Glycerol Stocks Research Articles

Interactions between the human milk oligosaccharide 2'-fucosyllactose and Bifidobacterium longum subspecies infantis in influencing systemic immune development and function in piglets.

The oligosaccharide 2'-fucosyllactose (2'-FL) is a predominant component of human milk, serving as a prebiotic for gut microbiota and influencing immune development in infants. Bifidobacterium longum subspecies infantis (B. infantis) is a commensal bacterium found in breastfed infants. Both 2'-FL and a specific strain of B. infantis, Bi-26™, are commercially available. This study investigates the potential synbiotic relationship between 2'-FL and Bi-26™ on immune development. Two-day-old piglets (n = 53) were randomized in a 2 × 2 design, receiving either a commercial milk replacer ad libitum without (CON) or with 1.0 g/L 2'-FL (FL). Piglets in each diet were further randomized to receive either glycerol stock alone or Bi-26™ (109 CFU) (BI and FLBI) orally once daily. On postnatal day (PND) 34/35, animals were euthanized, and blood was collected for serum cytokine analysis. Additionally, peripheral blood mononuclear cells (PBMCs) were isolated for ex vivo stimulation and flow cytometry analysis. Serum and ex vivo cytokines were analyzed using a multivariate model. All other outcomes were analyzed using a two-way ANOVA, considering prebiotic and probiotic fixed effects. The significance level was set at a p value <0.05, with trends reported for 0.05 < p < 0.1. Immune cell populations in PBMCs were unaffected by the experimental treatment. However, serum interleukin (IL)-1RA, IL-1β, IL-12, and IL-18 were all higher (p < 0.05) in the FL group than in the CON group. In isolated PBMCs, lipopolysaccharide (LPS) stimulation resulted in higher IL-1RA and a trend for higher IFN-γ secretion in the FL group vs. the CON group. 2'-FL stimulates a balanced cytokine profile in healthy piglets without changing immune cell populations. When immune cells are stimulated ex vivo with LPS, 2'-FL primes T-cells for a proinflammatory response, which is moderated by co-administration of Bi-26™.

Multi-Omics Analysis Unravels the Impact of Stool Sample Logistics on Metabolites and Microbial Composition.

Human health and the human microbiome are inevitably intertwined, increasing their relevance in clinical research. However, the collection, transportation and storage of faecal samples may introduce bias due to methodological differences, especially since postal shipping is a common practise in large-scale clinical cohort studies. Using four different Omics layer, we determined the structural (16S rRNA sequencing, cytometric microbiota profiling) and functional integrity (SCFAs, global metabolome) of the microbiota in relation to different easy-to-handle conditions. These conditions were storage at -20 °C, -20 °C as glycerol stock, 4 °C and room temperature with and without oxygen exposure for a maximum of one week. Storage time affected the microbiota on all Omics levels. However, the magnitude was donor-dependent, highlighting the need for purpose-optimized sample collection in clinical multi-donor studies. The effects of oxygen exposure were negligible for all analyses. At ambient temperature, SCFA and compositional profiles were stable for 24 h and 48 h, respectively, while at 4 °C, SCFA profiles were maintained for 48 h. The global metabolome was highly susceptible, already changing at 24 h in non-frozen conditions. Thus, faecal microbiota was best preserved on all levels when transported as a native sample frozen within 24 h, leading to the least biased outcomes in the analysis. We conclude that the immediate freezing of native stool samples for transportation to the lab is best suited for planned multi-Omics analyses that include metabolomics to extend standard sequencing approaches.

PSI-22 Evaluating colonization success of Bacteroidetes from different host origin in broiler chickens

