SRF-deficient astrocytes provide neuroprotection in mouse models of excitotoxicity and neurodegeneration.

Reactive astrogliosis is a common pathological hallmark of CNS injury, infection, and neurodegeneration, where reactive astrocytes can be protective or detrimental to normal brain functions. Currently, the mechanisms regulating neuroprotective astrocytes and the extent of neuroprotection are poorly understood. Here, we report that conditional deletion of serum response factor (SRF) in adult astrocytes causes reactive-like hypertrophic astrocytes throughout the mouse brain. These SrfGFAP-ERCKO astrocytes do not affect neuron survival, synapse numbers, synaptic plasticity or learning and memory. However, the brains of Srf knockout mice exhibited neuroprotection against kainic-acid induced excitotoxic cell death. Relevant to human neurodegenerative diseases, SrfGFAP-ERCKO astrocytes abrogate nigral dopaminergic neuron death and reduce ß-amyloid plaques in mouse models of Parkinson's and Alzheimer's disease, respectively. Taken together, these findings establish SRF as a key molecular switch for the generation of reactive astrocytes with neuroprotective functions that attenuate neuronal injury in the setting of neurodegenerative diseases.

Automated morphometric analysis with SMorph software reveals plasticity induced by antidepressant therapy in hippocampal astrocytes.

Nervous system development and plasticity involve changes in cellular morphology, making morphological analysis a valuable exercise in the study of nervous system development, function and disease. Morphological analysis is a time-consuming exercise requiring meticulous manual tracing of cellular contours and extensions. We have developed a software tool, called SMorph, to rapidly analyze the morphology of cells of the nervous system. SMorph performs completely automated Sholl analysis. It extracts 23 morphometric features based on cell images and Sholl analysis parameters, followed by principal component analysis (PCA). SMorph was tested on neurons, astrocytes and microglia and reveals subtle changes in cell morphology. Using SMorph, we found that chronic 21-day treatment with the antidepressant desipramine results in a significant structural remodeling in hippocampal astrocytes in mice. Given the proposed involvement of astroglial structural changes and atrophy in major depression in humans, our results reveal a novel kind of structural plasticity induced by chronic antidepressant administration.

SRF Is Required for Maintenance of Astrocytes in Non-Reactive State in the Mammalian Brain.

Astrocytes play several critical roles in the normal functioning of the mammalian brain, including ion homeostasis, synapse formation, and synaptic plasticity. Following injury and infection or in the setting of neurodegeneration, astrocytes become hypertrophic and reactive, a process termed astrogliosis. Although acute reactive gliosis is beneficial in limiting further tissue damage, chronic gliosis becomes detrimental for neuronal recovery and regeneration. Several extracellular factors have been identified that generate reactive astrocytes; however, very little is known about the cell-autonomous transcriptional mechanisms that regulate the maintenance of astrocytes in the normal non-reactive state. Here, we show that conditional deletion of the stimulus-dependent transcription factor, serum response factor (SRF) in astrocytes (SrfGFAPCKO) results in astrogliosis marked by hypertrophic morphology and increased expression of GFAP, vimentin, and nestin. These reactive astrocytes were not restricted to any specific brain region and were seen in both white and gray matter in the entire brain. This astrogliosis persisted throughout adulthood concomitant with microglial activation. Importantly, the Srf mutant mouse brain did not exhibit any cell death or blood brain barrier (BBB) deficits suggesting that apoptosis and leaky BBB are not the causes for the reactive phenotype. The mutant astrocytes expressed more A2 reactive astrocyte marker genes and the SrfGFAPCKO mice exhibited normal neuronal numbers indicating that SRF-deficient gliosis astrocytes are not neurotoxic. Together, our findings suggest that SRF plays a critical role in astrocytes to maintain them in a non-reactive state.

In vivo safety assessment of Lactobacillus fermentum strains, evaluation of their cholesterol-lowering ability and intestinal microbial modulation.

BACKGROUND: Despite the growing importance of probiotics apparent health benefits, an impediment to the use of new probiotic cultures is their safety. Hence there is a need to strictly examine the biosafety as well as health benefits of probiotics in in vivo model systems. RESULTS: In this study, two lactic acid bacterial (LAB) cultures Lactobacillus fermentum NCMR 2826 and FIX proven for their in vitro probiotic properties were investigated for their in vivo safety in Wistar rats. An acute toxicity study (14 days) with a high dose of biomass (1016 colony-forming units (CFU) mL-1 ) followed by a subchronic test for 13 weeks with oral feeding of the probiotic cultures in three different doses (107 , 108 and 1010 CFU mL-1 ) on a daily basis revealed the safety of the L. fermentum cultures. The probiotic feeding had no toxic effects on survival, body weight and food consumption with any of the dosages used throughout the treatment period. No statistically significant changes in relative organ weights and serum biochemical and hematological indices were found between the control and the probiotic fed animals. In addition to the safety attributes, the L. fermentum culture fed rats showed reduced serum cholesterol levels, macrovesicular steatosis and hepatocyte ballooning compared with control animals. Further, quantification of intestinal microbiota using real-time polymerase chain reaction (PCR) analysis from animal feces indicated a significant increase and stability of Lactobacillus and Bifidobacterium counts but a decrease of Escherichia coli numbers. CONCLUSION: This study of safety and beneficial features highlights the use of the two native L. fermentum isolates as potential probiotic food supplements. © 2019 Society of Chemical Industry.

