Systematic optimization of fermentation conditions for in vitro fermentations with fecal inocula

In vitro fermentation strategies with fecal inocula are considered cost-effective methods to gain mechanistic insights into fecal microbiota community dynamics. However, all in vitro approaches have their limitations due to inherent differences with respect to the in vivo situation mimicked, introducing possible biases into the results obtained. Here, we aimed to systematically optimize in vitro fermentation conditions to minimize drift from the initial inoculum, limit growth of opportunistic colonizers, and maximize the effect of added fiber products (here pectin) when compared to basal medium fermentations. We evaluated the impact of varying starting cell density and medium nutrient concentration on these three outcomes, as well as the effect of inoculation with fresh vs. stored fecal samples. By combining GC–MS metabolite profiling and 16 s rRNA gene-based amplicon sequencing, we established that starting cell densities below 1010 cells/ml opened up growth opportunities for members the Enterobacteriaceae family. This effect was exacerbated when using fecal samples that were stored frozen at −80°C. Overgrowth of Enterobacteriaceae resulted in lowered alpha-diversity and larger community drift, possibly confounding results obtained from fermentations in such conditions. Higher medium nutrient concentrations were identified as an additional factor contributing to inoculum community preservation, although the use of a less nutrient dense medium increased the impact of fiber product addition on the obtained metabolite profiles. Overall, our microbiome observations indicated that starting cell densities of 1010 cells/ml limited opportunities for exponential growth, suppressing in vitro community biases, whilst metabolome incubations should preferably be carried out in a diluted medium to maximize the impact of fermentable substrates

Benchmarking microbiome transformations favors experimental quantitative approaches to address compositionality and sampling depth biases

While metagenomic sequencing has become the tool of preference to study host-associated microbial communities, downstream analyses and clinical interpretation of microbiome data remains challenging due to the sparsity and compositionality of sequence matrices. Here, we evaluate both computational and experimental approaches proposed to mitigate the impact of these outstanding issues. Generating fecal metagenomes drawn from simulated microbial communities, we benchmark the performance of thirteen commonly used analytical approaches in terms of diversity estimation, identification of taxon-taxon associations, and assessment of taxon-metadata correlations under the challenge of varying microbial ecosystem loads. We find quantitative approaches including experimental procedures to incorporate microbial load variation in downstream analyses to perform significantly better than computational strategies designed to mitigate data compositionality and sparsity, not only improving the identification of true positive associations, but also reducing false positive detection. When analyzing simulated scenarios of low microbial load dysbiosis as observed in inflammatory pathologies, quantitative methods correcting for sampling depth show higher precision compared to uncorrected scaling. Overall, our findings advocate for a wider adoption of experimental quantitative approaches in microbiome research, yet also suggest preferred transformations for specific cases where determination of microbial load of samples is not feasible

The effect of a diet with fructan-rich chicory roots on intestinal helminths and microbiota with special focus on Bifidobacteria and Campylobacter in piglets around weaning

The restrictions on the use of antibiotic and anthelmintic treatments in organic pig farming necessitate alternative non-medical control strategies. Therefore, the antibiotic and parasite-reducing effect of a fructan-rich (prebiotic) diet of dried chicory was investigated in free-ranging piglets. Approximately half of 67 piglets from 9 litters were experimentally infected with Ascaris suum and Trichuris suis in the suckling period (1 to 7 weeks of age) and 58 of the piglets were challenged daily with E. coli O138:F8 for 9 days after weaning to induce weaning diarrhoea. The litters were fed either chicory (30% DM) or a control diet. The effect of chicory on intestinal helminths, intestinal microbiota, especially Bifidobacteria and Campylobacter spp., and E. coli post-weaning diarrhoea was assessed. The weight gain of the piglets was not impaired significantly by chicory. The intestinal A. suum worm burden was reduced by 64% (P=0.034) in the chicory-fed piglets, whereas these same piglets had 63% more T. suis worms (P=0.016). Feeding with chicory elicited no changes among the main bacterial groups in ileum according to terminal restriction fragment length polymorphism (T-RFLP) analysis. However, the terminal-restriction fragment (T-RF) 208 bp, which may belong to Lachnospiraceae, was stimulated by the chicory feed (P=0.03), and T-RF 370 bp that matches Enterobacter belonging to the Enterobacteria was reduced (P=0.004). Additionally, chicory increased the level of Bifidobacteria (P=0.001) and the faecal Campylobacter excretion level was transitorily reduced in chicory-fed piglets at 7 weeks of age (P=0.029). Unfortunately, it was not possible to assess the effect of chicory on post-weaning diarrhoea as it did not develop. In conclusion, feeding piglets chicory around the time of weaning caused complex changes of the microbiota and parasite communities within the intestinal tract, and feeding piglets chicory may therefore serve as an animal-friendly strategy to control pathogens

