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Background Characterizing SARS-CoV-2 outbreaks on university campuses is critical for informed public health measures and understanding transmission dynamics. Figure 1.Dropbox and Kiosk Samples Collected September 10, 2021 to April 23, 2022. Methods Faculty, staff, and students at a major public university in Seattle, WA, USA were enrolled in a COVID-19 testing study. Individuals could test using observed self-swabs at on-campus kiosks or unobserved self-swabs using a kit and returning it to a dropbox on campus. Sample collection volume for observed self-swabs was limited by staffing and space. All samples were returned to the laboratory and tested for SARS-CoV-2 by qRT-PCR. Results From September 10, 2021 to April 23, 2022, 38,400 individuals were enrolled in the study. Of these individuals, 5,089 used dropboxes only, 14,421 used kiosks only, and 5,820 used both. A total of 21,653 dropbox swabs and 75,493 observed self-swabs were collected. Median age was similar between individuals using dropboxes and observed self-swabs (20 vs. 22 years). A greater proportion of dropbox users were students compared to faculty and staff (students made up 83% of dropbox only population, 75% of kiosk only, and 86% of both, χ² p-value< 0.0001). Symptom data was reported for 65,349 swabs. Dropbox users were less likely to have symptoms compared to observed self-swab users (24% of swabs vs. 54%, χ² p-value< 0.0001). SARS-CoV-2 positivity was slightly lower for dropboxes compared to kiosks (4% vs. 5%; p=0.001). Dropboxes were highly utilized during periods of increased testing demand, including after academic breaks and variant emergence (Figure 1). Of the total tests distributed for use, a greater proportion of dropbox kits were unable to be resulted (6%) compared to observed self-swab kits (0.02%). Conclusion Dropboxes provided a flexible, high-volume collection method at times of increased testing demand. Individuals who used dropboxes were less likely to report symptoms and slightly less likely to test positive, suggesting a role for dropbox utilization in high-risk asymptomatic individuals during periods of high community transmission on a university campus. Disclosures Geoffrey S. Gottlieb, MD, PhD, Abbott Molecular Diagnostics: Grant/Research Support|Alere Technologies: Grant/Research Support|BMGF: Grant/Research Support|BMS: Grant/Research Support|Cerus Corp.: Grant/Research Support|Gilead Sciences: Grant/Research Support|Janssen Pharmaceutica: Grant/Research Support|Merck & Co: Grant/Research Support|Roche Molecular Systems: Grant/Research Support|THERA Technologies/TaiMed Biologics: Grant/Research Support|ViiV Healthcare: Grant/Research Support Helen Y. Chu, MD, MPH, Cepheid: Reagents|Ellume: Advisor/Consultant|Gates Ventures: Grant/Research Support|Merck: Advisor/Consultant|Pfizer: Advisor/Consultant.

Novel variants continue to emerge in the SARS-CoV-2 pandemic. University testing programs may provide timely epidemiologic and genomic surveillance data to inform public health responses. We conducted testing from September 2021 to February 2022 in a university population under vaccination and indoor mask mandates. A total of 3,048 of 24,393 individuals tested positive for SARS-CoV-2 by RT-PCR; whole genome sequencing identified 209 Delta and 1,730 Omicron genomes of the 1,939 total sequenced. Compared to Delta, Omicron had a shorter median serial interval between genetically identical, symptomatic infections within households (2 versus 6 days, P = 0.021). Omicron also demonstrated a greater peak reproductive number (2.4 versus 1.8), and a 1.07 (95% confidence interval: 0.58, 1.57; P

BackgroundOral cholera vaccines (OCVs) are an important tool for reduction of the worldwide cholera burden, but some individuals who receive an OCV do not develop protective immune responses. The gut microbiota is a potential explanation for these differences. Components of the gut microbiota associated with differences in OCV response have not been identified.ResultsWe used metagenomic sequencing to identify predicted protein-coding genes in the gut microbiota at the time of OCV administration, and then measured immune responses to vaccination. Vaccine recipients were classified as OCV “responders” if they developed a post­vaccination increase in memory B cell populations that produce IgA or IgG specific for cholera toxin and the V. cholerae O-specific polysaccharide. We next analyzed microbial genes seen at similar abundances across individual samples and classified these into co-abundant gene groupings (CAGs), and correlated CAGs with OCV responses. Next, to identify specific bacterial strains associated with OCV responses, we mapped CAGs to bacterial genomes and generated a “priority score” for each strain detected in the study population. This score reflects both the number of CAGs aligning to a specific bacterial genome and the strength of the association between the CAGs and the vaccine response. This strain-level analysis revealed relationships between the gut microbiota and immune response to OCV that were not detected at the genus or species level. Bacterial strains which produce short-chain fatty acids and those with sphingolipid-containing cell membranes were correlated with more robust immune responses to vaccination.ConclusionOur study demonstrates a method for translating metagenomic sequencing data into strain-specific results associated with a biological outcome. Using this approach, we identified strains for the study of bacterial-derived molecules or metabolites associated with immune responses; such agents might have potential utility as vaccine adjuvants.

Vibrio cholerae can cause a range of symptoms, from severe diarrhea to asymptomatic infection. Previous studies using whole-genome sequencing (WGS) of multiple bacterial isolates per patient showed that V. cholerae can evolve modest genetic diversity during symptomatic infection. To further explore the extent of V. cholerae within-host diversity, we applied culture-based WGS and metagenomics to a cohort of both symptomatic and asymptomatic cholera patients from Bangladesh. While metagenomics allowed us to detect more mutations in symptomatic patients, WGS of cultured isolates was necessary to detect V. cholerae diversity in asymptomatic carriers, likely due to their low V. cholerae load. Using both metagenomics and isolate WGS, we report three lines of evidence that V. cholerae hypermutators evolve within patients. First, we identified nonsynonymous mutations in V. cholerae DNA repair genes in 5 out of 11 patient metagenomes sequenced with sufficient coverage of the V. cholerae genome and in 1 of 3 patients with isolate genomes sequenced. Second, these mutations in DNA repair genes tended to be accompanied by an excess of intrahost single nucleotide variants (iSNVs). Third, these iSNVs were enriched in transversion mutations, a known hallmark of hypermutator phenotypes. While hypermutators appeared to generate mostly selectively neutral mutations, nonmutators showed signs of convergent mutation across multiple patients, suggesting V. cholerae adaptation within hosts. Our results highlight the power and limitations of metagenomics combined with isolate sequencing to characterize within-patient diversity in acute V. cholerae infections, while providing evidence for hypermutator phenotypes within cholera patients. IMPORTANCE Pathogen evolution within patients can impact phenotypes such as drug resistance and virulence, potentially affecting clinical outcomes. V. cholerae infection can result in life-threatening diarrheal disease or asymptomatic infection. Here, we describe whole-genome sequencing of V. cholerae isolates and culture-free metagenomic sequencing from stool of symptomatic cholera patients and asymptomatic carriers. Despite the typically short duration of cholera, we found evidence for adaptive mutations in the V. cholerae genome that occur independently and repeatedly within multiple symptomatic patients. We also identified V. cholerae hypermutator phenotypes within several patients, which appear to generate mainly neutral or deleterious mutations. Our work sets the stage for future studies of the role of hypermutators and within-patient evolution in explaining the variation from asymptomatic carriage to symptomatic cholera.

