Clinical and Genomic Characterization of a Cohort of Patients With Klebsiella pneumoniae Bloodstream Infection.

BACKGROUND: The clinical and microbial factors associated with Klebsiella pneumoniae bloodstream infections (BSIs) are not well characterized. Prior studies have focused on highly resistant or hypervirulent isolates, limiting our understanding of K. pneumoniae strains that commonly cause BSI. We performed a record review and whole-genome sequencing to investigate the clinical characteristics, bacterial diversity, determinants of antimicrobial resistance, and risk factors for in-hospital death in a cohort of patients with K. pneumoniae BSI. METHODS: We identified 562 patients at Massachusetts General Hospital with K. pneumoniae BSIs between 2016 and 2022. We collected data on comorbid conditions, infection source, clinical outcomes, and antibiotic resistance and performed whole-genome sequencing on 108 sequential BSI isolates from 2021 to 2022. RESULTS: Intra-abdominal infection was the most common source of infection accounting for 34% of all BSIs. A respiratory tract source accounted for 6% of BSIs but was associated with a higher in-hospital mortality rate (adjusted odds ratio, 5.4 [95% confidence interval, 2.2-12.8]; P < .001 for comparison with other sources). Resistance to the first antibiotic prescribed was also associated with a higher risk of death (adjusted odds ratio, 5.2 [95% confidence interval, 2.2-12.4]; P < .001). BSI isolates were genetically diverse, and no clusters of epidemiologically and genetically linked cases were observed. Virulence factors associated with invasiveness were observed at a low prevalence, although an unexpected association between O-antigen type and the source of infection was found. CONCLUSIONS: These observations demonstrate the versatility of K. pneumoniae as an opportunistic pathogen and highlight the need for new approaches for surveillance and the rapid identification of patients with invasive antimicrobial-resistant K. pneumoniae infection.

Seroprevalence of Vibrio cholerae in Adults, Haiti, 2017.

In Haiti in 2017, the prevalence of serum vibriocidal antibody titers against Vibrio cholerae serogroup O1 among adults was 12.4% in Cerca-la-Source and 9.54% in Mirebalais, suggesting a high recent prevalence of infection. Improved surveillance programs to monitor cholera and guide public health interventions in Haiti are necessary.

Seroincidence of Enteric Fever, Juba, South Sudan.

We applied a new serosurveillance tool to estimate typhoidal Salmonella burden using samples collected during 2020 from a population in Juba, South Sudan. By using dried blood spot testing, we found an enteric fever seroincidence rate of 30/100 person-years and cumulative incidence of 74% over a 4-year period.

Correlates of Protection for Cholera.

A correlate of protection (CoP) is a measured adaptive immune response to vaccination or infection that is associated with protection against disease. However, the degree to which a CoP can serve as a surrogate end point for vaccine efficacy should depend on the robustness of this association. While cholera toxin is a dominant target of the human antibody response to Vibrio cholerae infection, antitoxin responses are not associated with long-term immunity, and are not effective CoPs for cholera. Instead, protection appears to be mediated by functional antibodies that target the O-polysaccharide coated V. cholerae outer membrane. Vibriocidal antibodies, which are complement-dependent bactericidal antibodies, remain the most accepted CoP for cholera and are used as surrogate end points in some vaccine studies. However, the association between vibriocidal antibody titers and immunity is not absolute, and they are unlikely to reflect a mechanistic correlate of protection against cholera.

Predicting Vibrio cholerae Infection and Disease Severity Using Metagenomics in a Prospective Cohort Study.