PEMBINAAN NARAPIDANA YANG MENGALAMI GANGGUAN JIWA DI DALAM LEMBAGA PEMASYARAKATAN

PEMBINAAN NARAPIDANA YANG MENGALAMI GANGGUAN JIWADI LEMBAGA PEMASYARAKATAN Agusriadi Dahlan Ali Suhaimi ABSTRAKLembaga Pemasyarakatan sebelumnya disebut Penjara adalah tempat orang-orang yang melakukan kriminalitas dan pelanggaran hukum lainnya agar mereka dapat menyadari kesalahannya dan mempertanggungjawabkan apa yang telah mereka perbuat. Hukuman yang mereka terima sebagai balasan yang setimpal terhadap perbuatan mereka, meskipun nilai-nilai kemanusiaan beserta hak asasinya kurang diperhatikan. Lembaga Pemasyarakatan merupakan tempat yang stressfull atau menekan yang dapat berpengaruh terhadap fisik dan kejiwaan (psikologi) seseorang ditambah kecemasan yang berlebihan sehingga muncul depresi yang mengakibatkan seseorang melakukan bunuh diri. Akibat stress melahirkan depresi dan depresi biasanya akan terjadi kegoncangan kejiwaan yang luar biasa yang dapat mengakibatkan seseorang menjadi tidak waras (gila). Undang-Undang No.12/1995 tentang Lembaga Pemasyarakatan maupun Peraturan Pemerintah No. 99 Tahun 2012 tentang Perubahan Kedua Atas Peraturan Pemerintah Nomor 32 Tahun 1999 tentang Syarat dan Tata Cara Pelaksanaan Hak Warga Binaan Pemasyarakatan tidak menjelaskan ketentuan terhadap narapidana yang gila di dalam lapas sebagaimana yang pernah terjadi di Lapas Kelas II-A Lambaro, Aceh Besar dan Rutan Klas I Tanjung Gusta, Medan. Selanjutnya yang bersangkutan dikirim ke Lapas Kelas Klass II-B Meulaboh tanggal 27 Januari 2014. Selama tujuh hari di Lapas Klass II-B Meulaboh Zabir bin Ilyas kemudian di titipkan di Rumah Sakit Jiwa Banda Aceh karena penyakitnya sudah mencapai stadium empat untuk mendapatkan perawatan dan penyembuhan kejiwaannya. Bagaimanakah pengaturan dan pembinaan terhadap narapidana yang mengalami gangguan kejiwaan dalam Lembaga Pemasyarakatan. Kemudian bagaimanakah kepastian hukum bagi narapidana yang sedang mengalami gangguan kejiwaan, dan bagaimana status hukum bagi narapidana yang sembuh dari gangguan kejiwaan. Penelitian dan pengkajian ini bertujuan untuk mengetahui pengaturan dan pembinaan terhadap narapidana yang mengalami gangguan kejiwaan dalam Lapas dan mengidentifikasi kepastian hukum bagi narapidana yang sedang mengalami gangguan kejiwaan serta mengetahui status hukum bagi narapidana yang sembuh dari gangguan kejiwaan.Metode yang digunakan adalah metode penelitian yuridis-normatif dengan menggunakan jenis penelitian deskriptis analisis dalam rangka untuk menemukan aturan hukum, prinsip-prinsip hukum, norma maupun doktrin-doktrin hukum dengan pendekatan undang-undang, kasus. Sumber data adalah data sekunder berupa bahan hukum primer, bahan hukum sekunder, dan bahan hukum tersier.Hasil penelitian menunjukkan bahwa Pengaturan tentang perawatan narapidana yang mengalami gangguan kejiwaan dalam Lembaga Pemasyarakatan di Indonesia belum diatur, baik dalam Undang-Undang Nomor 12 Tahun 1995 tentang Lembaga Pemasyarakatan maupun PP No. 99 Tahun 2012 tentang PP No. 99 Tahun 2012 tentang Perubahan Kedua Atas PP No.32 Tahun 1999 tentang Syarat dan Tata Cara Pelaksanaan Hak Warga Binaan Pemasyarakatan. Namun bila narapidana sakit atau gangguan kesehatan lainnya yang tidak termasuk gangguan kejiwaan ada aturan tentang perawatan medis. Adapun mengenai narapidana yang mengalami gangguan kejiwaan di Lembaga Pemasyarakatan di Indonesia hingga kini belum ada kepastian hukum karena kasus seperti ini sangat langka terjadi di Indonesia. Pihak otoritas hanya menggunakan hak diskresi yang dimilikinya untuk menyelesaikan kasus tersebut sebagaimana terjadi di Lapas Porong Surabaya. Tetapi di Aceh kasus ini baru pertama kali terjadi yang dialami Zabir bin Ilyas pada tahun 2014. Pimpinan Lapas memutuskan memasukkan Zabir bin Ilyas ke Rumah Sakit Jiwa Banda Aceh. Terkait status hukum narapidana yang sembuh dari gangguan kejiwaan masih tetap sebagai tersangka (ditahan). Tidak ada keringanan hukuman apalagi sampai dibebaskan sebelum masa hukuman habis dijalankan. Hingga kini tidak ada satupun aturan yang mengatur baik secara implisit maupun ekplisit yang mengatur tentang narapidana yang sembuh dari penyakit kejiwaan (gila).Disarankan kepada Pemerintah agar merevisi Undang-Undang Nomor 12 Tahun 1995 tentang Lembaga Pemasyarakatan dan memasukkan salah satu pasal yang mengatur mengenai narapidana yang mengalami gangguan kejiwaan di dalam Lembaga Pemasyarakatan. Agar terwujudnya kepastian hukum di Indonesia mengenai narapidana yang mengalami gangguan kejiwaan dalam lebaga pemasyarakatan untuk membuat regulasi legal baik berupa undang-undang khusus atau melalui Peraturan Pemerintah. Kemudian disarankan agar narapidana yang mengalami gangguan kejiwaan dalam Lembaga Pemasyarakatan dapat dibebaskan dan dikembalikan ke keluarganya atau dimasukkan ke rumah sakit jiwa hingga sembuh.Kata Kunci : Pembinaan, Narapidana, Gangguan Jiwa, Lembaga Pemasyarakatan.Banda Ace

