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

['Jay Vornhagen', 'Department Of Pathology', 'University Of Michigan', 'Ann Arbor', 'Mi', 'United States Of America', 'Department Of Microbiology', 'Immunology', 'Christine M. Bassis', 'Department Of Internal Medicine Infectious Diseases Division']

Date: 2021-09

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 (TeO 3 -2 )-resistance (ter) operon as strongly associated with infection. However, TeO 3 -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.

The bacterial pathogen Klebsiella pneumoniae is of substantial public health concern due to its ability to cause serious antibiotic-resistant infections. These infections frequently occur in healthcare settings, especially in patients with detectable gut colonization by K. pneumoniae. Importantly, infectious K. pneumoniae strains are often detected in the gut of patients with K. pneumoniae disease, indicating that the gut is a reservoir of infectious K. pneumoniae. Our previous work interrogating the genetic underpinnings of K. pneumoniae disease in colonized patients identified a strong association between K. pneumoniae infection and the presence of an enigmatic genetic locus known as the ter operon. We found that this operon is not needed for pneumonia and bacteremia, and therefore, we explored the importance of the ter operon in the gut. K. pneumoniae lacking ter function was at a disadvantage in the gut, thus explaining the connection between the ter operon and infection in hospitalized patients. Interestingly, the advantage conferred by the ter operon in the gut was associated with the presence of specific indigenous gut microbiota and the presence of short-chain fatty acids, which are metabolized by the host and gut microbiota. This work demonstrates that the ter operon is a microbiome-dependent gut fitness factor and suggests that indigenous gut bacteria may limit colonization by infectious K. pneumoniae.

In Kp, the ter operon is found on pK2044-like plasmids that encode multiple virulence genes characteristic of hypervirulent Kp strains (hvKp) [ 35 , 36 ]. This suggests the association between the presence of the ter operon and Kp disease could be due to genetic linkage with plasmid-encoded virulence genes. Moreover, the ter operon was identified as a point of recombination for the Kp hypervirulence plasmid and a carbapenemase encoding plasmid [ 37 ], suggesting it can both enhance fitness and enable the convergence of two worrying Kp pathotypes. To distinguish linkage with virulence genes from an inherent function of the ter operon in pathogenesis, we performed comparative genomic studies on a broad collection of Kp plasmids and assessed fitness of isogenic ter mutants of a hypervirulent strain in a model of gut colonization in two distinct microbial communities. Collectively, these data reveal that the ter operon, highly associated with human infection, likely acts early in pathogenesis as a horizontally transferrable fitness factor promoting robust gut colonization in the presence of the indigenous microbiota. Analyses of these indigenous microbiota revealed a role for short-chain fatty acid (SCFA) metabolism in the reduction of gut fitness for Kp lacking a functional ter operon.

Comparing infected to asymptomatically colonized patients, we have previously identified the tellurium resistance operon, known as the ter operon, as highly associated with Kp pneumonia and bacteremia (OR = 11.3, 95% CI = 1.6–80.0 after adjustment for clinical variables). This enigmatic operon is found in many diverse bacteria, archaea, and some eukaryotes wherein it bestows resistance to the toxic compound tellurite oxide (TeO 3 -2 ) [ 21 ]. The antibacterial property of TeO 3 -2 was first described by Sir Alexander Fleming in 1932 [ 22 ], and the reduction of TeO 3 -2 to Te 0 in bacterial cells underlying that antimicrobial property was discovered even earlier in 1914 [ 23 ]. Resistance to TeO 3 -2 has long been used for clinical detection of Corynebacterium diphtheriae and other pathogens [ 23 – 26 ]. There are several distinct genetic loci (ter, teh, tel, kil, others) involved in TeO 3 -2 resistance whose gene products are predicted to be mechanistically divergent [ 21 , 27 ]. Of these, the ter operon is least understood. It is highly unlikely that the physiological function of the ter operon is to resist TeO 3 -2 , as this compound is exceedingly rare in the environment, and not present in humans. Previous mechanistic studies of the ter operon in non-Kp bacteria suggest a pleiotropic function, with evidence for resistance to oxidative, genotoxic, heavy metal, proton motive force, cell wall, membrane, phage, and protein synthesis stress [ 21 , 28 , 29 ], as well as a role for intracellular survival in macrophages [ 30 , 31 ]. Moreover, previous studies have suggested that the ter operon is transcriptionally regulated by the OxyRS system [ 29 , 32 ], further suggesting a connection between the ter operon and stress. Interestingly, this operon is found in several other pathogenic Enterobacterales, such as Yersinia pestis, Proteus mirabilis, and enterohemorrhagic Escherichia coli [ 29 , 31 , 33 , 34 ]. The association with infection, and the unique biology of TeO 3 -2 and the ter operon collectively imply that this operon is spuriously annotated based on historical in vitro findings, warranting further investigation into its true physiological function during Kp infection.

