Bacterial Cellular Fatty Acids Research Articles

Profiling of bacterial cellular fatty acids by pyrolytic derivatization to 3-pyridylcarbinol esters

Pyrolytic derivatization to 3-pyridylcarbinol esters coupled with gas chromatography-mass spectrometry (GC-MS) was applied to determine cellular fatty acid (CFA) profiles of Escherichia coli, Aerobacter aerogenes, Pseudomonas fluorescens, Bacillus subtilis and Clostridium perfringens. The structures of bacterial CFAs were inferred from the interpretation of mass spectra with electron ionization of the corresponding 3-pyridylcarbinol esters. In the method used, the entire bacterial cells are pyrolyzed in the presence of a derivatizing reagent, and CFA isolation prior to the GC-MS analysis is not required. The method is argued to be a cheap, fast and accurate alternative to classical esterification to 3-pyridylcarbinol esters, while still providing more detailed results than methods based on esterification to methyl esters. Significat reduction in required minimum bacterial mass allows for fast identification of the microorganisms, in particular slow growing bacteria species.

Profiling of Bacterial Cellular Fatty Acids by Pyrolytic Derivatization to 3-Pyridylcarbinol Esters

Profiling of Bacterial Cellular Fatty Acids by Pyrolytic Derivatization to 3-Pyridylcarbinol Esters

Dynamics of gut colonization and source of intestinal flora in healthy newborn infants

The aim of the study was to evaluate the dynamics of gut colonization and the main source of intestinal bacterial flora in infancy in a quantitative manner using computerized analysis of bacterial cellular fatty acid (CFA) profiles. Each stool was collected from 10 healthy newborn infants during their first 2-7 days of life and a follow-up sample at 6 months of age. Stool samples were collected from mothers and nurses for comparison. Gas-liquid chromatography of the 159 stool samples was used to produce bacterial cellular fatty acid (CFA) profiles by means of a previously developed computerized program. The CFA profiles for the infants fluctuated from hour to hour during the first days of life and resembled those for both the mothers and the nurses, doing so all the more in the case of the five infants examined 6 months after birth. Gut colonization fluctuated markedly from hour to hour in the perinatal period. The effect of the maternal flora on the initial gut colonization may be less than expected as the fecal flora of infants started to resemble both the fecal flora of the mother as well as that of the first nurse.

Impact of residual and therapeutic doses of ciprofloxacin in the human-flora-associated mice model

A study was conducted to evaluate the effects of therapeutic and residual doses of ciprofloxacin on the human intestinal flora implanted into germ-free mice. Ciprofloxacin was administered daily via drinking water at concentrations to provide doses of 0, 0.125, 1.25, and 12.5 mg/kg b.w. Changes in the intestinal flora composition, alteration in bacterial enzyme activities, fecal short chain fatty acid concentration and bacterial cellular fatty acid profiles, overgrowth of resistant bacteria, and disruption of the colonization barrier were the endpoints evaluated in the feces of human-flora-associated (HFA) mice. Ciprofloxacin at all tested doses decreased significantly the aerobic populations and particularly the population of Enterobacteriaceae. Selection of resistant Bacteroides fragilis group was noticed in HFA mice receiving 12.5 mg/kg b.w. In mice challenged with a Salmonella strain, exogenous Salmonella persisted in the feces of all treated mice indicating that the flora responsible for the colonization barrier effect was disturbed by the antibiotic treatment. None of the studied metabolic parameters of the flora were affected by ciprofloxacin at any dose level. Under the experimental conditions of the study, the no-observed-effect level of ciprofloxacin was found to be less than 0.125 mg/kg b.w.

