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Sverremark-Ekström, EvaORCID iD iconorcid.org/0000-0001-6271-8681
Alternative names
Publications (10 of 74) Show all publications
Badolati, I., van Der Heiden, M., Brodin, D., Zuurveld, M., Szilágyi, S., Björkander, S. & Sverremark-Ekström, E. (2023). Staphylococcus aureus-derived factors promote human Th9 cell polarization and enhance a transcriptional program associated with allergic inflammation. European Journal of Immunology, 53(3), Article ID 2250083.
Open this publication in new window or tab >>Staphylococcus aureus-derived factors promote human Th9 cell polarization and enhance a transcriptional program associated with allergic inflammation
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2023 (English)In: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 53, no 3, article id 2250083Article in journal (Refereed) Published
Abstract [en]

T helper (Th) 9 cells, characterized by robust secretion of IL-9, have been increasingly associated with allergic diseases. However, whether and how Th9 cells are modulated by environmental stimuli remains poorly understood. In this study, we show that in vitro exposure of human PBMCs or isolated CD4 T-cells to Staphylococcus (S.) aureus-derived factors, including its toxins, potently enhances Th9 cell frequency and IL-9 secretion. Furthermore, as revealed by RNA sequencing analysis, S. aureus increases the expression of Th9-promoting factors at the transcriptional level, such as FOXO1, miR-155, and TNFRSF4. The addition of retinoic acid (RA) dampens the Th9 responses promoted by S. aureus and substantially changes the transcriptional program induced by this bacterium, while also altering the expression of genes associated with allergic inflammation. Together, our results demonstrate a strong influence of microbial and dietary factors on Th9 cell polarization, which may be important in the context of allergy development and treatment.

Keywords
allergy, environmental stimuli, retinoic acid, Staphylococcus aureus, T helper 9 cells
National Category
Immunology in the medical area Respiratory Medicine and Allergy
Identifiers
urn:nbn:se:su:diva-215303 (URN)10.1002/eji.202250083 (DOI)000914727500001 ()36550071 (PubMedID)2-s2.0-85146465220 (Scopus ID)
Available from: 2023-03-06 Created: 2023-03-06 Last updated: 2023-04-25Bibliographically approved
Mata Forsberg, M., Arasa, C., van Zwol, W., Uzunçayır, S., Schönbichler, A., Regenthal, P., . . . Sverremark-Ekström, E. (2022). Activation of human γδ T cells and NK cells by Staphylococcal enterotoxins requires both monocytes and conventional T cells. Journal of Leukocyte Biology, 111(3), 597-609
Open this publication in new window or tab >>Activation of human γδ T cells and NK cells by Staphylococcal enterotoxins requires both monocytes and conventional T cells
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2022 (English)In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673, Vol. 111, no 3, p. 597-609Article in journal (Refereed) Published
Abstract [en]

Staphylococcal enterotoxins (SE) pose a great threat to human health due to their ability to bypass antigen presentation and activate large amounts of conventional T cells resulting in a cytokine storm potentially leading to toxic shock syndrome. Unconventional T- and NK cells are also activated by SE but the mechanisms remain poorly understood. In this study, the authors aimed to explore the underlying mechanism behind SE-mediated activation of MAIT-, γδ T-, and NK cells in vitro. CBMC or PBMC were stimulated with the toxins SEA, SEH, and TSST-1, and cytokine and cytotoxic responses were analyzed with ELISA and flow cytometry. All toxins induced a broad range of cytokines, perforin and granzyme B, although SEH was not as potent as SEA and TSST-1. SE-induced IFN-γ expression in MAIT-, γδ T-, and NK cells was clearly reduced by neutralization of IL-12, while cytotoxic compounds were not affected at all. Kinetic assays showed that unconventional T cell and NK cell-responses are secondary to the response in conventional T cells. Furthermore, co-cultures of isolated cell populations revealed that the ability of SEA to activate γδ T- and NK cells was fully dependent on the presence of both monocytes and αβ T cells. Lastly, it was found that SE provoked a reduced and delayed cytokine response in infants, particularly within the unconventional T and NK cell populations. This study provides novel insights regarding the activation of unconventional T- and NK cells by SE, which contribute to understanding the vulnerability of young children towards Staphylococcus aureus infections.

