Detection of Leptospira DNA among Post-Mortem Specimens during Covid-19 Pandemic

Intro: Leptospirosis is an emerging zoonosis with a global health concern. In Malaysia, leptospirosis incidence remains significant, since its first gazettement as a compulsorily notifiable disease in 2010. However, the prevalence of this disease among local forensic cases is unknown. Therefore, the present study aimed to determine the frequency of human leptospirosis among post-mortem specimens. Method(s): Archived forensic specimens referred to the Institute for Medical Research (IMR), Malaysia between January 2020 and December 2021 were retrieved. DNA from the specimens were extracted using an automated MagNA Pure 96 instrument and subjected to in-house qPCR targeting LipL32 gene and 16S rRNA gene of the pathogenic group of Leptospira spp. Amplification of RNaseP gene was included as internal amplification control (IAC). Finding(s): A total of 408 forensic specimens from 365 patients were received during the study period. Majority of the specimens were blood (n = 195, 47.8%), followed by tissue (n = 136, 33.3%) and liver (n = 59, 14.5%). Of the tested specimens, 2.2% (n = 9) were positive for leptospiral DNA. These positive specimens belonged to 9 different patients, of which the vast majority were male (n = 8, 88.9%), with an average age of 37.5 years. Conclusion(s): Albeit low detection of leptospiral DNA among forensic specimens in Malaysia, this study highlighted that majority of the positive patients were males of productive age.Copyright © 2023

The Association Between Stool Shedding of SARS-Cov-2 Microbiome Diversity and Intestinal Inflammation

Introduction: The transmission of the etiologic virus of COVID-19 (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) is thought to occur mainly via respiratory droplets even though limited evidence has shown the virus can be found in feces and involve the gastrointestinal (GI) tract. The aim of this study was to assess if patients with COVID-19 present with fecal shedding of SARS-CoV-2, intestinal inflammation or changes in their microbiota. Method(s): This was a prospective cohort study that included outpatients that presented with symptoms of COVID-19 and were tested using a nasopharyngeal PCR test (NPT). Two cohorts were selected: one with a (1) NPT and a control group with a (-) NPT. Stool and a clinical data were collected at baseline and then, days 14, 28 and 42. SARS-CoV-2 viral loads were measured in stool using PCR and stool microbiome was analyzed using 16S rRNA gene sequencing (V3/V4 region). Fecal calprotectin levels were also measured on each sample and used as a surrogate marker of intestinal inflammation. Result(s): 101 patients were recruited (410 total samples). Of those, 55 had a (1) COVID-19 NPT. Most patients with a (1) COVID-19 NPT PCR had a detectable fecal viral load (71%). Among these patients, 23 (55%) had detectable viral stool loads only at baseline, 12 through day 14, 6 through day 28 and 1 through day 42. One patient had a (-) NPT but detectable SARS-CoV-2 in the baseline stool sample. Subjects with (1) NPT presented more commonly with myalgias (p=0.02), dysgeusia (p=0.019) and anosmia (p=0.03) when compared to those with (-) NPT but there were no differences in any other symptoms including GI manifestations.Within the group with a (1) NPT, those patient with detectable SARS-CoV-2 in the stool were younger but no differences were seen in demographic, symptoms, or fecal calprotectin levels (Table). There was no correlation between fecal SARS-CoV-2 loads and fecal calprotectin levels (rho: 0.007 [p=0.95]). Patients with a (1) NPT PCR had higher evenness when compared to those that tested (-) for a NPT PCR. However, no differences were seen in other alpha or beta diversity (Figures 1A and 1B, respectively). Conclusion(s): Even though intestinal viral shedding of SARS-CoV-2 in patients with COVID-19 is common, these patients do not present with evidence of inflammation of the GI tract, a significantly disrupted gut microbiome or a higher incidence of GI symptoms when compared to patients with respiratory symptoms and no COVID-19.

Impact of Gut Microbiota on Immunogenicity to Covid-19 Vaccines in Plwh

Background: Immune responses to SARS-CoV-2 vaccines in people living with HIV (PLWH) have been the focus of several recent studies. As the gut microbiome can influence vaccine immunogenicity, in this study we are the first to investigate whether the baseline gut microbiota can predict immune responses to the BNT162b2 SARS-CoV-2 vaccine in people living with HIV (PLWH) and healthy controls (HC). Method(s): Fecal samples were collected from PLWH (n=68) and HC (n=75) at baseline, prior to the first vaccine dose, to extract DNA for 16S rRNA sequencing. The individuals were part of the COVAXID Clinical trial, where humoral and cellular responses to SARS-CoV-2 vaccine were evaluated on day 35 after the first dose. Comprehensive bioinformatic tools were used for bacterial identification to further reveal the associations between gut microbiota and SARS-CoV-2 antibody, spike CD4+ T cell responses, and clinical parameters such as age, gender, CD4/CD8 ratio, and length of antiretroviral (ART) treatment. Result(s): At day 35 post vaccination, HC showed significantly higher spike IgG titers than PLWH (p=0.0001). Interestingly, both phylogenetic and alpha-diversity were negatively correlated with antibody titers, in the whole cohort and within groups. Similarly, individuals with low alpha-diversity had higher levels of spike specific CD4+T-cell responses. Agathobacter, Lactobacillus, Bacteroides, and Lachnospira were positively correlated with both antibody levels and spike-specific CD4+ T-cell responses while Methanobrevibacter, Marvinbryantia, Cloacibacillus, and Succinivibrio have a negative one. Within the PLWH group, the gut microbiota taxa associated with CD4+ counts, such as Lachnospira (p=0.002), Oscillibacter (p=0.019) and Flavonifractor (p=0.017), were found to be positively correlated with spike IgG levels. Additionally, the length of ART treatment and CD4/CD8 ratio displayed a positive association with bacterial diversity. Notably, different microbiome profiles and immune status in PLWH, affect their immune responses to vaccination. Conclusion(s): Our results show potential associations between gut microbiota diversity and spike IgG responses after COVID-19 vaccination. These findings were consistent in the whole cohort, albeit group differences between the microbiome compositions in PLWH and HC were observed. Based on our findings, we propose that microbiome modulation could optimize immunogenicity to SARS-Cov-2 vaccines.