Gut colonization by Kp is consistently associated with subsequent Kp disease [ 10 – 14 ], and patients are predominantly infected with their colonizing strain [ 10 , 11 ]. However, colonizing strains likely vary in their potential to cause infection. Variation in virulence potential is likely mediated by the presence or absence of genes in the accessory genome of each isolate and may be important at any step between colonization maintenance in the gut to fitness at the eventual site of infection. As gut colonization often precedes infection [ 11 , 12 ], murine models of Kp colonization are needed that are relevant to human infection. Several studies have rapidly advanced our understanding of Kp gut colonization, including the relevance of the indigenous gut microbiota to Kp fitness during colonization [ 15 , 16 ]; however, the factors underlying these interactions remain unexplored. Importantly, previous studies have shown that gut microbiota differs by mouse vendor and by the room within each vendor facility (referred to as “barrier”) [ 17 ] and these differences can impact experimental output [ 18 – 20 ]. Therefore, any evaluation of fitness factors during colonization should account for variations in the microbiota and how different indigenous bacteria may interact with Kp directly or indirectly.

The emergence and spread of highly antibiotic-resistant bacteria have substantially complicated disease treatment and control. Enterobacteriaceae are a significant contributor to the burden of antibiotic-resistant infections through the production of extended-spectrum beta-lactamases (ESBL-) and carbapenemases (CP-). Within the Enterobacteriaceae family is Klebsiella pneumoniae (Kp), which is a substantial threat to human health, as it is the third leading cause of all hospital-acquired infections [ 1 , 2 ]. Infection with ESBL- and CP-Kp is associated with staggeringly high mortality (>50%) and excessive healthcare costs [ 3 , 4 ], leading the Centers for Disease Control and Prevention to categorize ESBL- and CP-Kp as serious and urgent threats, respectively. Further complicating this issue, strains of hypervirulent Kp (hvKp) have independently emerged in Southeast Asia. HvKp strains cause severe community-acquired infections that are associated with mortality rates as high as 31% [ 5 ]. Furthermore, hvKp and antibiotic-resistant strains are reported to be converging, leading to dangerous, highly antibiotic-resistance strains of hvKp, which have recently been detected outside of Southeast Asia [ 6 – 9 ]. As these dangerous strains circulate more widely and as the number of effective treatments dwindles, alternative interventions are necessary to diminish the threat posed by these bacteria.

To assess the effect of SCFAs on Kp gut fitness in vivo, we repeated our competitive gut colonization experiments with mice sourced from RB16 and RB07, but also included mice sourced from RB07 treated continuously with a cocktail of SCFAs via drinking water. As previously observed, ΔterC Kp were less fit than WT Kp in mice sourced from RB16 but were equally fit in mice sourced from RB07 ( Fig 5 ). Treatment of mice from RB07 with a SCFA cocktail led to significantly reduced fitness of the ΔterC mutant in the gut similar to that of what was observed in RB16 ( Fig 5 ). Finally, we measured the presence of SCFAs in the fecal pellets of mice sourced from both barriers, as well as mice sourced from RB07 treated with exogenous SCFAs. Fecal SCFA quantification revealed higher SCFA concentrations in the feces of mice sourced from RB16 compared to those from RB07 ( S28A Fig ); however, this difference did not reach statistical significance. Treatment of mice sourced from RB16 with antibiotics, which restored the ΔterC fitness defect, also significantly reduced fecal SCFA levels ( S29A Fig ), specifically acetate and butyrate ( S29C and S29D Fig ). These data indicate that the reduced fitness of the ΔterC mutant is due to gut SCFA levels, which is associated with differences in specific indigenous gut microbiota constituents between mice sourced from RB16 and RB07.

