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.     

Anaerobic Fish Spoilage by Bacteria I. Biochemical Changes in Herring Extracts

Water extracts of herring fillets were used as laboratory model substrates for the study of anaerobic bacterial spoilage of fish stored in bulk. The organisms studied ( Enterobacter sp., str. NTHC 151, Proteus sp., str. NTHC 153, and Aeromonas sp., str. NTHC 154) were selected for by an enrichment method designed to isolate the bacteria having the highest anaerobic growth yields in these herring extracts. The extracts were then inoculated with pure cultures of the bacteria and incubated anaerobically at 15°C. Chemical analyses of the extracts were carried out at the start and at the end of the incubation period. The compounds determined accounted for 88% of total carbon and 93% of total nitrogen of the sterile extracts. Ribose (free and bound), free hexoses and lactate were the main substrates for the growth of the bacteria. These compounds were converted to acetate and CO₂; hydrogen liberated by these processes was used for the quantitative reduction of trimethylamine oxide to trimethylamine. Fermentation balances support the contention that the biochemical changes observed represent the quantitative dominating chemical events in the herring extracts as a result of the development of the spoilage bacteria.     

Rapid Methods for Testing the Efficacy of Sterilization-Grade Filter Membranes

The validation of sterilization-grade membranes is integral to ensuring the efficient and safe use of microfiltration systems. Here validation refers to the production of sterile filtrate for sterilizing-grade membranes under challenge test conditions. Current validation methods require 48 h of culture for results to become available, which creates time delays within the manufacturing process and quality control (QC) backlogs. This work compares four methods for the production of filter challenge test data, to the desired test sensitivity, within 24 h using bioluminescent and fluorescent recombinant strains of the test organism Brevundimonas diminuta. These methods should provide a way to implement more rapid QC test regimens for filters.     

Micromethod System for Identification of Anaerobic Bacteria

A micromethod multitest system prepared by Analytab Products, Inc. and conventional tests employed at the Center for Disease Control for identification of anaerobes were compared. All procedures were conducted in an anaerobic glove box. A total of 104 cultures, including 18 reference strains and 86 diagnostic cultures, were examined. Ninety-one percent of the total tests performed with the two systems were in agreement. Greater than 90% agreement between the two systems was obtained with 12 of the 17 differential tests compared. The tests for nitrate reduction and H(2)S production gave the poorest agreement, 77.8 and 80.8%, respectively. Only 66% of the 86 diagnostic cultures could be presumptively identified with the micromethod system supplemented only with microscopy and colonial characteristics. However, when appropriate supplementary tests and gas-liquid chromatography were used with the micromethod system, 85% of the 86 strains could be identified. When Ehrlich reagent, instead of Kovac reagent, was used with the micromethod to test for indole, the agreement in identification was raised to 93%.     

Antibiotic Production by Anaerobic Bacteria

Soils from aerobic and anaerobic sources were investigated for the possible presence of bacteria which produce antibiotics under anaerobic conditions of growth. The screening techniques devised for this study yielded 157 soil bacteria which, during anaerobic growth, produced antibiotic activity against aerobic test bacteria.

Studies on choice of media, presence of oxygen, and changes in antibiotic activity during growth indicated that representative strains of these bacteria produced mixtures of antibiotics. The activity was heat labile.

     

Development of a Micromethod for Identification of Anaerobic Bacteria

A microprocedure was described for determining the carbohydrate fermentation patterns of 48 anaerobic bacteria at one time in microtiter plates. The cultures were transferred into agar-filled wells of microtiter plates with a replicator inside an anaerobic glove box. Fermentation was measured both with a colorimetric indicator and with a small pH electrode. The method was approximately 97% accurate. It would be most useful for laboratories that need to identify large numbers of anaerobes at one time.     