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-1β 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.

Recent studies indicate that the human intestinal microbiota could impact the outcome of infection by

BackgroundTesting programs have been utilized as part of SARS-CoV-2 mitigation strategies on university campuses, and it is not known which strategies successfully identify cases and contain outbreaks.ObjectiveEvaluation of a testing program to control SARS-CoV-2 transmission at a large university.DesignProspective longitudinal study using remote contactless enrollment, daily mobile symptom and exposure tracking, and self-swab sample collection. Individuals were tested if the participant was (1) exposed to a known case, developed new symptoms, or reported high-risk behavior, (2) a member of a group experiencing an outbreak, or (3) at baseline upon enrollment.SettingAn urban, public university during Autumn quarter of 2020ParticipantsStudents, staff, and faculty.MeasurementsSARS-CoV-2 PCR testing was conducted, and viral genome sequencing was performed.ResultsWe enrolled 16,476 individuals, performed 29,783 SARS-CoV-2 tests, and detected 236 infections. Greek community affiliation was the strongest risk factor for testing positive. 75.0% of positive cases reported at least one of the following: symptoms (60.8%), exposure (34.7%), or high-risk behaviors (21.5%). 88.1% of viral genomes (52/59) sequenced from Greek-affiliated students were genetically identical to at least one other genome detected, indicative of rapid SARS-CoV-2 spread within this group, compared to 37.9% (11/29) of genomes from non-Greek students and employees.LimitationsObservational study.ConclusionIn a setting of limited resources during a pandemic, we prioritized testing of individuals with symptoms and high-risk exposure during outbreaks. Rapid spread of SARS- CoV-2 occurred within outbreaks without evidence of further spread to the surrounding community. A testing program focused on high-risk populations may be effective as part of a comprehensive university-wide mitigation strategy to control the SARS-CoV-2 pandemic.

Vibrio cholerae O1 can cause life threatening diarrheal disease if left untreated. T cells can play critical roles in inducing B cell mediated immunity. As the mechanism of T cell dependent B cell maturation is not well established, we hypothesized that a specific population of T (follicular helper T, Tfh) cells, are involved in B cell maturation following cholera. We found flowcytometrically that V. cholerae infection induces significant increases in circulating Tfh cells expressing B cell maturation associated protein CD40L early in disease. The increased Tfh cells expressing CD40L recognize cholera toxin most prominently, with lessened responses to V. cholerae membrane preparation (MP) and V. cholerae cytolysin (VCC). We further showed that early induction of Tfh cells and CD40L was associated with later memory B cell responses to same antigens. Lastly, we demonstrated in vitro that Tfh cells isolated after cholera can stimulate class switching of co-cultured, isolated B cells from patients with cholera, leading to production of the more durable IgG antibody isotype colorimetrically. These studies were conducted on circulating Tfh cells; future studies will be directed at examining role of Tfh cells during cholera directly in gut mucosa of biopsied samples, at the single cell level if feasible.

Recent studies indicate that the human intestinal microbiota could impact the outcome of infection byVibrio cholerae, the etiological agent of the diarrheal disease cholera. A commensal bacterium,Paracoccus aminovorans, was previously identified in high abundance in stool collected from individuals infected withV. choleraewhen compared to stool from uninfected persons. However, if and howP. aminovoransinteracts withV. choleraehas not been experimentally determined; moreover, whether any association between this bacterium alters the behaviors ofV. choleraeto affect the disease outcome is unclear. Here we show thatP. aminovoransandV. choleraetogether form dual-species biofilm structures at the air-liquid interface, with previously uncharacterized novel features. Importantly, the presence ofP. aminovoranswithin the murine small intestine enhancesV. choleraecolonization in the same niche that is dependent on theVibrioexopolysaccharide (VPS) and other major components of matureV. choleraebiofilm. These studies illustrate that dual-species biofilm formation is a plausible mechanism used by a gut microbe to increase the virulence of the pathogen, and this interaction may alter outcomes in enteric infections.Significance StatementWhile ample evidence suggests that the outcome of some enteric infections can be affected by the intestinal microbiota, how specific gut microbes change the behaviors of a pathogen is unclear. Here we characterize the interaction betweenVibrio choleraeandParacoccus aminovorans, a gut microbe known to increase in abundance in the intestines during activeV. choleraeinfection in humans. These two bacteria form a dual-species biofilm structure at the air-liquid interface, and the gut microbe increases the host colonization efficiency ofV. cholerae. Importantly, our study identifies a previously unknown mechanism of gut microbe-pathogen interaction that has the potential to alter the disease outcome.

Vibrio cholerae can cause a range of symptoms, ranging from severe diarrhea to asymptomatic infection. Previous studies using whole genome sequencing (WGS) of multiple bacterial isolates per patient showed that V. cholerae can evolve modest genetic diversity during symptomatic infection. To further explore the extent of V. cholerae within-host diversity, we applied culture-based WGS and metagenomics to a cohort of both symptomatic and asymptomatic cholera patients from Bangladesh. While metagenomics allowed us to detect more mutations in symptomatic patients, WGS of cultured isolates was necessary to detect V. cholerae diversity in asymptomatic carriers, likely due to their low V. cholerae load. Using both metagenomics and isolate WGS, we report three lines of evidence that V. cholerae hypermutators evolve within patients. First, we identified nonsynonymous in V. cholerae DNA repair genes in five out of 11 patient metagenomes sequenced with sufficient coverage of the V. cholerae genome, and in one of three patients with isolate genomes sequenced. Second, mutations in DNA repair genes tended to be accompanied by an excess of intrahost single nucleotide variants (iSNVs). Third, these iSNVs were enriched in transversion mutations, a known hallmark of hypermutator phenotypes. While hypermutators appeared to generate mostly selectively neutral mutations, non-mutators showed signs of convergent mutation across multiple patients, suggesting V. cholerae adaptation within hosts. Our results highlight the power and limitations of metagenomics combined with isolate sequencing to characterize within-patient diversity in acute V. cholerae infections, while providing evidence for hypermutator phenotypes within cholera patients.IMPORTANCEPathogen evolution within patients can impact phenotypes such as drug resistance and virulence, potentially affecting clinical outcomes. V. cholerae infection can result in life-threatening diarrheal disease, or asymptomatic infection. Here we describe whole-genome sequencing of V. cholerae isolates and culture-free metagenomic sequencing from stool of symptomatic cholera patients and asymptomatic carriers. Despite the typically short duration of cholera, we found evidence for adaptive mutations in the V. cholerae genome that occur independently and repeatedly within multiple symptomatic patients. We also identified V. cholerae hypermutator phenotypes within several patients, which appear to generate mainly neutral or deleterious mutations. Our work sets the stage for future studies of the role of hypermutators and within-patient evolution in explaining the variation from asymptomatic carriage to symptomatic cholera.

