Dr Bashir Abdulkadir Departmental Seminar Series Presented Department

Dr Bashir Abdulkadir Departmental Seminar Series Presented @ Department of Microbiology Umaru Musa Yar’adua University Katsina Tuesday 24 th April 2018

v. Probiotics are live microbial supplements that colonize the gut and potentially exert health benefit to the host (1) v Lactobacillus spp. , Bifidobacterium spp. and Staphylocuccus spp. are the most common types of microbes used as Probiotics, though certain yeast and bacilli can also be used (2) v. Existing evidence suggests that probiotics significantly reduce the risk of necrotising enterocolitis (NEC) and late onset sepsis (LOS) in preterm infants (3) v. The routine use of probiotic prophylaxis in clinical practice is still insufficient; despite it has been robustly studied and reported in therapy of NEC (4) v probiotic species alter gut microbial communities during and after therapeutic administration (5) 1. Christian et al. 2015. B Beh Immun. Academic Press Inc. 2. Groer MW, et al. 2014. Bio. Med Central Ltd. Available from: http: //www. microbiomejournal. com/content/2/1/38 3. Stewart CJ et al. 2015. NPG; Available from: http: //www. pubmedcentral 4. Abdulkadir B. et al. 2016. Neonatology. Available from: http: //www. ncbi. nlm. nih. gov/pubmed/26859305 5. Abdulkadir B. et al. 2016. EHD ; Elsevier. Available from: http: //www. earlyhumandevelopment. com/article/S 0378378215300451

v. The gut bacterial community plays a vital role in human health with a diverse and complex composition (1) v. Neonates born prematurely are vulnerable to various infections due to their weak immune system and the immaturity of the gut (2) v. The preterm gut microbiota has important influences in the onset of necrotising enterocolitis (NEC) and late onset sepsis (LOS) (3) v. Microbial dysbiosis has been implicated on the NEC pathogenesis 1. Christian et al. 2015. B Beh Immun. Academic Press Inc. 2. Groer MW, et al. 2014. Bio. Med Central Ltd. Available from: http: //www. microbiomejournal. com/content/2/1/38 3. Stewart CJ et al. 2015. NPG; Available from: http: //www. pubmedcentral 4. Abdulkadir B. et al. 2016. Neonatology. Available from: http: //www. ncbi. nlm. nih. gov/pubmed/26859305 5. Abdulkadir B. et al. 2016. EHD ; Elsevier. Available from: http: //www. earlyhumandevelopment. com/article/S 0378378215300451

v. To explore the gut bacterial diversity in preterm neonates receiving the probiotics Infloran during their NICU stay compare to match controls v. To investigate the microbial load of the species presence in Infloran capsule and observe their long-term effects v. To observe the systematic functional changes associated with probiotics administration in gut metabolites

v. Microbial DNA extraction from stool samples v. V 3 PCR and DGGE v. Quantitative PCR (q. PCR) v. Analyses of their bacterial community using 16 S RNA gene profiling v. Metabolomics profiling using liquid chromatography mass spectrometry (LCMS)

Methods – Old School Denaturing Gradient Gel Electrophoresis (DGGE) LOW urea and formamide HIGH urea and formamide Muyzer G et al. 1993. Appl Environ Microbiol. A-T rich sequences G-C rich sequences

Metabolomics NGS & q. PCR Sample Eg Me. OH Stool DNA Extraction q. PCR Bioinformatics 16 S Sequencing

LCMS – Metabolite profiling

Methods – Next Generation 454 Pyrosequencing (Roche) Sequencing by synthesis (Illumina)

Preterm Disease • Leading cause of death in preterm infants • Acute inflammatory disease process of the intestine • +VE blood culture with signs suggestive of infection 1 • Higher susceptibility in preterm neonates due to 2 the immaturity of the immune system Stewart CJ et al. 2012. Acta paediatrica. 2 Stewart CJ 2013. Springer Encyclopedia of Metagenomics. empowher. com/files/ebsco/images/infant_sepsis. jpg • Multifactorial pathophysiology involving immaturity of intestine and abnormal bacterial colonisation 1 Late onset sepsis (LOS) • ~20% of VLBW infants develop LOS phil. cdc. gov/PHIL_Images/02051999/00021/20 G 0021_lores. jpg Necrotising Enterocolitis (NEC)

v. Probiotics are live Microorganisms when taken in an adequate amounts confer a health benefits to the host v. Increase the number and change the composition of the overall gut microbial community v. Probiotics reduce the morbidity and mortality rate during the neonatal period v Current studies revealed that gut development and protection in preterm infants is associated with increased in Probiotics intake 1 Abdulkadir B. et al. 2016. EHD ; Elsevier.

v Explore the bacterial diversity in the gut microbiota among preterm babies receiving probiotics versus non-probiotic (control group)

10 patients 85 Samples Probiotic 7 patients 57 Samples Non-Probiotic 3 patients 28 Samples

Results – DGGE Gels L Probiotic L Non-Probiotic L L

Results – PCA

Results – PLS-DA Probiotics Non-Probiotic

Results – Box Plot P = <0. 001

*Probiotics have the potentiality to alter the gut microbial community in preterm infants *There is significant reduction in diversity in infants receiving probiotics (P = <0. 001)

v. An increase in mucosa-associated bacterial density was associated with NEC development in preterm pigs (1) v To date, no research has been performed to determine if increased bacterial load of stool is associated with NEC nor how the bacterial load changes temporally before and after diagnosis (2) v Therefore we aimed to accurately quantify the bacterial copy number in stool samples from patients with NEC longitudinally in comparison to gestationally matched controls. 1 Cathrine et al. 2015. Neonatology. 2 Abdulkadir B. et al. 2016. EHD ; Elsevier.

q. PCR 20 Patients 72 samples NEC 10 patients 37 Samples 2 weeks Before 1 week Before Control 10 patients 35 Samples At Diagnosis 1 Week After 2 Weeks After

Abdulkadir B. et al. 2016. EHD; Elsevier.

v. No significant difference in the total bacterial load was found between samples at NEC diagnosis compared to matched controls or temporally within NEC v The only significant difference observed was a significant reduction (P = 0. 046) between the bacterial load of NEC samples and controls 1 week following diagnosis v We suggest that measuring stool bacterial load may not be a proxy measure for tissue bacterial load.