Keywords
MAIT cell, SEA, SEH, TSST-1, unconventional T cells
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:su:diva-195723 (URN)10.1002/JLB.3A1020-630RR (DOI)000659908900001 ()34114693 (PubMedID)2-s2.0-85107638406 (Scopus ID)
Available from: 2021-08-25 Created: 2021-08-25 Last updated: 2022-03-28Bibliographically approved
Pang, Y., Ermann Lundberg, L., Mata Forsberg, M., Ahl, D., Bysell, H., Pallin, A., . . . Roos, S. (2022). Extracellular membrane vesicles from Limosilactobacillus reuteri strengthen the intestinal epithelial integrity, modulate cytokine responses and antagonize activation of TRPV1. Frontiers in Microbiology, 13, Article ID 1032202.
Open this publication in new window or tab >>Extracellular membrane vesicles from Limosilactobacillus reuteri strengthen the intestinal epithelial integrity, modulate cytokine responses and antagonize activation of TRPV1
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2022 (English)In: Frontiers in Microbiology, E-ISSN 1664-302X, Vol. 13, article id 1032202Article in journal (Refereed) Published
Abstract [en]

Bacterial extracellular membrane vesicles (MV) are potent mediators of microbe-host signals, and they are not only important in host-pathogen interactions but also for the interactions between mutualistic bacteria and their hosts. Studies of MV derived from probiotics could enhance the understanding of these universal signal entities, and here we have studied MV derived from Limosilactobacillus reuteri DSM 17938 and BG-R46. The production of MV increased with cultivation time and after oxygen stress. Mass spectrometry-based proteomics analyses revealed that the MV carried a large number of bacterial cell surface proteins, several predicted to be involved in host-bacteria interactions. A 5′-nucleotidase, which catalyze the conversion of AMP into the signal molecule adenosine, was one of these and analysis of enzymatic activity showed that L. reuteri BG-R46 derived MV exhibited the highest activity. We also detected the TLR2 activator lipoteichoic acid on the MV. In models for host interactions, we first observed that L. reuteri MV were internalized by Caco-2/HT29-MTX epithelial cells, and in a dose-dependent manner decreased the leakage caused by enterotoxigenic Escherichia coli by up to 65%. Furthermore, the MV upregulated IL-1β and IL-6 from peripheral blood mononuclear cells (PBMC), but also dampened IFN-γ and TNF-α responses in PBMC challenged with Staphylococcus aureus. Finally, we showed that MV from the L. reuteri strains have an antagonistic effect on the pain receptor transient receptor potential vanilloid 1 in a model with primary dorsal root ganglion cells from rats. In summary, we have shown that these mobile nanometer scale MV reproduce several biological effects of L. reuteri cells and that the production parameters and selection of strain have an impact on the activity of the MV. This could potentially provide key information for development of innovative and more efficient probiotic products.

Keywords
extracellular membrane vesicles, Limosilactobacillus reuteri, microbe-host interaction, immune response, epithelial cells integrity, TRPV1 pain receptor, proteomics, probiotics
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-213530 (URN)10.3389/fmicb.2022.1032202 (DOI)000892749300001 ()36466671 (PubMedID)2-s2.0-85143301130 (Scopus ID)
Available from: 2023-01-09 Created: 2023-01-09 Last updated: 2024-01-17Bibliographically approved
Lasaviciute, G., Barz, M., van Der Heiden, M., Arasa, C., Tariq, K., Quin, J., . . . Sverremark-Ekström, E. (2022). Gut commensal Limosilactobacillus reuteri induces atypical memory-like phenotype in human dendritic cells in vitro. Gut microbes, 14(1), Article ID 2045046.
Open this publication in new window or tab >>Gut commensal Limosilactobacillus reuteri induces atypical memory-like phenotype in human dendritic cells in vitro
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2022 (English)In: Gut microbes, ISSN 1949-0976, E-ISSN 1949-0984, Vol. 14, no 1, article id 2045046Article in journal (Refereed) Published
Abstract [en]