SCFAs are known to have a wide variety of functions in the host, including increasing antimicrobial peptide production [ 61 ], intestinal epithelial barrier function [ 62 ], and acceleration of the immune response to Enterobacteriaceae [ 63 ]. Moreover, previous studies have identified a protective role for the SCFA acetate during Kp lung infection [ 64 ]. To determine if SCFA metabolism is responsible for the fitness defect exhibited by ΔterC, we first explored if SCFAs directly kill or inhibit the growth of Kp in a ter-dependent manner. Previous studies have shown that SCFAs are able to directly inhibit the growth of Kp under acidified conditions through disruption of respiration [ 65 ]. Thus, we grew WT Kp and ΔterC in the presence of SCFAs (acetate, butyrate, and propionate) in both neutral and acidified conditions. Similar to previous studies, high concentrations of SCFAs slightly inhibited growth of both strains in neutral conditions, but completely arrested growth in acidic conditions ( S27A Fig ). To determine if an individual SCFA was responsible for growth inhibition, WT Kp and ΔterC was grown in acetate, butyrate, and propionate individually and in combination in both neutral and acidified conditions. Acetate and butyrate had the largest impacts on Kp growth; however, growth inhibition was not dependent on the presence of terC ( S27B Fig ). Next, we assessed if growth inhibition occurs in a ter-dependent manner at lower concentrations of SFCAs. Again, titration of SCFAs in acidified media inhibited growth in a concentration-dependent manner, but not a ter-dependent manner ( S27C Fig ). To determine if WT Kp are able to antagonistically inhibit the growth of ΔterC in the presence of SCFAs, competitive growth assays were performed in the presence of SCFAs. No antagonism was observed between WT Kp and ΔterC in the presence of SCFAs ( S27D Fig ). Finally, we performed killing assays with the WT Kp and ΔterC strains to determine if SCFAs can kill Kp in a ter-dependent manner; however, no killing was observed for either strain ( S27E Fig ). Collectively, these data demonstrate that SCFAs can indeed inhibit Kp growth under acidified conditions as previously described [ 65 ], though not in a ter-dependent manner.

LEfSe was used to determine which specific metabolic pathways differentiated the predicted metagenomic profiles of the gut microbiota of mice sourced from RB16 and RB07 [ 51 ]. This analysis revealed several metabolic pathways that were enriched in the gut microbiota of mice sourced from RB16 on the day of inoculation and throughout the experiment, including gluconeogenesis, peptidoglycan biosynthesis, and fermentation of short-chain fatty acids ( S24 and S25 Figs, S4 Table and S3 Data ). The enrichment of SCFA metabolic pathways in the gut microbiota of mice sourced from RB16 may be explained by the presence of M. intestinale, which was strongly associated with the gut microbiota of mice sourced from RB16. In addition, antibiotic treatment of mice sourced from RB16 led to a decrease in OTUs that correspond to M. intestinale (0008, 0009, 0018, 0019), and a drastic reduction in the relative abundance of predicted SCFA metabolic pathways ( S26 Fig ). This corresponds to our 16S rRNA sequencing data, as SCFA pathways underpin the impact of M. intestinale on gut inflammatory homeostasis [ 20 , 54 – 57 ].

Next, we interrogated what biological processes may result in the reduced fitness of the ΔterC mutant. Specifically, we were interested in exploring biological processes that were characteristic of the microbiota constituents of mice sourced from RB16 associated with reduced fitness of the ΔterC mutant. To this end, the metagenomes of the fecal microbiota of mice sourced from RB16 and RB07 were predicted using PICRUSt2 [ 59 ]. The relative abundance of predicted metabolic pathways as annotated by MetaCyc [ 60 ] differed between the fecal microbiota of mice sourced from RB16 and RB07 ( S23 Fig and S3 Data ). A significant difference overall between predicted metabolic pathways of the fecal microbiota of RB16 and RB07 was detected on the day of inoculation and on multiple days post-inoculation although this was not readily detected in the first two principal components that account for the majority of variation in the data ( S23 Fig ). The subtlety of this finding was not surprising, as the microbiota of mice sourced from RB16 and RB07 are distinct based on specific OTUs ( Fig 4 ) but share a large number of gut constituents ( S9 Fig ). Thus, we expected that a limited number of predicted metabolic pathways would differentiate microbiota of mice sourced from RB16 and RB07, and that some of these pathways would correspond to the microbiota constituents of mice sourced from RB16 associated with the reduced the fitness of the ΔterC mutant.