Oral Microbiota in Children Who Started Antiretroviral Therapy at Young Ages

Background: Infancy is an important developmental period when the human microbiome is shaped. Given links between young age at antiretroviral treatment (ART) initiation and smaller persisting viral reservoirs, we hypothesized that earlier ART initiation may leave distinct microbial signatures in the oral cavity detectable in children living with HIV (CLWH). Method(s): Oral swab samples were collected from 477 CLWH and 123 children without HIV at two sites in Johannesburg, South Africa. CLWH had started ART < 2 years of age with 60% starting < 6 months of age. Most were wellcontrolled on ART at a median of 10 years of age when the swab was collected. Controls were age-matched and recruited from the same communities. Sequencing of the V4 amplicon of the 16S rRNA gene was done using established protocols. DADA2, decontam, and phyloseq were used for sequence inference, contaminant removal, and subsequent analyses. All p-values were adjusted for multiple testing using Benjamini-Hochberg false discovery rate method. Statistical analyses were performed with R. Result(s): CLWH had lower alpha diversity than uninfected children (Shannon index p< 0.0001). Genus-level abundances of Granulicatella, Streptococcus and Gemella were greater and Neisseria and Haemophilus were less abundant among CLWH compared to uninfected children. Associations were strongest among boys. There was no evidence of attenuation of associations with earlier ART initiation. In fact, decreased bacterial diversity and differences in taxa abundances in CLWH versus controls were consistent regardless of whether ART was started before or after 6 months of age. Shifts in genus-level taxa abundances relative to uninfected controls were most marked in children on regimens containing lopinavir/ritonavir;with few shifts seen if on regimens containing efavirenz. Conclusion(s): A distinct profile of less diverse oral bacterial taxa was observed in school-age CLWH on ART versus uninfected age-matched children suggesting persisting interference of HIV and its treatments on microbiota in the mouth. Any effects of earlier ART initiation were not detectable at this age. Studies of treated adults with HIV have observed similar shifts in taxa abundances. Oral microbiota have been linked to salivary cytokine levels with associations between Granulicatella and IL-8 and Neisseria and IL-6. Declines in Neisseria abundances in oral samples have been associated with more severe outcomes in influenza and COVID-19.

Advances in molecular diagnostic tests for community-acquired pneumonia

The use of a timely and appropriate antibiotic therapy, which requires early and accurate microorganisms' detection in pneumonia. Currently, the identification of microorganisms in pneumonia is limited by the low sensitivity and long response time of standard culture-based diagnostic tools. For this reason, treatment in pneumonia is empirical. An inadequate empirical treatment is related to poor outcomes in patients with pneumonia. The microbiological diagnosis is key to improve the outcomes in patient with pneumonia. Over the past years there was a significant advance in the molecular diagnosis of infectious diseases including pneumonia. Also the impact of the COVID-19 pandemic has impacted the development and application of these new molecular techniques. This review summarizes the advances in molecular diagnosis of community-acquired pneumonia.Copyright © 2022 EDIZIONI MINERVA MEDICA.

Airway microbiota profiles associated with total lung capacity after severe COVID-19

Background: SARS-CoV-2 infection induces disturbed airway microbiota during the acute phase of infection that may contribute to persistence of long-term pulmonary sequelae. To date it is unclear if the presence of a disrupted microbiota following severe disease is linked to long-term pulmonary function impairment. This one-year follow-up study investigated the association between airway microbiota and lung function after severe COVID-19. Method(s): In the Swiss COVID Lung study (NCT04581135), we conducted 16S rRNA sequencing on upper respiratory tract specimen obtained by oropharyngeal swabs 3 to 12 months after hospitalisation from 72 subjects (total samples n = 169) with severe COVID-19. Subjects underwent 1 - 3 follow-up visits during which lung function testing was performed to investigate correlation with the richness and composition of airway microbiota. Result(s): Total lung capacity (TLC) was negatively correlated with bacterial richness (p = 0.0081). Recovered COVID-19 subjects with ongoing respiratory impairment (TLC < 80%) showed low phylum heterogeneity with a majority of the dominant taxa being Bacteroidetes (64% of the 50 most abundant taxa in the group). In contrast, the phylum with the largest number of dominant taxa in subjects with TLC >= 80% was Firmicutes (48% of the 50 most abundant taxa in the group). Conclusion(s): Patients with impaired total lung capacity between 3 and 12 months after severe COVID-19 have a distinct oropharyngeal microbiota from those with restored total lung capacity. Future studies need to assess the contribution of microbiota to lung function impairment after severe COVID-19, as airway microbiota analysis may assist monitoring of sequelae and recovery.

