Response of populations of human faecal bacteria to viscosity in vitro

The effects of viscosity on cultures of human faecal bacteria in vitro were studied. The composition of the microbial populations which developed during in vitro incubation were affected by viscosity. The proportion of Bacteroides species was decreased at high viscosities and it is concluded that the 'viscotolerance' of a bacterial population contributes towards its composition. Viscosity alters the enzyme activity of cultures. Cell-associated total, and specific, beta-galactosidase activities of cultures of human faecal bacteria were decreased at high viscosity.     

In vitro fermentation of chitosan derivatives by mixed cultures of human faecal bacteria

Stirred, pH controlled batch cultures were carried out with faecal inocula and various chitosans to investigate the fermentation of chitosan derivatives by the human gut flora. Changes in bacterial levels and short chain fatty acids were measured over time. Low, medium and high molecular weight chitosan caused a decrease in bacteroides, bifidobacteria, clostridia and lactobacilli. A similar pattern was seen with chitosan oligosaccharide (COS). Butyrate levels also decreased. A three-stage fermentation model of the human colon was used for investigation of the metabolism of COS. In a region representing the proximal colon, clostridia decreased while lactobacilli increased. In the region representing the transverse colon, bacteroides and clostridia increased. Distally a small increase in bacteroides occurred. Butyrate levels increased. Under the highly competitive conditions of the human colon, many members of the microflora are unable to compete for chitosans of low, medium or high molecular weight. COS were more easily utilised and when added to an in vitro colonic model led to increased production of butyrate, but some populations of potentially detrimental bacteria also increased.     

Quantification of anammox populations enriched in an immobilized microbial consortium with low levels of ammonium nitrogen and at low temperature

The prebiotic effect of a pectic oligosaccharide-rich extract enzymatically derived from bergamot peel was studied using pure and mixed cultures of human faecal bacteria. This was compared to the prebiotic effect of fructo-oligosaccharides (FOS). Individual species of bifidobacteria and lactobacilli responded positively to the addition of the bergamot extract, which contained oligosaccharides in the range of three to seven. Fermentation studies were also carried out in controlled pH batch mixed human faecal cultures and changes in gut bacterial groups were monitored over 24 h by fluorescent in situ hybridisation, a culture-independent microbial assessment. Addition of the bergamot oligosaccharides (BOS) resulted in a high increase in the number of bifidobacteria and lactobacilli, whereas the clostridial population decreased. A prebiotic index (PI) was calculated for both FOS and BOS after 10 and 24 h incubation. Generally, higher PI scores were obtained after 10 h incubation, with BOS showing a greater value (6.90) than FOS (6.12).     

Enrichment of bifidobacteria from human gut contents by oligofructose using continuous culture

Abstract Chemostat cultures of human faecal bacteria were used to determine the bifidogenic effect of oligofructose, a fermentable carbohydrate found in a number of plants. In single stage continuous culture, oligofructose preferentially enriched for bifidobacteria, in comparison to sucrose and inulin. This stimulatory effect was enhanced at a high dilution rate, high substrate concentration and low pH. These parameters are likely to approximate to those that occur in the proximal colon. Studies with a three-stage continuous culture model of the large intestine confirmed the bifidogenic effect of oligofructose. These in vitro data indicate that an increase in the concentration of fructose-based oligosaccharides in the diet may alter the balance of the gut microflora towards bifidobacteria, a purported health-promoting genus.     

