Clinical Features and Genomic Epidemiology of Bloodstream Infections due to Enterococcal Species Other Than Enterococcus faecalis or E. faecium in Patients With Cancer.

Background: Non-Enterococcus faecium, non-E. faecalis (NFF) enterococci are a heterogeneous group of clinically pathogenic enterococci that include species with intrinsic low-level vancomycin resistance. Patients with cancer are at increased risk for bacteremia with NFF enterococci, but their clinical and molecular epidemiology have not been extensively described. Methods: We conducted a retrospective review of all patients (n = 70) with NFF bacteremia from 2016 to 2022 at a major cancer center. The main outcomes assessed were 30-day mortality, microbiological failure (positive blood cultures for ≥4 days), and recurrence of bacteremia (positive blood culture <14 days after clearance). Whole-genome sequencing was performed on all available NFF (n = 65). Results: Patients with hematological malignancies made up 56% of the cohort (77% had leukemia). The majority of solid malignancies (87%) were gastrointestinal in origin. The majority of infections (83%) originated from an intra-abdominal source. The most common NFF species were E. gallinarum (50%) and E. casseliflavus (30%). Most (61%) patients received combination therapy. Bacteremia recurred in 4.3% of patients, there was a 30-day mortality of 23%, and 4.3% had microbiological failure. E. gallinarum and E. casseliflavus isolates were genetically diverse with no spatiotemporal clustering to suggest a single strain. Frequencies of ampicillin resistance (4.3%) and daptomycin resistance (1.9%) were low. Patients with hematologic malignancy had infections with NFF enterococci that harbored more resistance genes than patients with solid malignancy (P = .005). Conclusions: NFF bacteremia is caused by a heterogeneous population of isolates and is associated with significant mortality. Hematological malignancy is an important risk factor for infection with NFF resistant to multiple antibiotics.

A randomized, non-comparative phase 2 study of neoadjuvant immune-checkpoint blockade in retroperitoneal dedifferentiated liposarcoma and extremity/truncal undifferentiated pleomorphic sarcoma.

Based on the demonstrated clinical activity of immune-checkpoint blockade (ICB) in advanced dedifferentiated liposarcoma (DDLPS) and undifferentiated pleomorphic sarcoma (UPS), we conducted a randomized, non-comparative phase 2 trial ( NCT03307616 ) of neoadjuvant nivolumab or nivolumab/ipilimumab in patients with resectable retroperitoneal DDLPS (n = 17) and extremity/truncal UPS (+ concurrent nivolumab/radiation therapy; n = 10). The primary end point of pathologic response (percent hyalinization) was a median of 8.8% in DDLPS and 89% in UPS. Secondary end points were the changes in immune infiltrate, radiographic response, 12- and 24-month relapse-free survival and overall survival. Lower densities of regulatory T cells before treatment were associated with a major pathologic response (hyalinization > 30%). Tumor infiltration by B cells was increased following neoadjuvant treatment and was associated with overall survival in DDLPS. B cell infiltration was associated with higher densities of regulatory T cells before treatment, which was lost upon ICB treatment. Our data demonstrate that neoadjuvant ICB is associated with complex immune changes within the tumor microenvironment in DDLPS and UPS and that neoadjuvant ICB with concurrent radiotherapy has significant efficacy in UPS.

LiaX is a surrogate marker for cell envelope stress and daptomycin non-susceptibility in Enterococcus faecium.

Daptomycin (DAP) is often used as a first-line therapy to treat vancomycin-resistant Enterococcus faecium infections, but emergence of DAP non-susceptibility threatens the effectiveness of this antibiotic. Moreover, current methods to determine DAP minimum inhibitory concentrations (MICs) have poor reproducibility and accuracy. In enterococci, DAP resistance is mediated by the LiaFSR cell membrane stress response system, and deletion of liaR encoding the response regulator results in hypersusceptibility to DAP and antimicrobial peptides. The main genes regulated by LiaR are a cluster of three genes, designated liaXYZ. In Enterococcus faecalis, LiaX is surface-exposed with a C-terminus that functions as a negative regulator of cell membrane remodeling and an N-terminal domain that is released to the extracellular medium where it binds DAP. Thus, in E. faecalis, LiaX functions as a sentinel molecule recognizing DAP and controlling the cell membrane response, but less is known about LiaX in E. faecium. Here, we found that liaX is essential in E. faecium with an activated LiaFSR system. Unlike E. faecalis, E. faecium LiaX is not detected in the extracellular milieu and does not appear to alter phospholipid architecture. We further postulated that LiaX could be used as a surrogate marker for cell envelope activation and non-susceptibility to DAP. For this purpose, we developed and optimized a LiaX enzyme-linked immunosorbent assay (ELISA). We then assessed 86 clinical E. faecium bloodstream isolates for DAP MICs and used whole genome sequencing to assess for substitutions in LiaX. All DAP-resistant clinical strains of E. faecium exhibited elevated LiaX levels. Strikingly, 73% of DAP-susceptible isolates by standard MIC determination also had elevated LiaX ELISAs compared to a well-characterized DAP-susceptible strain. Phylogenetic analyses of predicted amino acid substitutions showed 12 different variants of LiaX without a specific association with DAP MIC or LiaX ELISA values. Our findings also suggest that many E. faecium isolates that test DAP susceptible by standard MIC determination are likely to have an activated cell stress response that may predispose to DAP failure. As LiaX appears to be essential for the cell envelope response to DAP, its detection could prove useful to improve the accuracy of susceptibility testing by anticipating therapeutic failure.