Cholera is a severe dehydrating illness of humans caused by Vibrio cholerae. V. cholerae is a highly motile bacterium that has a single flagellum covered in lipopolysaccharide (LPS) displaying O-specific polysaccharide (OSP), and V. cholerae motility correlates with its ability to cause disease. The mechanisms of protection against cholera are not well understood; however, since V. cholerae is a noninvasive intestinal pathogen, it is likely that antibodies that bind the pathogen or its products in the intestinal lumen contribute to protection from infection. Here, we demonstrate that OSP-specific antibodies isolated from humans surviving cholera in Bangladesh inhibit V. cholerae motility and are associated with protection against challenge in a motility-dependent manner., The mechanism of protection against cholera afforded by previous illness or vaccination is currently unknown. We have recently shown that antibodies targeting O-specific polysaccharide (OSP) of Vibrio cholerae correlate highly with protection against cholera. V. cholerae is highly motile and possesses a flagellum sheathed in OSP, and motility of V. cholerae correlates with virulence. Using high-speed video microscopy and building upon previous animal-related work, we demonstrate that sera, polyclonal antibody fractions, and OSP-specific monoclonal antibodies recovered from humans surviving cholera block V. cholerae motility at both subagglutinating and agglutinating concentrations. This antimotility effect is reversed by preadsorbing sera and polyclonal antibody fractions with purified OSP and is associated with OSP-specific but not flagellin-specific monoclonal antibodies. Fab fragments of OSP-specific polyclonal antibodies do not inhibit motility, suggesting a requirement for antibody-mediated cross-linking in motility inhibition. We show that OSP-specific antibodies do not directly affect V. cholerae viability, but that OSP-specific monoclonal antibody highly protects against death in the murine cholera model. We used in vivo competitive index studies to demonstrate that OSP-specific antibodies impede colonization and survival of V. cholerae in intestinal tissues and that this impact is motility dependent. Our findings suggest that the impedance of motility by antibodies targeting V. cholerae OSP contributes to protection against cholera.

Background Skilled nursing facilities (SNFs) are high-risk settings for SARS-CoV-2 transmission. Infection rates among employees are infrequently described. Objective To describe SARS-CoV-2 rates among SNF employees and residents during a non-outbreak time period, we measured cross-sectional SARS-CoV-2 prevalence across multiple sites in the Seattle area. Design SARS-CoV-2 testing was performed for SNF employees and residents using quantitative real-time reverse transcription polymerase chain reaction. A subset of employees completed a sociodemographic and symptom questionnaire. Participants Between March 29 and May 13, 2020, we tested 1583 employees and 1208 residents at 16 SNFs for SARS-CoV-2. Main Measure SARS-CoV-2 testing results and symptom report among employees and residents. Key Results Eleven of the 16 SNFs had one or more resident or employee test positive. Overall, 46 (2.9%) employees had positive or inconclusive testing for SARS-CoV-2, and among those who completed surveys, most were asymptomatic and involved in direct patient care. The majority of employees tested were female (934, 73%), and most employees were Asian (392, 30%), Black (360, 28%), or white (360, 28%). Among the 1208 residents tested, 110 (9.1%) had positive or inconclusive results. There was no association between the presence of positive residents and positive employees within a SNF (p = 0.62, McNemar’s test). Conclusions In the largest study of SNFs to date, SARS-CoV-2 infections were detected among both employees and residents. Employees testing positive were often asymptomatic and involved in direct patient care. Surveillance testing is needed for SNF employees and residents during the pandemic response.

Both the host microbiome and the microbiome of the built environment can have profound impacts on human health. While prior studies have suggested that the variability introduced by DNA extraction method is less than typical biologic variation, most studies have focused on 16S rRNA amplicon sequencing or on high biomass fecal samples. Shotgun metagenomic sequencing provides advantages over amplicon sequencing for surveying the microbiome, but is a challenge to perform in lower microbial biomass samples with high human DNA content such as sputum or vacuumed dust. Here we systematically evaluate the impact of four different extraction methods (phenol:choloroform, and three high-throughput kit-based approaches, the Promega Maxwell gDNA, Qiagen MagAttract PowerSoil DNA, and ZymoBIOMICS 96 MagBead). We report the variation in microbial community structure and predicted microbial function assessed by shotgun metagenomics sequencing in human stool, sputum, and vacuumed dust obtained from ongoing cohort studies or clinical trials. The same beadbeating protocol was used for all samples to focus our evaluation on the impact of kit chemistries on sequencing results. DNA yield was overall highest in the phenol:choloroform and Promega approaches. Only the phenol:choloroform approach showed evidence of contamination in negative controls. Bias was evaluated using mock community controls, and was noted across all extraction methods, although Promega exhibited the least amount of bias. The extraction method did not impact the proportion of human reads, although stool had the lowest proportion of human reads (0.1%) as compared to dust (44.1%) and sputum (80%). We calculated Bray-Curtis dissimilarity and Aitchison distances to evaluate the impact of extraction method on microbial community structure by sample type. Extraction method had the lowest impact in stool (extraction method responsible for 3.0-3.9% of the variability), the most impact in vacuumed dust (12-16% of the variability) and intermediate values for sputum (9.2-12% variability). Similar differences were noted when evaluating microbial community function. Our results will inform investigators planning microbiome studies using diverse sample types in large clinical studies. A consistent DNA extraction approach across all sample types is recommended, particularly with lower microbial biomass samples that are more heavily influenced by extraction method.

BackgroundSusceptibility to Vibrio cholerae infection is impacted by blood group, age, and pre-existing immunity, but these factors only partially explain who becomes infected. A recent study used 16S rRNA amplicon sequencing to quantify the composition of the gut microbiome and identify predictive biomarkers of infection with limited taxonomic resolution.MethodsTo 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.ResultsUsing 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 also correlated with protection.ConclusionOur 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.SUMMARYCholera infection and disease severity can be predicted using metagenomic sequencing of the gut microbiome pre-infection in a prospective cohort, and suggests potentially protective bacterial species and genes.