Effects of different digestible carbohydrates on bile acid metabolism and SCFA production by human gut micro-flora grown in an in vitro semi-continuous culture

The main source of carbon in the human large intestine comes from carbohydrates like starches and oligosaccharides which remain unchanged by gastric digestion. These polysaccharides are metabolised in the colon by saccharolytic bacteria whose composition is dependent upon the substrate availability. Among the metabolites produced, the short-chain fatty acids (SCFA) are important for colon function and to prevent diseases. In particular, butyrate affects several cellular functions (proliferation, membrane synthesis, sodium absorption), and it has been shown to be protective against colorectal cancer. In addition, faecal bacteria are responsible for the conversion of primary bile acids (BA) to secondary BA, which are considered tumor promoters. In this study we investigated the in vitro effect of different substrates (CrystaLean starch, xylo-oligosaccharides, corn starch) supplied to human faecal micro-flora, on the SCFA production, on the bowel micro-flora composition and on the primary BA conversion rate. In addition, with corn starch as substrate, we considered the effect of enriching normal human faecal micro-flora with lactobacilli and bifidobacteria, on the above reported parameters.     

In vitro measurement of the impact of human milk oligosaccharides on the faecal microbiota of weaned formula-fed infants compared to a mixture of prebiotic fructooligosaccharides and galactooligosaccharides

Aims: To investigate the impact of human milk oligosaccharides (HMOs) from a single donor (SO), HMOs from multiple donors (PO), a fructooligosaccharides and galactooligosaccharides mixture (FG) on the composition of a batch culture inoculated with faecal microbiota from formula‐fed infants. Methods and Results: Three substrates were compared using 24‐h pH‐controlled anaerobic batch cultures inoculated with infant faecal slurries. Changes in bacterial populations, short‐chain fatty acids (SCFA) production and bacterial 16S rRNA gene profiles were determined. All three substrates significantly increased numbers of bifidobacteria, bacteroides and those aligning with the clostridial cluster XIVa. Neither the FG nor the HMOs substrates supported the growth of the Clostridium perfringens–histolyticum group. SCFA production corresponded to changes observed in bacterial populations. Denaturing gradient gel electrophoresis fingerprint analysis showed a distinct profile of faecal bacteria present in each infant. Conclusions: HMOs modulated infant faecal culture composition in a similar manner to the prebiotic mixture FG in vitro. Significance and Impact of the Study: This is the first demonstration of the impact of pure HMOs on the mixed culture of infant faecal bacteria. HMOs induced the growth of several saccharolytic bacterial groups and may thus play a role in the health‐promoting attributes of human breast milk and have an extended significance in infant diet during/after weaning.     

Unsaturated oligogalacturonic acids are generated by in vitro treatment of pectin with human faecal flora.

Pectins with a degree of esterification (DE) of 95, 66, 34 and 0%, respectively, were incubated in vitro with human faecal flora (pH 7.8). The concentration and composition of oligogalacturonic acids (oligoGalA) generated were determined using high-performance thin-layer chromatography (HPTLC) with UV and colorimetric detection. In the first period of the anaerobic degradation, the pectin macromolecules were fragmented into unsaturated oligoGalA as intermediate products by the action of bacterial pectate lyases. Depending on the incubation time and the DE of pectin, the amount of unsaturated oligoGalA having different degrees of polymerization changed continuously. These oligoGalA were present in the cultures for some hours. Mixtures of unsaturated di-, tri- and tetraGalA were the end products of a pectate lyase action. Later, the oligoGalA disappear as a result of their further fermentation by the gastrointestinal microflora under formation of short-chain fatty acids (SCFA). Low-esterified pectins were depolymerized and fermented faster than the highly esterified by the human faecal flora in vitro. Furthermore, a mixture of unsaturated oligoGalA prepared from pectic acid by the action of pectate lyase from Erwinia carotovora was completely fermented by human faecal flora.     

In vivo activity of nifurzide and nifuroxazide in intestinal bacteria in man and gnotobiotic mice

Although Gram-negative enteropathogenic bacteria are the target strains of nifurzide and nifuroxazide treatments, neither drug affected faecal counts of in vitro -susceptible Enterobacteriaceae in healthy volunteers. This absence of activity was shown to be due to the poor solubility of the drugs tested. Therefore, effect of high doses of nifurzide was investigated in gnotobiotic mice. Activity against in vitro susceptible enteropathogens was then observed. Normal bacterial cells were replaced in the faeces by elongated, nonseptate and unflagellated mutants. Moreover, the resistance to colonization by enterotoxigenic Escherichia coli and Shigella flexeri of an anaerobic flora of human origin was sharply decreased.     