Genetic determinants underlying the progressive phenotype of ß-lactam/ß-lactamase inhibitor resistance in Escherichia coli.

IMPORTANCE: The increased feasibility of whole-genome sequencing has generated significant interest in using such molecular diagnostic approaches to characterize difficult-to-treat, antimicrobial-resistant (AMR) infections. Nevertheless, there are current limitations in the accurate prediction of AMR phenotypes based on existing AMR gene database approaches, which primarily correlate a phenotype with the presence/absence of a single AMR gene. Our study utilized a large cohort of cephalosporin-susceptible Escherichia coli bacteremia samples to determine how increasing the dosage of narrow-spectrum ß-lactamase-encoding genes in conjunction with other diverse ß-lactam/ß-lactamase inhibitor (BL/BLI) genetic determinants contributes to progressively more severe BL/BLI phenotypes. We were able to characterize the complexity of the genetic mechanisms underlying progressive BL/BLI resistance including the critical role of ß-lactamase encoding gene amplification. For the diverse array of AMR phenotypes with complex mechanisms involving multiple genomic factors, our study provides an example of how composite risk scores may improve understanding of AMR genotype/phenotype correlations.

LiaX is a surrogate marker for cell-envelope stress and daptomycin non-susceptibility in Enterococcus faecium.

Daptomycin (DAP) is often used as a first line therapy to treat vancomycin-resistant Enterococcus faecium (VR Efm ) infections but emergence of DAP non-susceptibility threatens the effectiveness of this antibiotic. Moreover, current methods to determine DAP MICs have poor reproducibility and accuracy. In enterococci, DAP resistance is mediated by the LiaFSR cell membrane stress response system and deletion of liaR encoding the response regulator results in hypersusceptibility to DAP and antimicrobial peptides. The main genes regulated by LiaR are a cluster of three genes, designated liaXYZ . In Enterococcus faecalis , LiaX is surface exposed with a C-terminus that functions as a negative regulator of cell membrane remodeling and an N-terminal domain that is released to the extracellular medium where it binds DAP. Thus, in E. faecalis , LiaX functions as a sentinel molecule recognizing DAP and controlling the cell membrane response, but less is known about LiaX in E. faecium . Here, we found that liaX is essential in E. faecium ( Efm ) with an activated LiaFSR system. Unlike E. faecalis , Efm LiaX is not detected in the extracellular milieu and does not appear to alter phospholipid architecture. We further postulated that LiaX could be used as a surrogate marker for cell envelope activation and non-susceptibility to DAP. For this purpose, we developed and optimized a LiaX ELISA. We then assessed 86 clinical E. faecium BSI isolates for DAP MICs and used whole genome sequencing to assess for substitutions in LiaX. All DAP-R clinical strains of E. faecium exhibited elevated LiaX levels. Strikingly, 73% of DAP-S isolates by standard MIC determination had elevated LiaX ELISAs above the established cut-off. Phylogenetic analyses of predicted amino acid substitutions showed 12 different variants of LiaX without a specific association with DAP MIC or LiaX ELISA values. Our findings also suggest that many Efm isolates that test DAP susceptible by standard MIC determination are likely to have an activated cell stress response that may predispose to DAP failure. As LiaX appears to be essential for the cell envelope response to DAP, its detection could prove useful to improve the accuracy of susceptibility testing by anticipating therapeutic failure.

Young-onset Rectal Cancer: Unique Tumoral Microbiome and Correlation With Response to Neoadjuvant Therapy.

