Anaerobic Bacteriology in 75 Cases of Thoracic Empyema in Sofia,Bulgaria

The aim of this study was to evaluate the prevalence of anaerobes in patients with thoracic empyema over a period of 30 months and to assess the susceptibility of the isolates to penicillin, clindamycin and metronidazole. Seventy-nine pleural fluid specimens were obtained from 75 adult patients with empyema. Anaerobic isolates were identified by Crystal anaerobes identification system and routine methods. Susceptibility testing was conducted using broth microdilution method and limited agar dilution test. Anaerobic bacteria were found in 50 (66.7%) of the patients and included 96 isolates representing 16 genera. The predominant Gram-positive anaerobes were Peptostreptococcus species (19 isolates) and Streptococcus intermedius (10), and the commonest Gram-negative species were Fusobacterium nuleatum (13),Fusobacterium necrophorum (6) and Prevotella inermedia (3). From two to four anaerobes per specimen were present in 57.4% of the specimens yielding anaerobic bacteria. The susceptibility of the Gram-negative anaerobic isolates to penicillin and that of the Gram-positive anaerobes to clindamyin and metronidazole were unpredictable. The variable resistance patterns among anaerobes and the predominance of mixed anaerobic infections highlight the role of the anaerobic dignostics in case of serious pleuropulmonary diseases.     

Comparison of Methods for Isolation of Anaerobic Bacteria from Clinical Specimens

Five different anaerobic culture methods and several different media were compared for their ability to recover anaerobes from clinical specimens. Specimens were obtained from patients with documented infections, avoiding contamination with normal flora, and immediately placed in an anaerobic transporter. Each specimen was cultured by all methods and on all the various media. The comparative data indicate that anaerobic jars (GasPak and evacuation-replacement types) are just as effective in the recovery of clinically significant anaerobes as the more complex roll-tube and chamber methods employing prereduced media. Liquid media were disappointing as a "back-up" system but chopped-meat glucose was superior to two thioglycolate formulations. Growth of all anaerobes was poorer on selective media, but these media were very helpful in the workup of specimens containing mixed growth of anaerobic and facultative organisms. A variety of different anaerobes was isolated, but no very fastidious or extremely oxygen-sensitive organisms were recovered. This suggests that such organisms may not play a significant role in causing clinical infections.     

United States National Hospital Survey of anaerobic culture and susceptibility methods, II

To assess the status of clinical anaerobic bacteriology in the United States, we surveyed (by means of a questionnaire) 120 hospitals selected at random with bed capacities of 200-1000, and we received responses from 78 (65%), all of which performed some degree of clinical anaerobic microbiology. Separate anaerobic blood culture bottles were used by 73 labs (94%) (median, 450 specimens/mo): 56% used Bactec 7, 27 or 37; 15% used 'BacT-Alert'; 11% used Columbia broth; 5% used thioglycolate and 'lytic'; 3% each used, Dupont Isolator, Supplemented peptone or other media. Selective media was used for primary anaerobe isolation by 89% labs which included: LKV, 76%; PEA, 53%; BBE, 31%; CNA, 28%; 'CDC', 12%. Sixty labs (78%) stored anaerobes after isolation (median 7 days), most using blood agar plates (31%), chopped meat (26%) or thioglycolate broth (27%) either for further identification (30 out of 78) or susceptibility testing (33 out of 78), if clinically indicated. Only 23% performed routine anaerobic susceptibility testing of clinical isolates. Of the 77% that do not perform susceptibility studies, 59% would not even perform them upon physician request; 30% relied on published surveys; 68% did not publish results of anaerobic susceptibility in annual summaries. When susceptibility testing was performed, the test agents selected were related to availability on a commercial system (21), NCCLS recommendation (20), hospital formulary (15) or hospital committee input (20). Nine of 78 labs (12%) had discussed stopping or decreasing the performance of both anaerobic bacteriology and susceptibility testing. Despite educational and published guidelines, clinical anaerobic bacteriology is not uniformly practiced and could be improved. In addition, an educational effort must be made in order to stress the relevance and increase performance of anaerobic bacteriology.     