BACKGROUND: Susceptibility to Vibrio cholerae infection is affected by blood group, age, and preexisting immunity, but these factors only partially explain who becomes infected. A recent study used 16S ribosomal RNA amplicon sequencing to quantify the composition of the gut microbiome and identify predictive biomarkers of infection with limited taxonomic resolution. METHODS: To achieve increased resolution of gut microbial factors associated with V. cholerae susceptibility and identify predictors of symptomatic disease, we applied deep shotgun metagenomic sequencing to a cohort of household contacts of patients with cholera. RESULTS: Using machine learning, we resolved species, strains, gene families, and cellular pathways in the microbiome at the time of exposure to V. cholerae to identify markers that predict infection and symptoms. Use of metagenomic features improved the precision and accuracy of prediction relative to 16S sequencing. We also predicted disease severity, although with greater uncertainty than our infection prediction. Species within the genera Prevotella and Bifidobacterium predicted protection from infection, and genes involved in iron metabolism were also correlated with protection. CONCLUSION: Our results highlight the power of metagenomics to predict disease outcomes and suggest specific species and genes for experimental testing to investigate mechanisms of microbiome-related protection from cholera.

Gut Microbiota and Development of Vibrio cholerae-Specific Long-Term Memory B Cells in Adults after Whole-Cell Killed Oral Cholera Vaccine.

Cholera is a diarrheal disease caused by Vibrio cholerae that continues to be a major public health concern in populations without access to safe water. IgG- and IgA-secreting memory B cells (MBC) targeting the V. cholerae O-specific polysaccharide (OSP) correlate with protection from infection in persons exposed to V. cholerae and may be a major determinant of long-term protection against cholera. Shanchol, a widely used oral cholera vaccine (OCV), stimulates OSP MBC responses in only some people after vaccination, and the gut microbiota is a possible determinant of variable immune responses observed after OCV. Using 16S rRNA sequencing of feces from the time of vaccination, we compared the gut microbiota among adults with and without MBC responses to OCV. Gut microbial diversity measures were not associated with MBC isotype or OSP-specific responses, but individuals with a higher abundance of Clostridiales and lower abundance of Enterobacterales were more likely to develop an MBC response. We applied protein-normalized fecal supernatants of high and low MBC responders to THP-1-derived human macrophages to investigate the effect of microbial factors at the time of vaccination. Feces from individuals with higher MBC responses induced significantly different IL-1ß and IL-6 levels than individuals with lower responses, indicating that the gut microbiota at the time of vaccination may "prime" the mucosal immune response to vaccine antigens. Our results suggest the gut microbiota could impact immune responses to OCVs, and further study of microbial metabolites as potential vaccine adjuvants is warranted.

Seroprevalence of Severe Acute Respiratory Syndrome Coronavirus 2 IgG in Juba, South Sudan, 20201.

Relatively few coronavirus disease cases and deaths have been reported from sub-Saharan Africa, although the extent of its spread remains unclear. During August 10-September 11, 2020, we recruited 2,214 participants for a representative household-based cross-sectional serosurvey in Juba, South Sudan. We found 22.3% of participants had severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain IgG titers above prepandemic levels. After accounting for waning antibody levels, age, and sex, we estimated that 38.3% (95% credible interval 31.8%-46.5%) of the population had been infected with SARS-CoV-2. At this rate, for each PCR-confirmed SARS-CoV-2 infection reported by the Ministry of Health, 103 (95% credible interval 86-126) infections would have been unreported, meaning SARS-CoV-2 has likely spread extensively within Juba. We also found differences in background reactivity in Juba compared with Boston, Massachusetts, USA, where the immunoassay was validated. Our findings underscore the need to validate serologic tests in sub-Saharan Africa populations.

Antimicrobial-resistant bacteria in international travelers.

PURPOSE OF REVIEW: Antimicrobial resistance (AMR) in bacteria poses a major risk to global public health, with many factors contributing to the observed increase in AMR. International travel is one recognized contributor. The purpose of this review is to summarize current knowledge regarding the acquisition, carriage and spread of AMR bacteria by international travelers. RECENT FINDINGS: Recent studies have highlighted that travel is an important risk factor for the acquisition of AMR bacteria, with approximately 30% of studied travelers returning with an acquired AMR bacterium. Epidemiological studies have shown there are three major risk factors for acquisition: travel destination, antimicrobial usage and travelers' diarrhea (TD). Analyses have begun to illustrate the AMR genes that are acquired and spread by travelers, risk factors for acquisition and carriage of AMR bacteria, and local transmission of imported AMR organisms. SUMMARY: International travel is a contributor to the acquisition and dissemination of AMR organisms globally. Efforts to reduce the burden of AMR organisms should include a focus on international travelers. Routine genomic surveillance would further elucidate the role of international travel in the global spread of AMR bacteria.

