Extrachromosomal systems and gene transmission in anaerobic bacteria

Obligately anaerobic bacteria are important in terms of their role as medical pathogens as well as their degradative capacities in a variety of natural ecosystems. Two major anaerobic genera, Bacteroides and Clostridium, are examined in this review. Plasmid elements in both genera are reviewed within the context of conjugal transfer and drug resistance. Genetic systems that facilitate the study of these anaerobic bacteria have emerged during the past several years. In large part, these developments have been linked to work centered on extrachromosomal genetic systems in these organisms. Conjugal transfer of antibiotic resistance has been a central focus in this regard. Transposable genetic elements in the Bacteroides are discussed and the evolution and spread of resistance to lincosamide antibiotics are considered at the molecular level. Recombinant DNA systems that employ shuttle vectors which are mobilized by conjugative plasmids have been developed for use in Bacteroides and Clostridium. The application of transmission and recombinant DNA genetic systems to study these anaerobes is under way and is likely to lead to an increased understanding of this important group of procaryotes.     

Comparative study of bacterial groups within the human cecal and fecal microbiota

The composition of the human cecal microbiota is poorly known because of sampling difficulties. Samples of cecal fluid from eight subjects were collected via an intestinal tube. Feces were also collected. Total anaerobes, facultative anaerobes, bifidobacteria, and Bacteroides were enumerated by culture methods, and the predominant phylogenetic groups were quantified by molecular hybridization using a set of six rRNA-targeted probes. The numbers of strict anaerobes, bifidobacteria, Bacteroides, and members of the Clostridium coccoides group and Clostridium leptum subgroup were lower in the cecum. Facultative anaerobes represented 25% of total bacteria in the cecum versus 1% in the feces.     

Advances in development of a genetic system for Thermoanaerobacterium spp.: expression of genes encoding hydrolytic enzymes, development of a second shuttle vector, and integration of genes into the chromosome

Despite recent success in transforming various thermophilic gram-type-positive anaerobes with plasmid DNA, use of shuttle vectors for the expression of genes other than antibiotic resistance markers has not previously been described. We constructed new vectors in order to express heterologous hydrolytic enzymes in our model system, Thermoanaerobacterium saccharolyticum JW/SL-YS485. Transformed Thermoanaerobacterium expressed active enzyme, indicating that this system may function as an alternate expression host, especially for genes with a thermophilic origin. To develop further the genetic system for T. saccharolyticum JW/SL-YS485, two improved Escherichia coli-Thermoanaerobacterium shuttle vectors, pRKM1 and pRUKM, were constructed. Furthermore, the kanamycin resistance cassette alone and the kanamycin resistance cassette plus the cellobiohydrolase gene (cbhA) from Clostridium thermocellum JW20 were integrated into the xylanase gene (xynA) region of the Thermoanaerobacterium chromosome via homologous recombination using pUC-based suicide vectors pUXK and pUXKC.     

Metabolic engineering of Clostridium acetobutylicum: recent advances to improve butanol production

The biosynthesis of the solvents 1-butanol and acetone is restricted to species of the genus Clostridium, a diverse group of Gram-positive, endospore forming anaerobes comprising toxin-producing strains as well as terrestrial non-pathogenic species of biotechnological impact. Among solventogenic clostridia, Clostridium acetobutylicum represents the model organism and general but yet important genetic tools were established only recently to investigate and understand the complex life cycle-accompanied physiology and its regulatory mechanisms. Since clostridial butanol production regained much interest in the past few years, different metabolic engineering approaches were conducted--although promising and in part successful strategies were employed, the major breakthrough to generate an optimum phenotype with superior butanol titer, yield and productivity still remains to be expected.     

Identification, distribution, and toxigenicity of obligate anaerobes in polluted waters.

A seasonal occurrence of obligately anaerobic bacteria, predominantly of the genera Bacteroides and Clostridium, in a polluted water site has been observed. The number of anaerobes varied from 1.8 X 10(3) cells/ml in the warmer months to 10 cells/ml in winter. Several isolates were toxigenic, indicating a potential human health hazard.     