Bacterial composition of murine fecal microflora is indigenous and genetically guided

The gastrointestinal tract and the microbes colonizing it form a complex ecosystem that has various effects on the well-being of the host. In addition to acute infections, the composition of the gastrointestinal microbiota has been suspected to influence the etiopathogenesis of many chronic diseases, such as rheumatoid arthritis and inflammatory bowel diseases. It has been suggested that the bacterial colonization of the gastrointestinal tract is genetically determined. Using gas–liquid chromatography of bacterial cellular fatty acids we show in this study that modulation of the microbiota by a course of antibiotics is followed by regeneration of the murine intestinal flora depending on the genotype of the host. The mice used in our study were acclimatized to identical living conditions before treatment with ciprofloxacin and clindamycin for 1 week via drinking water. Within a few days of finishing the antibiotic course, the cellular fatty acid profiles of fecal samples resembled those of the pre-course community, showing a considerable indigenous recovery potential. Colonization of the gastrointestinal tract appeared to be genetically regulated since differences in communities between the mouse strains were observed. Our results are in harmony with earlier observations, indicating that the gut community is not established by chance and that it is influenced by host-derived factors.

Intestinal Microflora, Short Chain and Cellular Fatty Acids, Influence of a ProbioticSaccharomyces boulardii

The aim of our investigation was to study whether there are significant changes concerning intestinal flora, short chain fatty acids and cellular fatty acids produced by Saccharomyces boulardii (Ultra-Levure ® from Biocodex) when given orally (1 g daily, 6 consecutive days) to healthy volunteers and to patients with enteral nutrition. These patients were reported to have diarrhoeal diseases improved by S. boulardii treatment. There were no changes for the facultative bacteria with treatment by S. boulardii , however, there were significant changes concerning the gram-positive anaerobes, some clostridia dropping and bifidobacteria appearing with treatment with this probiotic. Concerning the short chain fatty acids there was a significant increase of acetate, butyrate and propionate in the faeces of patients versus the controls after treatment with this probiotic ( p =0.02). Under treatment the acetic acid increased for the controls, 78.31 versus 66.58 mmol kg −1 before treatment, and also for the patients, 87.17 versus 69.88 mmol kg −1 . With treatment for the patients there was a significant increase of butyric acid (16 ± 4.4 vs 10.1 ± 2.9 mmol kg −1 , p =0.004), and propionic acid (26.2 ± 3.8 vs 20.9 ± 3.7 mmol kg −1 , p=0.04). On the other hand the supplementation with this probiotic allowed the amount of bacterial cellular fatty acids to become normal for the patients with enteral nutrition and almost similar to the amount seen for controls. Before treatment palmitic acid was at 6.35 mg kg −1 for patients versus 10.17 mg kg −1 for controls and reach 9.61 mg kg −1 under treatment versus 8.17 kg −1 for controls. Stearic acid was at 6.09 mg kg −1 for patients and reached 8.41 mg kg −1 under treatment (almost similar to the rate of controls). The 18:1 9 trans -9 cis -11 octadecanoate was at 4.65 mg kg −1 and reached 8.07 mg kg −1 under treatment for patients, higher than for controls. This role of S. boulardii had not been described before, possibly because the dosage used in this study is higher than in the preceding studies and is possibly responsible for beneficial effects of this probiotic in prevention of diarrhoeal diseases induced by enteral nutrition. Keywords: intestinal microflora, short chain fatty acids, bacterial cellular fatty acids, Saccharomyces boulardii .

Effect of Smoking on Oral and Faecal Microbial Flora Studied by Gas–Liquid Chromatography of Bacterial Cellular Fatty Acids

Because smoking negatively affects oral health, we investigated whether this could be due to adverse effects of smoking per se on human oral microflora. Faecal microflora was also analysed for comparison. Samples of whole saliva, dental plaque, pharynx and faeces were collected from 48 healthy young military conscripts, 24 smokers and 24 non-smokers, and studied by gas–liquid chromatography of bacterial cellular fatty acids. The method has proved to be fast and sensitive in detecting microfloral differences and their magnitudes. The results showed significant differences in microflorae between the different sample types (saliva, dental plaque, pharyngeal or faecal). However, there were no significant differences within any sample type between smokers and non-smokers. When oral health is good, smoking history short (mean 5.4 years) and groups otherwise uniform, moderate daily smoking does not seem to change human microbial florae. Keywords: smoking, gas–liquid chromatography, bacterial fatty acids, microbial flora.

