Detection of anaerobic wound infection by analysis of pus swabs for volatile fatty acids by gas-liquid chromatography.

Swabs were able to absorb enough extractable volatile fatty acids from broth cultures of anaerobic organisms for detection and analysis by gas-liquid chromatography (GLC). Similarly, volatile fatty acids were often detected in swabs dipped into liquid pus. Fifty-three liquid pus specimens were then investigated fully to determine if GLC analysis of swab samples gave the same result as microbial culture of the specimens and GLC analysis of the liquid pus. Anaerobic bacteria failed to grow from 36 and volatile fatty acids were not extracted from swabs of 31 of these pus samples but were extracted from swabs of five. Anaerobic bacteria were isolated from 17 of the specimens, and in 15, volatile fatty acids were also detected in the swab samples; in two, volatile fatty acids were absent from both swab samples and liquid pus. In this study, results by culture and GLC analysis of swabs were similar in 87% of specimens.     

Use of prototype automated blood culture system and gas-liquid chromatography for the analysis of continuous ambulatory peritoneal dialysis associated infection.

AIMS: (1) To compare the recovery of organisms from continuous ambulatory peritoneal dialysis (CAPD) effluent fluid obtained from patients with clinical evidence of peritonitis, with an automated system (AS) and the Septichek blood culture system; (2) to evaluate the times to detection of organisms with the two systems; (3) to identify anaerobes from CAPD samples by extended anaerobic culture and gas-liquid chromatography (GLC). METHODS: 168 CAPD effluent fluid samples were studied, representing 157 episodes of peritonitis in 97 patients. CAPD samples were inoculated into two AS bottles-one anaerobic, one aerobic-and a Septichek bottle; samples were also examined for cell count, Gram stain, and direct culture. Culture bottles were then subcultured onto various media, and any organisms isolated were identified. After routine culture, GLC was performed on culture fluid in the anaerobic AS and Septichek bottles. When volatile fatty acids were detected, the broths were cultured anaerobically on specialised medium for a further five days. RESULTS: 147 organisms were isolated from the 168 samples: 96 (57%) yielded growth of significant organisms by direct culture, as compared to 129 (76.8%) by both AS and Septichek. There was no significant difference in isolation rates between AS and Septichek, but time to detection was more rapid with the AS system (p < 0.002). GLC showed volatile fatty acid in 15 specimens; of these, 14 subsequently grew anaerobic organisms. CONCLUSIONS: AS was comparable to Septichek for numbers of isolations. Speed to detection was faster with the AS, which may be an advantage in management of patients with CAPD peritonitis. GLC showed anaerobes in several cases which would not have been detected without prolonged anaerobic culture; thus anaerobic cultures are recommended for patients who are unresponsive to antimicrobials or who have evidence of bowel perforation.     

Agar medium for gas-liquid chromatography of anaerobes

This study evaluates a method of performing gas-liquid chromatography (GLC) by direct extraction of fatty acids from agar for identification of clinically significant anaerobic bacteria. The potential use of agar cultures for GLC was studied by comparing chromatograms of 117 clinically isolated anaerobes grown in peptone yeast glucose broth and chopped meat carbohydrate broth, and on enriched brucella blood agar. For 98 of 117 anaerobes, fatty acid patterns from agar cultures were similar to those in broth. Significant differences were only found with Streptococcus intermedius, Clostridium perfringens, Clostridium tertium, and Actinomyces species, which produced less of certain fatty acids on agar than in broth. Results of this study indicate that GLC of short chain fatty acids produced on agar medium by anaerobes, combined with simple tests such as Gram's stain and colonial morphology, may allow fir direct presumptive genus identification from an initial pure agar culture.     

Applications of cellular fatty acid analysis.

More than ever, new technology is having an impact on the tools of clinical microbiologists. The analysis of cellular fatty acids by gas-liquid chromatography (GLC) has become markedly more practical with the advent of the fused-silica capillary column, computer-controlled chromatography and data analysis, simplified sample preparation, and a commercially available GLC system dedicated to microbiological applications. Experience with applications in diagnostic microbiology ranges from substantial success in work with mycobacteria, legionellae, and nonfermentative gram-negative bacilli to minimal involvement with fungi and other nonbacterial agents. GLC is a good alternative to other means for the identification of mycobacteria or legionellae because it is rapid, specific, and independent of other specialized testing, e.g., DNA hybridization. Nonfermenters show features in their cellular fatty acid content that are useful in identifying species and, in some cases, subspecies. Less frequently encountered nonfermenters, including those belonging to unclassified groups, can ideally be characterized by GLC. Information is just beginning to materialize on the usefulness of cellular fatty acids for the identification of gram-positive bacteria and anaerobes, despite the traditional role of GLC in detecting metabolic products as an aid to identification of anaerobes. When species identification of coagulase-negative staphylococci is called for, GLC may offer an alternative to biochemical testing. Methods for direct analysis of clinical material have been developed, but in practical and economic terms they are not yet ready for use in the clinical laboratory. Direct analysis holds promise for detecting markers of infection due to an uncultivable agent or in clinical specimens that presently require cultures and prolonged incubation to yield an etiologic agent.     

