Microflora of the posterior pharyngeal wall, larynx and postoperative wounds in patients after laryngectomy]

The study group were persons with risk factors of colonization by pathogenic strains and included smokers, patients suffering from paradontosis, and patients with visibly neglected oral cavity and teeth. We isolated and classified to the species or genera 488 microorganisms. Of posterior pharyngeal wall flora, 61% of were Gram-negative bacteria, represented predominantly by Haemophilus and anaerobic rods and aerobic cocci belonging to the Neisseria and Moraxella (Branhamella) genera. In the group of Gram-positive cocci (34% of the total number of microorganisms), oral streptococci, Stomatococcus mucilaginosus and Streptococcus pneumoniae were isolated most frequently. The affected by neoplastic lesions larynx, was colonized by similar bacterial groups. However, the incidence of Gram-positive cocci was higher. The main etiologic factor of purulent post-operative wound inflammations were methicillin-resistant Staphylococcus aureus strains (MRSA), which had been absent among the bacteria isolated from patients on admission.     

口腔未培养微生物分离培养策略的研究进展

口腔微生物是人体微生物组的重要组成部分,其群落组成丰富且独特。现有研究显示,口腔微生物与龋病、牙周炎等口腔健康问题有直接的联系,因而具有重要的研究价值。随着高通量测序技术的发展,人们对口腔中未培养微生物多样性的认识不断加深,这进一步催生对微生物分离培养技术需求的增加。为此,本文将围绕口腔未培养微生物及其分离培养策略的研究进展,首先介绍口腔中未培养微生物的研究现状;其次分析口腔微生物分离培养中可能的限制因素;最后综述微生物分离培养技术发展及其在口腔未培养微生物研究中的应用。全文旨在为口腔未培养微生物的分离培养提供思路和技术参考。     

In Vitro Culture of Previously Uncultured Oral Bacterial Phylotypes

Around a third of oral bacteria cannot be grown using conventional bacteriological culture media. Community profiling targeting 16S rRNA and shotgun metagenomics methods have proved valuable in revealing the complexity of the oral bacterial community. Studies investigating the role of oral bacteria in health and disease require phenotypic characterizations that are possible only with live cultures. The aim of this study was to develop novel culture media and use an in vitro biofilm model to culture previously uncultured oral bacteria. Subgingival plaque samples collected from subjects with periodontitis were cultured on complex mucin-containing agar plates supplemented with proteose peptone (PPA), beef extract (BEA), or Gelysate (GA) as well as on fastidious anaerobe agar plus 5% horse blood (FAA). In vitro biofilms inoculated with the subgingival plaque samples and proteose peptone broth (PPB) as the growth medium were established using the Calgary biofilm device. Specific PCR primers were designed and validated for the previously uncultivated oral taxa Bacteroidetes bacteria HOT 365 and HOT 281, Lachnospiraceae bacteria HOT 100 and HOT 500, and Clostridiales bacterium HOT 093. All agar media were able to support the growth of 10 reference strains of oral bacteria. One previously uncultivated phylotype, Actinomyces sp. HOT 525, was cultivated on FAA. Of 93 previously uncultivated phylotypes found in the inocula, 26 were detected in in vitro-cultivated biofilms. Lachnospiraceae bacterium HOT 500 was successfully cultured from biofilm material harvested from PPA plates in coculture with Parvimonas micra or Veillonella dispar/parvula after colony hybridization-directed enrichment. The establishment of in vitro biofilms from oral inocula enables the cultivation of previously uncultured oral bacteria and provides source material for isolation in coculture.     

The anammox case-a new experimental manifesto for microbiological eco-physiology

The anaerobic ammonium oxidation process is a new process for ammonia removal from wastewater. It is also a new microbial physiology that was previously believed to be impossible. The identification of Candidatus Brocadia anammoxidans and its relatives as the responsible bacteria was only possible with the development of a new experimental approach. That approach is the focus of this paper. The approach is a modernisation of the Winogradsky/Beyerinck strategy of selective enrichment and is based on the introduction of the molecular toolbox and modern bioreactor engineering to microbial ecology. It consists of five steps: (1) postulation of an ecological niche based on thermodynamic considerations and macro-ecological field data; (2) engineering of this niche into a laboratory bioreactor for enrichment culture; (3) black-box physiological characterisation of the enrichment culture as a whole; (4) phylogenetic characterisation of the enriched community using molecular tools; (5) physical separation of the dominant members of the enrichment culture using gradient centrifugation and the identification of the species of interest in accordance with Koch's postulates; (6) verification of the in situ importance of these species in the actual ecosystems. The power of this approach is illustrated with a case study: the identification of the planctomycetes responsible for anaerobic ammonium oxidation. We argue that this was impossible using molecular ecology or conventional ‘cultivation based techniques’ alone. We suggest that the approach might also be used for the microbiological study of many interesting microbes such as anaerobic methane oxidisers. This revised version was published online in August 2006 with corrections to the Cover Date.     