Distinct patterns of neonatal gut microflora in infants in whom atopy was and was not developing

Background: Improved hygiene has altered early microbial exposure by reducing childhood infections, which has been suggested as a cause for the continuously rising prevalence of atopic diseases. On the basis of both intensity and timing of stimulus, it has been hypothesized that exposure to commensal microflora may represent another key protective modulator of immunity against atopy and subsequent atopic diseases. Objective: We sought to investigate whether differences in early gut microflora precede the later development of atopic sensitization. Methods: Intestinal microflora from 76 infants at high risk of atopic diseases were analyzed at 3 weeks and 3 months of age by using conventional bacterial cultivation and 2 culture-independent methods, gas-liquid chromatography of bacterial cellular fatty acids and quantitative fluorescence in situ hybridization of bacterial cells. Infants evincing at least one positive skin prick reaction at 12 months were grouped as atopic subjects, and those without positive reactions were grouped as nonatopic subjects. Results: Atopic sensitization was observed in 22 (29%) of 76 children. At 3 weeks, the bacterial cellular fatty acid profile in fecal samples differed significantly between infants in whom atopy was and was not developing (P = .005). By using fluorescence in situ hydridization, atopic subjects had more clostridia (geometric mean [95% confidence interval]: 9.3 × 107 [3.8-22.9 × 107] vs 3.3 × 107 [1.8-6.1 × 107], P = .04) and tended to have fewer bifidobacteria (1.8 × 109 [0.4-7.6 × 109] vs 6.1 × 109 [2.5-14.6 × 109], P = .11) in their stools than nonatopic subjects, resulting in a reduced ratio of bifidobacteria to clostridia (P = .03). The differences were not detected by bacterial cultivation. Conclusion: Differences in the neonatal gut microflora precede the development of atopy, suggesting a crucial role of the balance of indigenous intestinal bacteria for the maturation of human immunity to a nonatopic mode. (J Allergy Clin Immunol 2001;107:129-34.)

Effect of Smoking on Oral and Faecal Microbial Flora Studied by Gas-Liquid Chromatography of Bacterial Cellular Fatty Acids

Effect of Smoking on Oral and Faecal Microbial Flora Studied by Gas-Liquid Chromatography of Bacterial Cellular Fatty Acids

The effect of food restriction on the composition of intestinal microflora in rats.

The effect of a food-restricted diet on the fecal microflora of rats was studied by determining total anaerobic bacteria, bacterial cellular fatty acids, and the predominant intestinal bacteria shown by polymerase chain reaction (PCR) primers specific for the 16S rRNA gene sequences of 12 bacterial species. Twenty-four female Fischer 344 rats, 57 days old were divided into two groups and maintained on an NIH-31 diet. One group was fed ad libitum while the other group received 60% of ad libitum food intake (40% food restriction supplemented with vitamins and minerals equal to the ad libitum animals). After 2, 10, and 20 weeks on this dietary regimen, groups of four animals were sacrificed and the intestinal contents analyzed for changes in the bacterial flora. The anaerobic population for two-week (short-term) food-restricted rats was 3.2 x 10(8) per gram, slightly less than the 9.1 x 10(8) per gram found in the ad libitum-fed rats. The anaerobic populations in 20-week food restricted and ad libitum fed rats were 1.9 x 10(9) and 2.7 x 10(9) per gram, respectively. The total anaerobic population did not change significantly in either group during the 20-week study. No statistically significant differences were observed in the bacterial cellular fatty acid profiles between the two groups as determined by gas-liquid chromatography. PCR analysis of the intestinal contents indicated no significant shifts in the predominant flora due to dietary changes. The results, using three different methods to detect changes in the rat intestinal microflora, suggest that long-term dietary restriction had little effect on the microflora of female Fischer 344 rats.