The aerobic and anaerobic microbiology of pustular acne lesions

Specimens from 32 pustular acne lesions that were inoculated on media supportive for the growth of aerobic and anaerobic bacteria showed bacterial growth. Only aerobic or facultative bacteria were recovered in 15 (47%) specimens, only anaerobic bacteria in 11 (34%) specimens, and mixed aerobic and anaerobic bacteria in 6 (18%) specimens. A total of 57 isolates, 31 anaerobes (1.0 per specimen) and 26 aerobes (0.8 per specimen) were recovered. The predominant isolates were Staphylococcus sp. (19 isolates), Peptostreptococcus sp. (15), and Propionibacterium sp. (10). Twelve (37.5%) of the comedones yielded only one organism. This retrospective study highlighted the polymicrobial nature of over two-thirds of culture positive pustular acne lesions and suggests the potential for pathogenic role of aerobic and anaerobic organisms other than P. acnes and Staphylococcus sp. in acne vulgaris.     

National hospital survey of anaerobic culture and susceptibility methods: III

To assess the current status of anaerobic bacteriology in the United States, we surveyed, by means of a questionnaire, 150 hospitals selected at random with bed capacities of 200-1000 and we received responses from 98 (65%). Ninety-eight percent processed anaerobic culture specimens with 21% sending them to reference laboratories. Almost all these hospitals processed blood and wound cultures for anaerobes and all used selective media for identification, including BBE (52%), LKV (77%), and PEA (53%) agars. All hospital laboratories attempted identification of blood culture isolates including 80% that attempted speciation. Wound cultures for anaerobic bacteria and sterile site cultures were also processed for anaerobes by almost all labs. Identification of B. fragilis group species to species level was performed only in 56% of labs always and 37% sometimes. Preformed enzyme kits were used by 66% of labs and 30% used special potency disks for identification. Susceptibility testing was performed in-house by 21% of hospital labs and sent out to reference labs an additional 20%. Susceptibility testing was attempted for all blood culture isolates by both hospital (21% of total labs) and reference laboratories, but only performed by 17% for sterile body site and 14% of the time for wound isolates. Etest was used most often followed by broth microdilution. No labs used the agar dilution or disk elution methods. The antimicrobials most often tested in hospital labs, predicated on the commercial panel used, were penicillin/ampicillin and clindamycin (15/18; 83%; 15% of total labs), metronidazole (16/18; 89%; 16% of total labs) and cefotetan and ampicillin/sulbactam (12/18; 67%; 12% of total labs), piperacillin/tazobactam (7/18; 39%; 7% of total labs), cefoxitin (9/18; 50%), imipenem (8/18; 44%), and chloramphenicol (6/18; 33%). Our current survey suggests that while many labs are processing anaerobic cultures, especially blood cultures, the identification of isolates and the performance of antimicrobial susceptibility testing of isolates are in disarray and in dire need of improvement.     

Evaluation of culture results of specimens from patients with suspected anaerobic infection.

In order to establish the etiological agents of anaerobic infections, 154 clinical specimens collected from patients with suspected anaerobic infection were cultured under both aerobic and anaerobic conditions. Bacteria were isolated from 111 (72%) of these specimens. Only aerobes were recovered from 48 (43%), only anaerobes from 31 (28%) and both aerobes and anaerobes from 32 (29%) of the 111 specimens. A total 83 anaerobic bacteria including Bacteroides fragilis group (28%), Porphyromonas (19%), Peptostreptococci (10%), Prevotella (17%), Clostridia (12%), Fusobacteria (10%), Veillonella (2%) and Eubacteria (2%) were identified. Anaerobes were most commonly isolated from peritoneal fluid followed by joint fluid, abscess and endometrial materials, blood, soft-tissue biopsy and draining material.     