Clinical sensitivity and interpretation of PCR and serological COVID-19 diagnostics for patients presenting to the hospital.

The diagnosis of COVID-19 requires integration of clinical and laboratory data. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostic assays play a central role in diagnosis and have fixed technical performance metrics. Interpretation becomes challenging because the clinical sensitivity changes as the virus clears and the immune response emerges. Our goal was to examine the clinical sensitivity of two most common SARS-CoV-2 diagnostic test modalities, polymerase chain reaction (PCR) and serology, over the disease course to provide insight into their clinical interpretation in patients presenting to the hospital. We conducted a single-center, retrospective study. To derive clinical sensitivity of PCR, we identified 209 PCR-positive SARS-CoV-2 patients with multiple PCR test results (624 total PCR tests) and calculated daily sensitivity from date of symptom onset or first positive test. Clinical sensitivity of PCR decreased with days post symptom onset with >90% clinical sensitivity during the first 5 days after symptom onset, 70%-71% from Days 9 to 11, and 30% at Day 21. To calculate daily clinical sensitivity by serology, we utilized 157 PCR-positive patients with a total of 197 specimens tested by enzyme-linked immunosorbent assay for IgM, IgG, and IgA anti-SARS-CoV-2 antibodies. In contrast to PCR, serological sensitivity increased with days post symptom onset with >50% of patients seropositive by at least one antibody isotype after Day 7, >80% after Day 12, and 100% by Day 21. Taken together, PCR and serology are complimentary modalities that require time-dependent interpretation. Superimposition of sensitivities over time indicate that serology can function as a reliable diagnostic aid indicating recent or prior infection.

Cholera: Immunity and Prospects in Vaccine Development.

Vibrio cholerae is a prototypical noninvasive mucosal pathogen, yet infection generates long-lasting protection against subsequent disease. Vibriocidal antibody responses are an imperfect but established correlate of protection against cholera following both infection and vaccination. However, vibriocidal antibody responses are likely a surrogate marker for longer-lasting functional immune responses that target the O-polysaccharide antigen at the mucosal surface. While the current bivalent inactivated oral whole cell vaccine is being increasingly used to prevent cholera in areas where the disease is a threat, the most significant limitation of this vaccine is it offers relatively limited direct protection in young children. Future strategies for cholera vaccination include the development of cholera conjugate vaccines and the further development of live attenuated vaccines. Ultimately, the goal of a multivalent vaccine for cholera and other childhood enteric infections that can be incorporated into a standard immunization schedule should be realized.

Human Gut Microbiota Predicts Susceptibility to Vibrio cholerae Infection.

Background: Cholera is a public health problem worldwide, and the risk factors for infection are only partially understood. Methods: We prospectively studied household contacts of patients with cholera to compare those who were infected to those who were not. We constructed predictive machine learning models of susceptibility, using baseline gut microbiota data. We identified bacterial taxa associated with susceptibility to Vibrio cholerae infection and tested these taxa for interactions with V. cholerae in vitro. Results: We found that machine learning models based on gut microbiota, as well as models based on known clinical and epidemiological risk factors, predicted V. cholerae infection. A predictive gut microbiota of roughly 100 bacterial taxa discriminated between contacts who developed infection and those who did not. Susceptibility to cholera was associated with depleted levels of microbes from the phylum Bacteroidetes. By contrast, a microbe associated with cholera by our modeling framework, Paracoccus aminovorans, promoted the in vitro growth of V. cholerae. Gut microbiota structure, clinical outcome, and age were also linked. Conclusion: These findings support the hypothesis that abnormal gut microbial communities are a host factor related to V. cholerae susceptibility.