Computerised gas-liquid chromatography of bacterial cellular fatty acids in analysis of bacterial mixtures

Gas-liquid chromatography (GLC) of bacterial cellular fatty acids (CFA) was tested as a method to obtain species-specific information directly from multimicrobial samples. The CFA spectra of 145 mixtures containing 3, 4, 6 or 8 microbial species were produced by GLC. The idea of direct GLC of multibacterial samples is based on the assumption that the CFA composition of a sample reflects all the bacteria present in the sample. Correlation and cluster analyses of the spectra were performed and the results were presented as dendrograms. When all the CFAs in the samples were employed in the analysis the microbial species in the samples could not be identified. When repeated correlation and cluster analyses were performed employing at a time only the CFAs specific for one microbial species present in the mixtures (index species), the identification of the samples containing the index species was invariably correct in the mixtures containing 3 microbial species. In the mixtures of 4 species the sensitivity of detection was 98.9% and the specificity 94%. When various statistical approaches were used, the identification of the samples with the index species was also possible in the mixtures with 6 species, although the relative similarity of the CFA spectra of certain index species emerged then as a limiting factor in the identification. We conclude that species-specific information could be obtained from all the mixtures by employing species-specific CFAs in the analyses. The identification of a species present in a mixture was successful when the amount of the species was ≥25% of the bacterial mass.

Intestinal Microflora in Colicky and Noncolicky Infants

Summary: To find out whether intestinal microflora in colicky infants is different from that in noncolicky controls, stool samples were collected from colicky infants during colic (n = 55) and at the age of 3 months (n = 46) and compared with samples from age‐matched controls (n = 49 and n = 45, respectively). The samples were cultured on several selective and unselective aerobic and anaerobic culture agars, and gas‐liquid chromatography of bacterial cellular fatty acids was used to produce fatty‐acid profiles of the stool samples. In quantitative bacterial cultures, no differences were found between the colicky and control groups in the amounts of each bacterium. The colicky infants were more frequently colonized with Clostridium difficile during the time of colic than were the age‐matched controls. This difference disappeared by age 3 months. The fatty‐acid profiles did not differ between the colicky and control groups as a whole at the time of colicky symptoms. At age 3 months, a difference in fatty‐acid profiles was found between the colicky infants who had suffered from severe colic and the control infants. The fatty‐acid profiles were also influenced by the age of the infant, the mode of delivery, antimicrobial drugs taken by the mother during delivery, and breast‐feeding and type of feeding. In conclusion, no difference in intestinal microflora was found between the colicky infants at the time of colic and the controls. However, a difference in bacterial cellular fatty‐acid profiles at the age of 3 months was found that correlated with severe infantile colic. This difference may contribute to the cause(s) of colic, or it may be secondary to the colic, which may influence the microbial environment of the intestine.

Intestinal Microflora in Colicky and Noncolicky Infants

To find out whether intestinal microflora in colicky infants is different from that in noncolicky controls, stool samples were collected from colicky infants during colic (n = 55) and at the age of 3 months (n = 46) and compared with samples from age-matched controls (n = 49 and n = 45, respectively). The samples were cultured on several selective and unselective aerobic and anaerobic culture agars, and gas-liquid chromatography of bacterial cellular fatty acids was used to produce fatty-acid profiles of the stool samples. In quantitative bacterial cultures, no differences were found between the colicky and control groups in the amounts of each bacterium. The colicky infants were more frequently colonized with Clostridium difficile during the time of colic than were the age-matched controls. This difference disappeared by age 3 months. The fatty-acid profiles did not differ between the colicky and control groups as a whole at the time of colicky symptoms. At age 3 months, a difference in fatty-acid profiles was found between the colicky infants who had suffered from severe colic and the control infants. The fatty-acid profiles were also influenced by the age of the infant, the mode of delivery, antimicrobial drugs taken by the mother during delivery, and breast-feeding and type of feeding. In conclusion, no difference in intestinal microflora was found between the colicky infants at the time of colic and the controls. However, a difference in bacterial cellular fatty-acid profiles at the age of 3 months was found that correlated with severe infantile colic. This difference may contribute to the cause(s) of colic, or it may be secondary to the colic, which may influence the microbial environment of the intestine.