OBJECTIVE: External exposures, the host, and the microbiome interact in oncology. We aimed to investigate tumoral microbiomes in young-onset rectal cancers (YORCs) for profiles potentially correlative with disease etiology and biology. BACKGROUND: YORC is rapidly increasing, with 1 in 4 new rectal cancer cases occurring under the age of 50 years. Its etiology is unknown. METHODS: YORC (<50 y old) or later-onset rectal cancer (LORC, ≥50 y old) patients underwent pretreatment biopsied of tumor and tumor-adjacent normal (TAN) tissue. After whole genome sequencing, metagenomic analysis quantified microbial communities comparing tumors versus TANs and YORCs versus LORCs, controlling for multiple testing. Response to neoadjuvant therapy (NT) was categorized as major pathological response (MPR, ≤10% residual viable tumor) versus non-MPR. RESULTS: Our 107 tumors, 75 TANs from 37 (35%) YORCs, and 70 (65%) LORCs recapitulated bacterial species were previously associated with colorectal cancers (all P <0.0001). YORC and LORC tumoral microbiome signatures were distinct. After NT, 13 patients (12.4%) achieved complete pathologic response, whereas MPR occurred in 47 patients (44%). Among YORCs, MPR was associated with Fusobacterium nucleaum , Bacteroides dorei, and Ruminococcus bromii (all P <0.001), but MPR in LORC was associated with R. bromii ( P <0.001). Network analysis of non-MPR tumors demonstrated a preponderance of oral bacteria not observed in MPR tumors. CONCLUSIONS: Microbial signatures were distinct between YORC and LORC. Failure to achieve an MPR was associated with oral bacteria in tumors. These findings urge further studies to decipher correlative versus mechanistic associations but suggest a potential for microbial modulation to augment current treatments.

Contribution of the Oral and Gastrointestinal Microbiomes to Bloodstream Infections in Leukemia Patients.

Bloodstream infections (BSIs) pose a significant mortality risk for acute myeloid leukemia (AML) patients. It has been previously reported that intestinal domination (>30% relative abundance [RA] attributed to a single taxon) with the infecting taxa often precedes BSI in stem cell transplant patients. Using 16S rRNA amplicon sequencing, we analyzed oral and stool samples from 63 AML patients with BSIs to determine the correlation between the infectious agent and microbiome composition. Whole-genome sequencing and antimicrobial susceptibilities were performed on all BSI isolates. Species-level detection of the infectious agent and presence of antibiotic resistance determinants in the stool (blaCTX-M-15, blaCTX-M-14, cfrA, and vanA) were confirmed via digital droplet PCR (ddPCR). Individuals with Escherichia coli (stool P < 0.001), Pseudomonas aeruginosa (oral P = 0.004, stool P < 0.001), and viridans group streptococci (VGS) (oral P = 0.001) bacteremia had a significantly higher relative abundance of those respective genera than other BSI patients, which appeared to be site specific. Although 78% of patients showed presence of the infectious genera in the stool and/or saliva, only 7 exhibited microbiome domination. ddPCR confirmed species specificity of the 16S data and detected the antibiotic resistance determinants found in the BSI isolates within concurrent stools. Although gastrointestinal (GI) domination by an infecting organism was not present at the time of most BSIs in AML, the pathogens, along with AMR elements, were detectable in the majority of patients. Thus, rapid genetic assessment of oral and stool samples for the presence of potential pathogens and AMR determinants might inform personalized therapeutic approaches in immunocompromised patients with suspected infection. IMPORTANCE A major cause of mortality in hematologic malignancy patients is BSI. Previous studies have demonstrated that bacterial translocation from the GI microbiome is a major source of BSIs and is often preceded by increased levels of the infectious taxa in the GI (>30% abundance by 16S rRNA sequencing). In this study, we sought to better understand how domination and abundance levels of the oral and gut microbiome relate to bacteremia occurrence in acute myeloid leukemia patients. We conclude that analyses of both oral and stool samples can help identify BSI and antimicrobial resistance determinants, thus potentially improving the timing and tailoring of antibiotic treatment strategies for high-risk patients.

Multisite Detection of Tn1549-Mediated vanB Vancomycin Resistance in Multidrug-Resistant Enterococcus faecalis ST6 in Texas and Florida.

In the United States, vanB-mediated resistance in enterococci is rare. We characterized three sequence type (ST) 6, vancomycin-resistant Enterococcus faecalis isolates causing bacteremia in unique patients in spatiotemporally distinct settings. Isolates were recovered between 2018 and 2020 in two cities in the United States (Houston, TX; Miami, FL). The isolates harbored the vanB operon on a chromosomally located Tn1549 transposon, and epidemiological data suggested multiple introductions of the vanB gene cluster into ST6 E. faecalis.

Oral and Stool Microbiome Coalescence and Its Association With Antibiotic Exposure in Acute Leukemia Patients.