Heterogeneity of macrolide-lincosamide-streptogramin phenotype & conjugal transfer of erm(B) in Pediococcus pentosaceus.

Background & objectives: Pediococcus pentosaceus has been reported to cause clinical infections while it is being promoted as probiotic in food formulations. Antibiotic resistance (AR) genes in this species are a matter of concern for treating clinical infections. The present study was aimed at understanding the phenotypic resistance of P. pentosaceus to macrolide-lincosamide-streptogramin B (MLSB) antibiotics and the transfer of AR to pathogens. Methods: P. pentosacues isolates (n=15) recovered from fermented foods were screened for phenotypic resistance to MLSBantibiotics using disc diffusion and microbroth dilution methods. Localization and transferability of the identified resistance genes, erm(B) and msr(C) were evaluated through Southern hybridization and in vitro conjugation methods. Results: Four different phenotypes; sensitive (S) (n=5), macrolide (M) (n=7), lincosamide (L) (n=2) and constitutive (cMLSB) (n=1) were observed among the 15 P. pentosaceus isolates. High-level resistance (>256 µg/ml) to MLSBwas observed with one cMLSBphenotypic isolate IB6-2A. Intermediate resistance (8-16 µg/ml) to macrolides and lincosamides was observed among M and L phenotype isolates, respectively. Cultures with S phenotype were susceptible to all other antibiotics but showed unusual minimum inhibitory concentration (MIC) values of 8-16 µg/ml for azithromycin. Southern hybridization studies revealed that resistance genes localized on the plasmids could be conjugally transferred to Enterococcus faecalis JH2-2. Interpretation & conclusions: The study provides insights into the emerging novel resistance patterns in P. pentosaceus and their ability to disseminate AR. Monitoring their resistance phenotypes before use of MLS antibiotics can help in successful treatment of Pediococcal infections in humans.

Conjugal transfer of erm(B) and multiple tet genes from Lactobacillus spp. to bacterial pathogens in animal gut, in vitro and during food fermentation.

Three strains of Lactobacillus comprising Lactobacillus salivarius (CHS-1E and CH7-1E) and Lactobacillus reuteri (CH2-2) previously isolated from chicken meat were analyzed for their transferability of antibiotic resistance (AR) genes to pathogenic strains under in vivo, in vitro, and during food fermentation. For in vivo model, Albino Wistar rats were inoculated with 1010 CFU/g/ml of Enterococcus faecalis JH2-2 (recipient). After 7 days, either of two donors L. salivarius CH7-1E or L. reuteri [harbouring erythromycin and tetracycline resistance genes] were introduced at a concentration of 109 CFU/ml daily for 1 week. Two days after donor introduction, there was a stable increase in the number of transconjugants in the animal faeces from 102 to 103 CFU/g and presented erm(B), tet(M), tet(L) and tet(W) in their genome like donor strains. Similar observations were made with in vitro filter mating between CHS-1E, CH2-2 and CH7-1E and E. faecalis JH2-2 with transfer frequencies of 1 × 10-4, 3.8 × 10-3 and 2 × 10-3 per donor cell respectively. With the results obtained in vivo and in vitro, the AR transferability of donor strains was estimated during food fermentation (chicken sausage, fermented milk or idli batter) with pathogenic recipient strains added as contaminants. At the end of mating period, phenotypic resistance to erythromycin and tetracycline in Listeria monocytogenes and Yersinia enterocolitica strains was observed. This study showed the ability of food borne Lactobacillus in diffusing their AR traits in diverse natural environments increasing their concern of AR dissemination in the food chain when used as food additives and/or probiotics.

Presence of erythromycin and tetracycline resistance genes in lactic acid bacteria from fermented foods of Indian origin.

Lactic acid bacteria (LAB) resistant to erythromycin were isolated from different food samples on selective media. The isolates were identified as Enterococcus durans, Enterococcus faecium, Enterococcus lactis, Enterococcus casseliflavus, Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus fermentum, Pediococcus pentosaceus and Leuconostoc mesenteroides. Of the total 60 isolates, 88 % harbored the ermB gene. The efflux gene msrA was identified in E. faecium, E. durans, E. lactis, E. casseliflavus, P. pentosaceus and L. fermentum. Further analysis of the msrA gene by sequencing suggested its homology to msrC. Resistance to tetracycline due to the genes tetM, tetW, tetO, tetK and tetL, alone or in combination, were identified in Lactobacillus species. The tetracycline efflux genes tetK and tetL occurred in P. pentosaceus and Enterococcus species. Since it appeared that LAB had acquired these genes, fermented foods may be a source of antibiotic resistance.

Acquired resistance to macrolide-lincosamide-streptogramin antibiotics in lactic Acid bacteria of food origin.

Antibiotic resistance is a growing problem in clinical settings as well as in food industry. Lactic acid bacteria (LAB) commercially used as starter cultures and probiotic supplements are considered as reservoirs of several antibiotic resistance genes. Macrolide-lincosamide-streptogramin (MLS) antibiotics have a proven record of excellence in clinical settings. However, the intensive use of tylosin, lincomysin and virginamycin antibiotics of this group as growth promoters in animal husbandry and poultry has resulted in development of resistance in LAB of animal origin. Among the three different mechanisms of MLS resistance, the most commonly observed in LAB are the methylase and efflux mediated resistance. This review summarizes the updated information on MLS resistance genes detected and how resistance to these antibiotics poses a threat when present in food grade LAB.