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。     

Practical Method for Isolation of Anaerobic Bacteria in the Clinical Laboratory

An inexpensive but practical and simple method is described for providing an environment suitable for the maintenance and survival of clinically significant anaerobic bacteria, consistent with the needs of a large-volume laboratory. This modification of the GasPak procedure is based on two concepts. One involved the storage of "freshly prepared" plating medium under a constant stream of carbon dioxide. The other concept was to use an anaerobe jar, continuously flushed with oxygen-free carbon dioxide, as a "holding receptacle" for the inoculated media Experience with this modification has shown that the number of isolations of anaerobic bacteria from clinical material can be increased significantly.     

Rifampin-Blood-Agar as a Selective Medium for the Isolation of Certain Anaerobic Bacteria

A selective medium which allows detection of relatively small numbers of Fusobacterium varium in fecal specimens is described. Blood-agar containing 50 mug of rifampin per ml inhibits the growth of many species of Bacteriodes and of F. fusi-forme/nucleatum but allows good growth of F. varium and most strains of F. mortiferum. Quantitative cultures of 11 fecal specimens were done on rifampin and other selective and nonselective media. F. varium was recovered in counts of 10(6) and 10(7) per gram from two specimens on rifampin only. A third specimen yielded 10(10)F. varium on several media, including rifampin. Some Eubacterium and Clostridium species also grew on rifampin, and these ordinarily were distinguished from the Fusobacterium by colony morphology. This medium is of value in fecal flora studies and should be useful with other kinds of specimens where mixtures of organisms are common.     

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.     

Isolation, Culture Characteristics, and Identification of Anaerobic Bacteria from the Chicken Cecum

Studies on the anaerobic cecal microflora of the 5-week-old chicken were made to determine a suitable roll-tube medium for enumeration and isolation of the bacterial population, to determine effects of medium components on recovery of total anaerobes, and to identify the predominant bacterial groups. The total number of microorganisms in cecal contents determined by direct microscope cell counts varied (among six samples) from 3.83 x 10(10) to 7.64 x 10(10) per g. Comparison of different nonselective media indicated that 60% of the direct microscope count could be recovered with a rumen fluid medium (M98-5) and 45% with medium 10. Deletion of rumen fluid from M98-5 reduced the total anaerobic count by half. Colony counts were lower if chicken cecal extract was substituted for rumen fluid in M98-5. Supplementing medium 10 with liver, chicken fecal, or cecal extracts improved recovery of anaerobes slightly. Prereduced blood agar media were inferior to M98-5. At least 11 groups of bacteria were isolated from high dilutions (10(-9)) of cecal material. Data on morphology and physiological and fermentation characteristics of 90% of the 298 isolated strains indicated that these bacteria represented species of anaerobic gram-negative cocci, facultatively anaerobic cocci and streptococci, Peptostreptococcus, Propionibacterium, Eubacterium, Bacteroides, and Clostridium. The growth of many of these strains was enhanced by rumen fluid, yeast extract, and cecal extract additions to basal media. These studies indicate that some of the more numerous anaerobic bacteria present in chicken cecal digesta can be isolated and cultured when media and methods that have been developed for ruminal bacteria are employed.     

Anaerobic Mineralization of Quaternary Carbon Atoms: Isolation of Denitrifying Bacteria on Dimethylmalonate

The microbial capacity to degrade simple organic compounds with quaternary carbon atoms was demonstrated by enrichment and isolation of five denitrifying strains on dimethylmalonate as the sole electron donor and carbon source. Quantitative growth experiments showed a complete mineralization of dimethylmalonate. According to phylogenetic analysis of the complete 16S rRNA genes, two strains isolated from activated sewage sludge were related to the genus Paracoccus within the α-Proteobacteria (98.0 and 98.2% 16S rRNA gene similarity to Paracoccus denitrificans^T), and three strains isolated from freshwater ditches were affiliated with the β-Proteobacteria (97.4 and 98.3% 16S rRNA gene similarity to Herbaspirillum seropedicae^T and Acidovorax facilis^T, respectively). Most-probable-number determinations for denitrifying populations in sewage sludge yielded 4.6 × 10⁴ dimethylmalonate-utilizing cells ml⁻¹, representing up to 0.4% of the total culturable nitrate-reducing population.     

Improved Rapid Plate Method for the Isolation of Bacteriophages from Lysogenic Bacteria

A modification of a recently reported rapid plate method for the isolation of bacteriophages from lysogenic bacteria is described. The velveteen replica plate technique was used for inoculation of mitomycin C-induced colonies onto agar plates, and tetrazolium chloride was used to enhance detection of phage activity on replicated indicator plates.     

Anaerobic Roll Tube Media for Nonselective Enumeration and Isolation of Bacteria in Human Feces

Medium 10 (M10), developed for rumen bacteria and containing small amounts of sugars, starch, volatile fatty acids, hemin, Trypticase, yeast extract, cysteine, and sulfide, plus agar, minerals and CO(2)-HCO(3)-buffer, was used with the Hungate anaerobic method as a basal medium to evaluate the efficacy of various ingredients. Three-day-old colony counts from adults on normal diets (17 samples) were 0.55 x 10(11) to 1.7 x 10(11) per g (mean, 1.15 x 10(11)) for M10. Single deletion of volatile fatty acids, Trypticase, yeast extract, or sulfide did not reduce counts. Deletion of hemin or both Trypticase and yeast extract significantly lowered counts. Addition of fecal extract, rumen fluid, 1% dehydrated Brain Heart Infusion (BHI) or 2 to 6% liver infusion did not increase counts; 1% dehydrated bile or 3.7% BHI markedly depressed them. Decreasing the gas-phase CO(2) concentration from 100 to 5% with N(2) and correspondingly lowering the HCO(3) had little effect. Counts in supplemented Brewer Thioglycollate (Difco), BHI, and Trypticase soy agar were similar or lower than in M10; ease in counting was best in M10. Comparison of features of 88 predominant strains of fecal bacteria randomly isolated indicated that M10 supported growth of as many or more species of bacteria as compared to supplemented BHI. The results suggest that predominant bacteria of human feces, in general, are not as nutritionally fastidious as rumen bacteria and indicate that media for counts or isolation containing large amounts of rich organic materials are neither necessary nor desirable when adequate anaerobic techniques are used.     

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。     

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.     

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.