Failure to maintain segregation of oral and gut microbial communities has been linked to several diseases. We sought to characterize oral-fecal microbiome community coalescence, ectopic extension of oral bacteria, clinical variables contributing to this phenomenon, and associated infectious consequences by analyzing the 16S rRNA V4 sequences of longitudinal fecal (n=551) and oral (n=737) samples from 97 patients with acute myeloid leukemia (AML) receiving induction chemotherapy (IC). Clustering observed in permutation based multivariate analysis of variance (PERMANOVA) of Bray-Curtis dissimilarity and PCoA plot of UniFrac distances between intra-patient longitudinal oral-stool sample pairs suggested potential oral-stool microbial community coalescence. Bray-Curtis dissimilarities and UniFrac distances were used to create an objective definition of microbial community coalescence. We determined that only 23 of the 92 patients exhibited oral-stool community coalescence. This was validated through a linear mixed model which determined that patients who experienced coalescence had an increased proportion of shared to unique OTUs between their oral-stool sample pairs over time compared to non-coalesced patients. Evaluation of longitudinal microbial characteristics revealed that patients who experienced coalescence had increased stool abundance of Streptococcus and Stenotrophomonas compared to non-coalesced patients. When treated as a time-varying covariate, each additional day of linezolid (HR 1.15, 95% CI 1.06 - 1.24, P <0.001), meropenem (HR 1.13, 95% CI 1.05 - 1.21, P = 0.001), metronidazole (HR 1.13, 95% CI 1.05 - 1.21, P = 0.001), and cefepime (HR 1.10, 95% CI 1.01 - 1.18, P = 0.021) increased the hazard of oral-stool microbial community coalescence. Levofloxacin receipt was associated with a lower risk of microbiome community coalescence (HR 0.75, 95% CI 0.61 - 0.93, P = 0.009). By the time of neutrophil recovery, the relative abundance of Bacteroidia (P<0.001), Fusobacteria (P=0.012), and Clostridia (P=0.013) in the stool were significantly lower in patients with oral-gut community coalescence. Exhibiting oral-stool community coalescence was associated with the occurrence of infections prior to neutrophil recovery (P=0.002), as well as infections during the 90 days post neutrophil recovery (P=0.027). This work elucidates specific antimicrobial effects on microbial ecology and furthers the understanding of oral/intestinal microbial biogeography and its implications for adverse clinical outcomes.

Contemporary Clinical and Molecular Epidemiology of Vancomycin-Resistant Enterococcal Bacteremia: A Prospective Multicenter Cohort Study (VENOUS I).

BACKGROUND: Vancomycin-resistant enterococci (VRE) are major therapeutic challenges. Prospective contemporary data characterizing the clinical and molecular epidemiology of VRE bloodstream infections (BSIs) are lacking. METHODS: The Vancomycin-Resistant Enterococcal BSI Outcomes Study (VENOUS I) is a prospective observational cohort of adult patients with enterococcal BSI in 11 US hospitals. We included patients with Enterococcus faecalis or Enterococcus faecium BSI with ≥1 follow-up blood culture(s) within 7 days and availability of isolate(s) for further characterization. The primary study outcome was in-hospital mortality. Secondary outcomes were mortality at days 4, 7, 10, 12, and 15 after index blood culture. A desirability of outcome ranking was constructed to assess the association of vancomycin resistance with outcomes. All index isolates were subjected to whole genome sequencing. RESULTS: Forty-two of 232 (18%) patients died in hospital and 39 (17%) exhibited microbiological failure (lack of clearance in the first 4 days). Neutropenia (hazard ratio [HR], 3.13), microbiological failure (HR, 2.4), VRE BSI (HR, 2.13), use of urinary catheter (HR, 1.85), and Pitt BSI score ≥2 (HR, 1.83) were significant predictors of in-hospital mortality. Microbiological failure was the strongest predictor of in-hospital mortality in patients with E faecium bacteremia (HR, 5.03). The impact of vancomycin resistance on mortality in our cohort changed throughout the course of hospitalization. Enterococcus faecalis sequence type 6 was a predominant multidrug-resistant lineage, whereas a heterogeneous genomic population of E faecium was identified. CONCLUSIONS: Failure of early eradication of VRE from the bloodstream is a major factor associated with poor outcomes.

Population Genomics of emm4 Group A Streptococcus Reveals Progressive Replacement with a Hypervirulent Clone in North America.

