Optimization of Small Peptide Feed from Milk Thistle Residue by Synergistic Fermentation of Multiple Strains and Proteases

In order to improve the utilization rate of the milk thistle residue, this study used the synergistic fermentation of multiple strains and proteases to increase the small peptide content of the fermented feed produced by the milk thistle residue. Taking the small peptide content of the milk thistle residue fermented feed as an indicator, the optimal fermentation process was obtained by single-factor optimization experiments and the response surface methodology. The optimal fermentation process was as follows: fermentation time of 7 days, inoculum size of 15%, inoculation ratio of aerobic strains: anaerobic strains = 1: 2, solid-state fermentation water content of 66%, fermentation temperature of 36℃, and amount of protease was 0.25% acid protease+0.25% bromelain. Under the above process, the small peptide content of the fermented feed from milk thistle residue was greatly improved to 57.86%. These results inferred that the added proteases were beneficial to the growth of fermentative microorganisms, the secretion of protease and the increase of the small peptide content.

Diversity of Human-Associated Bifidobacterial Prophage Sequences

Members of Bifidobacterium play an important role in the development of the immature gut and are associated with positive long-term health outcomes for their human host. It has previously been shown that intestinal bacteriophages are detected within hours of birth, and that induced prophages constitute a significant source of such gut phages. The gut phageome can be vertically transmitted from mother to newborn and is believed to exert considerable selective pressure on target prokaryotic hosts affecting abundance levels, microbiota composition, and host characteristics. The objective of the current study was to investigate prophage-like elements and predicted CRISPR-Cas viral immune systems present in publicly available, human-associated Bifidobacterium genomes. Analysis of 585 fully sequenced bifidobacterial genomes identified 480 prophage-like elements with an occurrence of 0.82 prophages per genome. Interestingly, we also detected the presence of very similar bifidobacterial prophages and corresponding CRISPR spacers across different strains and species, thus providing an initial exploration of the human-associated bifidobacterial phageome. Our analyses show that closely related and likely functional prophages are commonly present across four different species of human-associated Bifidobacterium. Further comparative analysis of the CRISPR-Cas spacer arrays against the predicted prophages provided evidence of historical interactions between prophages and different strains at an intra- and inter-species level. Clear evidence of CRISPR-Cas acquired immunity against infection by bifidobacterial prophages across several bifidobacterial strains and species was obtained. Notably, a spacer representing a putative major capsid head protein was found on different genomes representing multiple strains across B. adolescentis, B. breve, and B. bifidum, suggesting that this gene is a preferred target to provide bifidobacterial phage immunity.

Atorvastatin does not display an antimicrobial activity on its own nor potentiates the activity of other antibiotics against Acinetobacter baumannii ATCC17978 or A. baumannii AB030

With the current arsenal of antibiotics increasingly becoming ineffective against bacteria, there is an increasing interest in the possibility of using previously approved non-antibiotic drugs as antimicrobials. Statins have recently been investigated for their antimicrobial activity and their ability to potentially synergize with current treatment options. Atorvastatin had been shown previously to be the most promising candidate for effectivity against Acinetobacter baumannii ATCC17978. In this study, we tested atorvastatin for its activity against an extensively drug-resistant (XDR) strain A. baumannii AB030. However, our data show that atorvastatin has no effect A. baumannii AB030. Intriguingly, atorvastatin was also ineffective against our laboratory’s A. baumannii ATCC17978. This lack of atorvastatin activity against A. baumannii ATCC17978 cannot be attributed to RND efflux pumps as a strain deficient in the three most clinically relevant RND efflux systems in A. baumannii showed no change in susceptibility compared to its parent strain ATCC17978. Further, atorvastatin failed to potentiate the activity of tobramycin and ciprofloxacin. While it is not clear to us why atorvastatin is not active against A. baumannii ATCC17978 used in our study, our study shows that evaluation of compounds for their antibacterial activity should involve multiple strains to account for strain-to-strain variation.