Effects of short-chain fatty acids on in vitro bacterial growth of Bacteroides fragilis and Escherichia coli

Short-chain fatty acids (SCFA) are produced in the large bowel of nonruminant mammals by bacterial anaerobic fermentation. The aim of the present study was to investigate the effects of SCFA on the in vitro growth of Bacteroides fragilis and Escherichia coli. B. fragilis and E. coli isolated from fresh human clinical samples and a reference strain for each species were incubated in a meat infusion broth with increasing amounts of SCFA and grown under anaerobic conditions at a temperature of 37 degrees C. Bacterial growth was estimated by spectrophotometry. Rate of growth was calculated from the logarithmic growth phase. SCFA, in concentrations normally found in the human colon, had a significant (p<0.01) inhibitory effect of the in vitro growth rate for E. coli, while they were without effect on the in vitro growth rate of B. fragilis. It may be concluded that under in vitro conditions SCFA had growth-inhibitory effects on E. coli, while they had no effect on B. fragilis.     

Detection of Bacteroides fragilis in clinical specimens by PCR.

The direct detection of Bacteroides fragilis from clinical specimens was examined by using the PCR method for amplifying a specific fragment of the glutamine synthetase gene from B. fragilis. By this method, all five B. fragilis strains tested were detected, but DNAs from anaerobic bacteria of 24 other species tested, from aerobic bacteria of 12 species tested, and from human leukocytes were not amplified. Using the nested PCR method, we were able to detect as little as one bacterial cell or 100 fg of chromosomal DNA of B. fragilis. A total of 39 clinical specimens, which consisted of 19 bronchial aspirates, 10 percutaneous lung aspirates, 2 transtracheal aspirates, 6 pleural fluid specimens, and 2 pus specimens, were tested. All four culture-positive samples, of which two were bronchial aspirates, one was pleural fluid, and one was pus, were positive by PCR. Among 35 culture-negative samples, 2 bronchial aspirates were positive by PCR. One was from a patient whose two previous samples were positive by both culture and PCR. It had been submitted for culture several hours after collection, and clindamycin had been administered to the patient before collection of the specimen. The other bronchial aspirate positive by PCR was from a pneumonia patient who had also been administered clindamycin. We believe that B. fragilis was present in these two specimens but that either it was dead, it was below the level detectable by culture, or the process of anaerobic culture was unsuccessful. Thus, the PCR method may be considered useful for the sensitive and rapid detection of anaerobes in clinical specimens.     

Aerobic and anaerobic microbiology of suppurative sialadenitis

Aspirates of pus from acute suppurative sialadenitis were investigated for the presence of aerobic and anaerobic bacteria. A total of 47 specimens, 32 from parotid, 9 from submandibular and 6 from sublingual glands yielded bacterial growth. Fifty five isolates, 25 aerobic and 30 anaerobic, were isolated from parotid infection: anaerobic bacteria only were detected in 13 (41%) specimens, aerobic or facultative bacteria only in 11 (34%) and mixed aerobic and anaerobic bacteria in 8 (25%). Of a total of 17 isolates, 8 aerobic and 9 anaerobic, from submandibular gland infection: anaerobic bacteria only were detected in 3 (33%) specimens, aerobic or facultative bacteria only in 4 (44%) and mixed aerobic and anaerobic bacteria in 2 (22%). Ten isolates, 5 aerobic and 5 anaerobic, were from sublingual gland infection: anaerobic bacteria only were detected in 2 (33%) specimens, aerobic or facultative bacteria only in 2 (33%) and mixed aerobic and anaerobic bacteria in 2 (33%). The predominant aerobes were Staphylococcus aureus and Haemophilus influenzae while the predominant anaerobes were gram-negative bacilli (including pigmented Prevotella and Porphyromonas spp., and Fusobacterium spp.) and Peptostreptococcus spp. The study highlights the polymicrobial nature and importance of anaerobic bacteria in acute suppurative sialadenitis.     