Clonal replacement is a major driver for changes in bacterial disease epidemiology. Recently, it has been proposed that episodic emergence of novel, hypervirulent clones of group A Streptococcus (GAS) results from acquisition of a 36-kb DNA region leading to increased expression of the cytotoxins Nga (NADase) and SLO (streptolysin O). We previously described a gene fusion event involving the gene encoding the GAS M protein (emm) and an adjacent M-like protein (enn) in the emm4 GAS population, a GAS emm type that lacks the hyaluronic acid capsule. Using whole-genome sequencing of a temporally and geographically diverse set of 1,126 isolates, we discovered that the North American emm4 GAS population has undergone clonal replacement with emergent GAS strains completely replacing historical isolates by 2017. Emergent emm4 GAS strains contained a handful of small genetic variations, including the emm-enn gene fusion, and showed a marked in vitro growth defect compared to historical strains. In contrast to other previously described GAS clonal replacement events, emergent emm4 GAS strains were not defined by acquisition of exogenous DNA and had no significant increase in transcript levels of nga and slo toxin genes via RNA sequencing and quantitative real-time PCR analysis relative to historic strains. Despite the in vitro growth differences, emergent emm4 GAS strains were hypervirulent in mice and ex vivo growth in human blood compared to historical strains. Thus, these data detail the emergence and dissemination of a hypervirulent acapsular GAS clone defined by small, endogenous genetic variation, thereby defining a novel model for GAS strain replacement. IMPORTANCE Severe invasive infections caused by group A Streptococcus (GAS) result in substantial morbidity and mortality in children and adults worldwide. Previously, GAS clonal strain replacement has been attributed to acquisition of exogenous DNA leading to novel virulence gene acquisition or increased virulence gene expression. Our study of type emm4 GAS identified emergence of a hypervirulent GAS clade defined by variation in endogenous DNA content and lacking augmented toxin gene expression relative to replaced strains. These findings expand our understanding of the molecular mechanisms underlying bacterial clonal emergence.

Alterations of the Oral Microbiome and Cumulative Carbapenem Exposure Are Associated With Stenotrophomonas maltophilia Infection in Patients With Acute Myeloid Leukemia Receiving Chemotherapy.

BACKGROUND: Stenotrophomonas maltophilia is increasingly common in patients with acute myeloid leukemia (AML). Little is known about factors that drive S. maltophilia infection. We evaluated the microbiome and cumulative antibiotic use as predictors of S. maltophilia infection in AML patients receiving remission induction chemotherapy (RIC). METHODS: Subanalysis of a prospective, observational cohort of patients with AML receiving RIC between September 2013 and August 2015 was performed. Fecal and oral microbiome samples collected from initiation of RIC until neutrophil recovery were assessed for the relative abundance of Stenotrophomonas via 16S rRNA gene quantitation. The primary outcome, microbiologically proven S. maltophilia infection, was analyzed using a time-varying Cox proportional hazards model. RESULTS: Of 90 included patients, 8 (9%) developed S. maltophilia infection (pneumonia, n = 6; skin-soft tissue, n = 2); 4/8 (50%) patients were bacteremic; and 7/8 (88%) patients with S. maltophilia infection had detectable levels of Stenotrophomonas vs 22/82 (27%) without infection (P < .01). An oral Stenotrophomonas relative abundance of 36% predicted infection (sensitivity, 96%; specificity, 93%). No association of S. maltophilia infection with fecal relative abundance was found. Cumulative meropenem exposure was associated with increased infection risk (hazard ratio, 1.17; 95% confidence interval, 1.01-1.35; P = .03). CONCLUSIONS: Here, we identify the oral microbiome as a potential source for S. maltophilia infection and highlight cumulative carbapenem use as a risk factor for S. maltophilia in leukemia patients. These data suggest that real-time monitoring of the oral cavity might identify patients at risk for S. maltophilia infection.

IS26-mediated amplification of blaOXA-1 and blaCTX-M-15 with concurrent outer membrane porin disruption associated with de novo carbapenem resistance in a recurrent bacteraemia cohort.

BACKGROUND: Approximately half of clinical carbapenem-resistant Enterobacterales (CRE) isolates lack carbapenem-hydrolysing enzymes and develop carbapenem resistance through alternative mechanisms. OBJECTIVES: To elucidate development of carbapenem resistance mechanisms from clonal, recurrent ESBL-positive Enterobacterales (ESBL-E) bacteraemia isolates in a vulnerable patient population. METHODS: This study investigated a cohort of ESBL-E bacteraemia cases in Houston, TX, USA. Oxford Nanopore Technologies long-read and Illumina short-read sequencing data were used for comparative genomic analysis. Serial passaging experiments were performed on a set of clinical ST131 Escherichia coli isolates to recapitulate in vivo observations. Quantitative PCR (qPCR) and qRT-PCR were used to determine copy number and transcript levels of ß-lactamase genes, respectively. RESULTS: Non-carbapenemase-producing CRE (non-CP-CRE) clinical isolates emerged from an ESBL-E background through a concurrence of primarily IS26-mediated amplifications of blaOXA-1 and blaCTX-M-1 group genes coupled with porin inactivation. The discrete, modular translocatable units (TUs) that carried and amplified ß-lactamase genes mobilized intracellularly from a chromosomal, IS26-bound transposon and inserted within porin genes, thereby increasing ß-lactamase gene copy number and inactivating porins concurrently. The carbapenem resistance phenotype and TU-mediated ß-lactamase gene amplification were recapitulated by passaging a clinical ESBL-E isolate in the presence of ertapenem. Clinical non-CP-CRE isolates had stable carbapenem resistance phenotypes in the absence of ertapenem exposure. CONCLUSIONS: These data demonstrate IS26-mediated mechanisms underlying ß-lactamase gene amplification with concurrent outer membrane porin disruption driving emergence of clinical non-CP-CRE. Furthermore, these amplifications were stable in the absence of antimicrobial pressure. Long-read sequencing can be utilized to identify unique mobile genetic element mechanisms that drive antimicrobial resistance.