BackgroundAntibiotic use as growth promoters for livestock is presumed to be a major contributor to the acquisition of antimicrobial resistance (AMR) genes in humans, yet data evaluating AMR patterns in the setting of animal exposure are limited to observational studies that do not capture data from prior to livestock introduction.MethodsWe performed a feasibility study by recruiting a subset of women in a delayed-start randomized controlled trial of small-scale chicken farming in order to examine the prevalence of clinically-relevant AMR genes. Stool samples were obtained at baseline and one year from five intervention women who received chickens at the start of the study, six control women who did not receive chickens until the end of the study, and from chickens provided to the control group at the end of the study. Stool was screened for 87 clinically significant AMR genes using a commercially available qPCR array (Qiagen).ResultsChickens harbored 23 AMR genes from classes also found in humans as well as vancomycin and additional β-lactamase resistance genes. After one year of exposure to chickens, six new AMR genes were detected in controls and seven new AMR genes were detected in the intervention group. Women who had direct contact with the chickens sampled in the study had greater similarities in AMR resistance gene patterns to chickens than those who did not have direct contact with chickens sampled (p = 0.006). There was a trend towards increased similarity in AMR gene patterns with chickens at one year (p = 0.12).ConclusionsChickens and humans in this study harbored AMR genes from many antimicrobial classes at both baseline and follow up timepoints. Studies designed to evaluate human AMR genes in the setting of animal exposure should account for high baseline AMR rates, and consider collecting concomitant animal samples, human samples, and environmental samples over time to determine the directionality and source of AMR genes.Trial registration: ClinicalTrials.gov Identifier: NCT02619227

Vibrio cholerae O1, the major causative agent of cholera, remains a significant public health threat. Although there are available vaccines for cholera, the protection provided by killed whole-cell cholera vaccines in young children is poor. An obstacle to the development of improved cholera vaccines is the need for a better understanding of the primary mechanisms of cholera immunity and identification of improved correlates of protection. Considerable progress has been made over the last decade in understanding the adaptive and innate immune responses to cholera disease as well as V. cholerae infection. This review will assess what is currently known about the systemic, mucosal, memory, and innate immune responses to clinical cholera, as well as recent advances in our understanding of the mechanisms and correlates of protection against V. cholerae O1 infection.

An episode of cholera provides better protection against reinfection than oral cholera vaccines, and the reasons for this are still under study. To better understand this, we compared the immune responses of human cells exposed to live Vibrio cholerae with those of cells exposed to heat-killed V. cholerae (similar to the contents of oral cholera vaccines). We also compared the effects of active cholera toxin and the inactive cholera toxin B subunit (which is included in some cholera vaccines). One key immune signaling molecule, IL-23, was uniquely produced in response to the combination of live bacteria and active cholera holotoxin. Stimulation with V. cholerae that did not produce the active toxin or was killed did not produce an IL-23 response. The stimulation of IL-23 production by cholera toxin-producing V. cholerae may be important in conferring long-term immunity after cholera., Vibrio cholerae infection provides long-lasting protective immunity, while oral, inactivated cholera vaccines (OCV) result in more-limited protection. To identify characteristics of the innate immune response that may distinguish natural V. cholerae infection from OCV, we stimulated differentiated, macrophage-like THP-1 cells with live versus heat-inactivated V. cholerae with and without endogenous or exogenous cholera holotoxin (CT). Interleukin 23A gene (IL23A) expression was higher in cells exposed to live V. cholerae than in cells exposed to inactivated organisms (mean change, 38-fold; 95% confidence interval [95% CI], 4.0 to 42; P

Staphylococcus aureus prosthetic valve endocarditis (PVE) remains among the most morbid bacterial infections, with mortality estimates ranging from 40% to 80%. The proportion of PVE cases due to methicillin-resistant Staphylococcus aureus (MRSA) has grown in recent decades, to account for more than 15% of cases of S. aureus PVE and 6% of all cases of PVE. Because no large studies or clinical trials for PVE have been published, most guidelines on the diagnosis and management of MRSA PVE rely upon expert opinion and data from animal models or related conditions (e.g., coagulase-negative Staphylococcus infection). We performed a review of the literature on MRSA PVE to summarize data on pathogenic mechanisms and updates in epidemiology and therapeutic management and to inform diagnostic strategies and priority areas where additional clinical and laboratory data will be particularly useful to guide therapy. Major updates discussed in this review include novel diagnostics, indications for surgical management, the utility of aminoglycosides in medical therapy, and a review of newer antistaphylococcal agents used for the management of MRSA PVE.

Background Vibrio cholerae, the causative agent of cholera, is a major cause of diarrhea worldwide. Children under the age of 5 have the highest disease burden of cholera. Vibriocidal antibody responses following natural infection and oral cholera vaccination (OCV) are associated with protective immunity, but whether this holds uniformly true in young children is not known. Methods Household contacts of cholera patients are at high risk of V cholerae infection. We measured the association between baseline vibriocidal titer and the subsequent risk of infection in 50 household contacts

Background Ceftaroline fosamil is a cephalosporin approved for treating skin and soft tissue infections (SSTIs), including those caused by methicillin-resistant Staphylococcus aureus and community-acquired pneumonia (CAP). Objectives We aimed to study ceftaroline use and associated adverse drug reactions (ADRs), including hypersensitivity reactions (HSRs), among inpatients. Methods We performed a retrospective electronic health record review of inpatients from Massachusetts General Hospital and Brigham and Women's Hospital who received ceftaroline between May 2012 and February 2015. ADRs diagnosed by clinical providers during the course of clinical care were subsequently verified and classified. Risk factors for ADRs were identified. Results Among 96 patients (median age, 57 years; 54% females) who received a median of 28 (interquartile range, 6-63) ceftaroline doses, 54% were being treated for methicillin-resistant Staphylococcus aureus and treatment indications other than SSTI and CAP comprised 59% of care. There were 31 ADRs observed in 20 (21%) patients; hematologic (n = 15) and cutaneous (n = 9) findings were most common. Observed HSRs included rash with mucosal lesions (n = 1), rash with skin desquamation (n = 1), and possible organ-specific HSRs (n = 2). Patients who suffered an ADR received more doses of ceftaroline (median, 46 vs 21; P = .013). There was no increased risk of ceftaroline ADR among patients with reported beta-lactam allergy history ( P > .5). Conclusions Ceftaroline is used to treat a range of infections beyond SSTI and CAP. We observed a high rate of ADRs from ceftaroline, including signs of severe HSRs. More data are needed to understand the frequency and predictors of ceftaroline ADRs and HSRs.