Surface Properties and Adherence of Bradyrhizobium diazoefficiens to Glycine max Roots Are Altered When Grown in Soil Extracted Nutrients

Soybean roots are colonized and nodulated by multiple strains of compatible nitrogen-fixing rhizobia primarily belonging to the Genus Bradyrhizobium. Motility towards the root and attachment to root hairs are key determinants of competitive colonization and subsequent nodulation. Bacterial surface properties and motility are known to vary with chemical composition of the culture medium, and root adhesion and nodulation occur in a soil environment rather than laboratory medium. We asked whether the nodulation-promoting factors motility, surface hydrophobicity and surface adhesion of Bradyrhizobium are affected by growth in a soil nutrient environment. B. diazoefficiens USDA 110, 126, 3384, and B. elkanii USDA 26 were grown in mineral salt medium with peptone, yeast extract and arabinose (PSY), and in a soil extracted soluble organic matter (SESOM) medium. Surface hydrophobicity was determined by partitioning into hydrocarbon, motility by transition through soft agar, and surface-exposed saccharides by lectin profiling, followed by biofilm formation and soybean root adhesion capacity of populations. SESOM-grown populations were generally less motile and more hydrophobic. They bound fewer lectins than PSY-grown populations, indicating a simpler surface saccharide profile. SESOM populations of USDA 110 did not form detectable biofilm, but showed increased binding to soy roots. Our results indicate that growth in a soil environment impacts surface properties, motility, and subsequent soy root adhesion propensity. Hence, evaluation of Bradyrhizobium for nodulation efficiency should be performed using soil from the specific field where the soybeans are to be planted, rather than laboratory culture media.

Pangenome evolution in environmentally transmitted symbionts of deep-sea mussels is governed by vertical inheritance

Microbial pangenomes vary across species; their size and structure are determined by genetic diversity within the population and by gene loss and horizontal gene transfer (HGT). Many bacteria are associated with eukaryotic hosts where the host colonization dynamics may impact bacterial genome evolution. Host-associated lifestyle has been recognized as a barrier to HGT in parentally transmitted bacteria. However, pangenome evolution of environmentally acquired symbionts remains understudied, often due to limitations in symbiont cultivation. Using high-resolution metagenomics, here we study pangenome evolution of two co-occurring endosymbiont populations inhabiting individual Bathymodiolus brooksi mussels from a single cold seep. The symbionts, sulfur-oxidizing (SOX) and methane-oxidizing (MOX) gamma-proteobacteria, are environmentally acquired at an early developmental stage and individual mussels may harbor multiple strains of each species. We found differences in the accessory gene content of both symbionts across individual mussels, which are reflected by differences in symbiont strain composition. Compared to core genes, accessory genes are enriched in functions involved in genome integrity maintenance. We found no evidence for recent horizontal gene transfer between both symbionts. A comparison between the symbiont pangenomes revealed that the MOX population is less diverged and contains fewer accessory genes, supporting the view that the MOX association with B. brooksi is more recent than that of SOX. Our results show that the pangenomes of both symbionts evolved mainly by vertical inheritance. We conclude that association with individual hosts over their lifetime leads to genetically isolated symbiont subpopulations, constraining the frequency of HGT in the evolution of environmentally transmitted symbionts.

995. A Murine Model of Klebsiella pneumoniae Gastrointestinal Colonization with Parenteral Vancomycin Administration

Background As a leading cause of nosocomial infections, Klebsiella pneumoniae poses a significant threat due to its propensity to acquire resistance to many classes of antibiotics, including carbapenems. Gastrointestinal (GI) colonization by K. pneumoniae is a risk factor for subsequent infection as well as transmission to other patients. To study this crucial step in pathogenesis, we developed a mouse model of K. pneumoniae GI colonization using a clinically relevant parenteral antibiotic regimen. Methods To improve the clinical relevance of our model, we elected to use intraperitoneal injections of vancomycin, one of the most highly utilized antibiotics in the United States. Results To optimize dosage in C57bl/6 mice, we injected 20mg/kg, 350mg/kg, or vehicle (PBS) for three days prior to gastric gavage with 108 colony forming units (CFU) of a low-resistance strain of K. pneumoniae. The mice who received 350mg/kg (a mouse equivalent of a human dose of 1g/day calculated through the FDA guidelines for estimating safe dosing) shed about 107 CFU/g of feces at Day 7 while those receiving the lower dose or vehicle shed 104 CFU/g. Next, we compared 3- or 5-day pre-treatments with vancomycin prior to inoculation with an ST258 (epidemic carbapenem-resistant) strain. At Day 7 post-inoculation, mice who received 5 days shed 1010 CFU/g feces while those who received vancomycin for 3 days or vehicle for 5 days (PBS) shed 106 or 104 CFU/g feces respectively. Thus, we chose 5 days of 350mg/kg vancomycin injection as our regimen for inducing robust GI colonization in mice. Finally, we tested the durability of colonization by following fecal shedding in mice up to Day 60 post-inoculation with a second ST258 strain. Shedding during the first 7 days occurs at about 1010 CFU/g feces, and from day 14 to day 60 fecal loads are stable around 107 CFU/g feces. Results are comparable between male and female mice. Conclusion In conclusion, we have developed a mouse model of robust, prolonged GI colonization with multiple strains of K. pneumoniae using controlled dosing of a clinically relevant antibiotic. This model may be used to study a key step in K. pneumoniae pathogenesis and infection prevention in the future. Disclosures All Authors: No reported disclosures