Memory-like responses in innate immune cells confer nonspecific protection against secondary exposures. A number of microbial agents have been found to induce enhanced or diminished recall responses in innate cells, however, studies investigating the ability of probiotic bacteria to trigger such effects are lacking. Here, we show that priming of human monocytes with a secretome from the gut probiotic bacterium Limosilactobacillus (L.) reuteri induces a mixed secondary response phenotype in monocyte-derived dendritic cells (mo-DCs), with a strong IL-6 and IL-1β response but low TNFα, IL-23 and IL-27 secretion. Instead, blood DC priming with L. reuteri-secretome resembles a tolerant state upon secondary exposure. A similar pattern was found in conventional and gut-like (retinoic acid exposed) DCs, although retinoic acid hampered TNFα and IL-6 production and enrichment of histone modifications in L. reuteri-secretome primed mo-DC cultures. Further, we show that the memory-like phenotype of mo-DCs, induced by priming stimuli, is important for subsequent T helper (Th) cell differentiation pathways and might determine the inflammatory nature of Th cells. We also show enhanced recall responses characterized by robust inflammatory cytokines and lactate production in the gut-like mo-DCs derived from β-glucan primed monocytes. Such responses were accompanied with enriched histone modifications at the promoter of genes associated with a trained phenotype in myeloid cells. Altogether, we demonstrate that a gut commensal-derived secretome prompts recall responses in human DCs which differ from that induced by classical training agents such as β-glucan. Our results could be beneficial for future therapeutic interventions where T cell responses are needed to be modulated.

Keywords
Limosilactobacillus reuteri, dendritic cells, T helper cells, innate immune memory, epigenetics
National Category
Biological Sciences Immunology in the medical area
Identifiers
urn:nbn:se:su:diva-203478 (URN)10.1080/19490976.2022.2045046 (DOI)000765991300001 ()35258405 (PubMedID)2-s2.0-85125981330 (Scopus ID)
Available from: 2022-04-05 Created: 2022-04-05 Last updated: 2022-05-09Bibliographically approved
van der Heiden, M., Nopp, A., Brandström, J., Carvalho-Queiroz, C., Nilsson, C. & Sverremark-Ekström, E. (2021). A pilot study towards the immunological effects of omalizumab treatment used to facilitate oral immunotherapy in peanut-allergic adolescents. Scandinavian Journal of Immunology, 93(4), Article ID e13005.
Open this publication in new window or tab >>A pilot study towards the immunological effects of omalizumab treatment used to facilitate oral immunotherapy in peanut-allergic adolescents
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2021 (English)In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 93, no 4, article id e13005Article in journal (Refereed) Published
Abstract [en]

Anti-IgE treatments, such as omalizumab, have shown promising effects in allergy treatment. Our previous work has shown that individualized omalizumab treatment (OT) allows a safe initiation and rapid up-dosing of peanut oral immunotherapy (OIT) in peanut-allergic adolescents. However, the broader immunological effects of this OT are incompletely understood. In this pilot study, we longitudinally followed the total B- and T-cell immunity during OT, using flow cytometry, ELISpot and ELISA. Peripheral blood mononuclear cells (PBMCs) and plasma were collected from participants (n = 17) at several timepoints during treatment, before starting OT (baseline), prior to starting OIT during OT (start OIT) and at maintenance dose OIT prior to OT reduction (maintenance). OT did not affect the total B-cell compartment over treatment time, but our results suggest an association between the OT dosage scheme and the B-cell compartment. Further, in vitro polyclonal T-cell activation at the different timepoints suggests a cytokine skewing towards the Th1 phenotype at the expense of Th2- and Th9-related cytokines during treatment. No differences in the frequencies or phenotype of regulatory T cells (Tregs) over treatment time were observed. Finally, plasma chemokine levels were stable over treatment time, but suggest elevated gut homing immune responses in treatment successes during the treatment as compared to treatment failures. The novel and explorative results of this pilot study help to improve our understanding on the immunological effects of OT used to facilitate OIT and provide guidance for future immunological investigation in large clinical trials.