The development of new approaches to cholera control relies on an accurate understanding of cholera epidemiology. However, most information on cholera incidence lacks laboratory confirmation and instead relies on surveillance systems reporting medically attended acute watery diarrhea. If recent infections could be identified using serological markers, cross-sectional serosurveys would offer an alternative approach to measuring incidence. Here, we used 1569 serologic samples from a cohort of cholera cases and their uninfected contacts in Bangladesh to train machine learning models to identify recent Vibrio cholerae O1 infections. We found that an individual’s antibody profile contains information on the timing of V. cholerae O1 infections in the previous year. Our models using six serological markers accurately identified individuals in the Bangladesh cohort infected within the last year [cross-validated area under the curve (AUC), 93.4%; 95% confidence interval (CI), 92.1 to 94.7%], with a marginal performance decrease using models based on two markers (cross-validated AUC, 91.0%; 95% CI, 89.2 to 92.7%). We validated the performance of the two-marker model on data from a cohort of North American volunteers challenged with V. cholerae O1 (AUC range, 88.4 to 98.4%). In simulated serosurveys, our models accurately estimated annual incidence in both endemic and epidemic settings, even with sample sizes as small as 500 and annual incidence as low as two infections per 1000 individuals. Cross-sectional serosurveys may be a viable approach to estimating cholera incidence.

Vibrio choleraeO1 can cause life threatening diarrheal disease if left untreated. A long lasting immune response, producing 3-5 years of protection from subsequent, symptomatic disease following natural infection, is mediated by B cell mediated humoral immunity. T cells can play critical roles in inducing such immunity. However, the mechanism of T cell dependent B cell maturation and whether a key sub-population of T cells are involved is not well established in cholera. We hypothesized that a specific population of T cells, follicular helper T (Tfh) cells, are involved in B cell maturation following cholera; we used flow cytometry, culture and colorimetric assays to address this question. We found thatV. choleraeinfection induces significant increase in circulating Tfh cells expressing B cell maturation associated protein CD40L early in disease. The increased Tfh cells expressing CD40L recognize cholera toxin most prominently, with lessened responses to two antigens tested,V. choleraemembrane preparation (MP) andVibrio choleraecytolysin (VCC). We further showed that early induction of Tfh cells and CD40L was associated with later memory B cell responses to same antigens. Lastly, we demonstratedin vitrothat Tfh cells isolated after cholera can stimulate class switching of cocultured, isolated B cells from patients with cholera, leading to production of the more durable IgG antibody isotype. These studies were conducted on circulating Tfh cells; future studies will be directed at examining role of Tfh cells during cholera directly in the gut mucosa of biopsied samples, at the single cell level if feasible.

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.

Cholera is a severe, water-borne diarrhoeal disease caused by toxin-producing strains of the bacterium Vibrio cholerae. Comparative genomics has revealed ‘waves’ of cholera transmission and evolution, in which clones are successively replaced over decades and centuries. However, the extent of V. cholerae genetic diversity within an epidemic or even within an individual patient is poorly understood. Here, we characterized V. cholerae genomic diversity at a micro-epidemiological level within and between individual patients from Bangladesh and Haiti. To capture within-patient diversity, we isolated multiple (8 to 20) V. cholerae colonies from each of eight patients, sequenced their genomes and identified point mutations and gene gain/loss events. We found limited but detectable diversity at the level of point mutations within hosts (zero to three single nucleotide variants within each patient), and comparatively higher gene content variation within hosts (at least one gain/loss event per patient, and up to 103 events in one patient). Much of the gene content variation appeared to be due to gain and loss of phage and plasmids within the V. cholerae population, with occasional exchanges between V. cholerae and other members of the gut microbiota. We also show that certain intra-host variants have phenotypic consequences. For example, the acquisition of a Bacteroides plasmid and non-synonymous mutations in a sensor histidine kinase gene both reduced biofilm formation, an important trait for environmental survival. Together, our results show that V. cholerae is measurably evolving within patients, with possible implications for disease outcomes and transmission dynamics.

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.

Cholera is a severe, waterborne diarrheal disease caused by toxin-producing strains of the bacteriumVibrio cholerae. Comparative genomics has revealed “waves” of cholera transmission and evolution, in which clones are successively replaced over decades and centuries. However, the extent ofV. choleraegenetic diversity within an epidemic or even within an individual patient is poorly understood. Here, we characterizedV. choleraegenomic diversity at a micro-epidemiological level within and between individual patients from Bangladesh and Haiti. To capture within-patient diversity, we isolated multiple (8 to 20)V. choleraecolonies from each of eight patients, sequenced their genomes and identified point mutations and gene gain/loss events. We found limited but detectable diversity at the level of point mutations within hosts (zero to three single nucleotide variants within each patient), and comparatively higher gene content variation within hosts (at least one gain/loss event per patient, and up to 103 events in one patient). Much of the gene content variation appeared to be due to gain and loss of phage and plasmids within theV. choleraepopulation, with occasional exchanges betweenV. choleraeand other members of the gut microbiota. We also show that certain intra-host variants have phenotypic consequences. For example, the acquisition of aBacteroidesplasmid and nonsynonymous mutations in a sensor histidine kinase gene both reduced biofilm formation, an important trait for environmental survival. Together, our results show thatV. choleraeis measurably evolving within patients, with possible implications for disease outcomes and transmission dynamics.Author SummaryVibrio choleraeis the etiological agent of cholera, a severe diarrheal disease endemic to Bangladesh and responsible for global outbreaks, including one ongoing in Haiti. Certain bacterial pathogens can evolve and diversify within the human host, often altering virulence and antibiotic resistance. However, most examples of within-host evolution have come from chronic infections, in which the pathogen has sufficient time to mutate and diversify, and little attention has been paid to more acute infections such as the one caused byV. cholerae. The goal of this study was to measure the extent of within-host evolution ofV. choleraewithin individual infected patients. By sequencing multiple bacterial isolates_from each of eight patients from Bangladesh and Haiti, we found that cholera patients can harbor a diverse population ofV. cholerae. As expected for an acute infection, this diversity is limited, ranging from zero to three point mutations (single nucleotide variants) per patient. However, gene gain/loss events are more prevalent than point mutations, occurring in every single patient, and sometimes involving the transfer of dozens of genes on plasmids. Even if rare, point mutations and gene gain/loss events may be maintained by natural selection, and can alter clinically-and environmentally-relevant phenotypes such as biofilm formation. Therefore, within-patient evolution has the potential to impact clinical and epidemiological outcomes. Together, our results demonstrate that within-patient evolution may be a general feature of both acute and chronic infections, and that gene gain/loss may be an important but under-appreciated feature of within-host evolution.