Production of Extracellular Lipase from Aspergillus niger by Solid-State Fermentation

Proizvodnja lipaze u soju plijesni Aspergillus niger J-1 ispitana je submerznim uzgojem i površinskim uzgojem na čvrstoj podlozi od pšeničnih posija. Provedeno je optimiranje podloge za oba postupka uzgoja. Maksimalna aktivnost lipaze od 1,46 IU/mL postignuta je submerznim uzgojem u podlozi s 2 % glukoze i 2 % maslinova ulja pri 1 vvm i 450 m-1. Medutim, 9,14 IU/g suhe tvari ekvivalentne 4,8 IU/mL aktivnosti lipaze postignuto je površinskim uzgojem na čvrstoj podlozi s 0,75 % amonijeva sulfata i 0,34 % uree. Optimalni pH i temperatura za aktivnost enzima bili su pH=6 i 40 °C. Enzim je pokazao 80 % od početne aktivnosti u neutralnom i blago kiselom mediju i pri temperaturi od 20 i 30 °C tijekom 24 sata.Lipase production in Aspergillus niger J-1 was tested using both submerged fermentation (SmF) and solid-state fermentation (SSF) on a mineral culture medium and wheat bran, respectively. The optimization of the culture medium was carried out for both SmF and SSF. The maximum lipase activity, 1.46 IU/mL, was obtained during the submerged fermentation in a medium containing glucose at 2 % and olive oil at 2 % under conditions of 1 vvm and 450 m–1. However, 9.14 IU/g of dry solid substrate equivalent to 4.8 IU/mL of lipase activity was reached using solid-state fermentation process with a medium containing 0.75 % of ammonium sulphate and 0.34 % of urea. The optimum pH and temperature for enzymatic activity were pH=6 and 40 °C, respectively. The enzyme also exhibited 80 % of its initial activity in neutral and mildly acid media and at temperatures between 20 and 30 °C for a period of 24 hours

Maximizing efficiency of rumen microbial protein production.

Rumen microbes produce cellular protein inefficiently partly because they do not direct all ATP toward growth. They direct some ATP toward maintenance functions, as long-recognized, but they also direct ATP toward reserve carbohydrate synthesis and energy spilling (futile cycles that dissipate heat). Rumen microbes expend ATP by vacillating between (1) accumulation of reserve carbohydrate after feeding (during carbohydrate excess) and (2) mobilization of that carbohydrate thereafter (during carbohydrate limitation). Protozoa account for most accumulation of reserve carbohydrate, and in competition experiments, protozoa accumulated nearly 35-fold more reserve carbohydrate than bacteria. Some pure cultures of bacteria spill energy, but only recently have mixed rumen communities been recognized as capable of the same. When these communities were dosed glucose in vitro, energy spilling could account for nearly 40% of heat production. We suspect that cycling of glycogen (a major reserve carbohydrate) is a major mechanism of spilling; such cycling has already been observed in single-species cultures of protozoa and bacteria. Interconversions of short-chain fatty acids (SCFA) may also expend ATP and depress efficiency of microbial protein production. These interconversions may involve extensive cycling of intermediates, such as cycling of acetate during butyrate production in certain butyrivibrios. We speculate this cycling may expend ATP directly or indirectly. By further quantifying the impact of reserve carbohydrate accumulation, energy spilling, and SCFA interconversions on growth efficiency, we can improve prediction of microbial protein production and guide efforts to improve efficiency of microbial protein production in the rumen