Keywords
B cells, gut homing, OIT, Omalizumab, T cells, Tregs
National Category
Immunology
Identifiers
urn:nbn:se:su:diva-188996 (URN)10.1111/sji.13005 (DOI)000598661900001 ()33244763 (PubMedID)
Available from: 2021-01-17 Created: 2021-01-17 Last updated: 2022-02-25Bibliographically approved
Martí, M., Spreckels, J. E., Ranasinghe, P. D., Wejryd, E., Marchini, G., Sverremark-Ekström, E., . . . Abrahamsson, T. (2021). Effects of Lactobacillus reuteri supplementation on the gut microbiota in extremely preterm infants in a randomized placebo-controlled trial. Cell Reports Medicine, 2(3), Article ID 100206.
Open this publication in new window or tab >>Effects of Lactobacillus reuteri supplementation on the gut microbiota in extremely preterm infants in a randomized placebo-controlled trial
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2021 (English)In: Cell Reports Medicine, E-ISSN 2666-3791 , Vol. 2, no 3, article id 100206Article in journal (Refereed) Published
Abstract [en]

Extremely low birth weight (ELBW) infants often develop an altered gut microbiota composition, which is related to clinical complications, such as necrotizing enterocolitis and sepsis. Probiotic supplementation may reduce these complications, and modulation of the gut microbiome is a potential mechanism underlying the probiotic effectiveness. In a randomized, double-blind, placebo-controlled trial, we assessed the effect of Lactobacillus reuteri supplementation, from birth to post-menstrual week (PMW)36, on infant gut microbiota. We performed 16S amplicon sequencing in 558 stool samples from 132 ELBW preterm infants at 1 week, 2 weeks, 3 weeks, 4 weeks, PMW36, and 2 years. Probiotic supplementation results in increased bacterial diversity and increased L. reuteri abundance during the 1st month. At 1 week, probiotic supplementation also results in a lower abundance of Enterobacteriaceae and Staphylococcaceae. No effects were found at 2 years. In conclusion, probiotics may exert benefits by modulating the gut microbiota composition during the 1st month in ELBW infants.

National Category
Pediatrics
Identifiers
urn:nbn:se:su:diva-194533 (URN)10.1016/j.xcrm.2021.100206 (DOI)000642329200001 ()33763652 (PubMedID)
Available from: 2021-08-03 Created: 2021-08-03 Last updated: 2023-08-25Bibliographically approved
Rahman Qazi, K., Jensen, G. B., van Der Heiden, M., Björkander, S., Marchini, G., Jenmalm, M. C., . . . Sverremark-Ekström, E. (2021). Extreme prematurity and sepsis strongly influence frequencies and functional characteristics of circulating gamma delta T and natural killer cells. Clinical & Translational Immunology (CTI), 10(6), Article ID e1294.
Open this publication in new window or tab >>Extreme prematurity and sepsis strongly influence frequencies and functional characteristics of circulating gamma delta T and natural killer cells
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2021 (English)In: Clinical & Translational Immunology (CTI), E-ISSN 2050-0068, Vol. 10, no 6, article id e1294Article in journal (Refereed) Published
Abstract [en]

Objectives. Extremely low gestational age neonates with extremely low birthweight (ELGAN/ELBW) are highly susceptible to infection. This is linked to their relatively immature immune system which is not yet fully compatible with an extra-uterine environment. Here, we performed a longitudinal characterisation of unconventional T and natural killer (NK) cells in ELGAN/ELBW during their first months of life. Methods. Peripheral blood mononuclear cells were collected from 97 ELGAN/ELBW at 14 and 28 days of life and at a time point corresponding to postmenstrual week 36 + 0. gamma delta T-cell, NKT-cell, mucosa-associated invariant T-cell and NK cell frequencies and characteristics were analysed by flow cytometry. As control, cells from 14-day-old full-term (FT) infants were included. Results. Extreme prematurity had significant bearing on gamma delta T-cell and NK cell frequencies and characteristics. ELGAN/ELBW had significantly higher proportions of gamma delta T cells that were skewed towards effector and effector memory phenotypes, characteristics that were maintained throughout the study period. Expression of the gut homing receptor CCR9 was also more common in gamma delta T cells from ELGAN/ELBW. Conversely, NK cell frequencies were markedly lower and skewed towards a cytotoxic phenotype in the ELGAN/ELBW group at 14 days of age. Culture-proven sepsis with an onset during the first 14 days after birth further manifested these differences in the gamma delta T- and NK cell populations at 14 days of age. Conclusion. Prematurity strongly influences the levels of gamma delta T and NK cells, in particular in cases where sepsis debuts during the first 2 weeks of life.