Multiple Vibrio cholerae infections within the same household are common. Household contacts of patients with cholera were observed with daily clinical assessments and collection of rectal swab cultures for nine days after presentation of the index case. During the follow-up period, 71 (24%) of 294 household contacts developed a positive V. cholerae rectal swab, signifying bacterial shedding. The average length of bacterial shedding was 2.0 days (95% confidence interval 1.7–2.4). However, 16 (5%) of 294 contacts shed V. cholerae for ≥ 4 days. In a multivariate analysis, malnutrition was predictive of long-term shedding (odds ratio = 1.4, 95% confidence interval = 1.3–13, P = 0.02). High rates of V. cholerae infection and bacterial shedding among household contacts of cholera patients represent an opportunity for intervention to reduce V. cholerae transmission.

Weil, A., Cameron, C., Soumerai, J., Dierberg, K., Mouwon, A., Kraemer, D., Lewy, D., Lee, P., Kraemer, J., & Siedner, M. (2014). Alcohol use and health care utilization in rural Liberia: Results of a community-based survey for basic public health indicators. The International Journal Of Alcohol And Drug Research, 3 (2), 169-181. doi: http://dx.doi.org/10.7895/ijadr.v3i2.147 Aim: To measure the association between alcohol use and health-seeking behavior in post-conflict Liberia. Design: Cross-sectional survey. Setting: A community in rural southeast Liberia, from January 11 to January 16, 2010 Participants: 600 heads-of-household. Measures: Logistic regression models for estimation of associations between alcohol use and indicators of healthcare utilization. Frequent alcohol use was defined as drinking more than seven days out of the last two weeks. Findings: Frequent alcohol use was reported by 14.9% of participants. These respondents were less likely to attend clinic for chronic cough (Adjusted Odds Ratio, AOR 0.40, 95% CI 0.18-0.87), to have had an HIV test ( AOR 0.39, 95% CI 0.19-0.77), and to have accessed prenatal care ( AOR = 0.26, 95% CI 0.12-0.54). Approximately 25% of all respondents had no access to latrines, and half reported going to sleep hungry in the past week. Conclusions: Within households in post-conflict Liberia, there is an association between reduced health care utilization and frequent alcohol use self-reported by a head of household or primary caregiver.

Vibrio cholerae O1 causes cholera, a dehydrating diarrheal disease. We have previously shown that V. cholerae -specific memory B cell responses develop after cholera infection, and we hypothesize that these mediate long-term protective immunity against cholera. We prospectively followed household contacts of cholera patients to determine whether the presence of circulating V. cholerae O1 antigen-specific memory B cells on enrollment was associated with protection against V. cholerae infection over a 30-day period. Two hundred thirty-six household contacts of 122 index patients with cholera were enrolled. The presence of lipopolysaccharide (LPS)-specific IgG memory B cells in peripheral blood on study entry was associated with a 68% decrease in the risk of infection in household contacts ( P = 0.032). No protection was associated with cholera toxin B subunit (CtxB)-specific memory B cells or IgA memory B cells specific to LPS. These results suggest that LPS-specific IgG memory B cells may be important in protection against infection with V. cholerae O1.

Vibrio cholerae O1 is a noninvasive enteric pathogen and serves as a model for studies of mucosal immunity. Although symptomatic V. cholerae infection induces durable protection against subsequent disease, vaccination with oral killed whole-cell V. cholerae stimulates less long-lasting protection against cholera. In this study, we demonstrated that cholera induces an early proinflammatory cellular immune response that results in priming of Th1- and Th17-type cytokine responses to ex vivo antigenic stimulation and an increase in the ratio of Th1 to Th2 CD4 + T-cell responses. Comparable priming of Th1 and Th17 responses, with an increased ratio of Th1 to Th2 CD4 + T-cell responses, was not observed in subjects who received two doses of the oral cholera vaccine Dukoral (a whole-cell cholera toxin B subunit containing [WC-CTB] vaccine). These findings suggest that natural V. cholerae infection induces an early, proinflammatory cellular immune response, despite the apparent lack of clinical signs of inflammation. The failure of the WC-CTB vaccine to activate equivalent, CD4 + T-cell responses is a potential explanation for the shorter duration of protection following immunization with this vaccine. Additional studies are needed to determine whether these early T-cell-mediated events predict the subsequent duration of immunologic memory.

Background Vibrio cholerae, the causative agent of cholera, is responsible for significant morbidity and mortality worldwide. Children less than 5 years old have the highest disease burden of cholera in endemic areas. While children develop serum vibriocidal antibody responses to cholera vaccines, they derive less protection from vaccination compared with adults. The aim of our study was to determine whether the vibriocidal immune responses to V. cholerae infection are equally accurate as markers of protection in all age groups. Methods Cholera patients and their household contacts, who are known to be at high risk of V. cholerae infection, were enrolled between 2001 and 2017 in Dhaka, Bangladesh. Baseline vibriocidal titers were measured at the time of enrollment of household contacts, and participants were followed prospectively for development of V. cholerae infection. Results We studied 50 contacts < 5 years old (“young children”), 228 contacts 5–16 years old (“older children”), and 548 contacts > 16 years old (“adults”). The baseline serum vibriocidal titer was higher in contacts who remained uninfected from all age groups than in contacts who developed cholera during the follow-up period (young children: P = 0.0092; older children: P = 0.0003, adults: P = 0.0012). Conclusion We found that higher vibriocidal antibody titers were associated with protection against V. cholerae infection across all three age categories. These findings may help increase our understanding of the protective immune response against V. cholerae infection and have importance for future vaccine development strategies. Acknowledgments: This research was supported by Massachusetts General Hospital training grant T32AI007061. Disclosures All authors: No reported disclosures.

Background Antibiotic use in livestock farming is thought to be a major contributor to the spread of antimicrobial resistance (AMR) genes in humans. However, quantitative data in this in this field are rare. To address this gap in the literature, we examined the prevalence of clinically important AMR genes before and after the introduction of chicken farming among women in rural Uganda. Methods We recruited a subset of women participating in a waitlist-randomized controlled trial of small-scale hybrid chicken farming in rural Uganda. Tetracycline is routinely administered to chicks during brooding. Stool samples before and one year after chicken introduction were obtained from six women randomized to the control arm, from five women randomized to the intervention arm, and from chickens. Microbial DNA was extracted from chicken and human stool and screened for 87 AMR genes using validated qPCR arrays (Qiagen). Results The median age was 35 years. At baseline, 10 of the women reported animal contact, most commonly goats (n = 8), free ranging village chickens (n = 7), cats (n = 4), and dogs (n = 4). During baseline testing of the women’s stool, we detected 18 genes conferring AMR to aminoglycosides, fluoroquinolones, macrolides, lincosamides, streptogramin B, Class A-C β-lactamases and tetracycline efflux pumps. Chickens harbored 23 AMR genes from the same classes as found in humans, and were also found to have vancomycin resistance genes (Van B and C) and Group D β-lactamases (OXA-58 and OXA-10). At one year, six new AMR genes emerged in controls, including one present in chickens; CTX-M-1, a Class A β-lactamase. In contrast, seven new AMR genes emerged in the intervention group, including four present in chickens: SHV, SHV(238G240E), (Class A β lactamases) and QnrS, QnrB-5 (fluoroquinolone resistance genes). Two AMR genes gained by both control and intervention groups were not present in chickens. Conclusion Women exposed to small-scale chicken farming acquired more AMR genes compared with unexposed participants. Chickens harbored many of the genes that emerged in humans. Introduction of antibiotic-treated animals may result in the transfer of AMR genes from animals to humans, even among humans exposed to a wide range of animals at baseline. Disclosures All authors: No reported disclosures.