743. Colonization with Extended-Spectrum Cephalosporin-Resistant Enterobacterales (ESCrE) in Healthcare and Community Settings in Botswana: An Antibiotic Resistance in Communities and Hospitals (ARCH) Study

Background Although ESCrE are a global challenge, data on ESCrE in low- and middle-income countries are limited. In particular, colonization data are critical for larger antibiotic resistance efforts. We characterized the colonization prevalence of ESCrE in various settings in Botswana. Methods This study was conducted in 3 hospitals and 6 clinics located in 3 districts in Botswana. In each hospital, we conducted surveillance of adult patients. Adult clinic patients were also randomly selected for participation. Finally, we enrolled community subjects by inviting each enrolled clinic subject to refer up to 3 adults. Each adult clinic or community subject was also allowed to refer their children. All subjects had rectal swabs obtained which were inoculated onto chromogenic media for preliminary identification of ESCrE. Final identification and susceptibility testing were performed using MALDI-TOF MS and VITEK-2, respectively. Genotyping was done for identification of extended-spectrum beta-lactamase (ESBL) genes. Results Enrollment occurred from 1/15/20-9/4/20 but paused from 4/2/20-5/21/20 due to a countrywide COVID lockdown. Of 5,088 subjects approached, 2,469 (49%) participated. Enrollment by subject type was: hospital – 469 (19%); clinic – 959 (39%); community adult – 477 (19%); and community child – 564 (23%). Of 2,469 subjects, the median (interquartile range) age was 32 years (19-44) and 1,783 (72%) were female. 759 (31%) subjects were colonized with at least one ESCrE; 130 subjects were colonized with multiple strains. E. coli (n=663) and K. pneumoniae (n=121) were most common. ESCrE colonization prevalence was 43% for hospital subjects, 31% for clinic subjects, 24% for adult community subjects, and 26% for child community subjects (p< 0.001)). ESCrE prevalence varied significantly across regions (Figure 1) and was significantly higher pre-lockdown vs post-lockdown (Figure 2). CTX-M was the most common ESBL gene (Figure 3). Figure 1. ESCrE Colonization - Study Sites Figure 2. ESCrE Colonization - Temporal Trends Figure 3. ESCrE Genotypic Analyses Conclusion ESCrE colonization was common in both healthcare and community settings in Botswana. Colonization prevalence varies by region and clinical setting and decreased following a countrywide lockdown. These findings provide important clues regarding potential drivers of ESCrE that might serve as targets for intervention. Disclosures Robert Gross, MD, MSCE, Pfizer (Other Financial or Material Support, Serve on DSMB for drug unrelated to HIV) Ebbing Lautenbach, MD, MPH, MSCE, Merck (Other Financial or Material Support, Member of Data and Safety Monitoring Board (DSMB))

733. Carbapenem-Resistant Enterobacterales (CRE) Colonization Prevalence in Botswana: an Antibiotic Resistance in Communities and Hospitals (ARCH) Study

