Animal Intestinal Microbiota Research Articles

Improved Quantitative Real-Time PCR Protocol for Detection and Quantification of Methanogenic Archaea in Stool Samples

Methanogenic archaea are an important component of the human and animal intestinal microbiota, and yet their presence is rarely reported in publications describing the subject. One of the methods of quantifying the prevalence of methanogens is quantitative real-time PCR (qPCR) of the methanogen-specific mcrA gene, and one of the possible reasons for detection failure is usually a methodology bias. Here, we refined the existing protocol by changing one of the primers and improving the conditions of the qPCR reaction. As a result, at the expense of a slightly lower yet acceptable PCR efficiency, the new assay was characterized by increased specificity and sensitivity and a wider linear detection range of 7 orders of magnitude. The lowest copy number of mcrA quantified at a frequency of 100% was 21 copies per reaction. The other validation parameters tested, such as reproducibility and linearity, also gave satisfactory results. Overall, we were able to minimize the negative impacts of primer dimerization and other cross-reactions on qPCR and increase the number of not only detectable but also quantifiable stool samples-or in this case, chicken droppings.

In vitro selection and characterization of probiotic properties in eight lactobacillus strains isolated from cocoa fermentation

Traditionally, probiotic microorganisms are isolated from human and animal intestinal microbiota. However, the demand for diversification of biofunctional products has driven the search for new sources of probiotic candidates, such as fermented foods and vegetables. The present study found that strains isolated from the fermentation of fine cocoa from southern Bahia have biotechnological potential for use as a probiotic, since they showed capacity for self-aggregation and co-aggregation, antimicrobial activity against intestinal pathogens and resistance to gastrointestinal transits. Scores of importance for each property were established in order to more accurately assess the probiotic potential of the strains. The tests carried out contemplate the criteria previously established for the selection of probiotic candidates.

Transcriptome modulation by in ovo delivered Lactobacillus synbiotics in a range of chicken tissues

Synbiotics are the bioactive compounds that synergistically combine effects of prebiotics and probiotics. In poultry, synbiotics can be used to reprogram animal's intestinal microbiota upon perinatal in ovo injection on day 12 of eggs incubation. Optimally composed synbiotic delivered in ovo efficiently stimulates the host's intestinal microflora, which in turn exerts beneficial effects on the host and improves its physiological functions. The aim of the study was to estimate long-term changes in the chicken transcriptome after a single in ovo administration of two different synbiotics. On day 12 of eggs incubation, 5850 eggs of broiler chicken were distributed to experimental groups and injected with synbiotic 1 (S1)- Lactobacillus salivarius with galactooligosaccharides (GOS) or synbiotic 2 (S2)– Lactobacillus plantarum with raffinose family oligosaccharides (RFO). On day 21 post-hatching cockerels were sacrificed and immunological (cecal tonsils and spleen), intestinal (jejunum) and metabolic (liver) tissues were collected (n = 5). Isolated RNA served as a template for the whole-transcriptome analysis using GeneChip Chicken Gene 1.1. ST Array Strip (Affymetrix). Data analysis was performed using Affymetrix Expression Console and Transcriptome Analysis Console software, Venn diagrams, DAVID and CateGOrizer. The highest number of Differentially Expressed Genes (DEG) was detected in cecal tonsils (160 DEG) after S1 in ovo injection, and in liver (159 DEG) after S2 injection. The influence of S1 on transcriptome modulation was demonstrated by a strong activation of the genes taking part in the pathways related to metabolism and immune response in cecal tonsils. S2 injection led to modulation of the gene expression associated with metabolic and developmental signaling pathways in the liver. Obtained results let us conclude that synbiotics delivered in ovo have significant impact on chicken transcriptome and their effect depends on the composition of the bioactive compound.

Prohibition of antibiotic growth promoters has affected the genomic profiles of Lactobacillus salivarius inhabiting the swine intestine.