There were several differences between the microbiota in mice sourced from RB16 and RB07. The diversity of the fecal microbiota of mice sourced from RB16 was significantly higher than mice sourced from RB07 on the day of inoculation ( Fig 4C ), and this difference was present throughout the experiment ( S15 Fig ). We next sought to determine if a bacterial family or families differentiated the fecal microbiota of mice sourced from RB16 and RB07 through the use of linear discriminant analysis (LDA) effect size (LEfSe) [ 51 ]. On the day of inoculation, 7 bacterial families were found to be differentially abundant, 2 of which were more abundant in mice sourced from RB16 and 5 of which were more abundant in mice sourced from RB07 ( Fig 4D and 4E ). Only unclassified Clostridiales, associated with mice sourced from RB07, remained differential throughout the entire experiment ( S16 and S17 Figs). We hypothesized that no family consistently distinguished mice sourced from RB16 from mice sourced from RB07 because variations in individual operational taxonomic unit (OTU) relative abundances obscured family-level analysis. As such, we used LEfSe to determine differentially abundant OTUs between these microbiotas. On the day of inoculation, 18 OTUs were found to be differentially abundant, 7 of which were more abundant in mice sourced from RB07, and 11 of which were more abundant in mice sourced from RB16 ( Fig 4F ). Intriguingly, 4 of the 11 OTUs associated with mice sourced from RB16 had 16S rRNA sequences most similar to Muribaculum intestinale ( Fig 4F ; 0008, 0009, 0018, 0019) [ 52 – 54 ]. These 4 OTUs, as well as 0030 which had 16S rRNA sequence most similar to Clostridium scindens, remained more abundant in mice sourced from RB16 than RB07 for the entire experiment ( S18 and S19 Figs). Notably, the Porphyromonadaceae family, of which M. intestinale was considered a member in our dataset, was also enriched in mice sourced from RB16 on days 1–3 post-inoculation ( S16 and S17 Figs). The identification of these species as differentially abundant in mice sourced from RB16 is notable, as M. intestinale belongs to family S24-7 which is suggested to play a role in gut inflammatory homeostasis [ 20 , 54 – 57 ], and C. scindens has been reported to be a clinically relevant probiotic candidate [ 58 ]. The only OTU that was more abundant in mice sourced from RB07 throughout the experiment is 0037, which is most similar to Olsenella profusa (Figs 4F and S18 and S19). OTUs associated with mice sourced from RB16 by LEfSe were mostly absent in mice sourced from RB07 ( Fig 4G ) throughout the course of the experiment ( S18 and S19 Figs). If these OTUs are tightly associated with the observed ΔterC mutant fitness defect, they should also be sensitive to the antibiotic treatment that ameliorates the defect. Indeed, differential OTUs, as well as corresponding families, were sensitive to antibiotic treatment ( S20 and S21 Figs) highlighting their potential role in reducing the fitness of the ΔterC mutant. Finally, we asked if the introduction of Kp impacted the relative abundance of these families and OTUs. At the family level, the microbiota of mice sourced from RB16 was only minimally modulated ( S22A Fig ) by Kp inoculation, and the only OTU that differentiated mice sourced from RB16 from RB07 that was modulated was 0045 (Mordavella massiliensis, S21B Fig ). Further analysis of the gut communities of mice sourced from RB16 and RB07 demonstrated that these communities remained stable through the course of the experiment in antibiotic-naïve mice but shift drastically following antibiotic treatment ( S22C Fig ). These data indicate that the microbiota constituents of mice sourced from RB16 reduce the fitness of the ΔterC mutant and are highly stable during Kp colonization.

To determine if the composition of the gut microbiota of mice sourced from RB16 and mice sourced from RB07 differed, we performed 16S rRNA gene sequence analysis from fecal DNA collected throughout the course of these experiments ( S9 Fig and S2 Data ). To compare the microbiota between all groups (mice sourced from RB16, RB07, RB16+Abx, RB07+Abx) and all time points, θ YC distances [ 47 ] were calculated, and principal coordinates analysis was used to visualize these distances. θ YC dissimilarity accounts for both the number of shared and unique species as well as differential species abundance in a single metric [ 47 ]. As expected, microbiota differed between male and female mice ( Fig 4A.i , axis 1, females cluster on left of graph) [ 48 – 50 ]. Despite sex-based differences, the fecal microbiota of RB16 and RB07 were significantly dissimilar on the day of inoculation ( Fig 4A.i , axis 2, AMOVA P = 0.001; Fig 4B ), suggesting that the results observed in Fig 3B–3C were attributable to differences in the microbiota of these mice. In addition, the fecal microbiota of antibiotic treated mice sourced from RB16 and RB07 were dissimilar from their untreated counterparts ( Fig 4A.ii and iii , AMOVA P < 0.001), but not from one another ( Fig 4A.iv , AMOVA P = 0.676). Assessment of the magnitude of dissimilarity indicated that intergroup dissimilarity was higher than intragroup dissimilarity, and antibiotic treatment resulted in the greatest dissimilarity ( Fig 4B , higher values indicate higher dissimilarity). These findings were consistent across all time points ( S10 – S14 Figs). These data demonstrate an association between ter-dependent fitness in the gut and the composition of the gut microbiota and suggest that an individual or group of gut microbiota constituents in the mice sourced from RB16 underlies the observed loss of fitness.