Bacteriology of human bite wound infections

This study was designed to define the bacteriology of infected soft-tissue wounds from human bites, and to compare this with the bacteriology of infected animal bites in humans as determined in previous studies. The specimens were collected from 57 patients presenting to emergency rooms at 12 locations around the country. Three hundred and eighty organisms were isolated (224 aerobes and 156 anaerobes), for an average of 6.6 per specimen. The most prevalent anaerobes recovered were Prevotella spp. (34%), while streptococci comprised 44% of all aerobic organisms, over half of which were in the "Streptococcus milleri" group, particularly S. anginosus. The study demonstrated that the pathogens in human bite infections differ considerably from those present in animal bites.     

Comparison of recovery of anaerobic bacteria using the Anoxomat, anaerobic chamber, and GasPak jar systems

The Anoxomat system provides an automated evacuation-replacement technique to create an anaerobic or microaerophilic environment in a jar. We evaluated the Anoxomat system for the growth of obligate anaerobes and for the recovery of anaerobic organisms from clinical specimens, and compared its performance to that of an anaerobic chamber and the GasPak System. Of the 54 stock strains tested, the Anoxomat, the chamber, and the GasPak recovered 95%, 95% and 93% at 24 h, respectively. On 29 occasions (51%), the colonies on the Anoxomat plates were slightly larger than those in the chamber and on 17 (30%) occasions larger than the colonies on the GasPak jar plates. At 48 h, the Anoxomat, the chamber, and the GasPak recovered 93.5%, 94.4% and 88.9%, respectively; of 108 anaerobes isolated from 31 clinical specimens. Methylene blue indicators became decolorized (average of 10 tests) within 2 h inside the Anoxomat jars, 2 h 10 min inside the anaerobic chamber, and 2 h 30 min inside the GasPak jars.     

The genus Prevotella in cystic fibrosis airways

Airway disease resulting from chronic bacterial colonization and consequential inflammation is the leading cause of morbidity and mortality in patients with Cystic Fibrosis (CF). Although traditionally considered to be due to only a few pathogens, recent re-examination of CF airway microbiology has revealed that polymicrobial communities that include many obligate anaerobes colonize lower airways. The purpose of this study was to examine Prevotella species in CF airways by quantitative culture and phenotypic characterization. Expectorated sputum was transferred to an anaerobic environment immediately following collection and examined by quantitative microbiology using a variety of culture media. Isolates were identified as facultative or obligate anaerobes and the later group was identified by 16S rRNA sequencing. Prevotella spp. represented the majority of isolates. Twelve different species of Prevotella were recovered from 16 patients with three species representing 65% of isolates. Multiple Prevotella species were often isolated from the same sputum sample. These isolates were biochemically characterized using Rapid ID 32A kits (BioMérieux), and for their ability to produce autoinducer-2 and β-lactamases. Considerable phenotypic variability between isolates of the same species was observed. The quantity and composition of Prevotella species within a patients’ airway microbiome varied over time. Our results suggest that the diversity and dynamics of Prevotella in CF airways may contribute to airway disease.     

厌氧菌预还原琼脂平板培养方法

为简化厌氧菌分离培养方法,使其在普通实验条件下于固体培养基上形成单菌落,本研究增加庖肉培养基无氧溶液体积,用作无氧倍比稀释液,在琼脂柱下进行倍比稀释,将皿盖带有胶塞孔的厌氧琼脂平板进行预还原,注射接种倍比稀释菌液,通过厌氧指示剂监测无氧效果,初步试用于肠道厌氧菌分离培养。结果显示,该方法整个操作过程厌氧效果良好,无需专门厌氧设备即可以分离纯化培养肠道乳酸杆菌,甚至无芽胞专性厌氧菌,如双歧杆菌和韦荣球菌。     