Background Cholera vaccines are recommended for use in outbreaks to prevent infections and reduce severity of disease. Variable immune responses are observed after administration of killed, whole-cell cholera vaccines, and limited data suggest that the gut microbiome may be one factor influencing immune responses to vaccination. Methods. We used microbial DNA sequencing of stool and serum vibriocidal titers to examine the gut microbiome and immune responses to vaccination at day 0, 7, 17 and 44 in adult vaccine recipients in Dhaka, Bangladesh. Using flow cytometry-based bacterial cell sorting, we identified IgA-coated gut microbes in stool before and after vaccination in a subset of patients. Results. Vibriocidal titer magnitude and kinetics were used to classify participants. Within 17 days of vaccination, 86/89 (96%) adults developed a four-fold rise in vibriocidal titer. Gut microbial diversity was not significantly changed after vaccination. Rate of seroconversion (four-fold increase in vibriocidal titer by Day 3 after vaccination) was faster in participants with increased bacteria from the genus Prevotella (multivariate analysis using linear models, q value 0.04). The gut microbes of participants with higher peak vibriocidal titers was characterized by increased Prevotella (3% vs. 3.5), particularly the species Prevotella copri (P < 0.001, unpaired t-test, linear discriminant analysis score >3.5). Lipopolysaccharide from Prevotella species is known to increase vaccination-associated antigen-specific antibody titers in animal models. Additionally, IgA coating of gut microbes in stool increased after vaccination, from 8.9% IgA coated at baseline to a peak level of 19% during follow-up (Wilcoxon signed rank test, P < 0.01). Conclusion. Certain microbiome profiles are correlated with greater immune responses to cholera vaccination, and IgA coating of gut bacteria indicates which commensal species may be participating in the mucosal immune response. The potential for modulation of mucosal immune responses based on gut microbial species warrants further study. Disclosures All authors: No reported disclosures.

Background. Multiple Vibrio cholerae infections in the same household are common. The objective of this study was to examine the incidence of V. cholerae infection and associated clinical symptoms in household contacts of patients with cholera and to identify risk factors for development of severe dehydration in this cohort. Methods. Household contacts of hospitalized patients with cholera were observed with frequent clinical assessments and collection of serum and rectal swab samples for culture for a period of 21 days after presentation of the index case. Results. One-half (460 of 944) of all contacts reported diarrhea during the study period, and symptoms most frequently began 2 days after presentation of the index case. Antibiotics were used by 199 (43%) of 460 contacts with diarrhea. Results of rectal swab cultures for V. cholerae were positive for 202 (21%) of 944 contacts, and 148 (73%) infected contacts experienced diarrhea. Significant dehydration developed in 26 contacts; predictors of dehydration included vomiting, each additional day of diarrhea, and blood group O status. Conclusions. In urban Bangladesh, the burden of diarrheal illness among household contacts of patients with cholera is higher than was previously estimated, and prophylactic intervention is feasible, because the majority of symptomatic cases of V. cholerae infection in contacts begin soon after presentation of the index case. Re-evaluation of targeted chemoprophylaxis for household contacts of patients with cholera may be warranted.

Background. Ceftaroline fosamil is a cephalosporin approved for treating skin and soft tissue infections (SSTIs), including those caused by methicillin-resistant Staphylococcus aureus (MRSA), and community acquired pneumonia. Given its broad-spectrum activity, its use has expanded. We studied indications for use, adverse drug reactions (ADRs), clinical outcomes, and tolerability in patients with prior report of β-lactam allergy at two large academic health centers.

Introduction: Cephalotheca foveolata is a saprobic fungus recently described among clinical isolates. Case presentation: A 59-year-old man presented with fevers, upper back pain and shortness of breath after gastric tube removal following an oesophagectomy, oesophagojejunostomy and gastrostomy for management of oesophogeal adenocarcinoma. He had a suspected oesophageal leak and polymicrobial empyema. Blood cultures grew only C. foveolata. He improved clinically on empiric antifungal and broad-spectrum antibacterial therapy. Conclusion: We report a probable case of C. foveolata bloodstream infection. This is a newly recognized pathogen requiring molecular diagnostic methods to make an accurate diagnosis.

Disability after childhood diarrhea is an important burden on global productivity. Recent studies suggest that gut bacterial communities influence how humans recover from infectious diarrhea, but we still lack extensive data and mechanistic hypotheses for how these bacterial communities respond to diarrheal disease and its treatment. Here, we report that after Vibrio cholerae infection, the human gut microbiota undergoes an orderly and reproducible succession that features transient reversals in relative levels of enteric Bacteroides and Prevotella. Elements of this succession may be a common feature in microbiota recovery from acute secretory diarrhea, as we observed similar successional dynamics after enterotoxigenic Escherichia coli (ETEC) infection. Our metagenomic analyses suggest that multiple mechanisms drive microbial succession after cholera, including bacterial dispersal properties, changing enteric oxygen and carbohydrate levels, and phage dynamics. Thus, gut microbiota recovery after cholera may be predictable at the level of community structure but is driven by a complex set of temporally varying ecological processes. Our findings suggest opportunities for diagnostics and therapies targeting the gut microbiota in humans recovering from infectious diarrhea., IMPORTANCE Disability after diarrhea is a major burden on public health in the developing world. Gut bacteria may affect this recovery, but it remains incompletely understood how resident microbes in the digestive tract respond to diarrheal illness. Here, we observed an orderly and reproducible succession of gut bacterial groups after cholera in humans. Genomic analyses associated the succession with bacterial dispersal in food, an altered microbial environment, and changing phage levels. Our findings suggest that it may one day be feasible to manage resident bacterial populations in the gut after infectious diarrhea.

Validated skin testing is lacking for many drugs, including ceftaroline. The cross-reactivity between ceftaroline and other β-lactam antibiotics is unknown. We report a case of a pregnant patient with cystic fibrosis and multiple drug allergies who required ceftaroline for methicillin-resistant Staphylococcus aureus pneumonia and underwent an uncomplicated empiric desensitization procedure.