Abstract Current broiler production practices that aim to maximize biosecurity have limited chick exposure to beneficial microbes. Previous research highlights a notable disparity in Bacteroidetes abundance and taxonomy between birds reared under intensive and extensive systems. Building upon this, we hypothesized that Bacteroidetes strains populating intensively raised birds may originate from human sources, potentially leading to varied colonization efficiencies. The objective of this study was to evaluate the colonization patterns of diverse Bacteroidetes strains within the chicken gastrointestinal tract. One day-old broiler birds (n = 63) were raised in individually ventilated isolators accommodating 3 birds per cage. The chicks were subjected to inoculation either via oral gavage with 150 µL of glycerol stock containing 1x104 colony forming units (CFU)/mL; or spray method (2 mL per cage) using chicken-derived, human-derived, and a combination of Bacteroidetes strains. Notably, birds administered via oral gavage displayed an increase in body weight gain by d 07 compared with their counterparts treated via spray and the control group (P &amp;lt; 0.01). Subsequent quantification of Bacteroidetes, Enterobacteriaceae, and total bacteria using qPCR revealed that oral gavage resulted in more successful Bacteroidetes colonization at d 14 (P &amp;lt; 0.01). Moreover, discernible variations were noted in total bacterial abundance across treatment cohorts. It can be concluded that the method of inoculation appears to influence the initial colonization patterns of Bacteroidetes in the chicken gut, or that inoculation in the environment requires a higher dose. Amplicon sequencing that is underway will reveal the success of the Bacteroidetes from different host origin and the most successful strains of chicken origin. Overall, enhanced growth coincided with more successful Bacteroidetes colonization, identifying them as potential targets for enhanced broiler performance. Further investigation is warranted to determine if Bacteroidetes from different sources demonstrate varying colonization efficiencies.

Developing stable, simplified, functional consortia from Brachypodium rhizosphere for microbial application in sustainable agriculture

The rhizosphere microbiome plays a crucial role in supporting plant productivity and ecosystem functioning by regulating nutrient cycling, soil integrity, and carbon storage. However, deciphering the intricate interplay between microbial relationships within the rhizosphere is challenging due to the overwhelming taxonomic and functional diversity. Here we present our systematic design framework built on microbial colocalization and microbial interaction, toward successful assembly of multiple rhizosphere-derived Reduced Complexity Consortia (RCC). We enriched co-localized microbes from Brachypodium roots grown in field soil with carbon substrates mimicking Brachypodium root exudates, generating 768 enrichments. By transferring the enrichments every 3 or 7 days for 10 generations, we developed both fast and slow-growing reduced complexity microbial communities. Most carbon substrates led to highly stable RCC just after a few transfers. 16S rRNA gene amplicon analysis revealed distinct community compositions based on inoculum and carbon source, with complex carbon enriching slow growing yet functionally important soil taxa like Acidobacteria and Verrucomicrobia. Network analysis showed that microbial consortia, whether differentiated by growth rate (fast vs. slow) or by succession (across generations), had significantly different network centralities. Besides, the keystone taxa identified within these networks belong to genera with plant growth-promoting traits, underscoring their critical function in shaping rhizospheric microbiome networks. Furthermore, tested consortia demonstrated high stability and reproducibility, assuring successful revival from glycerol stocks for long-term viability and use. Our study represents a significant step toward developing a framework for assembling rhizosphere consortia based on microbial colocalization and interaction, with future implications for sustainable agriculture and environmental management.

Preserving probiotic potency: A comparative study of storage methods for Lactobacillus fermentum MT308789 And Lactobacillus oris MT308790

Probiotics, comprising living microorganisms like Lactobacillus fermentum MT308789 and Lactobacillus oris MT308790, offer significant health benefits when consumed regularly. However, the efficacy of probiotics heavily relies on their viability and stability during storage. Various storage methods, including agar slant, glycerol stocks, lyophilization, and dry form, are utilized to preserve probiotic viability. In this study, we evaluated the efficacy of these storage methods in maintaining the viability of probiotic strains. Our findings demonstrate that lyophilization emerged as the most effective method, yielding the highest viabilities for both strains [77.51 %]. Glycerol stocks also showed promise for short-to-medium-term storage, while agar slants and dry form storage exhibited suboptimal viability. These results underscore the importance of selecting appropriate storage methods to ensure the delivery of viable and effective probiotic formulations to consumers.