Observational Cohort Study of Oral Mycobiome and Interkingdom Interactions over the Course of Induction Therapy for Leukemia.

Although the term "microbiome" refers to all microorganisms, the majority of microbiome studies focus on the bacteriome. Here, we characterize the oral mycobiome, including mycobiome-bacteriome interactions, in the setting of remission-induction chemotherapy (RIC) for acute myeloid leukemia (AML). Oral samples (n = 299) were prospectively collected twice weekly from 39 AML patients during RIC until neutrophil recovery. Illumina MiSeq 16S rRNA gene (V4) and internal transcribed spacer 2 (ITS2) sequencing were used to determine bacterial and fungal diversity and community composition. Intrakingdom and interkingdom network connectivity at baseline (T1) and at midpoint (T3) and a later time point (T6) were assessed via SPIEC-EASI (sparse inverse covariance estimation for ecological association inference). In this exploratory study, mycobiome α-diversity was not significantly associated with antibiotic or antifungal receipt. However, postchemotherapy mycobiome α-diversity was lower in subjects receiving high-intensity chemotherapy. Additionally, greater decreases in Malassezia levels were seen over time among patients on high-intensity RIC compared to low-intensity RIC (P = 0.003). A significantly higher relative abundance of Candida was found among patients who had infection (P = 0.008), while a significantly higher relative abundance of Fusarium was found among patients who did not get an infection (P = 0.03). Analyses of intrakingdom and interkingdom relationships at T1, T3, and T6 indicated that interkingdom connectivity increased over the course of IC as bacterial α-diversity diminished. In (to our knowledge) the first longitudinal mycobiome study performed during AML RIC, we found that mycobiome-bacteriome interactions are highly dynamic. Our study data suggest that inclusion of mycobiome analysis in the design of microbiome studies may be necessary to optimally understand the ecological and functional role of microbial communities in clinical outcomes.IMPORTANCE This report highlights the importance of longitudinal, parallel characterization of oral fungi and bacteria in order to better elucidate the dynamic changes in microbial community structure and interkingdom functional interactions during the injury of chemotherapy and antibiotic exposure as well as the clinical consequences of these interrelated alterations.

Gut Microbiome Signatures Are Predictive of Infectious Risk Following Induction Therapy for Acute Myeloid Leukemia.

BACKGROUND: The majority of studies that provide insights into the influence of the microbiome on the health of hematologic malignancy patients have concentrated on the transplant setting. Here, we sought to assess the predictive capacity of the gastrointestinal microbiome and its relationship to infectious outcomes in patients with acute myeloid leukemia (AML). METHODS: 16s rRNA-based analysis was performed on oral swabs and stool samples obtained biweekly from baseline until neutrophil recovery following induction chemotherapy (IC) in 97 AML patients. Microbiome characteristics were correlated with clinical outcomes both during and after IC completion. RESULTS: At the start of IC, higher stool Shannon diversity (hazard ratio [HR], 0.36; 95% confidence interval [CI], .18-.74) and higher relative abundance of Porphyromonadaceae (HR, 0.36; 95% CI, .18-.73) were associated with increased probability of remaining infection-free during neutropenia. A baseline stool Shannon diversity cutoff of <2 had optimal operating characteristics for predicting infectious complications during neutropenia. Although 56 patients received therapy >72 hours with a carbapenem, none of the patients had an infection with an extended spectrum ß-lactamase-producing organism. Patients who received carbapenems for >72 hours had significantly lower α-diversity at neutrophil recovery (P = .001) and were approximately 4 times more likely to have infection in the 90 days following neutrophil recovery (HR, 4.55; 95% CI, 1.73-11.93). CONCLUSIONS: Our results suggest that gut microbiome evaluation could assist with infectious risk stratification and that improved targeting of antibiotic administration during IC could decrease subsequent infectious complications in AML patients.Baseline microbiome diversity is a strong independent predictor of infection during acute myeloid leukemia induction chemotherapy (IC) among clinical and microbiome covariates. Higher baseline levels of Porphyromonadaceae appear protective against infection, while carbapenem use is associated with consequences to the microbiome and infection susceptibility post-IC.