Background Although CRE are a global threat, data in low- and middle-income countries are scarce. Colonization data are vital for informing antibiotic resistance strategies. We characterized the colonization prevalence of CRE in various settings in Botswana. Methods This study was conducted in 3 districts in Botswana (1 hospital and 2 clinics per district). Adult inpatients and clinic patients were randomly selected for enrollment. Community subjects were enrolled by inviting each enrolled clinic subject to refer up to 3 adults. Each adult clinic or community subject was also asked to refer their children. All subjects had rectal swabs obtained and inoculated on selective chromogenic media for preliminary identification of CRE. Final identification and susceptibility testing were performed using MALDI-TOF MS and VITEK-2, respectively. CRE underwent genotyping for carbapenemase genes. Results Subjects were enrolled from 1/15/20-9/4/20 with a pause from 4/2/20-5/21/20 due to a countrywide COVID lockdown. Of 5,088 subjects approached, 2,469 (49%) participated. Enrollment by subject type was: hospital – 469 (19%); clinic – 959 (39%); community adult – 477 (19%); and community child – 564 (23%). Of 2,469 subjects, the median (interquartile range) age was 32 years (19-44) and 1,783 (72%) were female. 42 (1.7%) subjects were colonized with at least one CRE; 10 subjects were colonized with multiple strains. E. coli (n=17), K. pneumoniae (n=20), and E. cloacae complex (n=11) were most common. CRE colonization prevalence was 6.8% for hospital subjects, 0.7% for clinic subjects, 0.2% for adult community subjects, and 0.5% for child community subjects (p< 0.001)). CRE prevalence varied across regions (Figure 1) and was significantly higher pre- vs post-lockdown (Figure 2). VIM and NDM were the most common carbapenemase genes (Figure 3). Figure 1. CRE Colonization - Study Sites Figure 2. CRE Colonization - Temporal Trends FIgure 3. CRE Genotypic Analyses Conclusion CRE colonization was significantly higher in hospital vs community settings in Botswana. CRE prevalence varied by region and decreased significantly following a countrywide lockdown. With CRE prevalence still modest, elucidating risk factors for CRE colonization holds promise in developing strategies to curb further emergence of CRE. Additional investigation of the CRE isolates without identified resistance genes is warranted. Disclosures Robert Gross, MD, MSCE, Pfizer (Other Financial or Material Support, Serve on DSMB for drug unrelated to HIV) Ebbing Lautenbach, MD, MPH, MSCE, Merck (Other Financial or Material Support, Member of Data and Safety Monitoring Board (DSMB))

Drug repositioning of Clopidogrel or Triamterene to inhibit influenza virus replication in vitro

Influenza viruses cause respiratory tract infections and substantial health concerns. Infection may result in mild to severe respiratory disease associated with morbidity and some mortality. Several anti-influenza drugs are available, but these agents target viral components and are susceptible to drug resistance. There is a need for new antiviral drug strategies that include repurposing of clinically approved drugs. Drugs that target cellular machinery necessary for influenza virus replication can provide a means for inhibiting influenza virus replication. We used RNA interference screening to identify key host cell genes required for influenza replication, and then FDA-approved drugs that could be repurposed for targeting host genes. We examined the effects of Clopidogrel and Triamterene to inhibit A/WSN/33 (EC50 5.84 uM and 31.48 uM, respectively), A/CA/04/09 (EC50 6.432 uM and 3.32 uM, respectively), and B/Yamagata/16/1988 (EC50 0.28 uM and 0.11 uM, respectively) replication. Clopidogrel and Triamterene provide a druggable approach to influenza treatment across multiple strains and subtypes.

A universal TLR7-nanoparticle adjuvant promotes broad immune responses against heterologous strains of Influenza and SARS-CoV-2

Fully effective vaccines for viruses such as Influenza and SARS-CoV-2 must elicit a diverse repertoire of antibodies against multiple drifted virus strains. However, how to achieve a diverse response has no general solution except to combine multiple strains, which risks diluting the response for all strains included. Here, we describe the synthesis of a universal, toll-like receptor 7 agonist (TLR7)-nanoparticle adjuvant, TLR7-NP, constructed from TLR7 agonist-initiated ring-opening polymerization of lactide and self-assembly with poly(ethylene glycol)-b-poly(lactic-co-glycolic acid). When mixed with Alum-adsorbed antigens, this TLR7-NP adjuvant elicited cross-reactive antibodies for both dominant and subdominant epitopes, as well as antigen-specific CD8+ T cell responses. TLR7-NPs adjuvanted influenza subunit vaccine successfully protected mice from heterologous viral challenge. TLR7-NPs also enhanced the antibody response to a SARS-CoV-2 subunit vaccine against multiple variants and revealed the mobilization of a virus-like response. We further demonstrate enhanced antigen-specific responses in human tonsil organoids with this novel adjuvant.