An improved mouse model of sepsis based on intraperitoneal injections of the enriched culture of cecum slurry

AimSepsis is a life-threatening clinical syndrome comprising multiorgan dysfunctions caused by a disproportionate body immune response. There are several animal sepsis models which are based on cecum ligation, cecal puncture, and cecum slurry injection. The major limitation of all current sepsis models is the high variability owing to the variable degree of ligation, puncture and inconsistent microbial composition used for sepsis initiation. The primary objective of this work is to demonstrate the feasibility of a standardized method for sepsis development. Materials and methodsThe cecal slurry bacterial culture was developed and preserved in glycerol stocks. Antibiotics aztreonam and vancomycin were used for generating several defined, enriched cecal slurry bacterial cultures. Mice survival was assessed until 48 hrs post injection, and the tissue samples were collected after 10 hrs from sepsis initiation. Key findingsThe results indicate that increasing polymicrobial load resulted in lower survival rates and was associated with the higher number of infiltrating immune cells and necrosis. H&E (haematoxylin & eosin) staining & serum markers revealed that septic mice exhibited increased inflammation and significant damage to the liver and kidneys. The defined Gram-negative and Gram-positive specific cecal slurry bacterial cultures were developed and their efficiency in inducing sepsis was characterized. SignificanceEnriched cecal slurry bacterial cultures can be stored in glycerol stocks at −80 °C. This has an ethical advantage of avoiding unnecessary animal euthanasia for each experiment and provides a standardization capability of sepsis development.

Construction of a versatile in vitro cultivation screening platform using human oral microbiota.

We developed a simulation model of human oral microbiota using Bio Palette oral medium (BPOM) containing 0.02% glucose and lower bacterial nitrogen sources, derived from saliva and dental plaque. By decreasing the concentration of Gifu anaerobic medium (GAM) from 30 to 10 g L-1 , we observed increased ratios of target pathogenic genera, Porphyromonas and Fusobacterium from 0.5% and 1.7% to 1.2% and 3.5%, respectively, in the biofilm on hydroxyapatite (HA) discs. BPOM exhibited the higher ratios of Porphyromonas and Fusobacterium, and amplicon sequence variant number on HA, compared with GAM, modified GAM and basal medium mucin. Mixing glycerol stocks of BPOM culture solutions from four human subjects resulted in comparable ratios of these bacteria to the original saliva. In this simulation model, sitafloxacin showed higher inhibitory effects on P. gingivalis than minocycline hydrochloride at a low dosage of 0.1 μg mL-1 . Probiotics such as Streptococcus salivarius and Limosilactobacillus fermentum also showed significant decreases in Porphyromonas and Fusobacterium ratios on HA, respectively. Overall, the study suggests that BPOM with low carbon and nutrients could be a versatile platform for assessing the efficacy of antibiotics and live biotherapeutics in treating oral diseases caused by Porphyromonas and Fusobacterium.

Growth and Storage Study of Streptococcus agalactiae as a Potential Target in Whole-Cell Based ‘Systematic Evolution of Ligands by Exponential Enrichment (SELEX)’ Procedure

Streptococcus agalactiae is a pathobiont that causes severe invasive infections, especially in fishponds or freshwater aquatic organisms. Streptococcus outbreak in cultured fishponds causes a negative impact on the production of fishes, especially tilapias. Therefore, it is essential to develop a reliable method for on-site monitoring of S. agalactiae in fishponds. This study aims to investigate a local S. agalactiae strain TP540K as a potential target for DNA aptamer in a whole-cell based ‘systematic evolution of ligands by exponential enrichment (SELEX)’ procedure. Before this procedure, the bacterial growth profile of S. agalactiae strain TP540K was studied. Besides, the viability of S. agalactiae strain TP540K was compared between different storage solutions (glycerol, phosphate buffer saline, distilled water) and temperatures (-20, 4 and 27 °C). In this study, S. agalactiae strain TP540K entered the log phase at 1.5 h of incubation time, thus the cells were collected at 2 h of incubation time with an OD600 value of 0.436 to proceed to SELEX process. Moreover, glycerol stock (20%, -20 °C) was the best storage condition for preservation of S. agalactiae strain TP540K until Day 84 with a cell viability of 2.48 x 107 CFU/mL. With the findings obtained, S. agalactiae strain TP540K could be utilised as a target for future study in DNA aptamer development through whole-cell based SELEX procedure, which could be useful for future investigations to overcome Streptococcus outbreak in the Malaysian aquaculture industry.

A group B Streptococcus indexed transposon mutant library to accelerate genetic research on an important perinatal pathogen.