To begin to characterize the effect of the indigenous microbiota on the ΔterC mutant, mice were treated with antibiotics and the experiment was repeated. Consistent with previous studies [ 15 ], treatment with antibiotics increased overall Kp colonization density in mice from both barriers ( S6C and S6D Fig ); however, this treatment also restored the fitness of ΔterC in mice sourced from RB16 (Figs 3D and S6C ). Conversely, antibiotic treatment of mice sourced from RB07 did not impact ΔterC fitness (Figs 3E and S6D ). These data indicate that an antibiotic-susceptible member or members of the microbiota of mice sourced from RB16 are involved in reducing the fitness of the ΔterC mutant, as opposed to the microbiota of mice sourced from RB07 enhancing ΔterC fitness. Furthermore, complementation of the ΔterC mutant by expression of terZ-F in trans ameliorated its fitness defect in mice sourced from RB16 ( Fig 3F ). This finding was confirmed with the fully sequenced ΔterC clone ( S1C Fig ). To determine if the ter operon is required for colonization, mono-colonization studies were performed. These results show that both the wild type and ΔterC mutant were able to colonize the gut ( S7 Fig ), although there was mouse-to-mouse variation that may obfuscate an advantage of the ter operon during mono-colonization. Consistent with previous studies [ 43 – 46 ], the mice exhibited mortality throughout the duration of the experiment. However, the increased bacterial load due to antibiotics ( S6 Fig ) was not associated with increased mortality, and both strains exhibited equal virulence in this model regardless of barrier ( S8 Fig ). Collectively, these data suggest that the ter operon is a microbiome-dependent gut fitness factor.

(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 5x10 6 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 5x10 6 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.

We previously reported a strong association between the ter operon and Kp infection (pneumonia and bacteremia) in Kp colonized patients, yet also found that terC is dispensable in a murine model of pneumonia [ 10 ]. To determine if the ter operon is important for bacteremia, WT Kp and ΔterC were competed in a peritoneal injection model of murine bacteremia. terC was dispensable in all tissues with the exception of a modest defect in the brain ( S5 Fig ). We did not explore this finding further, as a role in meningitis would not explain the correlation between the ter operon and infections observed in patients. We then hypothesized that the ter operon may be required during gut colonization, which precedes infection [ 11 , 12 ]. Exposure to antibiotics was not associated with Kp colonization or subsequent infection in our intensive care unit patient population [ 10 , 42 ], indicating that Kp must contend with the indigenous microbiota to colonize the gut and cause infection. Therefore, C57BL/6J mice were sourced from two different housing sites at The Jackson Laboratory (barriers RB16 and RB07) to control for natural variations in the gut microbiota induced by housing conditions [ 17 – 20 ]. Mice were orally gavaged with 100 μL of a mixture of wild-type and ΔterC Kp ( Fig 3A ). Intriguingly, a fitness defect (median 5.8-, 4.7-, 8.9-, and 4.0-fold-defect on days 1–4, respectively) was observed consistently for ΔterC in the mice sourced from RB16 over several days (Figs 3B and S6A ) but not from RB07, despite their genetic identity (Figs 3C and S6B ). These data suggest that the fitness defect exhibited by ΔterC is dependent on the gut microbiota.

To determine the geographical and ecological range of Klebsiella encoding the ter operon, 14,060 Klebsiella sp. genomes, including 1,989 containing terZ-F, and their associated metadata were extracted from the Pathosystems Resource Integration Center ( S1 Data ) [ 41 ]. These genomes were derived from Klebsiella sp. strains from 6 of 7 continents ( S4A Fig ), indicating a wide geographical range that corresponds to the environmental ubiquity of Klebsiella sp. Assessment of the specific source of Klebsiella sp. isolation indicates a wide variety of hosts, including both animals and plants, and a number of environmental sources ( S4B Fig ). Interestingly, we found that terZ-F containing isolates were evenly distributed amongst all isolation sources (~14% of all isolates, S4C Fig ); however, humans are the most represented isolation source, which is not surprising given the over-representation of human isolates in bacterial genome repositories. Many ter-containing Klebsiella sp. were isolated from human gut, blood, and respiratory samples ( S4B and S4D Fig ), which both supports a role for the ter operon in the human gut and comports with our previous studies where we originally identified a strong association between the ter operon and infection in Kp colonized patients [ 10 ]. Overall, relatively few ter-containing Klebsiella sp. isolates came from liver abscesses ( S4D Fig ), which are traditionally associated with hvKp [ 36 ], although they were enriched in this infection site ( S4D Fig ). Overall, ter-containing strains have a wide geographical and ecological range and are found across multiple sites of human infection.

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).