Physiological Properties and Phylogenetic Affiliations of Anaerobic Bacteria Isolated from Roots of Rice Plants Cultivated on a Paddy Field

Anaerobic bacteria associated with roots of rice plants cultivated on a paddy field were isolated, and their physiological properties and phylogenetic affiliations were investigated. The roots harbored culturable populations of anaerobic microorganisms at 10⁷ levels of viable counts (CFU/g dry roots), and the isolates were thought to represent numerically abundant populations of culturable anaerobic microorganisms present on the roots. Among 18 strains isolated in pure culture, five strains were obligately anaerobic and others were facultatively anaerobic. Eight strains including four obligately anaerobic strains were selected for further study. Of eight strains, seven strains were saccharolytic, and one strain was a non-saccharolytic sulfate-reducer. Glucose was fermented into ethanol and/or acetate by the saccharolytic strains, lactate, succinate or H₂ was also produced by some strains. Four facultatively anaerobic strains were saccharolytic and grew with the fermentative metabolism even under the oxic condition. Three facultatively anaerobic strains and one obligately anaerobic strain exhibited the Fe(III)-reducing ability. The comparative analysis of 16S rRNA gene sequences indicated that the sequence of any strain did not completely match to the sequences available in the database. The seven saccharolytic strains represented diverse phylogenetic groups: the classes ‘Alphaproteobacteria’ (two strains) and ‘Gammaproteobacteria’ (one strain), the family Bacteroidaceae (one strain), the orderActinomycetales (two strains), and the family Clostridiaceae. The sulfate-reducing strain was a close relative ofDesulfovibriodesulfuricans . At least five strains were considered to represent novel species. In particular, two strains were considered to represent novel lines of descent at the family level within the order ‘Rhizobiales’. These results suggested that phylogenetically different bacteria with a common physiological trait as the saccharolytic fermentative acidogen formed numerically most abundant populations of culturable anaerobes in the microbial community on rice roots.     

Bacteriology of the Gut and Its Clinical Implications

The bacteriology of the gastrointestinal tract is rapidly changing in laboratory techniques and clinical correlations. The flora is found to be very complex, predominantly anaerobic, and importantly dependent on diet. An etiologic role for colon bacteria in colon cancer is suggested by correlations between epidemiologic data and prevalent dietary patterns and stool culture findings. Cultures from aspiration pneumonia, subphrenic abscess, and other intra-abdominal sepsis all yield anaerobes, and for best results antibiotic therapy should combat them as well as aerobes.     

Level of redox potential as a possible contributing influence in the pathogenicity of oral anaerobes

Dental plaque anaerobes may be associated with the etiology of periodontal disease. This has created an interest in the potential pathogenicity of oral anaerobes. We compared the metabolic activity of anaerobic corynebacteria (C. parvum, C. anaerobium) and corresponding aerobic species (C. diphtheriae, C. xerosis). The anaerobes exhibited lower levels of growth rate, m-RNA half-life and ribosomal efficiency but much higher levels of RNA synthesis, ranging from 5 to 10 fold over the aerobes. We further examined these anaerobes, plusActinomyces naeslundi N16 (isolated from the anaerobic region of periodontally-diseased tissues), for the influence of redox potential on RNA level and antigenic function. Notable increases in RNA were found at specific Eh levels; the extent and direction of the changes varied with the different organisms. This environmental feature appeared to effect corresponding changes in agglutinability and PCA reactivity with antisera against the anaerobes cultured at different redox potentials. For example, while antisera against certain organisms (C. parvum, A. naeslundi) cultured under the most reduced conditions showed an intense PCA reaction, other antisera against the same organism cultured under less reduced conditions were non-reactive. Hence, alterations in redox potential may lead to altered metabolism and to altered antigenicity. Our results imply such a microbial response to environmental stress.     