Respiratory disease following viral lung infection alters the murine gut microbiota

Alterations in the composition of the gut microbiota have profound effects on human health. Consequently, there is great interest in identifying, characterizing, and understanding factors that initiate these changes. Despite their high prevalence, studies have only recently begun to investigate how viral lung infections have an impact on the gut microbiota. There is also considerable interest in whether the gut microbiota could be manipulated during vaccination to improve efficacy. In this highly controlled study, we aimed to establish the effect of viral lung infection on gut microbiota composition and the gut environment using mouse models of common respiratory pathogens respiratory syncytial virus (RSV) and influenza virus. This was then compared to the effect of live attenuated influenza virus (LAIV) vaccination. Both RSV and influenza virus infection resulted in significantly altered gut microbiota diversity, with an increase in Bacteroidetes and a concomitant decrease in Firmicutes phyla abundance. Although the increase in the Bacteroidetes phylum was consistent across several experiments, differences were observed at the family and operational taxonomic unit level. This suggests a change in gut conditions after viral lung infection that favors Bacteroidetes outgrowth but not individual families. No change in gut microbiota composition was observed after LAIV vaccination, suggesting that the driver of gut microbiota change is specific to live viral infection. Viral lung infections also resulted in an increase in fecal lipocalin-2, suggesting low-grade gut inflammation, and colonic Muc5ac levels. Owing to the important role that mucus plays in the gut environment, this may explain the changes in microbiota composition observed. This study demonstrates that the gut microbiota and the gut environment are altered following viral lung infections and that these changes are not observed during vaccination. Whether increased mucin levels and gut inflammation drive, or are a result of, these changes is still to be determined

Genomics and metagenomics of trimethylamine-utilizing Archaea in the human gut microbiome

International audienceThe biological significance of Archaea in the human gut microbiota is largely unclear. We recently reported genomic and biochemical analyses of the Methanomassiliicoccales, a novel order of methanogenic Archaea dwelling in soil and the animal digestive tract. We now show that these Methanomassiliicoccales are present in published microbiome data sets from eight countries. They are represented by five Operational Taxonomic Units present in at least four cohorts and phylogenetically distributed into two clades. Genes for utilizing trimethylamine (TMA), a bacterial precursor to an atherosclerogenic human metabolite, were present in four of the six novel Methanomassiliicoccales genomes assembled from ELDERMET metagenomes. In addition to increased microbiota TMA production capacity in long-term residential care subjects, abundance of TMA-utilizing Methanomassiliicoccales correlated positively with bacterial gene count for TMA production and negatively with fecal TMA concentrations. The two large Methanomassiliicoccales clades have opposite correlations with host health status in the ELDERMET cohort and putative distinct genomic signatures for gut adaptation

Effects of orange juice formulation on prebiotic functionality using an in vitro colonic model sytem

A three-stage continuous fermentative colonic model system was used to monitor in vitro the effect of different orange juice formulations on prebiotic activity. Three different juices with and without Bimuno, a GOS mixture containing galactooligosaccharides (B-GOS) were assessed in terms of their ability to induce a bifidogenic microbiota. The recipe development was based on incorporating 2.75g B-GOS into a 250 ml serving of juice (65°Brix of concentrate juice). Alongside the production of B-GOS juice, a control juice - orange juice without any additional Bimuno and a positive control juice, containing all the components of Bimuno (glucose, galactose and lactose) in the same relative proportions with the exception of B-GOS were developed. Ion Exchange Chromotography analysis was used to test the maintenance of bimuno components after the production process. Data showed that sterilisation had no significant effect on concentration of B-GOS and simple sugars. The three juice formulations were digested under conditions resembling the gastric and small intestinal environments. Main bacterial groups of the faecal microbiota were evaluated throughout the colonic model study using 16S rRNA-based fluorescence in situ hybridization (FISH). Potential effects of supplementation of the juices on microbial metabolism were studied measuring short chain fatty acids (SCFAs) using gas chromatography. Furthermore, B-GOS juices showed positive modulations of the microbiota composition and metabolic activity. In particular, numbers of faecal bifidobacteria and lactobacilli were significantly higher when B-GOS juice was fermented compared to controls. Furthermore, fermentation of B-GOS juice resulted in an increase in Roseburia subcluster and concomitantly increased butyrate production, which is of potential benefit to the host. In conclusion, this study has shown B-GOS within orange juice can have a beneficial effect on the fecal microbiota