Oxygen Sensitivity of Various Anaerobic Bacteria

Anaerobes differ in their sensitivity to oxygen, as two patterns were recognizable in the organisms included in this study. Strict anaerobes were species incapable of agar surface growth at pO(2) levels greater than 0.5%. Species that were found to be strict anaerobes were Treponema macrodentium, Treponema denticola, Treponema oralis n. sp., Clostridium haemolyticum, Selenomonas ruminatium, Butyrivibrio fibrisolvens, Succinivibrio dextrinosolvens, and Lachnospira multiparus. Moderate anaerobes would include those species capable of growth in the presence of oxygen levels as high as 2 to 8%. The moderate anaerobes could be exposed to room atmosphere for 60 to 90 min without appreciable loss of viability. Species considered as moderate anaerobes were Bacteroides fragilis, B. melaninogenicus, B. oralis, Fusobacteria nucleatum, Clostridium novyi type A, and Peptostreptococcus elsdenii. The recognition of at least two general types of anaerobes would seem to have practical import in regard to the primary isolation of anaerobes from source material.     

厌氧细菌L型的诱导

用苯唑青霉素、羧苄青霉素对脆弱类杆菌、多形类杆菌、核梭杆菌、产气荚膜杆菌和艰难杆菌等5种厌氧菌进行L型菌的诱导。通过诱导,可见到脆弱类杆菌、多形类杆菌和产气荚膜杆菌形成油煎蛋状或颗粒状菌落。革兰氏染色镜检,见到菌体肿胀,并有丝状体、圆球体和巨球体。细胞壁染色法可见到部分细菌因胞壁缺损而被结晶紫透入菌体着色。诱导后的少数细菌可转变成可滤过型而能通过0.4μm孔径的滤膜。核梭杆菌诱导后形态改变极显著而较难识别。艰难杆菌形态改变小。建设在临床标本细菌培养时,有时尚需增加L型厌氧菌培养,以提高检出率。     

Antibiotic Susceptibility of Anaerobic Ruminal Bacteria

This study demonstrated that 15 species of ruminal bacteria with no previous history of contact with antibiotics are susceptible to bacitracin, chloramphenicol, chlortetracycline, erythromycin, novobiocin, oleandomycin, oxytetracycline, penicillin, tetracycline, tylosin, and vancomycin. A number of the species were not inhibited by kanamycin, neomycin, polymyxin, and streptomycin. The data suggest that antibiotic-resistant cells occur within susceptible cultures of these species. Streptococcus bovis FD-10 and a nonruminal anaerobe, Bacteroides melaninogenicus BE-1, showed similar antibiotic susceptibilities.     

Cultivation of Anaerobic and Facultatively Anaerobic Bacteria from Spacecraft-Associated Clean Rooms

