Differential vaginalLactobacillusspecies metabolism of glucose, L and D-lactate by¹³C-nuclear magnetic resonance spectroscopy

Abstract

12Sheffield, Royal Hallamshire hospital, Glossop Road, Sheffield S10 2JF, UK. +44 114 215 9596, 13 steven.reynolds@sheffield.ac.uk 14 15 Acknowledgements: 16We are grateful to the University of Sheffield, UK, for providing support and the 9.4T MRS spectrometer with Abstract 28 Introduction 29 Cervicovaginal dysbiosis can lead to infection-associated spontaneous preterm birth.30 Objective 31 To determine whether vaginal Lactobacillus species, L. crispatus and L. jensenii, differentially metabolise glucose, 32 L-and/or D-lactate to propagate their survival/dominance.33 Methods 34Bacteria were incubated anaerobically for 24h at 37°C, with 13 Cu-glucose, 13 C3-D-lactate or 13 C3-L-lactate 35 (singularly or combined) for 24h. 13 C-spectra were acquired using a 9.4T NMR spectrometer. 36 Results 37L. crispatus and L. jensenii (n=6 each) metabolised 13 C-glucose to 13 C-lactate and 13 C-acetate. L. jensenii 38 converted more 13 C3-D-or 13 C3-L-lactate to 13 C-acetate than L. crispatus, p<0.001. 39 Conclusion 40Conversion of glucose and lactate to acetate by L. jensenii compared to L. crispatus, suggests a possibly important 41 pathomechanism of dysbiosis and infection-associated spontaneous preterm birth. 42In conjunction with the host vaginal habitat, the vaginal bacteria produce unique metabolic by-products. In a 55 healthy vagina, lactobacilli are the predominant species and have been linked to increased likelihood of term 56 delivery (Amabebe and Anumba, 2018; Stafford et al., 2017). The four major vaginal lactobacilli (L. crispatus, L. 57 jensenii, L. iners and L. gasseri) differentially produce L-and D-lactic acid that lowers the pH of the 58 cervicovaginal space, creating unfavourable conditions for other invading species. D-lactic acid is believed to be 59 more potent than L-lactic acid in relation to protection against colonisation by potentially pathogenic organisms 60 within the vagina and the accompanying inflammatory response (Witkin et al., 2013). Anaerobes are also 61 endogenous to the vagina and are associated with infection and preterm birth (PTB, i.e. delivery <37 weeks of 62 gestation) especially when lactobacilli are deficient (Amabebe and Anumba, 2018). 63Metabolism by cells can be tracked by 13 C-Nuclear Magnetic Resonance (NMR) to examine specific pathways 64 (Buescher et al., 2015). The use of 13 C labelled substrates means that they are metabolised identically to those 65 found naturally, with the cell viability maintained throughout the experiment. Additionally, strategic placement 66 of the 13 C label (Buescher et al., 2015) allows the activity of single or multiple metabolic pathways to be identified, 67 even if the end product is the same (Bruntz et al., 2017). 68Although, lactobacilli are known to thrive in the acidic condition of the vagina (pH < 4.5), the mechanism 69 underpinning how lactobacilli, and other anaerobes, interact with lactic acid remains unresolved (Amabebe and 70 Anumba, 2018). In this short communication, we report on the use of 13 C-NMR to examine how different 71 Lactob...