Next, we sought to determine if ter is genetically independent or a biomarker of hvKp, as has been previously reported [ 35 , 36 ]. To this end, we returned to the ter-encoding (ter+) Kp isolates from our previous study [ 10 ]. These isolates were highly diverse, as reflected by their sequence types ( Fig 2A ) and none were a hvKp sequence type previously associated with the ter operon. To determine virulence gene content, we sequenced ter-encoding plasmids from our patient isolates using long-read sequencing (Oxford Nanopore Technologies, Oxford, UK). ter-encoding plasmids were characteristically large (86.8–430.8 kb; S2 Fig ) and some were derived from plasmid fusions ( S2 Table ). These plasmids displayed substantial sequence variation outside the ter locus ( S3A and S3B Fig ), indicating that the ter operon is not a marker for a widely circulating, highly conserved, plasmid. Additionally, these ter-encoding plasmids do not contain virulence genes associated with Kp hypervirulence, nor was a single antibiotic resistance gene highly present on these plasmids ( Fig 2B and S2 and S3 Tables). This supports the premise that the ter operon is an independent fitness factor during infection, rather than a marker of a hypervirulence or antibiotic resistance-encoding plasmid. Finally, predicted open reading frames (ORFs) directly up- and downstream of the ter operon displayed minimal functional conservation ( Fig 2C ), further indicating that this operon is not tightly linked to a virulence factor. Next, we repeated this analysis using publicly available reference genomes of ter-encoding Kp isolates (n = 88). These isolates were not limited to hvKp sequence types associated with the ter operon ( Fig 2D ), and indeed, the ter+ plasmids displayed a high degree of sequence variability ( S3C Fig ). 42% of ter+ plasmids contained a rmpA/A2 homolog and an accessory iron acquisition system. The remaining 58% ter+ plasmids had no classical hypervirulence factor present ( Fig 2E and S2 and S3 Tables), and again, the predicted up- and downstream ORFs displayed little functional conservation except for transposase activity ( Fig 2F and S1 Table ). Together, these results indicate that ter is a genetically independent factor, and is likely playing a direct, but yet undescribed role in Kp disease.

(A) The ter locus is organized in two operons, a putative biosynthetic cluster and a TeO 3 -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 K 2 TeO 3 -2 to visualize inhibition of growth (dilution series 10 0 −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.

The complete ter locus consists of two distinct, but highly conserved, operons encoded on opposite DNA strands: a set of 14 individual genes with potential biosynthetic functionality that includes terXYW, and a tellurium resistance operon consisting of terZABCDEF (referred to as the “ter operon”). This is followed by a predicted haloacid dehydrogenase (HAD) that is highly conserved among ter-encoding Kp isolates ( Fig 1A and S1 Table ). Computational prediction of protein structure and function based on amino acid sequence using I-TASSER [ 38 – 40 ] indicates that many of the ter operon proteins are involved in response to stress ( S1 Table ), which comports with previous studies [ 21 ]. The deletion of terC, which is predicted to be involved in transmembrane transport ( S1 Table ), from the pK2044 plasmid renders hypervirulent Kp strain NTUH-K2044 (clone Kp2259) exquisitely sensitive to TeO 3 -2 ( Fig 1B ); however, terC expressed in trans in a ΔterC strain did not restore phenotypic resistance to TeO 3 -2 ( Fig 1B ). Sequencing of a second clone (Kp2257) did not reveal any spurious mutations and demonstrated insertion into the terC locus while replicating the sensitive to TeO 3 -2 phenotype ( S1A and S1B Fig ); thus, this phenotype is attributable to insufficient terC expression from the plasmid or polar effects of the mutation. The induction of TeO 3 -2 sensitivity via polar inactivation of terDEF via mutation of terC is consistent with previous studies that demonstrate that terBCDE are necessary for resistance to TeO 3 -2 [ 28 , 34 ]. Expression of terZ-F in trans in a ΔterC mutant fully restored TeO 3 -2 resistance ( Fig 1B ). Furthermore, the expression of terZ-F in trans was sufficient to confer TeO 3 -2 resistance to the Escherichia coli strain MG1655 ( Fig 1C ). These results indicate that an intact ter operon is necessary for TeO 3 -2 resistance in Kp NTUH-K2044.

Discussion

The work presented here advances our understanding of Kp colonization and the transition to infection by combining the study of both host and pathogen to describe a factor involved in this process: the ter operon. Our previous work demonstrated a role for ter during Kp infection [10], therefore indicating that ter is important in the infectious process. Given that prior gut colonization often precedes disease [11–13,66], our finding that the ter operon is necessary for gut fitness in a microbiome-dependent manner corroborates our previous studies and explains why the ter operon is dispensable in murine models of pneumonia [10] and bacteremia but strongly associated with human infection in these sites. The importance of the ter operon is validated by the demonstration that this operon is genetically independent, rather than a marker of the Kp hypervirulence plasmid. Finally, we have identified a set of microbes that impact the fitness of Kp strains in a ter-dependent manner, likely through SCFA metabolism. The role of ter in resisting SCFAs is based on findings that 1) M. intestinale is a primary fermenter capable of producing SCFAs and is uniquely present in the restrictive gut microbiota of mice sourced from RB16 and 2) administration of SCFAs to mice sourced from RB07, which lack M. intestinale, results in diminished ΔterC fitness. Moreover, the association between the antibiotic-mediated reduction of M. intestinale from the gut of mice from RB16 and a significant reduction in fecal SCFAs support this conclusion. Collectively, these results show how a bacterial factor can interact with its host’s microbiome to enhance colonization, which may increase risk of infection in humans.