A CASE OF EGGERTHELLA BACTEREMIA IN A PATIENT WITH COVID-19

SESSION TITLE: COVID-19 Co-Infections SESSION TYPE: Rapid Fire Case Reports PRESENTED ON: 10/19/2022 12:45 pm - 1:45 pm INTRODUCTION: We present a case of Eggerthella bacteremia in a patient with COVID-19. CASE PRESENTATION: A 69-year-old woman presented to the emergency room with chief complaint of cough, dyspnea, and malaise. After testing positive with a home COVID-19 test three days earlier, she continued to have worsening respiratory status and was brought in via ambulance. She was found to be tachycardic and hypoxic, requiring high-flow oxygen to maintain saturation in the emergency department. Chest X-ray showed bilateral patchy opacities consistent with multifocal COVID-19 pneumonia, and she was admitted to the intensive care unit for acute hypoxic respiratory failure. COVID-19 drug therapy was initiated, including baricitinib, remdesivir and decadron. Shortly after hospitalization, she began to endorse worsening abdominal pain. Physical exam elicited tenderness to palpation of her right lower quadrant. Abdominal CT scan showed distal ileum fluid collection concerning for possible bowel perforation. She underwent exploratory laparotomy which confirmed perforation, and a small bowel resection with anastomosis was performed. Blood cultures were positive for gram-positive bacilli, which were further identified as Eggerthella species. She required mechanical ventilation for worsening respiratory function post-surgery but remained unresponsive on the ventilator. The patient was administered vancomycin but continued to decline and eventually expired. DISCUSSION: Eggerthella is an anaerobic, gram-positive bacilli present in the gut microflora. Eggerthella infection has most often been reported in intra-abdominal infections. However, cases of bacteremia infection remain sparse. Most infections have been associated with other gastrointestinal processes including Crohn's disease, ulcerative colitis, appendicitis, and diverticulitis abscesses. Our case involved a patient with no significant gastrointestinal history admitted for COVID-19 pneumonia infection on baricitinib complicated by bowel perforation and bacteremia. Bowel perforation is a known risk factor of baricitinib use, and these risks should be discussed with the patient before beginning therapy. Overall mortality for Eggerthella species infection remains high, with some estimates as high as 31%. Much remains unknown about the impact on gut microbiome by SARS-CoV-2, however, early research suggests a higher rate of fungal co-infection in patients with COVID-19. As the literature on COVID-19 expands, more and more unusual pathogens such as Eggerthella may be found to contribute to the morbidity and mortality of patients being treated for COVID-19. CONCLUSIONS: Unusual pathogens such as Eggerthella may complicate a patient's hospital course while undergoing treatment for COVID-19. Reference #1: Alejandra Ugarte-Torres, Mark R Gillrie, Thomas P Griener, Deirdre L Church, Eggerthella lenta Bloodstream Infections Are Associated With Increased Mortality Following Empiric Piperacillin-Tazobactam (TZP) Monotherapy: A Population-based Cohort Study, Clinical Infectious Diseases, Volume 67, Issue 2, 15 July 2018. Reference #2: Gardiner BJ, Tai AY, Kotsanas D, et al. Clinical and microbiological characteristics of Eggerthella lenta bacteremia. J Clin Microbiol. 2015. Reference #3: Lau SK, Woo PC, Fung AM, Chan K-M, Woo GK, Yuen K-Y. Anaerobic, non-sporulating, gram-positive bacilli bacteraemia characterized by 16s rrna gene sequencing. Journal of medical microbiology. 2004. DISCLOSURES: No relevant relationships by Kristin Davis No relevant relationships by Charles Peng

COVID-19 VACCINATION RESPONSE IN IMMUNOSUPPRESSED PATIENTS WITH IBD IS ASSOCIATED WITH ALTERED GUT MICROBIOTA FUNCTION

Introduction Patients with inflammatory bowel disease (IBD) treated with anti-TNF therapy exhibit attenuated humoral immune responses to vaccination against SARS-CoV-2. The gut microbiota and its functional metabolic output, which are perturbed in IBD, play an important role in shaping host immune responses. We explored whether the gut microbiota and metabolome could explain variation in anti-SARS-CoV-2 vaccination responses in immunosuppressed IBD patients. Methods Faecal and serum samples were prospectively collected from patients with IBD established on infliximab therapy (for >12 weeks) who were undergoing vaccination against SARS-CoV-2. The Roche Elecsys Anti-SARS-CoV-2 spike (S) and nucleocapsid (N) immunoassays were used to measure antibody responses following two doses of either ChAdOx1 nCoV-19 or BNT162b2 vaccine. Seroconversion was defined by a cut-off anti-S concentration of 15 U/ml, which correlated with 20% viral neutralization;anti-S antibody concentration of < 380 U/ml was indicative of poor response to vaccination. Patients with serological evidence of prior SARS-CoV-2 infection were excluded from the analysis. Faecal calprotectin measurement, 16S rRNA gene amplicon sequencing, nuclear magnetic resonance (NMR) spectroscopy and bile acid profiling with ultra-performance liquid chromatography mass spectrometry (UPLC-MS) were performed on faecal samples. Results Forty-five infliximab-treated patients were recruited (median age 40 [range 19-67];32 Crohn's disease, 13 ulcerative colitis;28 with concomitant immunomodulator therapy;six with prior infection). 14 patients (35%) had seroconverted after one dose of vaccine and 37 (95%) seroconverted after two doses. 18 patients (46%) had a poor response after two doses of vaccine. There was no association between faecal calprotectin and vaccine response (p=0.41). No differences between satisfactory and poor vaccine responders were noted in alpha or beta diversity of the gut microbiota. The faecal metabolome of satisfactory responders was enriched in the microbial metabolite trimethylamine (q=0.03). Trends were noted linking the short chain fatty acid butyrate with satisfactory response (P=0.01) and succinate with poor response (P=0.06). No significant differences in primary or secondary bile acids were found to associate with vaccine response. The butyrate-producing genus Roseburia was positively correlated with faecal butyrate abundance (q=0.03). Conclusions Our data suggest an association between gut microbiota function and variable serological response to vaccination against SARS-CoV-2 in immunocompromised patients. Microbial metabolites including trimethylamine and butyrate may be important in mitigating anti-TNF-induced attenuation of the immune response.