In the course of this biodiversity study, the cultivable microbial community of European spacecraft-associated clean rooms and the Herschel Space Observatory located therein were analyzed during routine assembly operations. Here, we focused on microorganisms capable of growing without oxygen. Anaerobes play a significant role in planetary protection considerations since extraterrestrial environments like Mars probably do not provide enough oxygen for fully aerobic microbial growth. A broad assortment of anaerobic media was used in our cultivation strategies, which focused on microorganisms with special metabolic skills. The majority of the isolated strains grew on anaerobic, complex, nutrient-rich media. Autotrophic microorganisms or microbes capable of fixing nitrogen were also cultivated. A broad range of facultatively anaerobic bacteria was detected during this study and also, for the first time, some strictly anaerobic bacteria (Clostridium and Propionibacterium) were isolated from spacecraft-associated clean rooms. The multiassay cultivation approach was the basis for the detection of several bacteria that had not been cultivated from these special environments before and also led to the discovery of two novel microbial species of Pseudomonas and Paenibacillus.The major issue of planetary protection is to prevent the contamination of extraterrestrial environments by terrestrial biomolecules and life forms. Furthermore, reverse contamination of Earth by extraterrestrial material is also a fundamental concern (1). In order not to affect or even to confound future life detection missions on celestial bodies, which are of interest for their chemical and biological evolution, spacecraft are constructed in so-called clean rooms and are subject to severe cleaning processes and microbiological controls before launch (9). Therefore, these clean rooms are considered extreme environments for microorganisms (47).Detailed planetary protection protocols for missions to Mars were designed for the Viking missions, which were launched in 1975, and about 7,000 samples were taken from the two Viking spacecraft during prelaunch activities in order to determine the cultivable microbial load (37). Besides human-associated bacteria (pathogens and opportunistic pathogens), which were predominant among the microbes detected in these samples, aerobic spore-forming microorganisms (Bacillus) were found frequently on spacecraft and within the facilities.Spores are the resting states of bacteria and are often highly resistant to heat, desiccation, and other abiotic stresses. These multiresistance properties of such spore-forming microorganisms make them perfect candidates for surviving a space flight, and thus, the main focus of attention has been on them. Furthermore, only the detection of aerobic spore-forming bacteria is currently included in space agencies'' planetary protection protocols for the quantitative determination of microbial burden on spacecraft.The presence of extraordinarily (UV-) resistant spores in spacecraft facilities has been reported (31), but it also has been proven that vegetative microbial cells (e.g., Deinococcus radiodurans and Halobacterium sp. strain NRC-1) can resist very harsh conditions, such as extreme doses of (UV and ionizing) radiation and desiccation (8, 11). Recent culture-based and molecular studies have shown that the microbial diversity on spacecraft and within the clean rooms is extraordinarily high and does include extremotolerant bacteria and even archaea (25, 30).The atmospheres of most planets and bodies within the reach of human exploration contain only traces of oxygen (Mars contains 0.13%), probably not enough to support terrestrial aerobic life as we know it (26, 44). Even though Mars'' surface is highly oxidizing and radiation exposed, the Martian subsurface, as well as those of other planets and bodies (like, e.g., Titan), has been discussed as an anaerobic biotope for possible life (4, 40).Therefore, the lack of studies of the existence of anaerobically growing microorganisms in spacecraft-associated clean rooms is quite surprising. One possible reason for this discrepancy might be that the cultivation of anaerobes is challenging. Already in 1969, Hungate published a method for the cultivation of strictly anaerobic methanogenic Archaea (20). Although this technique has undergone a few simplifications during past decades, the cultivation of anaerobes requires specialized and expensive equipment (e.g., anaerobic glove boxes and gas stations), practical experience, and skills in specific methodology. Nevertheless, by the application of anaerobic cultivation strategies, many fascinating microorganisms—such as Nanoarchaeum equitans, the first representative of the new archaeal phylum Nanoarchaeota, or Thermotoga maritima, a hyperthermophilic bacterium growing at up to 90°C (17, 18)—have successfully been isolated from diverse and sometimes extreme biotopes.Generally, there are different types of anaerobic organisms. Facultative anaerobes (like Escherichia coli) are able to adapt their metabolism and can grow under conditions with or without oxygen but prefer aerobic conditions. Aerotolerant anaerobes do not need oxygen for their growth and show no preference, and strict anaerobes (e.g., methanogens) never require oxygen for their reproduction and metabolism. Even more, obligate (strict) anaerobes can be growth inhibited or even killed by oxygen.The presence of anaerobic microorganisms (enriched using the BD GasPak system) in surface samples from U.S. clean rooms has rarely been reported. Members of the facultatively anaerobic genera Paenibacillus and Staphylococcus have been isolated in the course of a study about extremotolerant microorganisms (25). During molecular surveys of U.S. clean rooms, the 16S rRNA genes from strictly anaerobic microorganisms, such as the spore-forming genus Clostridium, have already been detected (29). Nevertheless, the cultivation of these microbes has not yet been successful.With the ExoMars mission impending, the European Space Agency (ESA) is organizing and funding a biodiversity study of the ESA''s clean rooms and the spacecraft therein. The microbiology of these special environments is characterized in detail by a combination of standard procedures, new cultivation approaches, and molecular methods that shall illuminate the presence of planetary protection-relevant microorganisms in these facilities. At the date of sampling, all the clean rooms harbored the Herschel Space Observatory, a spacecraft to be launched together with the Planck satellite in spring 2009, as of this writing. Herschel will be fitted with the largest mirror ever built for a space mission (3.5 m in diameter), and its main goal will be the exploration of the cold universe, i.e., the formation and evolution of proto-galaxies (35). The Herschel Space Observatory does not demand planetary protection requirements, but all clean rooms were in a fully operating state during the construction work. This gave us the opportunity to sample the microbial diversity in these extreme environments without bioburden control but under strict contamination-controlled conditions, with respect to particulates and molecular contamination.This paper presents the results from our attempts to isolate anaerobic and facultatively anaerobic microorganisms from samples of spacecraft and surfaces in European spacecraft-associated clean rooms. For this purpose, we have successfully applied Hungate technology for anaerobic culturing and used an assortment of noncommercial media for the cultivation of a broad variety of microorganisms. Besides the capability of anaerobic growth, many of our isolates revealed special physiological capacities (e.g., nitrogen fixation and autotrophic metabolism) that might be relevant for further planetary protection considerations.     