Fecal Microbiome, Metabolites, and Stem Cell Transplant Outcomes: A Single-Center Pilot Study.

BACKGROUND: Accumulating evidence suggests that the intestinal microbiome may dramatically affect the outcomes of hematopoietic stem cell transplant (HSCT) recipients. Providing 16S ribosomal RNA based microbiome characterization in a clinically actionable time frame is currently problematic. Thus, determination of microbial metabolites as surrogates for microbiome composition could offer practical biomarkers. METHODS: Longitudinal fecal specimens (n = 451) were collected from 44 patients before HSCT through 100 days after transplantation, as well as 1-time samples from healthy volunteers (n = 18) as controls. Microbiota composition was determined using 16S ribosomal RNA V4 sequencing. Fecal indole and butyrate levels were determined using liquid chromatography tandem mass spectrometry. RESULTS: Among HSCT recipients, both fecal indole and butyrate levels correlated with the Shannon diversity index at baseline (P = .02 and P = .002, respectively) and directly after transplantation (P = .006 and P < .001, respectively). Samples with high butyrate levels were enriched for Clostridiales, whereas samples containing high indole were also enriched for Bacteroidales. A lower Shannon diversity index at the time of engraftment was associated with increased incidence of acute intestinal graft-vs-host disease (iGVHD) (P = .02) and transplant-related deaths (P = .03). Although fecal metabolites were not associated with acute iGVHD or overall survival, patients contracting bloodstream infections within 30 days after transplantation had significantly lower levels of fecal butyrate (P = .03). CONCLUSIONS: Longitudinal analysis of fecal microbiome and metabolites after HSCT identified butyrate and indole as potential surrogate markers for microbial diversity and specific taxa. Further studies are needed to ascertain whether fecal metabolites can be used as biomarkers of acute iGVHD or bacteremia after HSCT.

Associations of inflammation with symptom burden in patients with acute myeloid leukemia.

The aim of the study was to assess the association inflammation with symptom burden in patients with acute myeloid leukemia (AML), assessed before and during remission induction chemotherapy (IC). Patients with AML (n = 95) were followed from baseline (before IC) to the third week of IC for severity of self-reported somatic symptoms (assessed with the MD Anderson Symptom Inventory) and plasma levels of 13 inflammation-related biomarkers (n = 64). A composite symptom burden severity score was computed as the average score of the six most severe somatic symptoms i.e., fatigue, disturbed sleep, drowsiness, dry mouth, lack of appetite, and pain. Results from cross-sectional analyses showed that inflammation was weakly associated with symptom burden before IC (multiple regression model, explained variance = 10%) but this association grew stronger during IC (week 3 explained variance = 35%). About half of the sample showed a change over time in symptom severity. These changes were not reflected in similar changes over time in inflammatory markers, suggesting that it is the absolute concentration of a given inflammatory marker at a given time point rather than its relative change over time that is of importance. In conclusion, inflammation was strongly associated with symptom burden only during IC, possibly because expression of cytokines only then becomes relevant for regulation of symptom burden. While the current study does not allow for determination of causality, the results suggest that AML patients might benefit from anti-inflammatory interventions during treatment to alleviate somatic symptom experience.

Whole-Genome Sequencing Accurately Identifies Resistance to Extended-Spectrum ß-Lactams for Major Gram-Negative Bacterial Pathogens.

Background: There is marked interest in using DNA-based methods to detect antimicrobial resistance (AMR), with targeted polymerase chain reaction (PCR) approaches increasingly being incorporated into clinical care. Whole-genome sequencing (WGS) could offer significant advantages over targeted PCR for AMR detection, particularly for species where mutations are major drivers of AMR. Methods: Illumina MiSeq WGS and broth microdilution (BMD) assays were performed on 90 bloodstream isolates of the 4 most common gram-negative bacteria causing bloodstream infections in neutropenic patients. The WGS data, including both gene presence/absence and detection of mutations in an array of AMR-relevant genes, were used to predict resistance to 4 ß-lactams commonly used in the empiric treatment of neutropenic fever. The genotypic predictions were then compared to phenotypic resistance as determined by BMD and by commercial methods during routine patient care. Results: Of 133 putative instances of resistance to the ß-lactams of interest identified by WGS, only 87 (65%) would have been detected by a typical PCR-based approach. The sensitivity, specificity, and positive and negative predictive values for WGS in predicting AMR were 0.87, 0.98, 0.97, and 0.91, respectively. Using BMD as the gold standard, our genotypic resistance prediction approach had a significantly higher positive predictive value compared to minimum inhibitory concentrations generated by commercial methods (0.97 vs 0.92; P = .025). Conclusions: These data demonstrate the potential feasibility of using WGS to guide antibiotic treatment decisions for patients with life-threatening infections for an array of medically important pathogens.