Gut colonization is a critical first step for many pathogens that cause both intestinal and extra-intestinal infection. Kp, including ESBL-, CP-, and hvKp, can be isolated from multiple sites of colonization, including the gut, nasopharynx, and skin. While skin colonization is considered a transient event [67], colonization of mucosal sites is an important event preceding many cases of Kp disease [10–14]. Few factors have been identified that influence the transition from colonization to infection, either in a hypothesis-driven or systematic manner [15,43,68–71]. Notably, published systematic studies rely on the administration of antibiotics to permit gut colonization [69–71], and therein are identifying Kp gut colonization factors in the absence of an intact microbiota. Disruption of the gut microbiome can lead to the expansion of potential pathogens in the gut and increased susceptibility to infection [72–75]; however, our prior analysis of Kp colonization in over 2,400 patients found high rates of colonization (up to 17%) but no positive association between colonization and prior antibiotic exposure [42]. This indicates that Kp can colonize the gut in the absence of microbiome perturbation. Moreover, highly antibiotic-resistant bacteria have been isolated from otherwise healthy adults [76–80], suggesting that antibiotic-resistant bacteria are also able to invade intact microbiomes. An attractive hypothesis is that Kp circumvents microbiota-mediated colonization resistance by occupying newly accessible niches following antibiotic disruption of the gut microbiome. Yet, this fails to address how Kp invades the intact gut microbiome in the absence of antibiotic exposure. This study indicates an alternative means of circumventing microbiota-mediated colonization resistance, wherein specific Kp factors enhance fitness during invasion of the intact gut microbiome. In this case, the ter locus is needed for optimal fitness in the presence of certain indigenous gut microbes, suggesting that acquisition of ter+ plasmids can expand the host range of a pathogen by resisting the competitive pressures of these bacteria. This evasion of colonization resistance by horizontal gene transfer echoes the bacterial arms race seen in response to nutritional immunity and antibiotics.

To be fit in the gut environment, Kp must overcome the stress of direct interspecies competition, nutrient limitation, and anti-microbial stress induced by the indigenous microbiota (extensively reviewed in [81]). Kp has received increased notoriety for its ability to compete in stressful polymicrobial environments [82–84]; however, relatively little is known about how Kp gains an advantage over its competitors in the gut. The hvKp T6SS has been implicated in direct interspecies competition in the gut [43], and the trehalose-6-phosphate hydrolase [45] and cellobiose-specific PTS transporter CelB [46] have been suggested to play a role in gut nutrient acquisition when the indigenous microbiota is present. Additionally, the Sap (Sensitivity to antimicrobial peptides) transporter [85] and the acid-sensitive transcriptional regulator CadC [68] are important for optimal gut fitness in the presence of the indigenous microbiota. Our data indicates that the indigenous gut microbiota of mice sourced from RB16 create an environment that limits Kp fitness via SCFA metabolism when a functional ter operon is absent. Notably, many of the OTUs that differentiate the gut microbiota of mice sourced from RB16 from RB07 correspond to M. intestinale (also known as S24-7), which are associated with increased SCFA levels [86] and known to influence gut inflammatory homeostasis. Mice treated with a consortium of bacteria containing M. intestinale by oral gavage were more resistant to Salmonella typhimurium infection in an inflammation-dependent manner [20]. Additionally, depletion of the S24-7 family of bacteria, which includes M. intestinale, from the gut has been associated with lower expression of antimicrobial peptides [57], increased inflammatory cytokines [56], and increased gut permeability [56,57]. Interestingly, another study incidentally demonstrated an inverse relationship between family S24-7 abundance and Kp gut colonization density [15]. Thus, the ter operon enhances Kp fitness, likely by resisting stress induced by specific indigenous gut microbiota via SCFA metabolism.