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.     

Comparative Study of Bacterial Groups within the Human Cecal and Fecal Microbiota

The composition of the human cecal microbiota is poorly known because of sampling difficulties. Samples of cecal fluid from eight subjects were collected via an intestinal tube. Feces were also collected. Total anaerobes, facultative anaerobes, bifidobacteria, and Bacteroides were enumerated by culture methods, and the predominant phylogenetic groups were quantified by molecular hybridization using a set of six rRNA-targeted probes. The numbers of strict anaerobes, bifidobacteria, Bacteroides, and members of the Clostridium coccoides group and Clostridium leptum subgroup were lower in the cecum. Facultative anaerobes represented 25% of total bacteria in the cecum versus 1% in the feces.     

How obligatory is anaerobiosis?

Historically many bacteria have been classified as obligate anaerobes. They have been construed as wholly intolerant of oxygen, a feature that was originally ascribed to their lack of superoxide dismutases and catalases. Clostridial species were regarded as classic examples. We now know that this view is quite wrong: enzymes that scavenge superoxide, hydrogen peroxide and even oxygen itself abound in anaerobes. In the current issue of Molecular Microbiology, Hillmann et al. demonstrate that full production of these proteins can allow Clostridium acetobutylicum to survive and even grow in oxygenated culture medium. Evidently, oxidative defences in anaerobes can be robust. In all likelihood, they are critical for the movement of bacteria through aerobic environments to new anaerobic habitats.     

Comparative degradation of cellulose and sugar formation by three newly isolated mesophilic anaerobes and Clostridium thermocellum

Summary To develop a coculture for a single step conversion of cellulose to fuels and chemicals, the cellulose degradation by three newly isolated mesophilic anaerobes was compared with that of Clostridium thermocellum. Degradation of cellulose obtained from cotton linters, and delignified ball-milled pulp and steam-exploded aspen wood by mesophilic anaerobes was comparable to that by Cl. thermocellum. However mesophilic anaerobes produced larger amounts of sugars. In media containing 50 g/l of cellulose suspensions, the sugars produced by mesophilic anaerobes were 10.5 to 14.5 g/l, about 50% of the cellulose used.     

High prevalence and resistance rates to antibiotics in anaerobic bacteria in specimens from patients with chronic balanitis

Aim of the study was to assess both prevalence and antibiotic resistance in anaerobic bacteria from glans penis skin of 70 adults. Strain susceptibility was determined by breakpoint susceptibility test or E test. In 9 asymptomatic, 48 untreated and 13 treated symptomatic patients, anaerobes were found in 22.2%, 70.8% and 53.3%, respectively. Gram-positive strains (GPAs) were 2.2-fold more common than Gram-negative ones. Prevalent Gram-negative (GNAs) and GPAs were Prevotella spp. and anaerobic cocci, respectively. Clostridium difficile strain was found in an untreated patient. In GNAs, resistance rates to amoxicillin, metronidazole, clindamycin, tetracycline, levofloxacin, and amoxicillin/clavulanate were 42.1, 0, 52.6, 53.3, 86.7 and 5.2%, respectively. In GPAs, the resistance rates to metronidazole, clindamycin, tetracycline, levofloxacin and amoxicillin/clavulanate were 18.2, 34.1, 52.6, 36.8 and 0%, respectively. In conclusion, anaerobes were 1.6-fold more frequent in untreated symptomatic patients compared with other patients, suggesting their participation in development of chronic balanitis. GPAs were more common than GNAs. The resistance rates to amoxicillin, clindamycin, tetracycline, and levofloxacin were high. Most active agents were metronidazole and amoxicillin/clavulanate. Resistance in anaerobes varies according to sites of specimens and years of study.     