Group B Streptococcus (GBS) is a significant global cause of serious infections, most of which affect pregnant women, newborns, and infants. Studying GBS genetic mutant strains is a valuable approach for learning more about how these infections are caused and is a key step toward developing more effective preventative and treatment strategies. In this resource report, we describe a newly created library of defined GBS genetic mutants, containing over 1,900 genetic variants, each with a unique disruption to its chromosome. An indexed library of this scale is unprecedented in the GBS field; it includes strains with mutations in hundreds of genes whose potential functions in human disease remain unknown. We have made this resource freely available to the broader research community through deposition in a publicly funded bacterial maintenance and distribution repository.

Erratum: High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay.

Erratum: High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay.

High resolution ITS amplicon melting analysis as a tool to analyse microbial communities of household biofilms in ex-situ models

Biofilms are the most common growth types of microorganisms. These complex communities usually consist of different species and are embedded in an extracellular matrix containing polymers, proteins and DNA. This matrix offers protection against different (a)biotic environmental factors and generally increases resistances. Higher resistances against antibiotics are one of the main reasons why biofilms are often associated with healthcare settings. Nevertheless, they are also found in domestic settings, mostly in humid places with abundant nutrients like dishwashers or washing machines. Biofilms in these areas show individual compositions and are influenced for example by temperature, frequency of use or the age of the device.In this study, we introduce a model for the ex-situ cultivation of domestic biofilms from household appliances. Furthermore, we tested the ability of high resolution melting analysis (HRMA) as a tool for analysing these biofilms. Our goal was to maintain a high amount of complexity in the ex-situ biofilms that is characterized by the melting behavior of the contained DNA. Dishwasher and washing machine biofilms were sampled in private households and cultivated for 10 d. After DNA extraction, 16S rDNA was sequenced and melting behavior of the bacterial Internal Transcribed Spacer (ITS) region was analysed. Additionally, testing for independence of continuous new sampling, storage of cultivated biofilms in glycerol stocks and following recultivation of them was done up to three times.Our results show that a high level of complexity could be maintained in the ex-situ biofilms after 10 d of cultivation, although in general the bacterial diversity slightly decreased compared to the original biofilm in most cases. Recultivation of a similar biofilm from glycerol stocks was possible as well with some impact by various factors. Differences in the bacterial composition of biofilms could clearly made visible by HRMA although it was not possible to match peaks to a specific phylogenetic group. Still, HRMA proved to be a less costly and time consuming alternative to sequencing for the characterization of biofilms.

Isolation of Actinomycetes from Arachis hypogaea L. and Gossypium herbaceum L. for Screening Antibacterial and Antifungal Activities

The principal objective of the present study was to check the antimicrobial activity of Actinomycetes isolated from soil samples collected from the fields of Arachis hypogea L. and Gossipium herbaceum L. against different plant pathogenic strains. Various soil samples were isolated from fields located near the Junagadh district, Gujarat, India. Isolation was followed by a serial dilution process which was later plated on Actinomycete Isolation Agar (AIA) media. Potential colonies were subjected to screening, purification, and storage in glycerol stock. Morphological and Biochemical characterization of the isolates was performed. Isolated candidates were subjected to extraction for the production of the antimicrobial compound. The antimicrobial activity of the purified extract of isolates was tested. Total 30 actinomycete isolates were evaluated for antagonistic activity against pathogenic microorganisms.&#x0D; Isolates C-25, C-15, and G-1 showed the best results in the decreasing order of their potency against fungal pathogens, and C-5, C-25, C-14, and C-13 showed the best results in decreasing order of potency against bacterial pathogens. 3 isolates inhibited all 4 test fungi. 10 isolates inhibited 3 test fungi. 11 isolates inhibited 2 test fungi. 6 isolates did not inhibit any test fungi. 4 isolates show potent inhibition. 15 inhibited Macrophomina. C-10 showed a 1 cm inhibition zone &amp; G-1 showed a 0.8 cm zone of inhibition. 12 isolates gave 0.2-0.6 cm zone and 15 isolates gave negative results against Macrophomina. C-10 showed a very potent zone of inhibition of 0.7 cm. 9 isolates showed a 0.1-0.5 cm zone of inhibition. 20 isolates did not show inhibition against Fusarium. 1 isolate C-11(a) gave the 1cm potent zone of inhibition. 15 isolates gave the 0.7-0.2cm inhibition of the growth. 14 isolates gave negative results against Alternaria fungus. From these results, it was concluded that isolates had antibacterial and antifungal activities and could be used in the development of new antibiotics for pharmaceutical or agricultural purposes.