Two cases of subcutaneous infection due to Phaeoacremonium spp

We describe two cases in Brazil of human subcutaneous infections due to Phaeoacremonium spp. The first case was caused by Phaeoacremonium aleophilum. The patient presented with a unique fistulized nodule on the left ankle. The fungus was detected by direct microscopic examination and was isolated repeatedly from material collected from the lesion. This is the first reported case of human infection caused by this fungus. The second case was caused by Phaeoacremonium rubrigenum. The patient presented with multiple nodules around the left ankle and foot. The fungus was detected by direct examination of pus and histological sections of the nodules. It was repeatedly isolated from the clinical specimens. This is the second reported case of human infection caused by this species.     

Gas-liquid chromatography in the diagnosis of anaerobic infections: a three year experience.

Nearly two thousand clinical samples were examined by direct gas-liquid chromatography over a three year period. Absence of volatile fatty acids (VFAs) in the samples correlated well with negative culture results for anaerobic bacteria. In general the presence of acetic acid alone correlated well with the presence of aerobic organisms, whereas the presence of a mixture of VFAs correlated well with the presence of anaerobic organisms, either alone or in combination with aerobes. However a proportion of such VFA-positive samples gave no growth on culture. Swabs gave comparable results to samples of pus or exudates except that a higher proportion of the former were VFA-negative but culture positive.     

Incidence of anaerobic bacteria in respiratory tract infections

Anaerobic bacteria are predominant components of normal oral cavity, upper respiratory tract, gastrointestinal, genital and skin flora. They are involved in infections such as pneumonia, aspiration pneumonia, lung abscess and empyema. Laboratory diagnosis of anaerobic infections is based on recovering the etiological agents from clinical materials. Appropriatte specimens include: pus, purulent fluid, biopsy specimen of lung, transtracheal aspirates and bronchoalveolar lavage (BAL). Lower respiratory infections are usually either polymicrobial or mixed anaerobic-aerobic infections. Peptostreptococcus, Fusobacterium, Prevotella and Bacteroides are the most common anaerobes. Anaerobic bacteria are susceptible to metronidazole, tinidazole (exception of Gram-positive rods), amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin/tazobactam, imipenem and clindamycin. Treatment includes an antibiotics regimen with an agent active against anaerobic and aerobic bacteria (therapy with 2 or 3 antimicrobial drugs).     

Anaerobes survive in clinical specimens despite delayed processing.

Quantitative cultures were performed on 11 purulent specimens of at least 2 ml from mixed aerobic-anaerobic infections to determine the effect of prolonged exposure to air on the recovery of anaerobes. The specimens were processed immediately and after air exposure for periods of 10 min and 1, 4, and 24 h. There were at total of 37 anaerobic and 36 aerobic strains recovered from these specimens. Of the anaerobes, 26 were isolated with the initial processing and 22 were still present after air exposure for 24 h. The numerical concentrations of anaerobes showed little change with the sequential samplings. Eleven anaerobic strains were not detected in the initial culture but appeared sporadically in subsequent cultures. Using the types of specimens and method of processing employed in this study, most pathogenic anaerobes survived in purulent exudate despite extended periods of air exposure. The major cause of discrepent results with periodic cultures was attributed to vagaries in sampling.     

Rapid identification of non-sporing anaerobes using nuclear magnetic resonance spectroscopy and an identification strategy

PURPOSE: The non-sporing anaerobes cause a wide spectrum of infections. They are difficult to culture and their identification is tedious and time-consuming. Rapid identification of anaerobes is highly desirable. Towards this end, the potential of nuclear magnetic resonance (NMR) spectroscopy for providing a fingerprint within the proton spectrum of six genera belonging to anaerobes reflecting their characteristic metabolites has been investigated. METHODS: NMR analysis was carried out using Mercury plus Varian 300 MHz (7.05 T) NMR spectrophotometer on six different anaerobes. These included Bacteroides fragilis, Prevotella melaninogenica, Prevotella denticola, Fusobacterium necrophorum, Peptococcus niger and Peptostreptococcus spp. After the NMR analysis (256/512 scans), the different peaks were noted. The eight pus specimens, which yielded pure culture of anaerobe, also were analysed similarly. RESULTS: The major resonances of multiplex of amino acids/lipid at 0.9 ppm along with lactate/lipid at 1.3 ppm, acetate at 1.92 ppm and multiplex of lysine at 3.0 ppm remained constant to label the organism as an anaerobe. There was a difference found in the MR spectra of different genera and species. A simple algorithm was developed for the identification of the six different anaerobes studied. The MR spectra of the pure culture of the organism matched the MR spectra of pus from which the organism was isolated. CONCLUSIONS: MR-based identification was of value in the identification of anaerobes. However, a larger database of the peaks produced by anaerobes needs to be created for identification of all genera and species. It could then have the potential of diagnosing an anaerobic infection in vivo and thus expedite management of deep-seated abscesses.     