Discovery and development of surotomycin for the treatment of <Emphasis Type="Italic">Clostridium difficile</Emphasis>

The primary challenge for treating Clostridium difficile infections (CDI) is maintenance of clinical response after the end of treatment (sustained clinical response). Disease recurrence following a positive clinical response occurs in approximately 6–25 % of patients after the first episode and in up to 65 % for subsequent recurrences. Surotomycin, a novel cyclic lipopeptide antibiotic with a core derived by Streptomyces roseosporus fermentation, disrupts C. difficile cellular membrane activity in both logarithmic and stationary phases and minimally disturbs normal gastrointestinal microbiota because of its lack of activity against Gram-negative anaerobes and facultative anaerobes. Preclinical and clinical evidence indicate that surotomycin has low oral bioavailability, allowing gastrointestinal tract concentrations to greatly exceed its minimum inhibitory concentration for C. difficile. Surotomycin is well tolerated and effective in hamster models of CDI. Phase 2 clinical evidence suggests that surotomycin (250 mg twice daily) is an effective CDI treatment, with statistically lower recurrence rates than vancomycin.     

An Evaluation of the Gaspak System in the Culture of Anaerobic Bacteria

S ummary : Comparable results were obtained with a commercially available disposable system for the production of H₂ and CO₂ in an anaerobic jar (the Gaspak system) and with a standard anaerobic jar procedure. Both systems were used with the Baird & Tatlock anaerobic jar and a room temperature catalyst. The test organisms included strict anaerobes such as Clostridium oedematiens type D and Cl. tetani. Comparative tests were also made with Bacteroides species from laboratory stock cultures and with freshly isolated strains of Bacteroides. On occasion, higher recoveries were obtained with the standard system when relatively demanding models were set up but it has limitations, having been developed initially for research purposes. The Gaspak system probably gives more reliable and more reproducible results in a laboratory in which experience with strict anaerobes is limited or sporadic.     

红霉素生物合成的分子生物学

近年来,国外对大环内酯类抗生素生物合成和基因工程的研究非常迅速,不仅认识了许多抗生素生物合成的过程,而且利用基因工程技术改造抗生素生物合成基因,合成了100多种非天然的“天然”抗生素。抗生素生物合成的分子生物学是抗生素基因工程的基础。本全面介绍了五厌内酯类抗生素的代表－红霉素生物合成分子生物学的历史、现状及发展趋势。     

Advances in Development of a Genetic System for Thermoanaerobacterium spp.: Expression of Genes Encoding Hydrolytic Enzymes, Development of a Second Shuttle Vector, and Integration of Genes into the Chromosome

Despite recent success in transforming various thermophilic gram-type-positive anaerobes with plasmid DNA, use of shuttle vectors for the expression of genes other than antibiotic resistance markers has not previously been described. We constructed new vectors in order to express heterologous hydrolytic enzymes in our model system, Thermoanaerobacterium saccharolyticum JW/SL-YS485. Transformed Thermoanaerobacterium expressed active enzyme, indicating that this system may function as an alternate expression host, especially for genes with a thermophilic origin. To develop further the genetic system for T. saccharolyticum JW/SL-YS485, two improved Escherichia coli-Thermoanaerobacterium shuttle vectors, pRKM1 and pRUKM, were constructed. Furthermore, the kanamycin resistance cassette alone and the kanamycin resistance cassette plus the cellobiohydrolase gene (cbhA) from Clostridium thermocellum JW20 were integrated into the xylanase gene (xynA) region of the Thermoanaerobacterium chromosome via homologous recombination using pUC-based suicide vectors pUXK and pUXKC.     

Characterization of an antibiotic resistant Clostridium hathewayi strain from a continuous-flow exclusion chemostat culture derived from the cecal contents of a feral pig

The chemostat model has been an important tool in studying intestinal microflora. To date, several competitive exclusion products have been developed from such studies as prophylactic treatment against pathogenic bacteria. A continuous-flow chemostat model of a feral pig was developed using inocula from the cecal contents of a wild boar caught in East Texas. Several strains of antibiotic-sensitive bacteria were isolated including Bacteroides, Lactobacillus, Enterococcus and Clostridium sp. This study reports on the characterization of a multidrug-resistant Clostridium hathewayi strain that was isolated from this feral pig's cecal contents maintained in a continuous-flow chemostat system showing high resistance to carbapenems and macrolides (including the growth promoter tylosin). Clostridium hathewayi has been documented to be pathogenic to both humans and animals. Feral pigs may be an important source of pathogenic and antibiotic resistant bacteria and may pose potential risk to domestic species. Further work is needed to elucidate the prevalence of these reservoirs and assess the contribution these may play in the spread of disease and resistance.     

