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A plasmid locus associated with Klebsiella clinical infections encodes a microbiome-dependent gut fitness factor - PubMed

  • ️Fri Jan 01 2021

. 2021 Apr 30;17(4):e1009537.

doi: 10.1371/journal.ppat.1009537. eCollection 2021 Apr.

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A plasmid locus associated with Klebsiella clinical infections encodes a microbiome-dependent gut fitness factor

Jay Vornhagen et al. PLoS Pathog. 2021.

Abstract

Klebsiella pneumoniae (Kp) is an important cause of healthcare-associated infections, which increases patient morbidity, mortality, and hospitalization costs. Gut colonization by Kp is consistently associated with subsequent Kp disease, and patients are predominantly infected with their colonizing strain. Our previous comparative genomics study, between disease-causing and asymptomatically colonizing Kp isolates, identified a plasmid-encoded tellurite (TeO3-2)-resistance (ter) operon as strongly associated with infection. However, TeO3-2 is extremely rare and toxic to humans. Thus, we used a multidisciplinary approach to determine the biological link between ter and Kp infection. First, we used a genomic and bioinformatic approach to extensively characterize Kp plasmids encoding the ter locus. These plasmids displayed substantial variation in plasmid incompatibility type and gene content. Moreover, the ter operon was genetically independent of other plasmid-encoded virulence and antibiotic resistance loci, both in our original patient cohort and in a large set (n = 88) of publicly available ter operon-encoding Kp plasmids, indicating that the ter operon is likely playing a direct, but yet undescribed role in Kp disease. Next, we employed multiple mouse models of infection and colonization to show that 1) the ter operon is dispensable during bacteremia, 2) the ter operon enhances fitness in the gut, 3) this phenotype is dependent on the colony of origin of mice, and 4) antibiotic disruption of the gut microbiota eliminates the requirement for ter. Furthermore, using 16S rRNA gene sequencing, we show that the ter operon enhances Kp fitness in the gut in the presence of specific indigenous microbiota, including those predicted to produce short chain fatty acids. Finally, administration of exogenous short-chain fatty acids in our mouse model of colonization was sufficient to reduce fitness of a ter mutant. These findings indicate that the ter operon, strongly associated with human infection, encodes factors that resist stress induced by the indigenous gut microbiota during colonization. This work represents a substantial advancement in our molecular understanding of Kp pathogenesis and gut colonization, directly relevant to Kp disease in healthcare settings.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. The Kp terZ-F genes are sufficient for TeO3-2 resistance.

(A) The ter locus is organized in two operons, a putative biosynthetic cluster and a TeO3-2 resistance cluster. These sections are found on opposite DNA strands and are encoded bidirectionally. The representative ter locus from the hvKp strain NTUH-K2044 is shown. NTUH-K2044 containing the empty vector pACYC184, the isogenic ΔterC mutant (clone Kp2259) containing an empty vector, the pTerC, or the pTerZ-F plasmid (B), and the E. coli K12 strain MG1655 with or without the pTerZ-F plasmid (C) were grown on LB or LB containing 10 or 100 μM K2TeO3-2 to visualize inhibition of growth (dilution series 100−10−7 of overnight culture). Two representative clones (labeled #1 and #2) of NTUH-K2044ΔterC containing the pTerC or the pTerZ-F plasmid and MG1655 containing the pTerZ-F plasmid are shown.

Fig 2
Fig 2. The Kp ter operon is not exclusive to hypervirulence plasmids.

ter+ plasmids from Martin et al. mSystems, 2018 [10] (A-C) and reference strains from the NCBI database (D-F) were analyzed. (A,D) Relative frequencies of sequence types (ST) of Kp strains containing ter+ plasmids. HvKp sequence types previously associated with the ter operon are outlined in a dashed line. (B,E) Heat map of ter+ plasmid sequence similarity to genes known to influence infection and antibiotic resistance genes. Each row represents an individual plasmid in the order of S2 Table (Martin et al. mSystems, 2018 [10] index 1–14, NCBI reference strains index 15–102). The pK2044 hvKp plasmid is highlighted by the red box, and hypervirulent Kp sequence types (hvST) previously associated with the ter operon are indicated. (C,F) To determine if any neighboring gene was consistently associated with ter, the gene neighborhood of ter plasmids encoding the ter operon from Martin et al. mSystems, 2018 [10] was visualized (C) and the frequency of ORFs adjacent to the ter operon encoded on reference plasmids from the NCBI database was calculated (F).