Effects of Alternative Dietary Substrates on Competition between Human Colonic Bacteria in an Anaerobic Fermentor System

Duplicate anaerobic fermentor systems were used to examine changes in a community of human fecal bacteria supplied with different carbohydrate energy sources. A panel of group-specific fluorescent in situ hybridization probes targeting 16S rRNA sequences revealed that the fermentors supported growth of a greater proportion of Bacteroides and a lower proportion of gram-positive anaerobes related to Faecalibacterium prausnitzii, Ruminococcus flavefaciens-Ruminococcus bromii, Eubacterium rectale-Clostridium coccoides, and Eubacterium cylindroides than the proportions in the starting fecal inoculum. Nevertheless, certain substrates, such as dahlia inulin, caused a pronounced increase in the number of bacteria related to R. flavefaciens-R. bromii and E. cylindroides. The ability of three strictly anaerobic, gram-positive bacteria to compete with the complete human fecal flora was tested in the same experiment by using selective plating to enumerate the introduced strains. The Roseburia-related strain A2-183^F was able to grow on all substrates despite the fact that it was unable to utilize complex carbohydrates in pure culture, and it was assumed that this organism survived by cross-feeding. In contrast, Roseburia intestinalis L1-82^R and Eubacterium sp. strain A2-194^R survived less well despite the fact that they were able to utilize polysaccharides in pure culture, except that A2-194^R was stimulated 100-fold by inulin. These results suggest that many low-G+C-content gram-positive obligate anaerobes may be selected against during in vitro incubation, although several groups were stimulated by inulin. Thus, considerable caution is necessary when workers attempt to predict the in vivo effects of probiotics and prebiotics from their effects in vitro.     

一次性厌氧菌培养平板的制作与应用

用一次性厌氧菌培养平板,对１０属１７种６７株厌氧菌和３０份临床标本进行培养,同时用厌氧手套箱及厌氧罐作对照培养,其培养效果与厌氧手套箱一致,优于厌氧罐,且用一次性厌氧菌培养平板进行培养具有操作简便,宜于推广应用等特点。     

Understanding aerobic/anaerobic metabolism in Caldibacillus debilis through a comparison with model organisms

Caldibacillus debilis GB1 is a facultative anaerobe isolated from a thermophilic aero-tolerant cellulolytic enrichment culture. There is a lack of representative proteomes of facultative anaerobic thermophilic Bacillaceae, exploring aerobic/anaerobic expression. The C. debilis GB1 genome was sequenced and annotated, and the proteome characterized under aerobic and anaerobic conditions while grown on cellobiose. The draft sequence of C. debilis GB1 contains a 3,340,752 bp chromosome and a 5,386 bp plasmid distributed over 49 contigs. Two-dimensional liquid chromatography mass spectrometry/mass spectrometry was used with Isobaric Tags for Relative and Absolute Quantification (iTRAQ) to compare protein expression profiles, focusing on energy production and conversion pathways. Under aerobic conditions, proteins in glycolysis and pyruvate fermentation pathways were down-regulated. Simultaneously, proteins within the tricarboxylic acid cycle, pyruvate dehydrogenase, the electron transport chain, and oxygen scavenging pathways showed increased amounts. Under anaerobic conditions, protein levels in fermentation pathways were consistent with the generated end-products: formate, acetate, ethanol, lactate, and CO₂. Under aerobic conditions CO₂ and acetate production was consistent with incomplete respiration. Through a direct comparison with gene expression profiles from Escherichia coli, we show that global regulation of core metabolism pathways is similar in thermophilic and mesophilic facultative anaerobes of the Phylum Proteobacteria and Firmicutes.     