Routine analysis of short-chain fatty acids for anaerobic bacteria identification using capillary electrophoresis and indirect ultraviolet detection

The diagnosis of anaerobes can be difficult to perform, using classical biochemical tests. Characterization of metabolic end-products such as short-chain fatty acids (SCFA) was often used because of their reproducible biosynthesis. Despite this, SCFA are difficult to study using gas chromatography, due to their high volatility. Furthermore, the treatment of the samples are long and fastidious. Capillary electrophoresis and indirect UV detection (CE-indirect UV) is a well-known analytical method to study inorganic or organic anions. In this work, we validate the analysis of SCFA using CE-indirect UV detection. To do this, we studied the culture media of 98 anaerobic strains for the detection and quantitation of the following acids: succinic, pyruvic, acetic, lactic, propionic, 2-hydroxybutyric, butyric, 2-hydroxyvaleric, isovaleric, isocaproic, and 3-phenylpropionic. We verified that the CE-indirect UV detection analysis of SCFA for taxonomical data can be used as a mean for rapid identification for the study of anaerobes.     

Influence of the collection and transport of specimens on the recovery of bacteria from peritonsillar abscesses.

In 30 patients with peritonsillar abscesses, pus was obtained by aspiration and by taking a swab after incision; bacterial recovery was compared. Although processed in the laboratory within 2 h, swab speciments gave results comparable to syringe specimens in only 9 of 13 patients with beta-hemolytic streptococci and 7 of 25 patients with anaerobic bacteria. Both kinds of microorganisms were lost in some cases but appeared as additional flora in others. The poor results from the swab technique was ascribed to overgrowth of respiratory flora contaminating the sample after incision. In aspirated pus kept in the syringe, or transferred to anaerobic transporters, the microbial flora was unchanged for 24 to 48 h. Some anaerobes also survived on agar slants for 24 h, but specially designed anaerobic transporters are recommended.     

Differentiation of Peptococcus and Peptostreptococcus by gas-liquid chromatography of cellular fatty acids and metabolic products

Gas-liquid chromatographic (GLC) profiles of cellular fatty acids and metabolic products were useful in identifying strains of Peptococcus saccharolyticus, Peptococcus asaccharolyticus, Peptostreptococcus anaerobius, Peptostreptococcus micros, and Streptococcus intermedius. The GLC results supported the recent taxonomic decision to transfer aerotolerant Peptostreptococcus species to the genus Streptococcus. Because inconsistencies in the results prevented our differentiating Peptococcus prevotii. Peptococcus magnus, and Peptococcus variabilis by GLC, additional strains will have to been examined. These GLC techniques are amenable to routine use; however, for interlaboratory results to be meaningful, the classification and nomenclature of the anaerobic gram-positive cocci should be standardized.     

Direct detection of Prevotella intermedia and P. nigrescens in suppurative oral infection by amplification of 16S rRNA gene.

A specific 16S rDNA PCR and subsequent hybridisation reaction was designed to discriminate between strains of Prevotella intermedia (n = 15) and P. nigrescens (n = 15). This technique was then used to detect the presence of these two bacterial species in acute suppurative oral infection. A total of 36 pus samples aspirated from 26 peri-apical abscesses, three root canals, three periodontal abscesses, two cases of refractory periodontitis, one cyst and one haematoma was examined. A portion of the pus sample was processed by PCR and the remainder of the specimen was subjected to routine culture. The PCR-based technique gave an identical pattern of detection of P. intermedia or P. nigrescens to that obtained by culture for 30 of the 36 specimens. Either P. intermedia or P. nigrescens was present in 14 samples and neither species was detected in 16 samples. In the remaining six samples the PCR method indicated the presence of one (n = 3) or both (n = 3) of the Prevotella species but neither or only one species was isolated by culture. It is concluded that the presence of P. intermedia and P. nigrescens in pus can be detected rapidly and specifically by direct PCR amplification of 16S rDNA. P. nigrescens was detected more frequently than P. intermedia in suppurative peri-apical infection both by culture and PCR.     