UPPER RESPIRATORY TRACT BACTERIAL INFECTION FOR POST COVID-19 PATIENTS

This study was conducted in the College of Medicine, Wasit University Cooperation with the Al Zahraa Teaching Hospital, Al Kut Hospital laboratory in Wasit, Al-Karama hospital and private clinics of internal from the period of November 2020 to April 2021. It has been carried out on 150 samples of nasal and throat swabs from post COVID-19 patients who suffered from nasal and throat infection from both sex (male &female). The infections were in age group between (4-88) years. The results of throat swabs showed that 84(56%) were infected with bacteria and 66(44%) non-infected and the results of nasal swabs showed that 67(44.66%) were infected with bacteria and 83(55.33%) non-infected. The results of culture appeared that from 150 throat swab sample found that 66(44%) samples were no growth and 84(56%) were infected with bacteria. The results were Pseudomonas aeruginosa 12/150(8%), E. coli 9/150(6%), Enterobacter spp. 2/150(1.33), Pseudomonas spp. 2/ 150(1.33%), Klebsiella spp. 2/150(1.33%), Staphylococcus spp. 4/150(2.66%), Staphylococcus aureus 4/150(2.66%), Streptococcus viridans 43/150(28.66%) and mix of Staphylococcus spp. and Streptococcus viridans 6/150(4%). Out of 150 nasal swab sample found that 83/150(55.33%) sample were no growth and 67/150(44.67%) were infected with bacteria. The result were Pseudomonas aeruginosa 7/150(4.66%), E.coli 3/150(2%), Enterobacter spp. 2/150(1.33), Pseudomonas spp. 5/ 150(3.33%), Klebsiella spp. 6/150(4%), Staphylococcus spp. 12/150(8%), Staphylococcus aureus 3/150(2%), Streptococcus viridans 24/150(16%) and mix of Staphylococcus spp. and Streptococcus viridans 3/150(5.33%) and mix of E. coli and Pseudomonas spp. 2/150(1.33%). Antimicrobial sensitivity for Pseudomonas aeruginosa showed sensitivity to Amikacin (100%), levofloxacin (90%), Meropenem (90%), Cefipime (70%), Imipenem (60%), Aztreonam (30%), Chloramphenicol (5%), and don’t show sensitive to Tetracycline, Pipracillin, Ampicillin, Trimethoprim-Sulphamethoxazole and Clarithromycin. To facilitate species identification, used molecular methods (PCR analysis) by 16s rRNA primers gene for more predominant bacteria isolates (Pseudomonas aeruginosa) isolates studied were detected by 16S rRNA gene and there virulence factors based on multiplex polymerase chain reaction technique amplifying five virulence factors primer for Pseudomonas aeruginosa (aprA, filC, toxA, pilA, pslA). In this study, we concluded that the production of virulence factors genes in Pseudomonas aeruginosa is important to human infection especially (ToxA) gene and the PCR technique was very specific and fast method in detection virulence factor genes in Pseudomonas aeruginosa.

COVID-19: DIARRHEA, INFLAMMATION AND INTESTINAL MICROBIOTA

Introduction: Increased inflammatory cytokines has been observed in COVID-19 patients and there is evidence showing an alteration in gut-microbiota composition. SARS-CoV-2 can cause gastrointestinal symptoms, such as diarrhea. Evidence of an altered gut-microbiota composition and cytokines levels in COVID-19 diarrhea patients is lacking. Objectives: To compare serum cytokine levels and gut microbiota between COVID-19 diarrhea (D-COVID- 19) and non-diarrhea (NonD-COVID-19) patients and non- COVID-19 controls (HC). Material and methods: We included 143 hospitalized COVID-19 patients (positive quantitative reverse transcription PCR) in a single University Hospital, and 53 ambulatory HC (negative rapid serological test) were included. Blood and stool samples were collected at hospital admission in COVID-19 patients and at the time of HC recruitment. 27- pro and anti-inflammatory cytokines (Bio-Plex Pro™, Bio- Rad) were measured. Gut microbiota composition and diversity profiles were characterized by sequencing the 16S rRNA gene V3-V4 region amplified using DNA extracted from stool samples. Bioinformatics analysis was performed with QIIME2 software. First, we compare cytokine levels between COVID- 19 and HC and then COVID-19 with and without diarrhea. All comparisons were adjusted for age, sex, and BMI with linear regression. Results: The mean age in COVID-19 patients was 54 +/- 15 years (F=50%) and 52 +/- 8 (F=62%) for HC. Diarrhea was present in 19 (13.29%) of COVID-19 patients. COVID-19 patients had significative higher levels of: IL- 1ra, IL-2, IL-6, IL-7, IL-8, IL-13, IP-10 and PDGF-bb. Significant lower values of: IL-9, FGF -basic, MIP-1β, TNF-α were observed in D-COVID-19 compared to NonD-COVID-19. COVID-19 patients had a significant reduction of bacterial species (p=0.0001), and diversity and complexity of the bacterial community (Shannon's index) (p=0.0001) compared to the HC. There was no difference between D-COVID-19 and NonD-COVID-19. There were also changes in the composition of the microbiota associated with COVID-19. At the phylum level, COVID-19 patients showed a significant decrease in Actinobacteria and Firmicutes, and an increase in Bacteroidetes. At species level, an increase of 4 species of the genus Bacteroides was observed in COVID-19 patients. 31 very diverse bacterial species were found, all decreased in D-COVID-19. Conclusions: An alteration in serum cytokine levels was observed between COVID-19 and HC. D-COVID-19 had a decrease in some proinflammatory cytokines. A significant decrease in richness and species diversity of gutmicrobiota was observed in COVID-19 patients compared to HC, but no significant differences were observed between D-COVID-19 and NonD-COVID-19. However, in D-COVID- 19, a decrease in some bacterial species was observed.(Table Presented)(Figure Presented)