Analysis of the Human Mucosal Response to Cholera Reveals Sustained Activation of Innate Immune Signaling Pathways.

To better understand the innate immune response to Vibrio cholerae infection, we tracked gene expression in the duodenal mucosa of 11 Bangladeshi adults with cholera, using biopsy specimens obtained immediately after rehydration and 30 and 180 days later. We identified differentially expressed genes and performed an analysis to predict differentially regulated pathways and upstream regulators. During acute cholera, there was a broad increase in the expression of genes associated with innate immunity, including activation of the NF-κB, mitogen-activated protein kinase (MAPK), and Toll-like receptor (TLR)-mediated signaling pathways, which, unexpectedly, persisted even 30 days after infection. Focusing on early differences in gene expression, we identified 37 genes that were differentially expressed on days 2 and 30 across the 11 participants. These genes included the endosomal Toll-like receptor gene TLR8, which was expressed in lamina propria cells. Underscoring a potential role for endosomal TLR-mediated signaling in vivo, our pathway analysis found that interferon regulatory factor 7 and beta 1 and alpha 2 interferons were among the top upstream regulators activated during cholera. Among the innate immune effectors, we found that the gene for DUOX2, an NADPH oxidase involved in the maintenance of intestinal homeostasis, was upregulated in intestinal epithelial cells during cholera. Notably, the observed increases in DUOX2 and TLR8 expression were also modeled in vitro when Caco-2 or THP-1 cells, respectively, were stimulated with live V. cholerae but not with heat-killed organisms or cholera toxin alone. These previously unidentified features of the innate immune response to V. cholerae extend our understanding of the mucosal immune signaling pathways and effectors activated in vivo following cholera.

Plasma and Mucosal Immunoglobulin M, Immunoglobulin A, and Immunoglobulin G Responses to the Vibrio cholerae O1 Protein Immunome in Adults With Cholera in Bangladesh.

Background: Cholera is a severe dehydrating illness of humans caused by toxigenic strains of Vibrio cholerae O1 or O139. Identification of immunogenic V. cholerae antigens could lead to a better understanding of protective immunity in human cholera. Methods: We probed microarrays containing 3652 V. cholerae antigens with plasma and antibody-in-lymphocyte supernatant (ALS, a surrogate marker of mucosal immune responses) from patients with severe cholera caused by V. cholerae O1 in Bangladesh and age-, sex-, and ABO-matched Bangladeshi controls. We validated a subset of identified antigens using enzyme-linked immunosorbent assay. Results: Overall, we identified 608 immunoreactive V. cholerae antigens in our screening, 59 of which had higher immunoreactivity in convalescent compared with acute-stage or healthy control samples (34 in plasma, 39 in mucosal ALS; 13 in both sample sets). Identified antigens included cholera toxin B and A subunits, V. cholerae O-specific polysaccharide and lipopolysaccharide, toxin coregulated pilus A, sialidase, hemolysin A, flagellins (FlaB, FlaC, and FlaD), phosphoenolpyruvate-protein phosphotransferase, and diaminobutyrate-2-oxoglutarate aminotransferase. Conclusions: This study is the first antibody profiling of the mucosal and systemic antibody responses to the nearly complete V. cholerae O1 protein immunome; it has identified antigens that may aid in the development of an improved cholera vaccine.

Ensemble-based docking: From hit discovery to metabolism and toxicity predictions.

This paper describes and illustrates the use of ensemble-based docking, i.e., using a collection of protein structures in docking calculations for hit discovery, the exploration of biochemical pathways and toxicity prediction of drug candidates. We describe the computational engineering work necessary to enable large ensemble docking campaigns on supercomputers. We show examples where ensemble-based docking has significantly increased the number and the diversity of validated drug candidates. Finally, we illustrate how ensemble-based docking can be extended beyond hit discovery and toward providing a structural basis for the prediction of metabolism and off-target binding relevant to pre-clinical and clinical trials.