Selection of Affibody Affinity Proteins from Phagemid Libraries.

Herein, we describe a general protocol for the selection of target-binding affinity protein molecules from a phagemid-encoded library. The protocol is based on our experience with phage display selections of non-immunoglobulin affibody affinity proteins but can in principle be applied to perform biopanning experiments from any phage-displayed affinity protein library available in a similar phagemid vector. The procedure begins with an amplification of the library from frozen bacterial glycerol stocks via cultivation and helper phage superinfection, followed by a step-by-step instruction of target protein preparation, selection cycles, and post-selection analyses. The described procedures in this standard protocol are relatively conservative and rely on ordinary reagents and equipment available in most molecular biology laboratories.

Do-it-Yourself Mock Community Standard for Multi-Step Assessment of Microbiome Protocols.

Microbiomes provide critical functions that support animals, plants, and ecosystems. High-throughput sequencing (HTS) has become an essential tool for the cultivation-independent study of microbiomes found in diverse environments, but requires effective and meaningful controls. One such critical control is a mock microbial community, which is used as a positive control for nucleic acid extraction, marker gene amplification, and sequencing. While mock community standards can be purchased, they can be costly and often include only medically relevant microbial strains that are not expected to be major players in non-human microbiomes. As an alternative, it is possible to design and construct a do-it-yourself (DIY) mock community, which can then be used as a positive control that is specifically customized to the protocol needs of a particular study system. In this article, we describe protocols to select appropriate microbial strains for the construction of a mock community. We first describe the steps to verify the identity of community members via Sanger sequencing. Then, we provide guidance on assembling and storing the DIY mock community as viable whole cells. This includes steps to create standard growth curves referenced to plate counts for each member, so that the community members can be quantified and later compared in terms of their "expected versus returned" relative contributions after sequencing. We also describe appropriate methods for the cryostorage of the fully assembled mock community as viable whole cells, so that they can be used as a unit in a microbiome analysis, from the lysis and nucleic acid extraction steps onwards. Finally, we provide an example of returned data and interpretation of DIY mock community sequences, discussing how to assess possible contamination and identify protocol biases for particular members. Overall, DIY mock communities serve to determine success and possible bias in a cultivation-independent microbiome analysis. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Strain identification and verification using Sanger sequencing Basic Protocol 2: Creation of glycerol stocks of each mock community strain for long-term cryostorage Basic Protocol 3: Assessment of strain freezer viability without cryoprotectant Basic Protocol 4: Creation of standard curve to determine CFU/ml of a liquid culture as a function of optical density Basic Protocol 5: Full mock community assembly using community concentration calculations and standard curves.

Isolation, Screening, Characterization and Maintenance of Amylase Producing Bacteria from Soil

Abstract: Amylases are amongst most widely used enzymes in industries such as food, fermentation, starch processing, textile, and paper. In the present investigation, bacteria were isolated from soil samples of different areas of Padmashri Vikhe Patil College, Pravaranagar, Maharashtra, India screened for the production of amylase. Soil samples were serially diluted in normal saline and plated on sterile nutrient agar plates. The colonies obtained from higher dilutions were subjected to Gram staining, biochemical reactions, and starch hydrolysis test. Bacterial colony yielding positive starch hydrolysis test were subjected to Amylase activity test by DNSA method. Characteristic feature of the strain indicates that it belongs to the genus Bacillus and will be later used for further characterization. Maximum yield of amylase was obtained after 48hrs of incubation. The isolated amylase producing organism was maintain by using agar slant &amp; glycerol stock method.

Influence of 2'-Fucosyllactose and Bifidobacterium longum Subspecies infantis Supplementation on Cognitive and Structural Brain Development in Young Pigs.