Intestinal flora in early rheumatoid arthritis.

To study the role of intestinal flora in the pathogenesis of RA, we have applied computerized gas-liquid chromatography (GLC) for bacterial cellular fatty acids (CFAs) present in the stool. The CFA spectra represents the total composition of bacterial CFAs in a faecal sample. Correlation and cluster analysis of CFA spectra gathers samples with quantitatively and qualitatively similar bacterial flora into clusters, which then reveal the relationship of samples to each other. Stool samples were collected at the time of hospital admission from patients with early RA before any specific treatment. The CFA spectra in stool samples of RA patients were significantly different from those of non-RA controls. Patients with erosive RA formed a group most clearly different from the controls. Analyses based on the CFA composition of reference bacteria revealed that anaerobic bacteria are primarily responsible for the differences observed. These results suggest that intestinal bacteria play a role in the development of RA.

Effect of dietary fish powder on intestinal flora and development of arthritis in the pig.

In 1971 Månsson et al. reported that a 20% dry fish powder diet fed to young pigs induced arthritis and increases in faecal Clostridium perfringens counts. To confirm this interesting finding we have repeated their experiment by following their regimen as closely as possible. Twelve piglets of Finnish landrace were divided at 8 weeks of age into two groups according to litter, sex and initial weight. The control group received a cereal-based diet. In the experimental group 20% of cereal in the diet was replaced by fish meal. Stool samples were cultured for Cl. perfringens and subjected to direct gas-liquid chromatography (GLC) to provide bacterial cellular fatty acid profiles. Development of arthritis was monitored macroscopically, and synovial samples were taken at autopsy for microscopical examination. Faecal Cl. perfringens counts increased 10-40 times more in the fish diet group than in the control group. GLC revealed a significant difference in bacterial florae between the groups. No arthritis was observed and only slight synovial inflammation in four experimental pigs and in one control pig. We conclude that fish diet changed faecal flora significantly, but did not induce arthritis.

Direct Automatic Bacterial Analysis of Rat Stool Samples; The Effects of Diet and Medical Treatment Studied by Computerised Gas-liquid Chromatography of Bacterial Fatty Acids

Gas-liquid chromatography (GLC) of bacterial cellular fatty acids was used to produce fatty acid profiles of the stool samples of six different groups of rats, three with different medications, two with experimental diets and a control group with ordinary laboratory food. Such a profile represents all the bacterial cellular fatty acids in the sample and thus reflects its microflora. The profiles were then compared to each other in order to test the applicability of the GLC method for simple and rapid detection, clustering and measurement of faecal bacterial changes. The analysis of the profiles was performed automatically by using coniputerised fatty acid profile correlation analysis combined with a cluster analysis. The results showed the method able to detect identical samples and quantify the differences between the groups. The experimental diets, fish-powder and all-meat, and the anltibiotics, ciprofloxacin and clindamycin, markedly altered the intestinal flora, whereas a non-steroidal anti-inflammatory analgesic, diclofenac, did not. The results indicate that the GLC of stool samples combined with computerised fatty acid profile analysis is a fast and useful method to detect and quantify changes in faecal floras induced by various environmental factors. Keywords: Faecal flora; Diet; Antimicrobe effect; Computer analysis; Gas chromatography; Fatty acids.