Keywords
extreme preterm, gestational age, natural killer cells, neonatal immunity, sepsis, unconventional T cells
National Category
Basic Medicine Immunology in the medical area
Identifiers
urn:nbn:se:su:diva-196430 (URN)10.1002/cti2.1294 (DOI)000667244800009 ()34136218 (PubMedID)
Available from: 2021-09-08 Created: 2021-09-08 Last updated: 2022-12-09Bibliographically approved
Lasaviciute, G., Höbinger, A., Ujvari, D., Salamon, D., Yusuf, A., Sundin, M., . . . Saghafian-Hedengren, S. (2021). Human Bone Marrow Mesenchymal Stromal Cell-Derived CXCL12, IL-6 and GDF-15 and Their Capacity to Support IgG-Secreting Cells in Culture Are Divergently Affected by Doxorubicin. Hemato, 2(1), 154-166
Open this publication in new window or tab >>Human Bone Marrow Mesenchymal Stromal Cell-Derived CXCL12, IL-6 and GDF-15 and Their Capacity to Support IgG-Secreting Cells in Culture Are Divergently Affected by Doxorubicin
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2021 (English)In: Hemato, E-ISSN 2673-6357, Vol. 2, no 1, p. 154-166Article in journal (Refereed) Published
Abstract [en]

Various subsets of bone marrow mesenchymal stromal cells (BM MSCs), including fibroblasts, endothelial, fat and reticular cells, are implicated in the regulation of the hematopoietic microenvironment and the survival of long-lived antibody-secreting cells (ASCs). Nowadays it is widely acknowledged that vaccine-induced protective antibody levels are diminished in adults and children that are treated for hematological cancers. A reason behind this could be damage to the BM MSC niche leading to a diminished pool of ASCs. To this end, we asked whether cell cytotoxic treatment alters the capacity of human BM MSCs to support the survival of ASCs. To investigate how chemotherapy affects soluble factors related to the ASC niche, we profiled a large number of cytokines and chemokines from in vitro-expanded MSCs from healthy donors or children who were undergoing therapy for acute lymphoblastic leukemia (ALL), following exposure to a widely used anthracycline called doxorubicin (Doxo). In addition, we asked if the observed changes in the measured soluble factors after Doxo exposure impacted the ability of the BM niche to support humoral immunity by co-culturing Doxo-exposed BM MSCs with in vitro-differentiated ASCs from healthy blood donors, and selective neutralization of cytokines. Our in vitro results imply that Doxo-induced alterations in BM MSC-derived interleukin 6 (IL-6), CXCL12 and growth and differentiation factor 15 (GDF-15) are not sufficient to disintegrate the support of IgG-producing ASCs by the BM MSC niche, and that serological memory loss may arise during later stages of ALL therapy.

Keywords
human bone marrow mesenchymal stromal cells, anthracycline, plasma cell niche, cytokines, chemokines, antibody-secreting cells
National Category
Immunology Cell Biology
Identifiers
urn:nbn:se:su:diva-204446 (URN)10.3390/hemato2010009 (DOI)
Available from: 2022-05-06 Created: 2022-05-06 Last updated: 2023-10-30Bibliographically approved
Stone, V. M., Ringqvist, E. E., Larsson, P. G., Domsgen, E., Holmlund, U., Sverremark-Ekström, E. & Flodström-Tullberg, M. (2021). Inhibition of Type III Interferon Expression in Intestinal Epithelial Cells-A Strategy Used by Coxsackie B Virus to Evade the Host's Innate Immune Response at the Primary Site of Infection?. Microorganisms, 9(1), Article ID 105.
Open this publication in new window or tab >>Inhibition of Type III Interferon Expression in Intestinal Epithelial Cells-A Strategy Used by Coxsackie B Virus to Evade the Host's Innate Immune Response at the Primary Site of Infection?
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2021 (English)In: Microorganisms, E-ISSN 2076-2607, Vol. 9, no 1, article id 105Article in journal (Refereed) Published
Abstract [en]