Development of the gut-brain axis during early-life is an important contributor of brain structural and functional development. Human milk oligosaccharides and gut microbiota have potential beneficial effects on various aspects of development; however, the effects of 2′-fucosyllactose (2′-FL) and Bifidobacterium longum subsp. infantis Bi-26 (Bi-26) administration during infancy separately and combined are still not clear. Therefore, we investigated the effects of early administration of dietary 2′-FL and Bi-26 on brain structural and functional development in the young pig. From postnatal day (PND) 2–34 or 35, fifty-two intact male pigs were randomly assigned to treatment groups in a 2 × 2 factorial arrangement and provided ad libitum access to a nutritionally adequate milk replacer without or with 1.0 g of 2′-FL/L of reconstituted liquid. Pigs within each diet group were further stratified to receive a daily oral dose of glycerol stock without or with Bi-26 (109 CFU). Pigs were subjected to the novel object recognition (NOR) task from PND 27–31 to assess recognition memory and subsequently underwent magnetic resonance imaging procedures at PND 32 or 33 to assess brain macrostructure and microstructure. Pigs that received Bi-26 had smaller absolute brain volumes for 9 of 27 brain regions of interest, and smaller relative volumes for 2 regions associated with kinesthesia (P < 0.05). Synbiotic administration of 2′-FL and Bi-26 elicited interactive effects (P < 0.05) on several microstructural brain components, where dual supplementation negated the effects of each test article alone. Behavioral outcomes indicated that pigs did not express novelty preference, regardless of treatment group, demonstrating no effects of 2′-FL and Bi-26 on recognition memory when supplemented alone or in combination. Interactive effects (P < 0.05) were observed for the number of all object visits, latency to the first object visit, and number of familiar object visits. Pigs that did not receive Bi-26 supplementation exhibited less time interacting with the familiar object in total (P = 0.002) and on average (P = 0.005). In conclusion, supplementation of 2′-FL and/or Bi-26 elicited some alterations in object exploratory behaviors and macro/micro-structures of the brain, but changes in recognition memory were not observed. Specifically in brain microstructure, synbiotic administration of 2′-FL and Bi-26 appeared to negate effects observed when each dietary article was supplemented separately.

Improved live-cell PCR method for detection of organophosphates degrading opd genes and applications

Organophosphates are becoming an emerging pollutant due to their various applications, particularly as pesticides. In this study, an improved Colony (Live-cell) PCR method was developed for the detection of opd genes from bacteria encoding theorganophosphate hydrolase enzymes capable of degrading various organophosphates. The improved method does not require pre-heating or pre-lysis of bacterial cells as essential in the conventional colony PCR. The reaction volume was scaled down to 10µl by optimizing thePCR buffer and amplification conditions. The improved method was used for Gram positive and negative bacteria, glycerol stocks, liquid cultures, recombinant and mutant strains. Also, 16S rRNA gene was amplified from unknown environmental isolates and known E. coli strains. The amplified opd and 16S rRNA genes from the improved colony PCR method and by conventional PCR were sequenced, and similar results were obtained from both techniques. Thus, the improved method can be further explored in molecular biology or during biomarker studies. KEY POINTS: • Improved colony PCR method was developed for screening of opd genes from bacteria. • Method was validated for Gram positive/negative bacteria from solid as well as liquid media. • The improved method was rapid, efficient, and can be applied under various conditions.

Evaluation of 2'-Fucosyllactose and Bifidobacterium longum Subspecies infantis on Growth, Organ Weights, and Intestinal Development of Piglets.

Human milk is rich in oligosaccharides that influence intestinal development and serve as prebiotics for the infant gut microbiota. Probiotics and 2’-fucosyllactose (2’-FL) added individually to infant formula have been shown to influence infant development, but less is known about the effects of their synbiotic administration. Herein, the impact of formula supplementation with 2’-fucosyllactose (2’-FL) and Bifidobacterium longum subsp. infantis Bi-26 (Bi-26), or 2’-FL + Bi-26 on weight gain, organ weights, and intestinal development in piglets was investigated. Two-day-old piglets (n = 53) were randomized in a 2 × 2 design to be fed a commercial milk replacer ad libitum without (CON) or with 1.0 g/L 2’-FL. Piglets in each diet were further randomized to receive either glycerol stock alone or Bi-26 (109 CFU) orally once daily. Body weights and food intake were monitored from postnatal day (PND) 2 to 33/34. On PND 34/35, animals were euthanized and intestine, liver and brain weights were assessed. Intestinal samples were collected for morphological analyses and measurement of disaccharidase activity. Dry matter of cecum and colon contents and Bifidobacterium longum subsp. infantis abundance by RT-PCR were also measured. All diets were well tolerated, and formula intake did not differ among the treatment groups. Daily body weights were affected by 2’-FL, Bi-26, and day, but no interaction was observed. There was a trend (p = 0.075) for greater total body weight gain in CON versus all other groups. Jejunal and ascending colon histomorphology were unaffected by treatment; however, there were main effects of 2’-FL to increase (p = 0.040) and Bi-26 to decrease (p = 0.001) ileal crypt depth. The addition of 2’-FL and/or Bi-26 to milk replacer supported piglet growth with no detrimental effects on body and organ weights, or intestinal structure and function.