In vitro alterations in fecal short chain fatty acids and organic anions induced by the destruction of intestinal microflora under hypotonic and aerobic conditions

The pathogenesis of inflammatory bowel disease (IBD) remains unknown. It has been suggested that luminal factors such as the microflora, short chain fatty acids (SCFAs), food antigens and so on play important roles in the disease progression. Many reports have revealed alterations in the SCFA and organic acid concentration of the colon, especially increased lactate and decreased butyrate, in IBD patients. The mechanisms responsible for these alterations, however, remain unclear. Therefore, the effects of aerobic conditions on the alterations in the SCFA and organic anion levels in the feces was evaluated. Fecal specimens were collected from 5 healthy volunteers. Under aerobic condition, a mixture of feces and distilled water was incubated in 37 degrees C for 1 and 3 h. The pH, osmotic pressures, the concentrations of potassium, bicarbonate, SCFA and organic anion, and the activities of alpha-amylase and lactate dehydrogenase (LDH) in the mixture were then measured. We also examined any changes in the microscopic microflora under the hypotonic and aerobic conditions. The results showed the osmotic pressure, and the concentrations of lactate and SCFAs (formate, acetate, propionate and n-valerate) were progressively increased with longer incubation times, and reached a statistically significant difference. In particular, the ratios of lactate, succinate and n-valerate after 1 and 3 h of incubation increased remarkably. In contrast, the electrolyte levels and both alpha-amylase and LDH activities were not altered significantly. Microscopically, the microflora in the mixture decreased with prolonged incubation times. These data suggest that under these in vitro conditions, the organic anion and SCFA levels in the feces easily increased. It is probable that the alterations in the SCFA and organic anion levels in IBD patients may be partly due to intracellular components derived from microflora destroyed under hypotonic and aerobic conditions in the colonic lumen, for example caused by mucosal bleeding. These alterations may influence the pathogenesis and progression of IBD.     

Effect of temperature on survival of Bacteroides fragilis subsp. fragilis and Escherichia coli in pus.

Approximately 40 to 60% of Bacteroides fragilis subsp. fragilis in pus from experimental intra-abdominal abscesses lost their viability within 24 h when stored at refrigeration temperature (4 degrees C) either aerobically or anaerobically. No viability loss of B. fragilis was noted when pus was stored at 25 degrees C. Only slight loss of viaability of B. fragilis was observed at 15 degrees C. Escherichia coli coexisting in pus with B. fragilis increased several 100fold in 24 h when stored at 25 degrees C, but no significant growth occurred when they were kept at 15 degrees C. Approximately 20 to 40% of E. coli lost their viability when such pus was stored at 4 degrees C. We suggest that 15 degrees C may be an alternative temperature for storage of anaerobic specimens in laboratories where some delay in routine processing is unavoidable.     

Analysis of the conjugated bile acid distribution in human intestinal aspirates using gas liquid chromatography.

The conjugated bile acid pattern was evaluated in intestinal aspirates of five normal subjects and five patients known to have an abnormal bile acid distribution. The glycine/taurine (G/T) ratios for total bile acids were determined by gas liquid chromatography (GLC) and by enzymatic assay using 3-hydroxysteroid dehydrogenase (STDH). By both methods G/T ratios in normal samples approximated 3.0 as previously reported. A discrepancy between ratio values obtained by the two methods was found in patient samples. It is suggested that the presence of keto bile acids in patient aspirates correlates with this discrepancy. The G/T ratios for individual bile acids were obtained by GLC. The G/T ratio for cholic acid was higher than previously reported. Recovery studies showed that a loss of taurocholic acid in the extraction procedure employed did not account for the high G/T-cholic acid.     

Influence of short-chain fatty acids on ammonia absorption across the rumen wall in sheep.

The effects of short-chain fatty acids (SCFA) on ammonia net absorption from the sheep rumen in vivo and on ammonia transport across rumen wall mucosa in vitro were studied. Ammonia net absorption was directly, though in a non-linear manner, correlated with the SCFA concentration in the artificial rumen fluid. Almost 70% of total ammonia absorption was dependent upon the presence of SCFA when 12 mmol l-1 ammonia and 67.5 mmol l-1 SCFA were present. Lactic acid was ineffective. Incubation experiments showed that mucosal disappearance and serosal appearance of ammonia were reduced by 38% and 32%, respectively, when SCFA (63 mmol l-1) were replaced by lactic acid. The SCFA effect was independent of the type of SCFA used. In part of the experiments up to 54% of the ammonia taken up by the tissue was not recovered in the serosal incubation solution and must have been metabolized in the mucosa.