Extracellular Deoxyribonuclease Production by Anaerobic Bacteria

The production of extracellular deoxyribonuclease was examined with anaerobic organisms isolated from clinical specimens. Nuclease activity was extraordinarily common. All strains of Fusobacterium, including eight species, as well as Bacteroides fragilis and B. melaninogenicus, displayed enzyme activity. Whereas the gram-positive bacteria were generally less productive, all strains of Clostridium perfringens, Peptostreptococcus intermedius, and P. anaerobius specifically produced deoxyribonuclease. The test is taxonomically valuable, particularly in the characterization of gram-positive cocci, since a deoxyribonuclease-producing coccus indicates P. intermedius or P. anaerobius. Additionally, possession of the enzyme may prove to be a useful correlate of the potential pathogenicity of anaerobes.     

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。     

Standardized Antimicrobial Disc Susceptibility Testing of Anaerobic Bacteria. I. Susceptibility of Bacteroides fragilis to Tetracycline

A modified Bauer-Kirby-Sherris-Turck method for disc susceptibility testing of anaerobic bacteria is presented. When tetracycline was used against 100 strains of Bacteroides fragilis as a model, reasonably reproducible results were obtained after overnight incubation in both the GasPak atmosphere and an atmosphere achieved by adding 10% CO(2) to a mixture of 10% H(2) and 90% N(2). The minimal inhibitory concentration for the strains determined by the agar dilution technique correlated well with the results of disc tests performed in the GasPak atmosphere with 30-mug tetracycline discs. Among 63 strains isolated from 1970 to the present, only 24 (38.1%) were found to be susceptible to tetracycline.     

Method for Rapid Detection of Cyanogenic Bacteria

An agar plate method is described in which the production of hydrogen cyanide by as many as 50 microbial isolates per plate may be detected. Cyanide produced by the organisms reacts with copper(II) ethylacetoacetate and 4,4′-methylenebis-(N,N-dimethylaniline) in a paper disk suspended above the microbial colonies. Cell growth occurs in depressions in the agar surface, which allows separation of colonies and enhances sensitivity of hydrogen cyanide detection.     

临床厌氧菌的分离和鉴定

我们从口腔、胸部、腹部和盆腔等处采集了110份临床感染标本,以自制的输送培养基立即送实验室,在厌氧手套箱(霍尔玛厌氧系统1029型)或Gas-pak罐中,行厌氧菌的分离和培养,其中64份标本检出厌氧菌,阳性率为58％。临床标本中牙周炎标本厌氧菌检出率高达100％,牙髓炎标本为85％,阑尾脓肿和腹膜炎标本为83％,胆道标本为39％,脓胸标本为57％,盆腔标本为33％,早期单纯性阑尾炎和甲状腺囊肿合并感染的标本各5份,都未检出厌氧菌。从64份阳性分离的标本中共分离到厌氧菌370株,经鉴定分别属于11个菌属32个菌种(未定种的有74株),其中类杆菌最多占45.9％(类杆菌属中脆弱类杆菌占37.6％),次为梭杆菌属和消化链球菌属,各占15.1％。革兰氏阳性无芽胞厌氧菌占8％左右,而梭菌属为8.9％,其余是二氧化碳噬纤维菌属(2.9％)、韦荣氏球菌属(1.3％)、链球菌属和纤毛菌属(1.5％)等。在厌氧菌鉴定中,我们使用了微量生化直接酶测定技术和代谢产物的气相色谱分析技术,这些方法在厌氧菌鉴定中比常规方法敏感且有较大的价值。