Increasing evidence highlights the importance of the antiviral activities of the type III interferons (IFN lambda s; IL-28A, IL-28B, IL29, and IFN lambda 4) in the intestine. However, many viruses have developed strategies to counteract these defense mechanisms by preventing the production of IFNs. Here we use infection models, a clinical virus isolate, and several molecular biology techniques to demonstrate that both type I and III IFNs induce an antiviral state and attenuate Coxsackievirus group B (CVB) replication in human intestinal epithelial cells (IECs). While treatment of IECs with a viral mimic (poly (I:C)) induced a robust expression of both type I and III IFNs, no such up-regulation was observed after CVB infection. The blunted IFN response was paralleled by a reduction in the abundance of proteins involved in the induction of interferon gene transcription, including TIR-domain-containing adapter-inducing interferon-beta (TRIF), mitochondrial antiviral-signaling protein (MAVS), and the global protein translation initiator eukaryotic translation initiation factor 4G (eIF4G). Taken together, this study highlights a potent anti-Coxsackieviral effect of both type I and III IFNs in cells located at the primary site of infection. Furthermore, we show for the first time that the production of type I and III IFNs in IECs is blocked by CVBs. These findings suggest that CVBs evade the host immune response in order to successfully infect the intestine.

Keywords
Coxsackievirus (CVB), enterovirus, IFIH1, immune evasion, innate immunity, interferon, intestine, intestinal epithelial cells, poly I, C, type 1 diabetes
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-190999 (URN)10.3390/microorganisms9010105 (DOI)000610601700001 ()33466313 (PubMedID)
Available from: 2021-03-12 Created: 2021-03-12 Last updated: 2022-02-25Bibliographically approved
Spreckels, J. E., Wejryd, E., Marchini, G., Jonsson, B., de Vries, D. H., Jenmalm, M. C., . . . Abrahamsson, T. (2021). Lactobacillus reuteri Colonisation of Extremely Preterm Infants in a Randomised Placebo-Controlled Trial. Microorganisms, 9(5), Article ID 915.
Open this publication in new window or tab >>Lactobacillus reuteri Colonisation of Extremely Preterm Infants in a Randomised Placebo-Controlled Trial
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2021 (English)In: Microorganisms, E-ISSN 2076-2607, Vol. 9, no 5, article id 915Article in journal (Refereed) Published
Abstract [en]

Lactobacillus reuteri DSM 17938 supplementation reduces morbidities in very low birth weight infants (<1500 g), while the effect on extremely low birth weight infants (ELBW, <1000 g) is still questioned. In a randomised placebo-controlled trial (ClinicalTrials.gov ID NCT01603368), head growth, but not feeding tolerance or morbidities, improved in L. reuteri-supplemented preterm ELBW infants. Here, we investigate colonisation with the probiotic strain in preterm ELBW infants who received L. reuteri DSM 17938 or a placebo from birth to postmenstrual week (PMW) 36. Quantitative PCR was used on 582 faecal DNA samples collected from 132 ELBW infants at one, two, three, and four weeks, at PMW 36, and at two years of age. Human milk oligosaccharides were measured in 31 milk samples at two weeks postpartum. At least 86% of the ELBW infants in the L. reuteri group were colonised with the probiotic strain during the neonatal period, despite low gestational age, high antibiotic pressure, and independent of infant feeding mode. Higher concentrations of lacto-N-tetraose, sialyl-lacto-N-neotetraose c, and 6 '-sialyllactose in mother's milk weakly correlated with lower L. reuteri abundance. Within the L. reuteri group, higher L. reuteri abundance weakly correlated with a shorter time to reach full enteral feeding. Female sex and L. reuteri colonisation improved head growth from birth to four weeks of age. In conclusion, L. reuteri DSM 17938 supplementation leads to successful colonisation in ELBW infants.

Keywords
antibiotic, extremely low birth weight, feeding intolerance, human milk oligosaccharide, Lactobacillus reuteri, premature, probiotic, randomised controlled trial
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-196358 (URN)10.3390/microorganisms9050915 (DOI)000662354700001 ()33923278 (PubMedID)
Available from: 2021-09-07 Created: 2021-09-07 Last updated: 2022-02-25Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0001-6271-8681

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