981. An Investigation into Possible Nosocomial Clusters and On-Treatment Resistance Patterns in Candidemia

BackgroundCandidemia has significant clinical implications, due to high rates of mortality and increasing resistance to antifungal drugs. Candida auris shows person-to-person transmission and survival on fomites. We aimed to determine if similar hospital transmission of Candida species, other than C. auris, is occurring. We analyzed candidemia infections for species, geographical, and temporal clusters, and clonality. We also aimed to study resistance patterns in individual patients on antifungal treatment. Here we present our current data from December 2019 – March 2021. MethodsPatients with candidemia were identified with the help of the clinical microbiology lab serving an urban health system. Isolates were stored prospectively as glycerol stocks at -80 C. Data were collected by retrospective chart review and described in terms of frequency distributions and percentages. Patient locations within the hospital setting were traced by Infection Prevention. A cluster was defined as the same species being isolated from ≥ 2 patients in the same unit within a 90-day period. Genomic DNA was isolated and whole genome sequencing was performed. Genomic data were visualized using the Yeast Mapping Analysis Pipeline. The Minimum Inhibitory Concentration (MIC) was determined using broth microdilution. Results105 patients were identified from six hospitals (Table 1). Seven clusters of candidemia were identified (Table 2). Genome sequencing supported that all isolates from an individual patient were genetically related. No clonality was observed for isolates from different patients, including those representing two of the seven clusters (Figure 1). Loss of heterozygosity was detected in isolates collected 15 minutes apart in the same patient, indicating distinct populations. MICs remained the same at 0.5 ug/ml over 6 days in Patients l and m (Table 1). For Patient n, MICs remained at 0.5-1 ug/ml Day 1-8, but increased to 4 ug/ml on Day 9. No copy number variation was observed in these isolates. Table 1. Species and resistance patterns of candidemia Table 2. Clusters of candidemia Figure 1. Yeast Mapping Analysis Pipeline for Clusters 3 and 4 – Candida albicans ConclusionWe have not found evidence of hospital transmission of candida isolates in our investigations to date. We plan to evaluate clonality in the remaining 5 clusters. Future single nucleotide polymorphism analysis will determine if acquisition of point mutations is causing the increased MIC in Patient n.Disclosures All Authors: No reported disclosures

Listeria monocytogenes sensitivity to antimicrobial treatments depends on cell origin

In this study we investigated how cell origin could affect the efficacy of an antimicrobial treatment (mild heating combined with terpenoids) in Listeria monocytogenes Scott A, considering cells from: 1. single colony, 2. glycerol stock, 3. cold adapted culture, and 4. fresh culture in stationary phase. After treatment, culturability on BHI medium and viability assessed by flow cytometry were evaluated. Our results showed that the cell origin significantly impacted viability and culturability of L. monocytogenes towards antimicrobial treatment. The mild heat treatment combined or not with terpenoids mainly affected culturability rather than viability, although the culturability of cells from single colony was less impacted. Therefore, to mimic the worst scenario, these latter were selected to contaminate Gorgonzola rind and roast beef slices and we evaluated the ability of L. monocytogenes cells to recover their culturability (on ALOA agar medium) and to growth on the food matrix stored at 4 °C for 7 days. Our results suggest that only Gorgonzola rind allowed a partial recovery of the culturability of cells previously heated in presence or not of terpens. In conclusion, we found a connection between the cell history and sensitivity toward an antimicrobial treatment, underlying the importance to standardize the experimental procedures (starting from the cells to be used in the assay) in the assessment of cell sensitivity to a specific treatment. Finally, our study clearly indicated that VBNC cells can resuscitate under favorable conditions on a food matrix, becoming a threat for consumer’s health.