After the introduction of a ban on the use of antibiotic growth promoters (AGPs) for livestock, the feeding environment, including the composition of animal intestinal microbiota, has changed rapidly. We hypothesized that the microbial genomes have also been affected by this legal prohibition, and investigated an important member of the swine gut microbiota, Lactobacillus salivarius, with a pan-genomic approach. Here, we isolated 21 L. salivarius strains composed of 6 strains isolated before the AGP prohibition (SBPs) and 15 strains isolated after the AGP prohibition (SAPs) at an interval of a decade, and the draft genomes were generated de novo. Several genomic differences between SBPs and SAPs were identified, although the number and function of antibiotic resistance genes were not different. SBPs showed larger genome size and a higher number of orthologs, as well as lower genetic diversity, than SAPs. SBPs had genes associated with the utilization of L-rhamnose and D-tagatose for energy production. Because these sugars are also used in exopolysaccharide (EPS) synthesis, we tried to identify differences in biofilm formation-associated genes. The genes for the production of EPSs and extracellular proteins were different in terms of amino acid sequences. Indeed, SAPs formed dense biofilm and survived better than SBPs in the swine intestinal environment. These results suggest that SAPs have evolved and adapted to protect themselves from new selection pressure of the swine intestinal microenvironment by forming dense biofilms, adopting a distinct antibiotic resistance strategy. This finding is particularly important to understand the evolutionary changes in host-microbe interaction and provide detailed insight for the development of effective probiotics for livestock.

Application of genomic technologies to measure and monitor antibiotic resistance in animals.

One of the richest reservoirs of antibiotic-resistant bacteria and genes, animal intestinal microbiota contributes to the spread of antibiotic resistance in the environment and, potentially, to human pathogens. Both culture-based genomic technology and culture-independent metagenomics have been developed to investigate the abundance and diversity of antibiotic resistance genes. The characteristics, strengths, limitations, and challenges of these genomic approaches are discussed in this review in the context of antibiotic resistance in animals. We also discuss the advances in single-cell genomics and its potential for surveillance of antibiotic resistance in animals.

Characterization of Escherichia coli strains from red deer (Cervus elaphus) faeces in a Mexican protected natural area

Escherichia coli is a commensal bacterium from the human and animal intestinal microbiota; however, some strains may be pathogenic and can cause a wide range of intestinal and extraintestinal diseases. The presence of pathogenic strains in wild animals, such as deer, may constitute a risk for humans and other animals. Thus, the aim of this study was to determine the antimicrobial resistance (AMR), serotype, phylogenetic groups and virulence genes (VGs) of 22 E. coli isolates obtained from red deer faeces (Cervus elaphus). Results showed that most isolates (17/22) were susceptible to the antimicrobials assessed, whereas only five of them were resistant to some of the tested antimicrobials (mainly to β-lactamics, and to trimethoprim/sulphamethoxazole). Regarding the phylogenetic groups, 19 isolates belonged to the B1 group and three to the B2 group. The identified VGs corresponded to stx1 (17/22), stx2 (12/22), estA (12/22), bfpA (6/22), and eae (4/22). Interestingly, hybrid strains containing VGs that belonged to STEC and EPEC (stx1, stx2, bfpA (n = 2) and stx1, bfpA (n = 1)) and to STEC and ETEC (stx1, estA (n = 1), stx2, estA (n = 1), stx1, stx2, bfpA (n = 6) and eae, stx1, stx2, bfpA (n = 1)) pathotypes were found. The identified serotypes were O:H21, O70:NM, O91:NM, O5:NM, O:NM, O23:H16, O23:H25, O38:H25, O6:H34, O6:NT and O75:H9. This work represents the first insight of potentially pathogenic E. coli strains in red deer from Mexico, and it establishes the importance of deer strain characterization. Moreover, the high frequency (12/22) of hybrid strains found in this study, along with a presumably pathogenic potential profile, could represent an additional risk for humans and animals, therefore further investigations on this issue would be needed.