FAECAL MICROBIOTA TRANSPLANT IN ULCERATIVE COLITIS (FMTUC) – A RANDOMISED CLINICAL TRIAL FEASIBILITY STUDY

Background Dysbiosis of the gut microbiota may be responsible for the pathogenesis of ulcerative colitis (UC). Restoration of gut microbiota diversity by means of faecal microbiota transplantation (FMT) is of increasing interest as a therapeutic option in the management of UC. The aims of this phase II feasibility study are to estimate the magnitude of treatment response to FMT in treatment-naïve patients with newly diagnosed UC, evaluate donor and patient recruitment rates and determine optimal study conditions for phase III study (ISRCTN 58082603). Methods Treatment-naïve patients with histologically confirmed UC below the sigmoid were recruited. Subjects were randomised to single FMT enema, five daily enemas and control group. All groups received antibiotic for 10 days and bowel preparation 48 hours before the interventions. They were followed up for 12 weeks with quality of life (QOL) scores (IBDex, CUCQ-32) and 16S RNA study on faecal samples. Endoscopic (Mayo score) and histological assessments were performed at the baseline and week 12. The primary endpoints were endoscopic remission of UC and rate of persistent microbial engraftment at 12 weeks. Secondary endpoints included QoL and mucosal cytokine profiling with IL-10. Clinical remission was defined as Mayo score ≤ 2 with an endoscopic Mayo score of 0. Results Eighteen UC patients were recruited between July 2016 and February 2020 until the COVID-19 pandemic, of those five achieved clinical remission. One subject from the control group withdrew at week 4 due to worsening symptoms. 72% improved Mayo and QOL scores, and 44% avoided medical treatment. Clinical remission was more observed among subjects with lower baseline QoL and mild-moderate disease, although this did not reach statistical significance (P=0.173). No correlation between FMT dose, frequency and clinical remission were observed. The 16S evaluation of the faecal samples demonstrated successful engraftment of FMT and showed a similar faecal microbiota profile amongst the intervention groups, which was markedly different from the control group. Coprococcus was found to be much more abundant amongst subjects who responded to the FMT intervention. This study also suggested an inverse correlation between IL-10 and the severity of UC. Conclusions FMT intervention protocols were well adhered and 94% completion rate, though the recruitment period was much longer than the original plan due to some unforeseen interruptions. Yet, this feasibility study demonstrated potential for employing this method for a larger multicentre RCT to further evaluate FMT dose and frequency effects. The correlation between IL-10 and IL-10 producing microorganisms should be sought in the future study.

GUT MICROBIOME CHANGES ASSOCIATED WITH HIGHER AND LONGER-LASTING ANTIBODY TITERS FOLLOWING COVID-19 VACCINATION

Background: COVID-19 associated gut microbiome dysbiosis has been strongly linked to more severe disease and has most recently been shown to persist long after symptomatic recovery. While studies have correlated the depletion of gut commensals, like Faecalibacterium and Bifidobacterium, to disease severity, little is known about specific microbes that may be protective against disease, especially in the context of vaccination against SARS-CoV-2. We aimed to characterize changes in the gut microbiota following COVID-19 vaccination and associate them with antibody titers against SARS-CoV-2. Methods: We obtained paired stool samples from a cohort of 8 patients, the first sample taken within 10 days before the beginning of their COVID-19 mRNA vaccine series and the second taken within 10 days after their second vaccine. 16s rRNA gene sequencing and principal coordinate analysis were performed. In parallel, blood samples were also collected at 1, 3, 6, and 12 months to enumerate serum IgG antibody titers. Patients were stratified into 2 groups—medium and high—based on IgG titers following vaccination, wherein the `high' response group maintained significantly higher titers beyond 6 months follow-up. We used linear discriminant analysis effect size (LefSe) to estimate which microbes significantly differed at baseline between the 2 response groups. Results: The gut microbiome composition differed before and after vaccination for all patients, with medium responders showing significant differences by both Bray-Curtis dissimilarity (p = 0.04, pairwise PERMANOVA) and unweighted UniFrac (p = 0.03, pairwise PERMANOVA) beta diversity metrics (Figure 1), while differences within high responders were non-significant. The most abundant families present before vaccination in all subjects included Lachnospiraceae, Bacteroidaceae, Ruminococcaceae, and Enterobacteriaceae. Following vaccination, a stark contraction in the relative abundance of the family Bacteroidaceae occurred in all subjects, and in the majority of cases was accompanied by a concomitant increase in the abundance of Lachnospiraceae. The relative abundances of Ruminococcaceae, Bifidobacteriaceae, and Streptococcaceae were also increased in the majority of post-vaccination samples. Post-vaccination samples were also increased in community evenness and community richness for both response groups. LefSe analysis indicated that the orders Bifidobacteriales and Lactobacillales were significantly associated with high responders (Figure 2). Conclusion: Altogether, these findings demonstrate an association between the gut microbiota and COVID-19 immunity and highlight a potential link between specific taxa and the strength of humoral responses following vaccination. (Figure Presented) (Figure Presented)