The characterization of the ter operon as a microbiome-dependent gut fitness factor is of interest, as the biology of this operon has long been enigmatic. It is possible that the ter operon facilitates a general stress response and is not specific to TeO 3 -2.The antibacterial properties of TeO 3 -2 are attributable to its ability to disrupt membrane stability and strong oxidizing action [27,87–89]. The reduction of TeO 3 -2 results in the formation of superoxides, leading to abrogation of DNA synthesis, protein synthesis and exhaustion of cellular reductases [90,91]. The diversity of stress induced by TeO 3 -2, in combination with corresponding studies confirming these stresses, accounts for the pleotropic characterization of the ter operon; however, these studies fall short of concluding that the ter operon is necessary for response to a specific physiologically relevant stress. Further complicating this paradigm, other factors are often implicated in the response to TeO 3 -2-induced stress, such as superoxide dismutase [27] or 6-phosphogluconate dehydrogenase [92]. Studies aimed at understanding the regulation of the ter operon have provided some insight, as they have suggested transcriptional control by OxyR [29,32]. While these studies further connect the ter operon to oxidative stress, we were unable to identify an OxyR binding motif in the promoter region of the Kp ter operon. The chromosomal location of the ter operon in these studies [29,32], as opposed to the plasmid locality in Kp, may account for this difference. In lieu of a clear physiologically relevant stress or an obvious regulator, in silico studies have suggested the gene products of terZABCDEF form a membrane-associated complex with TerC acting as the transmembrane protein ([21] and S1 Table). This complex is thought to sense a soluble ligand or regulate membrane permeability, therein linking the extracellular and intracellular environments [21]. Our in silico data is consistent with many of the predicted functions of the ter operon gene products [21], and some experimental evidence exists supporting the formation of a membrane-associated TerC complex [93]. The putative biosynthetic cluster is yet more complicated and is predicted to synthesize a nucleoside-like metabolite or potentially be involved in DNA processing [21], though it is unclear how the TerC complex would functionally interact with this gene cluster or its products. In addition to TeO 3 -2, ter in non-Kp species has been implicated in resistance to phage and colicins that can be found in the gut [28], the latter being antibacterial proteins produced by bacteria to kill their competitors [94]. It may be the case that the presence of SCFAs or SCFA-producing bacteria create a gut environment where Kp encounters these stresses more frequently.

Despite their simplicity, SCFAs are one of the most important gut metabolites. In the gut, SCFAs are products of bacterial fermentation, the most common of which are acetate, butyrate, and propionate, which have many critical functions in colonization resistance. First, they are able to directly inhibit bacterial growth through intracellular acidification [65,95]. We observed a dose-dependent growth inhibition of Kp by SCFAs; however, this was independent of the presence of a functional ter operon, suggesting that SCFAs negatively impact ΔterC fitness in the gut by a different mechanism. Alternatively, the TerC protein is a predicted proton symporter, and therein may be able to sense intracellular pH, leading to regulation of other genes necessary for complete fitness in the gut in response to pH perturbations caused by SCFAs that would not be observable in vitro. Regardless, SCFAs are highly plastic in their function. Second, SCFAs are important for maintaining immune homeostasis (reviewed in [96]). This function is critical in the context of colonization resistance, as SCFAs reduce epithelial oxygenation [97,98] and stimulate antimicrobial peptide expression in the gut [61], leading to reduced pathogen colonization. Interestingly, SCFAs have been shown to accelerate the immune response to Enterobacteriaceae in the gut [63]. Interestingly, gut microbiota-influenced inflammatory homeostasis has been shown to influence Kp gut colonization via IL-36 signaling though this was not linked to SCFAs [16]. Notably, the SCFA that showed the largest difference between mice sourced from RB16 compared to those from RB07 was acetate. This is notable since M. intestinale is known to produce SCFAs, and specifically acetate [55]. Therefore, stimulation of immune pathways via microbiota derived SCFAs may reduce the fitness of the ΔterC mutant in the gut. Alternatively, Kp strains encoding the ter operon may antagonize Kp strains that lack the ter operon in the gut in an SCFA- or microbiome-dependent manner. Though we explored antagonism between the WT Kp and ΔterC strains, these experiments were performed under in vitro conditions and do not rule out the possibility of in vivo antagonism. Finally, SCFAs may induce production of metabolites from members of the indigenous gut microbiota that reduce ΔterC fitness in the gut. The varied impacts of SCFAs on gut homeostasis may explain why exogenous SCFA administration significantly reduces the fitness of the ΔterC strain while only subtle differences in fecal SCFA concentration were observed. Importantly, absorption and/or metabolism of these substrates by the host in the small intestine, cecum, proximal colon, or by the indigenous microbiota may alter gut homeostasis while simultaneously masking large differences in fecal SCFA concentrations [99,100]. In summary, the ter operon represents a novel, transferrable locus that enhances fitness of Kp NTUH-K2044 in the presence of specific gut microbiota and is associated with increased risk of infection in hospitalized patients. Given the breadth of genetic diversity exhibited by Kp [101], further studies with additional strains are necessary to determine the full impact of the interaction between SCFAs and the ter operon on Kp gut fitness. As interventions that modulate gut homeostasis, such as fecal microbiota transplants and administration of SCFA-producing probiotic bacteria, become more common, an understanding of how pathogens are able to overcome these barriers to colonization will be critical to ensure their success.

[END]

[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1009537

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