In vivo activity of nifurzide and nifuroxazide in intestinal bacteria in man and gnotobiotic mice

Although Gram-negative enteropathogenic bacteria are the target strains of nifurzide and nifuroxazide treatments, neither drug affected faecal counts of in vitro-susceptible Enterobacteriaceae in healthy volunteers. This absence of activity was shown to be due to the poor solubility of the drugs tested. Therefore, effect of high doses of nifurzide was investigated in gnotobiotic mice. Activity against in vitro susceptible enteropathogens was then observed. Normal bacterial cells were replaced in the faeces by elongated, nonseptate and unflagellated mutants. Moreover, the resistance to colonization by enterotoxigenic Escherichia coli and Shigella flexeri of an anaerobic flora of human origin was sharply decreased.     

Important impacts of intestinal bacteria on utilization of dietary amino acids in pigs

Bacteria in pig intestine can actively metabolize amino acids (AA). However, little research has focused on the variation in AA metabolism by bacteria from different niches. This study compared the metabolism of AA by microorganisms derived from the lumen and epithelial wall of the pig small intestine, aiming to test the hypothesis that the metabolic profile of AA by gut microbes was niche specific. Samples from the digesta, gut wall washes and gut wall of the jejunum and ileum were used as inocula. Anaerobic media containing single AA were used and cultured for 24 h. The 24-h culture served as inocula for the subsequent 30 times of subcultures. Results showed that for the luminal bacteria, all AA concentrations except phenylalanine in the ileum decreased during the 24-h in vitro incubation with a increase of ammonia concentration, while 4 AA (glutamate, glutamine, arginine and lysine) in the jejunum decreased, with the disappearance rate at 60–95 %. For tightly attached bacteria, all AA concentrations were generally increased during the first 12 h and then decreased coupled with first a decrease and then an increase of ammonia concentration, suggesting a synthesis first and then a catabolism pattern. Among them, glutamate in both segments, histidine in the jejunum and lysine in the ileum increased significantly during the first 12 h and then decreased at 24 h. The concentrations of glutamine and arginine did not change during the first 12 h, but significantly decreased at 24 h. Jejunal lysine and ileal threonine were increased for the first 6 or 12 h. For the loosely attached bacteria, there was no clear pattern for the entire AA metabolism. However, glutamate, methionine and lysine in the jejunum decreased after 24 h of cultivation, while glutamine and threonine in the jejunum and glutamine and lysine in the ileum increased in the first 12 h. During subculture, AA metabolism, either utilization or synthesis, was generally decreased with disappearance rate around 20–40 % for most of AA and negligible for branch chained AA (BCAA). However, the disappearance rate of lysine in each group was around 90 % throughout the subculture, suggesting a high utilization of lysine by bacteria from all three compartments. Analysis of the microbial community during the 24-h in vitro cultivation revealed that bacteria composition in most AA cultures varied between different niches (lumen and wall-adherent fractions) in the jejunum, while being relatively similar in the ileum. However, for isoleucine and leucine cultures, bacteria diversity was similar between the luminal fraction and tightly attached fraction, but significantly higher than in the loosely attached fraction. For glutamine and valine cultures, bacteria diversity was similar between the luminal and loosely attached fractions, but lower than that of tightly attached bacteria. After 30 subcultures, bacteria diversity in arginine, valine, glutamine, and leucine cultures varied between niches in the jejunum while being relatively stable in the ileum, consistent with those in the 24-h in vitro cultures. The findings may suggest that luminal bacteria tended to utilize free AA, while tightly attached adherent bacteria seemed in favor of AA synthesis, and that small intestinal microbes contributed little to BCAA metabolism.     