Impact of antibiotics used as growth promoters on bacterial resistance

For decades intensive husbandry has more or less been based on the use of antibiotics in sub-minimum inhibitory concentrations (sub-MIC) aimed at growth promotion. Continuous exposure of animal intestinal microbiota, including opportunistic zoonotic pathogens, to sub-MIC poses a pressure to selection and spread of bacteria strains with developed mechanism of antibiotic resistance. These bacteria may be transferred to people either by direct contact with farm animals or indirectly, via the food chain. Although in the EU a ban on the use of antibiotics as growth promoters was imposed in 2006, in many countries, including the largest producers and consumers of antibiotics in the world, it has yet to be done. Given that we are faced with a global problem of the loss of the efficacy of several antibiotic classes which are available for the treatment of human bacterial infections, it is unacceptable that antibiotic use in husbandry is not under global control. Reduction in antibiotic use in clinical practice in human medicine remains in dispute, but non-therapeutic use in husbandry remains a field in which much can be done to contribute substantially to the extension of antibiotic effectiveness and health care of future generations.

Enterotoxigenic and non-enterotoxigenic Bacteroides fragilis from fecal microbiota of children.

Enterotoxigenic Bacteroides fragilis (ETBF) is an important part of the human and animal intestinal microbiota and is commonly associated with diarrhea. ETBF strains produce an enterotoxin encoded by the bft gene located in the B. fragilis pathogenicity island (BfPAI). Non-enterotoxigenic B. fragilis (NTBF) strains lack the BfPAI and usually show two different genetic patterns, II and III, based on the absence or presence of a BfPAI-flanking region, respectively. The incidence of ETBF and NTBF strains in fecal samples isolated from children without acute diarrhea or any other intestinal disorders was determined. All 84 fecal samples evaluated were B. fragilis-positive by PCR, four of them harbored the bft gene, 27 contained the NTBF pattern III DNA sequence, and 52 were considered to be NTBF pattern II samples. One sample was positive for both ETBF and NTBF pattern III DNA sequences. All 19 B. fragilis strains isolated by the culture method were bft-negative, 9 belonged to pattern III and 10 to pattern II. We present an updated overview of the ETBF and NTBF incidence in the fecal microbiota of children from Sao Paulo City, Brazil.

A sensitive bacterial-growth-based test reveals how intestinal Bacteroides meet their porphyrin requirement.

BackgroundBacteroides sp. are dominant constituents of the human and animal intestinal microbiota require porphyrins (i.e., protoporphyrin IX or iron-charged heme) for normal growth. The highly stimulatory effect of porphyrins on Bacteroides growth lead us to propose their use as a potential determinant of bacterial colonization. However, showing a role for porphryins would require sensitive detection methods that work in complex samples such as feces.ResultsWe devised a highly sensitive semi-quantitative porphyrin detection method (detection limit 1-4 ng heme or PPIX) that can be used to assay pure or complex biological samples, based on Bacteroides growth stimulation. The test revealed that healthy colonized or non-colonized murine and human hosts provide porphyrins in feces, which stimulate Bacteroides growth. In addition, a common microbiota constituent, Escherichia coli, is shown to be a porphyrin donor, suggesting a novel basis for intestinal bacterial interactions.ConclusionsA highly sensitive method to detect porphyrins based on bacterial growth is devised and is functional in complex biological samples. Host feces, independently of their microbiota, and E. coli, which are present in the intestine, are shown to be porphryin donors. The role of porphyrins as key bioactive molecules can now be assessed for their impact on Bacteroides and other bacterial populations in the gut.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0616-0) contains supplementary material, which is available to authorized users.