Metabolomics profiling and molecular analysis of probiotics in the preterm gut Bashir Abdulkadir Faculty of Health and Life Sciences, Northumbria University, Newcastle upon Tyne, United Kingdom bashir. abdulkadir@northumbria. ac. uk Presented

v Explore the gut microbiome in preterm neonates receiving the probiotics (Infloran ® : Lactobacillus acidophilus NCIMB 701748 and Bifidobacterium bifidum -ATCC 15696) during their NICU stay compare to match controls v Determine the long-term effects of probiotic species following discharge v Observe the systematic functional changes associated with probiotics administration in gut metabolites

Abdulkadir B. et al. 2016. Neonatology. Available from: http: //www. ncbi. nlm. nih. gov/pubmed/26859305

Abdulkadir B. et al. 2016. Neonatology. Available: http: //www. ncbi. nlm. nih. gov/pubmed/26859305

PLS-DA of 16 S bacterial profiles

Table – Summary of the bacterial species Control NICU Probiotic Before During Acinetobacter 1. 38 0. 02 0. 00 Actinomyces 2. 36 0. 00 Anaerostipes 0. 00 Bacteroides 0. 00 0. 03 Bifidobacterium 4. 02 11. 06 15. 03 Clostridium 1 3. 84 0. 03 0. 02 Clostridium 2 0. 81 0. 00 Enterococcus 18. 41 18. 93 24. 38 Escherichia 11. 44 2. 06 19. 66 Klebsiella 37. 41 7. 93 16. 54 Lachnospiracea 0. 00 Lactobacillus 1 0. 00 1. 73 4. 81 Lactobacillus 2 0. 00 0. 77 5. 80 Lactobacillus 3 0. 00 0. 73 0. 20 Lactobacillus 4 0. 00 0. 06 0. 19 Proteus 0. 00 Pseudomonas 1. 58 1. 44 0. 49 Staphylococcus 5. 86 53. 49 9. 73 Streptococcus 1 3. 09 0. 01 Streptococcus 2 1. 00 0. 00 Veillonella 0. 00 select from OTUs PD After 0. 00 4. 37 0. 00 10. 36 19. 10 14. 16 0. 47 0. 00 0. 08 12. 04 1. 16 23. 77 14. 02 23. 92 12. 84 0. 00 5. 10 4. 77 0. 00 0. 08 0. 00 2. 32 0. 01 0. 24 0. 00 5. 53 0. 01 10. 52 0. 00 1. 04 0. 00 4. 47

PLS-DA of the metabolite profiles

v Significantly lower diversity occurred in during probiotic administration compared to controls and in all groups when compared to samples post discharge v The Infloran strains (Bifidobacterium and Lactobacillus) colonize the preterm gut with diverse abundance and they all proliferate with probiotics administration v Bifidobacterium demonstrates long-term effects during post discharge v Probiotic species reduce the abundance of taxa such as Clostridia and Klebsiella as potential pathogens associated with NEC or LOS v Probiotic strains play significantly to shape the systematic function in the preterm gut microbiome

v. Overall the bacterial load of stool samples is not associated with NEC in preterm infants v Probiotic species successfully colonise the preterm gut, reducing the relative abundance of potentially pathogenic bacteria associated with NEC and LOS v Bifidobacterium (but not Lactobacillus) has a long-term effects on the preterm gut v Probiotics supplementation causes metabolomics shifts in gut functioning in early infancy v. Further work is necessary to determine the contribution of these changes to health and how medical intervention can be tailored accordingly

v. Future studies should explore the functional metabolite changes further to better understand the mechanistic role of probiotic supplementation

Further Reading Some Important links to my published research articles on Preterm infants for further reading: ü http: //doi. org/10. 1159/000442936 ü http: //doi. org/10. 1016/j. earlhumdev. 2016. 018 ü https: //neonatalresearch. org/2016/05/06/necrotizing-enterocolitis-manipulating-the-microbiomepart-2/ ü https: //neonatalresearch. org/2016/05/09/necrotizing-enterocolitis-manipulating-the-microbiomepart-3/ ü Microbiome (2016) 4: 67 DOI 10. 1186/s 40168 -016 -0216 -8.

Citations in “who is who” My Online publications got cited in Research Gate with 777 Reads, 43 citations and total RG Score 8. 52. For details on my online research profile see the link below: https: //www. researchgate. net/profile/Bas hir_Abdulkadir. Google Scholar My research work has a Google scholar citation indices with 65 citations and hindex of 4, see the details in the following link: https: //scholar. google. com/citations? hl =en&user=Eg. Ao 0 CQAAAAJ.

Prof. Stephen Cummings Dr. Andrew Nelson Dr. Christopher Stewart Dr. Janet Berrington Prof. John Perry Dr. Nicholas Embleton Dr Emma Marrs Dr. Tom Skeath

Newcastle Neonatology Research Group

bashir. abdulkadir@umyu. edu. ng @bashirakadir MY ORCID ID orcid. org/0000 -0001 -8616 -6615