The Effect of Cadmium on Indicator Bacteria in Sewage

The effect of cadmium (Cd) on the number of different faecal indicator bacteria in sewage, and on species composition of different indicator bacteria, was studied. Different amounts of Cd were added to aliquots of a sewage sample, and after 0, 4% and 24 h of Cd exposure at 20°C conforms, faecal coliforms, faecal streptococci and Clostridium perfringens were enumerated by the membrane filter method. The Cd-induced reduction in the number of coliforms and faecal coliforms during exposure was found to be greater than the decrease in the number of faecal streptococci. In the case of C. perfringens the Cd concentrations used produced no observable effect on the cell number. The addition of Cd changed the faecal coliforms and faecal streptococci density relationship. Escherichia coli seems to be more resistant to Cd than other coliforms and Streptococcus faecalis var. liquefaciens and Streptococcus durans more resistant to Cd than other faecal streptococci. No influence of Cd on gas production by faecal coliforms was observed. Faecal streptococci and C. perfringens seem to be better indicator bacteria than coliforms and faecal coliforms in evaluating the hygienic quality of Cd polluted sewage.     

Metabolism of unsaturated bile acids and androstanes by human faecal bacteria

The metabolism of unsaturated bile acids and androstanes by mixed human faecal cultures has been studied. The reactions observed were mainly reductive. Unsaturated 4-ene-3-oxo and 1,4-diene-3-oxo bile acids were reduced in Ring A. 5 beta-3-Oxo bile acids were reduced to 5 beta-3-hydroxy bile acids. 4-Ene, 1,4-diene and 4,6-diene-3,17-dioxo-androstanes were reduced in Ring A with concomitant reduction of oxo groups to hydroxyl groups. The Gram-negative facultative anaerobic faecal bacteria are implicated in the reductive process, whilst the genus Clostridium does not appear to be important. Inclusion of menadione, a synthetic form of vitamin K, retards the reductive process.     

Production and metabolism of lignans by the human faecal flora

Lignans have, until recently, been found only in plants. Enterolactone and enterodiol are the major lignans present in the urine of humans and have a potential physiological protective role against cancer. It has been shown that these compounds can be formed in vitro by human faecal flora and that enterodiol is oxidized to enterolactone by bacteria that are present in stools at a concentration of up to 10(3)/g. It was also possible to produce both of these lignans in vitro from linseeds and from secoisolariciresinol, a precursor present in linseed, by bacteria present in stools, at a concentration of between 10(3) and 10(4)/g. Enterolactone was produced from matairesinol, a more abundant plant lignan than secoisolariciresinol, after incubation with a mixed faecal flora under both aerobic and anaerobic conditions. In each case conversion was dependent on the presence of viable bacteria. These findings indicate that a number of different pathways operate to produce enterolactone and enterodiol depending on the ingested dietary precursor.     

Characteristic faecal flora of NC mice

The composition of faecal flora of NC mice was compared with that of CF #1 mice. NC- and CF #1-germfree (GF) mice were cage-mated with NC- or CF #1-conventional (CV) mice in an isolator. The faecal flora of these ex-GF mice was dependent on the recipient mouse strain modifying colonization by the donor mouse bacteria. Although NC- and CF #1-pups removed by hysterectomy were fostered to different strains, almost all these mice at 8 weeks old had a strain characteristic pattern of faecal flora regardless of the foster strains. In GF mice mono-associated with a Lactobacillus strain or a Bifidobacterium strain isolated from faeces of CV mice, the numbers of these bacteria in the stomach and small intestine of NC mice were lower than those of CF #1 mice. In GF mice associated with chloroform-treated faeces of CV mice, and a Lactobacillus strain or a Bifidobacterium strain, the numbers of these bacteria in the stomach and all parts of the intestine of NC mice were considerably lower than those of CF #1 mice. These results suggested that the composition of faecal flora of NC mice were characteristic, i.e. the fact that the numbers of lactobacilli were low compared with CF #1 mice with ordinary faecal flora and the colonization of bifidobacteria, peptococcaceae and eubacteria on ES agar in NC mice intestine differed, was due to genetic factors.     