Protocol for Metatranscriptomic analysis of Intestinal Microbiota

AbstractThe objective of this publication is to provide the detailed protocol for metartancriptomisc studies of animal intestinal microbiota. The protocol describes isolation of high quality microbial community RNA from the mammalian intestinal content, subsequent mRNA enrichment, cDNA synthesis and sequencing. Twelve libraries were prepared, pooled in equimolar concentrations into a single library and sequenced on one GS Titanium 70 × 75 picotiter plate, following this protocol. The total number of reads obtained for 12 libraries was 1,155,062 (average 96,000 per library) and the combined size of 12 libraries was 521 million bases (average 43 million bases per library). The reported size of non-ribosomal RNA library fraction is ~15%, the fraction of non-ribosomal reads is ~17%. Hence we described a robust technique for metranscriptomic studies of animal intestinal microbiota. The double stranded cDNAs, prepared following this protocol, are suitable for pyrosequencing (454, Illumina), clone library construction or could be used to archive and store metaranscriptomic samples.

Protocol for Metatranscriptomic analysis of Intestinal Microbiota

The objective of this publication is to provide the detailed protocol for metartancriptomisc studies of animal intestinal microbiota. The protocol describes isolation of high quality microbial community RNA from the mammalian intestinal content, subsequent mRNA enrichment, cDNA synthesis and sequencing. Twelve libraries were prepared, pooled in equimolar concentrations into a single library and sequenced on one GS Titanium 70 × 75 picotiter plate, following this protocol. The total number of reads obtained for 12 libraries was 1,155,062 (average 96,000 per library) and the combined size of 12 libraries was 521 million bases (average 43 million bases per library). The reported size of non-ribosomal RNA library fraction is ~15%, the fraction of non-ribosomal reads is ~17%. Hence we described a robust technique for metranscriptomic studies of animal intestinal microbiota. The double stranded cDNAs, prepared following this protocol, are suitable for pyrosequencing (454, Illumina), clone library construction or could be used to archive and store metaranscriptomic samples.

Adherence and invasion of Bacteroidales isolated from the human intestinal tract

Members of the genera Bacteroides and Parabacteroides are important constituents of both human and animal intestinal microbiota, and are significant facultative pathogens. In this study, the ability of Bacteroides spp. and Parabacteroides distasonis isolated from both diarrhoeal and normal stools (n = 114) to adhere to and invade HEp-2 cells was evaluated. The presence of putative virulence factors such as capsule and fimbriae was also investigated. Adherence to HEp-2 cells was observed in 75.4% of the strains, which displayed non-localized clusters. Invasion was observed in 37.5% and 26% of the strains isolated from diarrhoeal and non-diarrhoeal stools, respectively. All strains displayed a capsule, whereas none of them showed fimbriae-like structures. This is the first report of the ability of Bacteroides spp. and P. distasonis to adhere to and invade cultured HEp-2 epithelial cells.

Ex-germfree mice harboring intestinal microbiota derived from other animal species as an experimental model for ecology and metabolism of intestinal bacteria.

Ex-germfree (GF) animals harboring intestinal microbiota derived from other animal species, e.g. human-flora-associated (HFA) and pig-flora-associated (PFA) mice, have been considered as a tool for studying the ecology and metabolism of intestinal bacteria of man and animals. Human fecal microbiota was transferred into the intestines of the mice with minor modification by inoculating GF mice with human fecal suspensions. Interestingly, bifidobacteria were eliminated from some of the HFA mouse groups, whereas other dominant bacterial groups remained constant. Elimination of bifidobacteria appeared to be dependent on the composition of microbiota in the inoculated sample. Human fecal microbiota established in the intestines of the HFA mice reproduced in the intestine of offspring of these HFA mice and of cage-mated ex-GF mice without any remarkable change in composition. Although the HFA mice could be used for studying the effects of diet on human intestinal microbiota, the metabolism of microbiota of HFA mice reflected that of human feces with respect to some metabolic activities but not others. PFA mice were also a good model for studying the ecosystem of pig fecal microbiota and the control of short chain fatty acids in pig intestines, but not for studying putrefactive products generated in pig intestines. In conclusion, HFA and PFA mice provide a stable and valuable tool for studying the ecosystem and metabolism of the human and animal intestinal microbiota, but they have some limitations as a model.