POOR RESPONSE TO ANTI-SARS-COV-2 VACCINATION IN IMMUNOSUPPRESSED INFLAMMATORY BOWEL DISEASE PATIENTS IS ASSOCIATED WITH ALTERED GUT MICROBIOTA FUNCTION

Introduction: Patients with inflammatory bowel disease (IBD) treated with anti-TNF therapy exhibit attenuated humoral immune responses to vaccination against SARS-CoV-2. The gut microbiota and its functional metabolic output, which are perturbed in IBD, play an important role in shaping host immune responses. We explored whether the gut microbiota and metabolome could explain variation in anti-SARS-CoV-2 vaccination responses in immunosuppressed IBD patients. Methods: Faecal and serum samples were prospectively collected from patients with IBD established on infliximab therapy (for >12 weeks) who were undergoing vaccination against SARS-CoV-2. The Roche Elecsys Anti-SARS-CoV-2 spike (S) and nucleocapsid (N) immunoassays were used to measure antibody responses following two doses of either ChAdOx1 nCoV-19 or BNT162b2 vaccine. Seroconversion was defined by a cut-off anti-S concentration of 15 U/ml, which correlated with 20% viral neutralization;anti-S antibody concentration of < 380 U/ml was indicative of poor response to vaccination. Patients with serological evidence of prior SARS-CoV-2 infection were excluded from the analysis. Faecal calprotectin measurement, 16S rRNA gene amplicon sequencing, nuclear magnetic resonance (NMR) spectroscopy and bile acid profiling with ultra-performance liquid chromatography mass spectrometry (UPLC-MS) were performed on faecal samples. Results: Forty-five infliximab-treated patients were recruited (median age 40 [range 19-67];32 Crohn's disease, 13 ulcerative colitis;28 with concomitant immunomodulator therapy;six with prior infection). 14 patients (35%) had seroconverted after one dose of vaccine and 37 (95%) seroconverted after two doses. 18 patients (46%) had a poor response after two doses of vaccine. There was no association between faecal calprotectin and vaccine response (p=0.41). No differences between satisfactory and poor vaccine responders were noted in alpha or beta diversity of the gut microbiota. The faecal metabolome of satisfactory responders was enriched in the microbial metabolite trimethylamine (q=0.03). Trends were noted linking the short chain fatty acid butyrate with satisfactory response (P=0.01) and succinate with poor response (P=0.06). No significant differences in primary or secondary bile acids were found to associate with vaccine response. The butyrate-producing genus Roseburia was positively correlated with faecal butyrate abundance (q=0.03). Conclusion: Our data suggest an association between gut microbiota function and variable serological response to vaccination against SARS-CoV-2 in immunocompromised patients. Microbial metabolites including trimethylamine and butyrate may be important in mitigating anti-TNF-induced attenuation of the immune response.

The Surgical Mask Microbiome Among Children with Asthma

Rationale: Exposure to respiratory pathogens, aeroallergens, and air pollution can lead to asthma exacerbations. The SarsCoV2 (COVID-19) pandemic led to widespread public health mandates including mask-wearing. We hypothesize that mask-wearing sequesters respiratory pathogens, leading to observed reduction in asthma exacerbations. The goal of this study was to characterize the bacterial microbiome from surgical masks in a cohort of school children with and without asthma. By identifying what is on both inside and outside the masks, we will be able to build a catalogue as a baseline for future analyses. Methods: We performed a cross-sectional study of children (4-18 years) attending an inner-city public school district. Students wore a surgical mask for a minimum of one school day. Parents completed a questionnaire about their child's demographics, respiratory history, and level of asthma impairment. To establish the protocol, we piloted the extraction and sequencing procedures among a sample of used masks from a hospital clinical personnel. DNA was extracted using a commercial DNA extraction kit on separated mask layers: inner, middle, and outer. 16S rRNA gene sequencing was then performed and then mapped against the most recent Greengene 16S rRNA gene database.Results: Recruitment and mask wearing occurred during an 8-week period (May 2021-July 2021). 34 students (18 with asthma;16 without asthma) from four schools were enrolled and completed the study. 74% of participants were in grades K-4, mean age was 8.4 years, and 53% identified as Hispanic/Puerto Rican. 59% of participants wore the mask one school day. 44% reported an asthma-related ED visit in their lifetime, while only 16% reported an ED visit in the past 12-months;53% of participants reported asthma symptoms with upper respiratory infections, however 77% reported zero respiratory infections in the past 12-months. In the masks worn by medical staff, bacterial genera including Staphyloccus, Haemophilus, Lawsonella, Streptococcus as well as Actinomyces, were identified similarly on inner and outer layers in the masks worn by clinicians. (Figure 1).Conclusions: We have demonstrated that recruiting and enrolling students from a medium-sized, inner-city public school district and obtaining facial mask samples is feasible. We demonstrate that self-reported rates of asthmarelated ED visits and respiratory infections differed pre-pandemic as compared to during. In addition, identifying the microbiome from surgical masks is possible. Bacteria genera identified were similar to known human nasal, oral and skin microbiomes. Current work is now in process characterizing and comparing the mask microbiomes among students with and without asthma.