Age and Microenvironment Outweigh Genetic Influence on the Zucker Rat Microbiome

Animal models are invaluable tools which allow us to investigate the microbiome-host dialogue. However, experimental design introduces biases in the data that we collect, also potentially leading to biased conclusions. With obesity at pandemic levels animal models of this disease have been developed; we investigated the role of experimental design on one such rodent model. We used 454 pyrosequencing to profile the faecal bacteria of obese (n = 6) and lean (homozygous n = 6; heterozygous n = 6) Zucker rats over a 10 week period, maintained in mixed-genotype cages, to further understand the relationships between the composition of the intestinal bacteria and age, obesity progression, genetic background and cage environment. Phylogenetic and taxon-based univariate and multivariate analyses (non-metric multidimensional scaling, principal component analysis) showed that age was the most significant source of variation in the composition of the faecal microbiota. Second to this, cage environment was found to clearly impact the composition of the faecal microbiota, with samples from animals from within the same cage showing high community structure concordance, but large differences seen between cages. Importantly, the genetically induced obese phenotype was not found to impact the faecal bacterial profiles. These findings demonstrate that the age and local environmental cage variables were driving the composition of the faecal bacteria and were more deterministically important than the host genotype. These findings have major implications for understanding the significance of functional metagenomic data in experimental studies and beg the question; what is being measured in animal experiments in which different strains are housed separately, nature or nurture?     

Microbial characterization during composting of municipal solid waste

This study investigates the prevailing physico-chemical conditions and microbial community; mesophilic bacteria, yeasts and filamentous fungi, bacterial spores, Salmonella and Shigella as well as faecal indicator bacteria: total coliforms, faecal coliforms and faecal Streptococci, present in a compost of municipal solid waste. Investigations were conducted in a semi-industrial pilot plant using a moderate aeration during the composting process. Our results showed that: (i) auto-sterilization induced by relatively high temperatures (60–55°C) caused a significant change in bacterial communities. For instance, Escherichia coli and faecal Streptococci populations decreased, respectively, from 2×10⁷ to 3.1×10³ and 10⁷ to 1.5×10³ cells/g waste dry weight (WDW); yeasts and filamentous fungi decreased from 4.5×10⁶ to 2.6×10³ cells/g WDW and mesophilic bacteria were reduced from 5.8×10⁹ to 1.8×10⁷ bacteria/g WDW. On the other hand, the number of bacterial spores increased at the beginning of the composting process, but after the third week their number decreased notably; (ii) Salmonella disappeared completely from compost by the 25th day as soon as the temperature reached 60°C; and (iii) the bacterial population increased gradually during the cooling phase. While Staphylococci seemed to be the dominant bacteria during the mesophilic phase and at the beginning of the thermophilic phase, bacilli predominated during the remainder of the composting cycle. The appearance of gram-negative rods (opportunistic pathogens) during the cooling phase may represent a serious risk for the sanitary quality of the finished product intended for agronomic reuse. Compost sonication for about 3 min induced the inactivation of delicate bacteria, in particular gram-negatives. By contrast, gram-positive bacteria, especially micrococcus, spores of bacilli, and fungal propagules survived, and reached high concentrations in the compost.     