Although an uncommon pathogen causing clinical disease, Nocardia species are ubiquitous microorganisms found in the environment with over 50 species identified [1]. The majority of Nocardia infections occur as pulmonary infections following inhalation in immunocompromised hosts, including patients with HIV, chronic corticosteroids, and persons taking immunosuppression after transplantation [2]. Less commonly, immunocompetent hosts can develop a cutaneous form of Nocardia infection after a traumatic percutaneous inoculum [3]. Disseminated disease often involves the brain, lungs and skin [4]. Despite the low incidence of Nocardia infection in both groups, several studies have demonstrated the need for a high degree of clinical suspicion in at-risk patients, where microbiologic diagnosis is delayed by slow bacterial growth in culture. Suspect patients require aggressive treatment given the high mortality rate associated with these infections [5–7]. The genus Nocardia contains relatively few species that have been shown to be truly pathogenic in humans [8]. Nocardiosis is often misdiagnosed because of its rarity and non-specific clinical picture. Some of the more common species causing human disease include N. nova, N. brasiliensis, N. farcinia and N. aesteroides, the causative organism in this case [2]. These are most commonly found in sand, soil, dust, stagnant water and decaying vegetation [2]. Nocardia are aerobic partially acid-fast Gram positive bacteria which form thin branching rods and can be cultured on routine culture media such as blood agar or chocolate agar [8]. These bacteria are notoriously difficult to identify in culture as they require a minimum of 48–72 h to grow before colonies are evident, and in some cases can take as long as 14 days to appear. If the distinctive Gram positive filamentous, branching rods characteristic of Nocardia are not seen on Gram stain of a clinical sample, the culture may be discarded before Nocardia can be grown and identified. With a high degree of clinical suspicion and prolonged incubation of the culture, the organism is more often correctly identified [7]. In this report, we present a patient on long term corticosteroid treatment for an ANCA associated small vessel vasculitis that developed a necrotizing soft tissue infection of the upper extremity with cultures demonstrating Nocardia aesteroides. To our knowledge, there have been few reported cases of a Nocardia infection of the hand and none that has presented as a necrotizing soft tissue infection.

We commend the authors of “Weight Gain After Fecal Microbiota Transplantation” for describing this case of obesity after a fecal microbiota transplant (FMT). Fecal microbiota transplant is a highly effective therapy for recurrent Clostridium difficile infection (r-CDI), and the authors discuss possible unintended consequences in a patient after successful FMT, which could be due to alteration of the gut microbiota. Strengths of this study include (1) detailed information about the clinical course of the patient receiving FMT for r-CDI and (2) characteristics of the FMT donor, the patient’s daughter. This experience led to a change in FMT donor policy at the authors’ institution. The authors do not identify the mechanism for transmission of the obese phenotype, directly demonstrate a causal relationship, or study the patient’s microbiome over time. They do identify several factors that may have contributed to weight gain after FMT in the patient. These include, but are not limited to, resolution of CDI, antibiotic use, treatment for Helicobacter pylori infection, stress related to illness, older age, and genetic factors. There is no discussion of eating patterns or changes in eating patterns in these related and presumably cohabitating individuals. These possibilities aside, there is a growing body of data exploring the relationship between host body mass index (BMI), host metabolism, and the gut microbiota. The transmission of an obese phenotype after therapeutic FMT for r-CDI has not been reported previously, to our knowledge. There are differences in the gut microbial structure between lean and obese individuals [1]. A complex relationship exists between BMI and gut microbes, and this association cannot be reduced to a correlation between specific bacterial taxonomic groups and host BMI [2, 3]. Studies of the gut microbiome in monozygotic twins with discordant BMIs have advanced the knowledge of the gut microbe-host BMI interaction by demonstrating transmissibility and reversibility of the obese phenotype. In studies by Ridaura et al [4], germ-free mice receiving a stool lavage from an obese twin developed significantly greater adiposity than mice infused with the lean twin’s microbiota, and this effect was lessened when the 2 groups were cohoused (mice are normally coprophagic). The expression of microbial genes important for detoxification and stress responses were more prominent in germ-free mice that received the obese twin microbiome compared with the lean twin flora [4]. There is also new evidence that host genetics influence the structure of the gut microbiota, and in a study of more than 400 twin pairs, specific bacterial taxa were found to be more “heritable” than others [5]. It is possible and perhaps even likely that the weight gain in the case reported was influenced not only by microbial communities transmitted during FMT, but also by genetic factors common to the FMT donor and recipient. In controlled, randomized, and doubleblinded human studies described by Vrieze et al [6], transfer of a leanmicrobiota to individuals with metabolic syndrome was associated with improved insulin sensitivity when compared with controls (individuals with metabolic syndrome who received an infusion of their own stool). In this study, butyrate-producing bacterial strains (known to activate intestinal gluconeogenesis) were increased in both lean stool samples and lean individual’s intestinal biopsies [6]. Animal studies have shown that a relative paucity of butyrate-producing bacteria in the gut is associated with increased insulin resistance, and butyrate supplementation can reverse insulin resistance in diet-induced obesity in mice [7, 8]. A reduction in butyrate-producing bacteria can be achieved using antimicrobial agents directed at Gram-positive organisms, and by this mechanism, a loss of butyrate-producing organisms has been suggested as an explanation for the increase in BMI observed in patients treated with vancomycin for treatment of infective endocarditis [9, 10]. Bacterial products from the gut have been shown to enter the serum and affect distal organs. A recent murine study showed that manipulating gut flora in germ-free maternal mice affected the Received 22 December 2014; accepted 14 January 2015. Correspondencer: Ana A. Weil, MD, MPH, Infectious Diseases, Massachusetts General Hospital, 55 Fruit St., GRJ 520, Boston, MA 02114 (aweil@partners.org). Open Forum Infectious Diseases © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of thework, in anymedium, provided theoriginalwork is not altered or transformed in anyway, and that thework is properly cited. For commercial re-use, please contact journals.permissions@oup. com. DOI: 10.1093/ofid/ofv005

Vibriocidal antibody is a marker of recent exposure to Vibrio cholerae O1 infection. We examined vibriocidal titers for 1 year after an episode of severe cholera in patients in Dhaka, Bangladesh; 16 of 53 (30%) patients had a fourfold or greater increase in vibriocidal titer between 6 and 12 months after an episode of severe cholera, suggesting reexposure to the organism. Among patients with rises in titers during follow-up, the patients initially infected with serotype Ogawa had earlier rises in titer than the patients initially infected with serotype Inaba. These data and others suggest that an episode of severe cholera protects against symptomatic disease for several years, but reexposure to the organism occurs frequently in an endemic area, with immunological boosts beginning as early as 6 months after severe disease. Repeated exposures to V. cholerae in endemic areas may be a necessary component for long-lasting protection against severe disease.