ALTERED GUT MICROBIOTA and RESPIRATORY DYSFUNCTION 3 MONTHS after SEVERE COVID-19

Background: Although COVID-19 is primarily a respiratory infection, mounting evidence suggests that the GI tract is involved in the disease, with gut barrier dysfunction and gut microbiota alterations being related to disease severity. Whether these alterations persist and associate with long-term respiratory dysfunction is unknown. Methods: From the NOR-Solidarity trial (n=181), plasma was collected during hospital admission and after three months, and analyzed for markers of gut barrier dysfunction and inflammation. At the three-month follow-up, pulmonary function was assessed by measuring diffusing capacity of the lungs for carbon monoxide (DLCO), and rectal swabs for gut microbiota analyses were collected (n= 97) and analysed by sequencing of the 16S rRNA gene. Results: Gut microbiota diversity was reduced in COVID-19 patients with respiratory dysfunction, defined as DLCO below lower limit of normal three months after hospitalization. These patients also had an altered global gut microbiota composition (Fig. 1), with reduced abundance of Erysipelotrichaceae UCG-003 and increased abundance of Flavonifractor and Veillonella, the latter potentially being linked to fibrosis. During hospitalization, increased plasma levels of lipopolysaccharide-binding protein (LBP) were strongly associated with respiratory failure, defined as pO2/fiO2-(P/F-ratio)<26.6 kPa. LBP levels remained elevated during and after hospitalization, and were associated with low-grade inflammation and respiratory dysfunction after three months. Figure 1 legend: Gut microbial composition in patients with respiratory dysfunction at the three-month follow-up (DLCO<="" div=""> Conclusion: Respiratory dysfunction after COVID-19 is associated with reduced biodiversity and gut microbiota alterations, along with persistently elevated LBP levels. Our results point to a potential gut-lung axis that should be further investigated in relation to long-term pulmonary dysfunction and long COVID.

Prenatal Stress and the Microbiome in the Time of COVID- A Prospective Cohort Study

Background: Given the emerging importance of the role of the gut microbiota-brain-axis in mediating prenatal stress-induced neurodevelopmental outcomes, a prospective cohort study was conducted. The COVID-19 Pandemic occurred halfway through study recruitment (n=35). The study aims to a) evaluate perceived stress across gestation, b) determine whether maternal microbiome composition changes with stress, and c) discern the influence of the COVID-19 pandemic on maternal stress, psychometric scores, and alterations in the microbiome. Methods: This longitudinal study design includes five time points across pregnancy and the post-partum period, at which biological samples were collected and psychometrics administered. Samples include maternal rectal and vaginal swabs. Psychometrics include measures of perceived stress, anxiety, depression, sleep, diet, and childhood adversity. Study participants identify as 62.9% White and 31.4% Black or African American. Finally, PacBio full-length 16S rRNA sequencing using SMRT Cell technology is used to identify the maternal rectal and vaginal microbial communities. Results: Participants delivering during the pandemic reporting greater perceived stress (p≤0.05). Of note, there were no significant differences in anxiety or depressive symptoms across gestation in the pre-pandemic participants as compared to participants during the pandemic. During the second trimester, increased depression associated with increased rectal alpha diversity, and increased perceived stress was associated with increased levels of Prevotella, Sneathia, and Gardnerella in the rectal samples. In contrast, participants with increased depressive symptoms during the third trimester had reduced vaginal alpha diversity measures at delivery. Conclusions: Findings suggests maternal perceived stress and depressive symptoms are associated with alterations in maternal microbiota Keywords: Gut Microbiome, Prenatal Maternal Stress, Gut-Brain Axis

Phenotypic and Genomic Analysis of Novel, Fastidious, Gram-negative Bacilli Isolated from Clinical Wound Specimens

Background. Animal bites are considered the thirteenth leading cause of nonfatal ED visits. Epidemiology studies have shown a rise in dog bites during the COVID-19 pandemic in the U.S. In Oct. 2020, we received a facultatively anaerobic, non-hemolytic Gram-negative rod (OL1) from a dog bite wound for identification. 16S rRNA gene sequencing showed OL1 was 95.9% identical to Ottowia pentelensis in the family Comamonadaceae. Our historical sequence database revealed 8 additional isolates (OL2-OL9) from hand wounds/abscesses (including 3 dog bites) since 2012 that had > 99.8% identity with OL1. Most other Ottowia sp. have been isolated from industrial and food sources, with no reports from patient samples. As these clinical isolates likely represent a novel Ottowia species, we aimed to characterize them using both phenotypic and genomic approaches. Methods. The OL isolates were tested in API 20 NE panels (8 conventional and 12 assimilation tests) for 4 d. Paired-end genomic DNA libraries (Nextera DNA Flex Library Prep, Illumina) were sequenced as 150 nt reads by Illumina NovaSeq. De novo assembly, annotation, functional prediction, and phylogenetic analyses were performed with Geneious, PATRIC, and web-prediction databases. Strain comparison was done with StrainTypeMer. Results. All 9 OL isolates were negative for indole, urea, arginine, esculin, PNPG, glucose fermentation and carbohydrate assimilation tests. Potassium gluconate assimilation and gelatin hydrolysis were positive for 5 and 4 isolates, respectively. StrainTypeMer showed the isolates from different patients were not closely related, but 2 from the same patient were indistinguishable. The estimated genome size was ~3.1 Mbp, with 66.1% G/C, and ~3523 coding genes. Potential virulence factors (BrkB and MviM), multidrug efflux systems (MdtABC-TolC and Bcr/CflA), and 1-2 intact prophages were identified. Genomic phylogenetic analysis with RAxML showed the OL isolates clustered separately from all known Ottowia spp. Conclusion. These OL isolates are fastidious, Gram-negative bacilli from clinical wound specimens, and are associated with dog bites. Genomic and 16S rRNA gene sequence analysis suggests these isolates constitute a novel species within the family Comamonadaceae.