Survival,ATP pool,and ultrastructural characterization of benthic foraminifera from Drammensfjord (Norway): response to anoxia

While much evidence indicates that certain benthic foraminifera are facultative anaerobes, little is known regarding the physiologic response of foraminifera to anoxia. In order to assess their response, specimens of four foraminiferal species, collected from a typically dysoxic area of Drammensfjord, Norway (45 m water depth), were incubated in seawater purged with nitrogen. Over a time course of > 3 weeks, the specimens were extracted for adenosine triphosphate (ATP) in a nitrogen-flushed glove bag to assess their survival and ATP reserve under such conditions. For comparative purposes, similar extractions were done on conspecifics one week after their collection from the seafloor, as well as on other conspecifics, obtained from the same site, incubated in aerated conditions. The survival rates of nitrogen-treatedAdercotryma glomeratum, Psammosphaera bowmanni, and Stainforthia fusiformis were not significantly lower than those of the control specimens. However, the ATP concentrations of nitrogen-incubated A. glomeratum and S. fusiformis were significantly lower than those of their aerated conspecifics, while there was no significant difference between the [ATP] of P. bowmanni from the two treatments. Both the survival rate and the ATP concentrations of nitrogen-incubated Bulimina marginata were significantly lower than those of control specimens. The ultrastructure of B. marginata and S. fusiformis incubated in N₂ for 18 days were compared with those of specimens fixed within 15 minutes of collection. For both species, the specimens that survived the experimental treatment had ultrastructures indistinguishable from those fixed just after field collection. However, the ultrastructure of B. marginata differed from that of S. fusiformis in that it lacked the numerous peroxisome-endoplasmic reticulum (ER) complexes and what appeared to be algal chloroplasts observed in S. fusiformis. Copious arrays of paracrystals were observed in both species from the experimental treatment as well as the shipboard-fixed specimens, suggesting that neither population had extensive pseudopodial networks. When considered in combination, our results indicate that the four species respond to and survive anoxia differently, with responses including dormancy and, as yet unidentified, anaerobic metabolic pathways.     

Inhibition of Vancomycin-Resistant <Emphasis Type="Italic">Enterococcus</Emphasis> by Continuous-Flow Cultures of Human Stool Microflora With and Without Anaerobic Gas Supplementation

A continuous-flow competitive exclusion (CFCE) culture model of human stool microflora was used to examine whether supplemental anaerobic gas is necessary for maintenance of anaerobes and inhibition of vancomycin-resistant Enterococcus (VRE). CFCE cultures of human stool microflora were maintained with supplemental nitrogen, without supplemental nitrogen, or with percolated room air. Cultures with or without supplemental nitrogen maintained >9 log₁₀ CFU mL^–1 of obligate anaerobes and eliminated 10⁶ CFU mL^–1 of VRE. When room air was percolated into the culture, anaerobes were detected at 2 log₁₀ CFU mL^–1, and the same VRE inoculum was not eliminated (P < 0.001). These data demonstrate that human stool CFCE cultures maintain high levels of obligate anaerobes and inhibit VRE without the addition of supplemental anaerobic gas.     

Bacterial Diversity within Feedlot Manure

In this study, we identified the predominant culturable anaerobic bacteria and enumerated the total culturable anaerobic bacterial population present in samples of feedlot manure from Southern Queensland. Sixteen bacterial isolates were cultured from feedlot pad material with species of Lactobacillus, Clostridium and Bacillus predominating. From a library of 123 clones, produced by the amplification, cloning and partial DNA sequencing of the 16S rRNA gene, only 3% were closely related to previously described species and 21% to known genera. Of the total clone library, 96% were apparently Gram-positive and fell within families whose members were generally anaerobes. The majority (71%) of the clone library was related to either members of the family Clostridiaceae or lactic acid-producing bacteria (Lactobacillus or Lactosphaera). It was concluded that Gram-positive clostridial and lactic acid-producing bacteria predominate in feedlot pad manure. The overwhelming majority of species are novel and have not been obtained in culture. It would appear that the most likely source of the sickly-sweet nuisance odours (particularly from butyric acid) that emanate from feedlots is the by-product of anaerobic fermentation by clostridia. Gut-inhabiting and Gram-negative bacteria do not appear to survive for lengthy periods of time under the environmental conditions present in feedlot manure.