38例慢性化脓性中耳炎厌氧菌分析

对广东省湛江农垦第二医院３８例慢性化脓性中耳炎患者脓性标本,进行厌氧菌和需氧菌培养,结果３２例培养阳性,总检出率８４．２％。其中单纯厌氧菌和需氧菌检出率,分别占阳性标本的２１８％和４０．６％。两者混合阳性占３７．５％。厌氧菌中以革兰氏阳性球菌多见４８．０％（１２／２５）,需氧菌以绿脓杆菌多见４２．８％（１２／２８）。并对感染机理,细菌种类和药敏试验进行了讨论。     

Bacterial metabolism of natural and synthetic sex hormones undergoing enterohepatic circulation

Steroids undergoing enterohepatic circulation are exposed to bacterial metabolism particularly by obligate anaerobes which account for 99.99% of the fecal flora. The most common transformation is hydrolysis of conjugated steroids. The glucuronidases are synthesized by Escherichia coli and Bacteroides species. The bacterial catabolism of unconjugated steroids may be considered under several headings: 1. Reduction of ring-A due to clostridia species synthesizing specific enzymes; C. paraputrificum, 3 alpha,5 beta-reductase; C. innocuum, 3 beta,5 beta-reductase; and a new species C.J-1, 3 beta,5 alpha-reductase. 2. Reduction of the delta 5 bond by human fecal flora. The specific strain(s) synthesizing the enzyme have not yet been identified. 3. Reduction of 17-keto estrogens by the above mentioned ring-A reducing clostridia and by Eubacterium lentum. 4. Reduction of 17-keto androstenes by Bacteroides fragilis. 5. Desmolase mediated side chain cleavage at C17-C20 position of 17 alpha-hydroxysteroids by two new species Clostridium scindens and Eubacterium desmolans isolated from human and cat fecal flora respectively and by Clostridium cadavaris isolated from New York City sewage. 6. And 16 alpha- and 21-dehydroxylase by E. lentum a normal inhabitant of the human gut; it is the only organism known to synthesize 16 alpha- or 21-dehydroxylases. Due to the high specificity of the enzymes and the simplicity of extracting the metabolites, biosynthesis of reference compounds and radioimmunoassay reagents is practical and inexpensive. The enzymes can also be used for titration of specific bacterial strains in fecal flora and as markers for bacterial identification in particular for the strains difficult to be defined by regular biochemical reactions.     

Antibiotic Resistance Among Anaerobes: What Does it Mean?

Antibiotic resistance among anaerobes is increasing, with significant resistance to clindamycin, cephalosporins, cephamycins, and penicillins noted at community hospitals and major medical centers. A total of 615 anaerobes isolated from various Chicago area hospitals in 1996 were tested against 13 antibiotics, and the resistance patterns compared with similar data from 1991. For the Bacteroides fragilis group anaerobes, the most effective antibiotics were the β-lactam/β-lactamase inhibitor combination agents, carbapenems, trovafloxacin and metronidazole. High levels of resistance to clindamycin, piperacillin, cefoxitin and ceftizoxime were seen 1996. For non- B. fragilis group anaerobes, resistance was mush lower, and was notable only in Clostridium spp. (clindamycin and cephamycins) and Prevotella spp. (clindamycin and piperacillin). Despite the prevalence of antibiotic resistance among anaerobes, the frequency of antimicrobial susceptibility testing of anaerobes is declining. There are a number of factors that account for this decline, including a general reduction in funding of hospital clinical laboratories, a concomitant loss of expertise at these institutions, a lack of automated testing for anaerobes, and a failure to consider resistance as important to clinicians. The case for increased susceptibility testing is built upon the changing patterns of resistance such as those reported in this paper, the identification and transfer of genetic determinants corresponding to antibiotic resistance, as well as the correlation of resistance and clinical outcome.