Characterization of oral and gut microbiome temporal variability in hospitalized cancer patients.

BACKGROUND: Understanding longitudinal variability of the microbiome in ill patients is critical to moving microbiome-based measurements and therapeutics into clinical practice. However, the vast majority of data regarding microbiome stability are derived from healthy subjects. Herein, we sought to determine intra-patient temporal microbiota variability, the factors driving such variability, and its clinical impact in an extensive longitudinal cohort of hospitalized cancer patients during chemotherapy. METHODS: The stool (n = 365) and oral (n = 483) samples of 59 patients with acute myeloid leukemia (AML) undergoing induction chemotherapy (IC) were sampled from initiation of chemotherapy until neutrophil recovery. Microbiome characterization was performed via analysis of 16S rRNA gene sequencing. Temporal variability was determined using coefficients of variation (CV) of the Shannon diversity index (SDI) and unweighted and weighted UniFrac distances per patient, per site. Measurements of intra-patient temporal variability and patient stability categories were analyzed for their correlations with genera abundances. Groups of patients were analyzed to determine if patients with adverse outcomes had significantly different levels of microbiome temporal variability. Potential clinical drivers of microbiome temporal instability were determined using multivariable regression analyses. RESULTS: Our cohort evidenced a high degree of intra-patient temporal instability of stool and oral microbial diversity based on SDI CV. We identified statistically significant differences in the relative abundance of multiple taxa amongst individuals with different levels of microbiota temporal stability. Increased intra-patient temporal variability of the oral SDI was correlated with increased risk of infection during IC (P = 0.02), and higher stool SDI CVs were correlated with increased risk of infection 90 days post-IC (P = 0.04). Total days on antibiotics was significantly associated with increased temporal variability of both oral microbial diversity (P = 0.03) and community structure (P = 0.002). CONCLUSIONS: These data quantify the longitudinal variability of the oral and gut microbiota in AML patients, show that increased variability was correlated with adverse clinical outcomes, and offer the possibility of using stabilizing taxa as a method of focused microbiome repletion. Furthermore, these results support the importance of longitudinal microbiome sampling and analyses, rather than one time measurements, in research and future clinical practice.

Use of a Phosphorylation Site Mutant To Identify Distinct Modes of Gene Repression by the Control of Virulence Regulator (CovR) in Streptococcus pyogenes.

Control of the virulence regulator/sensor kinase (CovRS) two-component system (TCS) serves as a model for investigating the impact of signaling pathways on the pathogenesis of Gram-positive bacteria. However, the molecular mechanisms by which CovR, an OmpR/PhoB family response regulator, controls virulence gene expression are poorly defined, partly due to the labile nature of its aspartate phosphorylation site. To better understand the regulatory effect of phosphorylated CovR, we generated the phosphorylation site mutant strain 10870-CovR-D53E, which we predicted to have a constitutive CovR phosphorylation phenotype. Interestingly, this strain showed CovR activity only for a subset of the CovR regulon, which allowed for classification of CovR-influenced genes into D53E-regulated and D53E-nonregulated groups. Inspection of the promoter sequences of genes belonging to each group revealed distinct promoter architectures with respect to the location and number of putative CovR-binding sites. Electrophoretic mobility shift analysis demonstrated that recombinant CovR-D53E protein retains its ability to bind promoter DNA from both CovR-D53E-regulated and -nonregulated groups, implying that factors other than mere DNA binding are crucial for gene regulation. In fact, we found that CovR-D53E is incapable of dimerization, a process thought to be critical to OmpR/PhoB family regulator function. Thus, our global analysis of CovR-D53E indicates dimerization-dependent and dimerization-independent modes of CovR-mediated repression, thereby establishing distinct mechanisms by which this critical regulator coordinates virulence gene expression.IMPORTANCEStreptococcus pyogenes causes a wide variety of diseases, ranging from superficial skin and throat infections to life-threatening invasive infections. To establish these various disease manifestations, Streptococcus pyogenes requires tightly coordinated production of its virulence factor repertoire. Here, the response regulator CovR plays a crucial role. As an OmpR/PhoB family member, CovR is activated by phosphorylation on a conserved aspartate residue, leading to protein dimerization and subsequent binding to operator sites. Our transcriptome analysis using the monomeric phosphorylation mimic mutant CovR-D53E broadens this general notion by revealing dimerization-independent repression of a subset of CovR-regulated genes. Combined with promoter analyses, these data suggest distinct mechanisms of CovR transcriptional control, which allow for differential expression of virulence genes in response to environmental cues.