Molecular Phenotyping and Genomic Characterization of a Novel Neuroactive Bacterium Strain, Lactobacillus murinus HU-1

Abstract

Over a century ago, Elie Metchnikoff observed unusual longevity among Bulgarian populations that consumed soured milk containing lactic acid bacteria (Metchnikoff and Mitchell, 1908). He theorized that the production of lactic acid by such bacteria prevented “intestinal putrefaction” and popularized the deliberate consumption of Lactobacilli-cultured milk for health purposes (Cavaillon and Legout, 2016). Since Metchnikoff’s time, the contributions of Lactobacilli to host health have been greatly expanded to include roles in immune homeostasis, production of key nutrients and vitamins, and even as a physical barrier against infection by pathogenic microorganisms (Macfarlane and Macfarlane, 2012). Recent investigations into the gut–brain axis revealed possible additional functions of lactic acid bacteria in regulating mood and cognition when ingested orally as a probiotic supplement. Indeed, many Lactobacillus sp. have been correlated with improved psychological outcomes, especially for neurodevelopmental, mood, stress, and anxiety disorders (Bravo et al., 2011; Buffington et al., 2016; Liu et al., 2019; Marotta et al., 2019; Sgritta et al., 2019). While the neurological mechanisms behind probiotic consumption have yet to be fully understood, current evidence suggests Lactobacilli likely confer mental health benefits through both direct and indirect pathways, such as vagal nerve signaling and T_reg regulation (Bravo et al., 2011; Wells, 2011). Gut bacterial production of known neurotransmitters, such as gamma-aminobutyric acid (GABA), serotonin, and glutamate (Lyte, 2011; Dienel, 2012; Steenbergen et al., 2015), as well as a newfound appreciation for neuroactive potential of common bacterial metabolites, such as lactate and short-chain fatty acids, further suggest additional pathways in which Lactobacilli may contribute to neurological health (Proia et al., 2016; Oleskin et al., 2017). Previously, we reported neuroprotective effects of Lactobacillus murinus HU-1, a mutant strain isolated from mouse, in preventing development of premature senescence in cortical microglia and social behavior deficits in murine offspring reared under antibiotics-driven maternal microbiome dysbiosis (Lebovitz et al., 2019). A key component of a complete and diverse gut microbiome, L. murinus represents a commensal gut bacterium naturally found in the gut of healthy mammals, including rodents, dogs, pigs, and poultry (Kurzak et al., 1998; Greetham et al., 2002; Gardiner et al., 2004). Compared to other Lactobacilli, L. murinus is a relatively understudied species that only recently gained attention as a probiotic candidate, including potential applications regarding neonatal necrotizing enterocolitis (Isani et al., 2018), antimicrobial production (Nardi et al., 2005), pathogen antagonism (Vasconcelos et al., 2003), intestinal barrier function (Delucchi et al., 2017), food allergy (Huang et al., 2016), type 1 diabetes (Sane et al., 2018), hypertension (Wilck et al., 2017), age-associated inflammation (Pan et al., 2018), and bacterial translocation (Ma et al., 1990). Here, we characterize the genome of a novel strain, L. murinus HU-1, and profile its molecular features in an effort to better understand its influence on host physiology and neurobehavior. Lactobacillus murinus HU-1 originally isolated from murine gut was maintained as frozen stock in 20% glycerol at −80 °C until needed. Frozen stock was directly cultured overnight in MRS broth or streaked onto MRS agar (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) at 37 °C, as described (Kragh et al., 2018). All mice were housed in an AAALAC accredited, virus/specific antigen-free facility with a 12 h light-dark cycle; food (Teklad 2918, Envigo, Huntingdon, UK) and water provided ad libitum. Outbred CD-1 IGS mice were purchased from Charles River (Strain code 022, Charles River Laboratories, Wilmington, MA, USA), and inbred B6.129P-Cx3cr1^tm1Litt/J mice (Stock no. 005582) were purchased from Jackson Laboratory (Jackson Laboratory, Bar Harbor, ME, USA). Experimental CD-1 mice were administered a single oral dose of L. murinus HU-1 (10⁹ CFU) and then maintained on an antibiotic cocktail of 0.4 mg/ml kanamycin, 850 U/ml colistin, 0.215 mg/ml metronidazole (Bio-World, Dublin, OH, USA), 0.035 mg/ml gentamicin (Vet One, Boise, ID, USA), and 0.045 mg/ml vancomycin (Hospira Inc., Lake Forest, 372 IL, USA) (ABX^HU-1) or the above antibiotic cocktail with an addition of 0.5 mg/ml amoxicillin/clavulanic acid (Zoetis, Parsippany, NJ, USA) (ABX^HU-1+AC). Antibiotics were administered via drinking water. All experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and conducted under the approval of the Virginia Tech Institutional Animal Care and Use Committee (IACUC; #17-043). Fresh fecal pellets from mice were collected into sterile 1.5 ml microcentrifuge tubes and submitted to the Virginia-Maryland College of Veterinary Medicine’s Animal Laboratory Services for identification of culturable bacteria and to undergo antibiotics susceptibility testing. In brief, murine fecal pellets were immediately cultured on MacConkey and chocolate agar overnight. Colony formations were scored and identified using Bruker Microflex Biotyper 3.1 MALDI-TOF (Bruker Daltonics, Billerica, MA, USA). Additional colonies were collected from pure cultures of identified bacteria and subjected to antibiotics susceptibility testing using Sensititre™ Complete Automated AST System (Thermo Fisher Scientific Solutions LLC, Waltham, MA, USA) according to manufacturer’s instructions. Genomic DNA was extracted and purified from L. murinus HU-1 isolates via kit (SKU D6010, Zymo Research, Irvine, CA, USA) and submitted to the...