Immunogenicity of the Bivalent Oral Cholera Vaccine Shanchol in Haitian Adults With HIV Infection.

We evaluated immune responses following bivalent oral cholera vaccination (Shanchol [Shantha Biotechnics]; BivWC) in a cohort of 25 human immunodeficiency virus (HIV)-infected adults in Haiti. Compared with adults without HIV infection, vaccination in HIV-infected individuals resulted in lower vibriocidal responses against Vibrio cholerae O1, and there was a positive relationship between the CD4(+) T-cell count and vibriocidal responses following vaccination. Nevertheless, seroconversion occurred at a rate of 65% against the Ogawa serotype and 74% against the Inaba serotype in adults with HIV infection. These results suggest that the vaccine retains substantial immunogenicity in adults with HIV infection and may benefit this population by protecting against cholera.

Comparative proteomic analysis reveals activation of mucosal innate immune signaling pathways during cholera.

Vibrio cholerae O1 is a major cause of acute watery diarrhea in over 50 countries. Evidence suggests that V. cholerae O1 may activate inflammatory pathways, and a recent study of a Bangladeshi population showed that variants in innate immune genes play a role in mediating susceptibility to cholera. We analyzed human proteins present in the small intestine of patients infected with V. cholerae O1 to characterize the host response to this pathogen. We collected duodenal biopsy specimens from patients with acute cholera after stabilization and again 30 days after initial presentation. Peptides extracted from biopsy specimens were sequenced and quantified using label-free mass spectrometry and SEQUEST. Twenty-seven host proteins were differentially abundant between the acute and convalescent stages of infection; the majority of these have known roles in innate defense, cytokine production, and apoptosis. Immunostaining confirmed that two proteins, WARS and S100A8, were more abundant in lamina propria cells during the acute stage of cholera. Analysis of the differentially abundant proteins revealed the activation of key regulators of inflammation by the innate immune system, including Toll-like receptor 4, nuclear factor kappa-light-chain-enhancer of activated B cells, mitogen-activated protein kinases, and caspase-dependent inflammasomes. Interleukin-12ß (IL-12ß) was a regulator of several proteins that were activated during cholera, and we confirmed that IL-12ß was produced by lymphocytes recovered from duodenal biopsy specimens of cholera patients. Our study shows that a broad inflammatory response is generated in the gut early after onset of cholera, which may be critical in the development of long-term mucosal immunity against V. cholerae O1.

Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of Vibrio cholerae.

We evaluated the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS) for the identification of Vibrio cholerae. MS identified all 42 isolates of V. cholerae O1 and O139 and 7 of 9 non-O1/O139 isolates. MS correctly discriminated between all Aeromonas and V. cholerae isolates. Overall, MS performed as well as or better than biochemical methods.

In vitro and in vivo antimicrobial efficacy of natural plant-derived compounds against Vibrio cholerae of O1 El Tor Inaba serotype.

In this study, we investigated antibacterial activities of 20 plant-derived natural compounds against Gram-negative enteric pathogens. We found that both flavonoids and non-flavonoids, including honokiol and magnolol, possess specific antibacterial activities against V. cholerae, but not against other species of Gram-negative bacterium which we tested. Using various antibacterial assays, we determined that there was a dose-dependent bactericidal and biofilm inhibitory activity of honokiol and magnolol against Vibrio cholerae. In addition to antibacterial activities, these molecules also induced an attenuating effect on reactive oxygen species (ROS) production and pro-inflammatory responses generated by macrophages in response to lipopolysaccharides (LPS). Additionally, Caenorhabditis elegans lethality assay revealed that honokiol and magnolol have an ability to extend a lifespan of V. cholerae-infected worms, contributing to prolonged survival of worms after lethal infection. Altogether, our data show for the first time that honokiol and magnolol may be considered as attractive protective or preventive food adjuncts for cholera.