Application of gas-liquid chromatographic analysis of cellular fatty acids for species identification and typing of coagulase-negative staphylococci

Gas-liquid chromatography (GLC) of bacterial cellular fatty acids was used to analyze 264 isolates of coagulase-negative staphylococci, of which 178 were Staphylococcus epidermidis. The presence and amounts of individual fatty acids were determined to generate fatty acid profiles for each of the seven coagulase-negative species tested. The fatty acid profiles were then analyzed by computerized correlation and cluster analysis to calculate mean correlation values between isolates belonging to the same or different species, as well as to establish cluster analysis dendrograms. These data ultimately allowed the clustering of individual samples into species-specific clusters. Species identification by the GLC clustering was highly consistent with species identification by biochemical assays; the results were similar in 92.4% of the cases. The GLC profile correlation analysis was further used to analyze multiple blood isolates from 60 patients in order to determine the usefulness of this methodology in establishing identity, as well as differences, between consecutive patient isolates. The correlation between those multiple S. epidermidis isolates determined to be identical by standard techniques (such as the antibiogram, biotype, and plasmid profile) was significantly (P less than 0.001) higher than that between random isolates of the same species. The correlation coefficient was greater than 97 for 40 (97.6%) of the 41 patients with multiple identical blood isolates, compared with less than 95 in all 19 (100.0%) patients with multiple nonidentical isolates. The successful use of the computerized GLC analysis in this study demonstrated its appropriate application for species identification and typing of coagulase-negative staphylococci.

Selectivity optimization for the capillary gas chromatographic analysis of bacterial cellular fatty acids

The importance of selectivity in the capillary gas chromatographic analysis of the cellular fatty acids of micro-organisms is underestimated. The analysis on apolar silicone phases can lead to erratic elucidation of the fatty acid structure. Qualitative errors have been detected in commercially available standards on which computer matching identification techniques are based. Using highly polar capillary columns of the cyanopropyl silicone type, the errors could be elucidated. The exact identification of the hydrolysis products of bacteria is a must for chemotaxonomic studies applying chromatographic techniques. The fatty acid methyl ester profiles can also contain other chemical components which are very important taxonomic markers. Fatty aldehydes, for example, are main components in someClostridium species. Fractionation techniques are described for selective enrichment of fatty aldehydes and hydroxy fatty acids.

Contribution to the Taxonomy of the Turkey Coryza Agent: Cellular Fatty Acid Analysis of the Bacterium

Gas-liquid chromatography was used to analyze bacterial cellular fatty acids to elucidate the relatedness of the turkey coryza (TC) bacterium to Alcaligenes spp., Bordetella spp., and other gram-negative bacteria. The results indicated that the TC bacterium is not closely related to Alcaligenes faecalis or any of the reference strains of Alcaligenes and Bordetella studied. Most urease-positive bacterial isolates obtained from the upper respiratory tract of turkeys were identified as Bordetella bronchiseptica. It is suggested that Bordetella avium is a suitable designation for the TC bacterium formally called Bordetella-"like" and A. faecalis type I. It is also suggested that the nonpathogenic bacterium previously identified as type II A. faecalis be designated B. avium-like until further taxonomic studies are available. Furthermore, it is proposed that the term turkey coryza be used to refer to the disease induced by this bacterium.

Injection principles in capillary gas chromatographic analysis of bacterial fatty acids

Principles for sample injection in fused silica capillary gas chromatographic analysis of bacterial cellular fatty acids are discussed. Comparative analyses were made of the methyl esters of fatty acids in a reference solution and in Legionella pneumophila serogroup 1, using both split and splitless injection. A splitless injection technique, in which the methyl esters are cold-trapped immediately after injection and consecutively released in accordance with temperature programming, gave sensitivity increase corresponding to the split ratio used in split injections, without diminishing the speed of analysis or separation efficiency. Splitless injection, utilizing cold-trapping, can be recommended for capillary gas chromatography in routine profiling of bacterial fatty acids.