Faecal Microbiota Transplant in Ulcerative Colitis (FMTUC) - A randomised clinical trial feasibility study

Background: Dysbiosis of the gut microbiota may be responsible for the pathogenesis of ulcerative colitis (UC). Restoration of gut microbiota diversity by means of faecal microbiota transplantation (FMT) is of increasing interest as a therapeutic option in the management of UC. The aims of this phase II feasibility study are to estimate the magnitude of treatment response to FMT in treatment-naïve patients with newly diagnosed UC, evaluate donor and patient recruitment rates and determine optimal study conditions for phase III study (ISRCTN 58082603). Methods: Treatment-naïve patients with histologically confirmed UC below the sigmoid were recruited. Subjects were randomised to three arms;single FMT enema, five daily enemas and control. All groups received antibiotic for 10 days and bowel preparation 48 hours before the interventions. They were followed up for 12 weeks with quality of life (QOL) scores (IBDex, CUCQ-32) and 16S RNA study on faecal samples. Endoscopic (Mayo score) and histological assessments were performed at the baseline and week 12. The primary endpoints were endoscopic remission of UC and rate of persistent microbial engraftment at 12 weeks. Secondary endpoints included QoL and mucosal cytokine profiling with IL-10. Clinical remission was defined as Mayo score ≤ 2 with an endoscopic Mayo score of 0. Results: Eighteen UC patients were recruited between July 2016 and February 2020 until the COVID-19 pandemic, of those five achieved Clinical remission. One subject from the control group withdrew at week 4 due to worsening symptoms. 72% improved Mayo and QOL scores, and 44% avoided medical treatment. Clinical remission was more observed among subjects with lower baseline QoL and mildmoderate disease, although this did not reach statistical significance (P=0.173). No correlation between FMT dose, frequency and clinical remission were observed. The 16S evaluation of the faecal samples demonstrated successful engraftment of FMT and showed a similar faecal microbiota profile amongst the intervention groups, which was markedly different from the control group. Coprococcus was found to be much more abundant amongst subjects who responded to the FMT intervention. This study also suggested an inverse correlation between IL-10 and the severity of UC. Conclusion: FMT intervention protocols were well adhered and achieved 94% completion rate, though the recruitment period was much longer than the original plan due to unforeseen interruptions. Yet, this feasibility study demonstrated potential for employing this method for a larger multicentre RCT to further evaluate FMT dose and frequency effects. The correlation between IL-10 and IL-10 producing microorganisms should be sought in the future study.

The Gut Microbiome Of Covid-19 Recovered Patients Returns To Uninfected Status In A Minority-Dominated United States Cohort

Introduction: Intestinal Microbiota Influences Both Susceptibility And Severity Of Bacterial And Viral-Induced Pathogenicity, Including Respiratory Diseases. In This Study, We Investigated The Relationship Between Intestinal Microbiota And Sars-Cov-2-Mediated Pathogenicity In The United States, Majority African American Cohort. Hypothesis: Intestinal Microbiota Is Modulated By Sars-Cov-2 Infection And Is Related To Symptom Severity And Recovery From The Disease. Methods: We Conducted A Single-Institution Study, Prospectively Collecting Fecal Samples From 50 Sars-Cov-2 Infected Patients Within 3 Days Of Icu Admission And 9 Sars-Cov-2 Recovered Patients Upon Testing Negative For The Virus. Feces Of 34 Uninfected Subjects At The Hospital With Unrelated Respiratory Medical Conditions Were Used As Controls. Total Fecal Rna/Dna Was Extracted And Microbiota Composition Was Determined Using 16s Rrna Gene Sequencing Of The V1-V3 Region. The 16s Rdna Sequencing Reads Were Processed Using Dada2 To Generate Amplicon Sequence Variants (Asv). Rt-Pcr On Fecal Rna Using Two Sets Of Validated Primer/Probes Was Performed To Establish The Presence Or Absence Of Sars-Cov-2 Viral Rna. Results: The Fecal Microbial Composition Was Found To Be Significantly Different Between Sars-Cov-2 Patients And Controls (Permanova Fdr-P=0.004), Independent Of Treatments Such As Antibiotic Exposure. Peptoniphilus, Corynebacterium And Campylobacter Were Identified As The Three Most Significantly Enriched Genera In Covid Patients Compared To Controls. Actively Infected Patients Were Also Found To Have A Different Gut Microbiota Than Recovered Patients (Permanova Fdr-P=0.003), And The Most Enriched Genera In The Covid-19 Patients Was Campylobacter, With Agathobacter Being Enriched In The Recovered Patients. No Difference In Microbial Community Structure Between Recovered Patients And Uninfected Controls Was Observed (Permanova Fdr-P=0.93), With Phocea Being The Top Genus Associated With Patients Who Recovered From Covid-19. Furthermore, No Difference In Alpha Diversity Between The Three Groups Was Noticed. More Importantly, 24 Of The 50 Covid-19 Patients (48%) Tested Positive Via Rt-Qpcr For Fecal Sars-Cov-2 Rna. A Significant Difference In Gut Microbial Composition Between Sars-Cov-2 Positive And Negative Samples Was Observed, With Klebsiella And Agathobacter Being Enriched In The Positive Cohort And Phocea In The Negative Cohort. No Significant Associations Between Microbiome Composition And Disease Severity Or Proton Pump Inhibitor Treatment Were Found. Conclusion: The Intestinal Microbiota Is Sensitive To The Presence Of Sars-Cov-2, With Increased Relative Abundance Of Genera (Campylobacter, Klebsiella) Associated With Gi Disease. Further Studies Are Needed To Investigate The Functional Impact Of Deleterious Bacterial Genera In Sars-Cov-2 On Gi Health.