased detergent susceptibility. (A) OD₆₀₀ ratios demonstrating that the ΔydgH mutant has decreased second- and third-generation cephalosporin susceptibility (including to cefoxitin, moxalactam, and ceftriaxone) but no large differences in susceptibility to first-generation cephalosporins, antipseudomonal cephalosporins, penicillins, or carbapenems. (B) OD₆₀₀ ratios demonstrating that the ΔydgH mutant has no large differences in non-beta-lactam antibiotic susceptibility. Cefoxitin ratio reproduced for reference. (C) OD₆₀₀ ratios demonstrating that the ΔydgH mutant has no large differences in bacitracin or polymyxin susceptibility, but has small but significant increases in susceptibility to rifampin, and more broadly to the detergents sodium dodecyl sulfate (SDS), benzethonium chloride, and benzalkonium chloride. Cefoxitin ratio reproduced for reference. (D) Viability experiments of the Wt and the ΔydgH mutant in ceftriaxone, benzethonium chloride (BTC), sodium dodecyl sulfate (SDS), and rifampin confirmed the results of the turbidity experiments. *, P ≤ 0.05; **, P ≤ 0.01; unpaired two-tailed t test (A, B, and C) or Wilcoxon rank-sum test (D), unadjusted.

FIG 6

YdgH deletion results in conserved phenotypes in Escherichia coli O157:H7 EDL933 and Enterobacter cloacae ATCC 13047. (A) The YdgH phylogeny was constructed based on amino acid substitutions by using the maximum-likelihood method and the JTT matrix-based model in MEGA X. The relevant higher-order families are indicated. (B) All 3 mutants have decreased susceptibility to ceftriaxone (E. cloacae has high intrinsic resistance to cefoxitin, so ceftriaxone was chosen), as well as increased susceptibility to benzethonium chloride. Growth of the E. cloacae ΔydgH mutant was decreased in SDS, although growth of E. coli O157:H7 was not. Due to inherent differences in Wt susceptibility, drug concentrations differed considerably between isolates and were as follows: ceftriaxone, 4 μg/ml for E. cloacae, 0.04 μg/ml for E. coli O157:H7, and 0.06 μg/ml for S. marcescens; benzethonium chloride (BTC), 10 μg/ml for E. cloacae, 18 μg/ml for E. coli O157:H7, and 52 μg/ml for S. marcescens); SDS, 0.8% for E. cloacae, 0.13% for E. coli O157:H7, and 5% for S. marcescens. *, P ≤ 0.05; **, P ≤ 0.01; unpaired two-tailed t test, unadjusted.