Soil: the environmental source of Escherichia coli and Enterococci in Guam's streams

We have previously documented that faecal indicator bacteria (Escherichia coli, faecal coliform, enterococci) recommended by the U.S. Environmental Protection Agency (USEPA) to establish recreational water quality standards are naturally found in high concentrations in the surface and subsurface of soils in Hawaii. Rain, the source of all streams in Hawaii, washes the soil sources of faecal bacteria into all the streams of Hawaii, at concentrations which consistently exceed the USEPA recreational water quality standards. The objective of this study was to test the hypothesis that faecal bacteria are able to establish themselves in the soil environments of tropical islands by conducting the same study in Guam, a tropical pacific island with warmer temperatures and higher humidity than Hawaii. The same methods and study design used in Hawaii was used in Guam. The results of the study conducted in Guam revealed that all streams contain consistently high concentrations of faecal coliform, E. coli, and enterocci which exceeded the old USEPA recreational water quality standard of 200 faecal coliform/100 ml as well as the new water quality standards of 126 E. coli/100 ml or 33 enterococci/100 ml. These same faecal indicator bacteria were recovered in high concentrations in surface and subsurface (18-36 cm depth) soil samples in Guam. Limited coastal water analysis showed that most coastal marine waters contain low concentrations of faecal bacteria but coastal waters impacted by stream run-off showed elevated levels of faecal bacteria. The results of this study support the hypothesis that environmental conditions in the tropical areas of the world can support the growth and establishment of populations of faecal bacteria in the soil. Thus, soil becomes an environmental, non-faecal source of faecal indicator bacteria. These results indicate that USEPA water quality standards may not be directly applicable to tropical island environments.     

Effects of chronic triclosan exposure upon the antimicrobial susceptibility of 40 ex-situ environmental and human isolates

BACKGROUND: Triclosan (TCS) exposure of Escherichia coli selects for tolerant clones, mutated in their enoyl-acyl carrier protein reductase (FabI). It has been inferred that this phenomenon is widespread amongst bacterial genera and might be associated with resistance to third party agents. METHODS: Ex-situ, low passage isolates of enteric, human axilla, human oral origin and bacteria isolated from a domestic drain, together with selected type cultures were exposed to escalating concentrations of TCS over 10 passages using a gradient plate technique. One fresh faecal isolate of E. coli was included as a positive control. TCS susceptibility was determined for all strains before and after exposure, whilst enteric isolates were additionally assessed for susceptibility towards chlorhexidine, tetracycline, chloramphenicol, nalidixic acid and ciprofloxacin, and the oral isolates towards chlorhexidine, tetracycline and metronidazole. RESULTS: Triclosan exposure of E. coli markedly decreased TCS susceptibility. TCS susceptibility also decreased for Klebsiella oxytoca, Aranicola proteolyticus and Stenotrophomonas maltophilia. Susceptibility of the remaining 35 strains to TCS and the other test agents remained unchanged. CONCLUSIONS: These data suggest that selection for high level resistance by TCS exposure is not widespread and appears to be confined to certain enteric bacteria, especially E. coli. Change in TCS susceptibility did not affect susceptibility towards chemically unrelated antimicrobials. SIGNIFICANCE AND IMPACT: Acquired high-level TCS resistance is not a widespread phenomenon.     

Rapid and bacteria-dependent in vitro hydrolysis of iodothyronine-conjugates by intestinal contents of humans and rats

Faecal suspensions from healthy humans, conventional (CV), germ-free (GF) and intestine-decontaminated (ID) rats were tested for the in vitro hydrolysis of 125I-labelled iodothyronine sulphates and 3,3',5-triiodothyronine glucuronide (T3G). Whereas 20-fold diluted human and CV rat faecal suspensions hydrolyzed up to 90% of the sulphates, no hydrolysis was observed in 5 times diluted faecal suspensions of GF and ID rats. These results add further weight to the assumption that intestinal iodothyronine sulphatase activity is of bacterial origin. Twenty times diluted human and CV rat faecal suspensions hydrolyzed approximately 80% of the T3G. In the 5 times diluted faecal suspensions of GF and ID rats up to 15% hydrolysis of T3G was still observed. It was concluded that the major part of the gastrointestinal iodothyronine glucuronidase activity is produced by bacteria. The remaining activity presumably originates from gastrointestinal mucosal cells.