Fig 3
Fig 3. TerC is a fitness factor during gut colonization.

(A) Three days prior to inoculation, male and female C57BL6/J mice sourced from barriers RB16 and RB07 were treated with 0.5 g/L ampicillin or regular drinking water. (B-E) NTUH-K2044 and the isogenic ΔterC mutant (clone Kp2259) were mixed 1:1 and approximately 5x106 CFU were orally gavaged into mice (n = 9–18 per group). A fresh fecal pellet was collected daily from each animal, CFUs were enumerated, and log competitive indices (mutant:WT) were calculated (median and IQR displayed, *P < 0.05, **P < 0.005, ***P < 0.0005, one-sample t test compared to a hypothetical value of 0). (F) NTUH-K2044 and the isogenic ΔterC mutant containing an empty vector or the pTerZ-F plasmid were mixed 1:1 and approximately 5x106 CFU were orally gavaged into mice sourced from barrier RB16 (n = 14–16). A fresh fecal pellet was collected 24 hours after inoculation, CFUs were enumerated, and log competitive indices (mutant:WT) were calculated (F, median and IQR displayed, ****P < 0.00005, one-sample t test compared to a hypothetical value of 0 or Student’s t test). Each data point represents an individual animal.

Fig 4
Fig 4. The fecal microbiota in which terC is (RB16) and is not (RB07) a fitness factor are distinct.

Fecal pellets collected from male and female C57BL6/J mice sourced from barriers RB16 and RB07 (n = 9–20 mice per group) on the day of Kp inoculation were subjected to 16S rRNA sequencing. Pairwise community dissimilarity values between the fecal microbiota communities of barriers RB16 and RB07 with or without three days treatment with 0.5 g/L ampicillin were visualized by Principal coordinates analysis (A, AMOVA) and individually (B, **P < 0.005, ****P < 0.00005, one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test). (C) Diversity of the fecal microbiota was summarized by inverse Simpson index (blue points: RB16+Abx, orange points: RB07+Abx, **P < 0.005, one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test). LEfSe was used to determine if specific bacterial families (D) or OTUs (F) were differentially abundant between the fecal microbiota of RB16 and RB07 (D, LDA ≥ 3.5 and P < 0.05 are shown). Differential bacterial families (E) or OTUs (G) relative abundance values were plotted (E, *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.00005, Student’s t test).

Fig 5
Fig 5. Exogenous treatment of mice with SCFAs results in a terC fitness defect.

Seven days prior to inoculation, male and female C57BL6/J mice sourced from barriers RB16 and RB07 were treated with a SCFA cocktail or regular drinking water (sham). NTUH-K2044 and the isogenic ΔterC mutant (clone Kp2257) were mixed 1:1 and approximately 5x106 CFU were orally gavaged into mice (n = 19 per group). A fresh fecal pellet was collected daily from each animal, CFUs were enumerated, and log competitive indices (mutant:WT) were calculated (median and IQR displayed, *P < 0.05, **P < 0.005, ***P < 0.0005, one-sample t test compared to a hypothetical value of 0 or Holm-Sidak multiple-comparison test following one-way ANOVA).

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References

    1. Magill SS, Edwards JR, Bamberg W, Beldavs ZG, Dumyati G, Kainer MA, et al.. Multistate Point-Prevalence Survey of Health Care–Associated Infections. New England Journal of Medicine. 2014;370(13):1198–208. 10.1056/NEJMoa1306801 . - DOI - PMC - PubMed
    1. Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clinical microbiology reviews. 1998;11(4):589–603. Epub 1998/10/10. - PMC - PubMed
    1. CDC. The Direct Medical Costs of Healthcare-Associated Infections in U.S. Hospitals and the Benefits of Prevention. 2009.
    1. O’Neill. Tackling Drug-Resistant Infections Globally: final report and recommendations 2016. [updated May 19]. Available from: https://amr-review.org.
    1. Ko WC, Paterson DL, Sagnimeni AJ, Hansen DS, Von Gottberg A, Mohapatra S, et al.. Community-acquired Klebsiella pneumoniae bacteremia: global differences in clinical patterns. Emerging infectious diseases. 2002;8(2):160–6. Epub 2002/03/19. 10.3201/eid0802.010025 - DOI - PMC - PubMed

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