Microbial diversity of the brine-seawater interface of the Kebrit Deep, Red Sea, studied via 16S rRNA gene sequences and cultivation methods.

The brine-seawater interface of the Kebrit Deep, northern Red Sea, was investigated for the presence of microorganisms using phylogenetic analysis combined with cultivation methods. Under strictly anaerobic culture conditions, novel halophiles were isolated. The new rod-shaped isolates belong to the halophilic genus Halanaerobium and are the first representatives of the genus obtained from deep-sea, anaerobic brine pools. Within the genus Halanaerobium, they represent new species which grow chemoorganotrophically at NaCl concentrations ranging from 5 to 34%. The cellular fatty acid compositions are consistent with those of other Halanaerobium representatives, showing unusually large amounts of Delta7 and Delta11 16:1 fatty acids. Phylogenetic analysis of the brine-seawater interface sample revealed the presence of various bacterial 16S rRNA gene sequences dominated by cultivated members of the bacterial domain, with the majority affiliated with the genus Halanaerobium. The new Halanaerobium 16S rRNA clone sequences showed the highest similarity (99.9%) to the sequence of isolate KT-8-13 from the Kebrit Deep brine. In this initial survey, our polyphasic approach demonstrates that novel halophiles thrive in the anaerobic, deep-sea brine pool of the Kebrit Deep, Red Sea. They may contribute significantly to the anaerobic degradation of organic matter enriched at the brine-seawater interface.     

Taxonomy of halophilic Archaea: current status and future challenges

Several groups of Archaea, all Euryarchaeota, develop in hypersaline environments (from >10 % salt up to saturation). The cultured diversity of halophilic Archaea includes the family Halobacteriaceae of aerobic or facultative anaerobic, generally red-pigmented species (47 genera and 165 species as of February 2014) and seven representatives of four genera of methanogens, most of which obtain energy from methylated amines under anaerobic conditions. Metagenomic studies have identified an additional deep lineage of Archaea in salt lakes and ponds with brines approaching NaCl saturation. Genomic information is now available for representatives of these ‘Nanohaloarchaea’, but no members of this lineage have yet been cultured. Multilocus sequence analysis is becoming increasingly popular in taxonomic studies of the Halobacteriaceae, and such studies have demonstrated that recombination of genetic traits occurs at an extremely high frequency at least in some genera. Metagenomic studies in an Antarctic lake showed that large identical regions of up to 35 kb in length can be shared by members of different genera living together in the same environment. Such observations have important implications not only for the taxonomy of the Halobacteriaceae, but also for species concepts and questions on taxonomy and classification for prokaryotic microorganisms in general.     

Bacterial stress enrichment enhances anaerobic hydrogen production in cattle manure sludge

Methodology was evaluated to selectively enrich hydrogen-producing species present in biological sludge produced during organic wastewater treatment. The influence of bacterial stress enrichment on anaerobic hydrogen-producing microorganisms was investigated in batch tests using serum bottles. Enrichment conditions investigated included application of acute physical and chemical stresses: wet heat, dry heat and desiccation, use of a methanogen inhibitor, freezing and thawing, and chemical acidification with and without preacidification of the sludge at pH 3. For each enrichment sample, cultivation pH value was set at an initial value of 7. After application of selective enrichment (by bacterial stress), hydrogen production was significantly higher than that of untreated original sludge. Hydrogen production from the inocula with bacterial stress enrichment was 1.9–9.8 times greater when compared with control sludge. Chemical acidification using perchloric acid showed the best hydrogen production potential, irrespective of preacidification. Enhancement is due to the selective capture of hydrogen-producing sporeformers, which induces altered anaerobic fermentative metabolism.     

Isolation and diversity of culturable rhizobacteria associated with economically important crops and uncultivated plants in Québec,Canada

Plants are chronically associated with microorganisms, residing all tissues. Holonomic analysis of diversity of established rhizobacteria in uncultivated plants is scarce. Thus, the present study was conducted to access the root-associated bacterial diversity of 6 crops (maize, canola, soybean, reed canarygrass, alfafa, and miscanthus) and 20 uncultivated plant species in the region of Sainte-Anne-de-Bellevue, Québec, Canada, using pure-culture methods. Based on 16S rRNA gene sequence analysis, 446 bacterial isolates were distributed onto four phyla (Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes), 32 families and 90 genera. Proteobacteria constituted the largest group of isolates (240), 40% of ectophytic and 61% of endophytic bacteria. Representatives of the genera Bacillus and Pseudomonas dominated in rhizosphere soil; Microbacterium and Pseudomonas were the predominant endophytes. Some genera were associated with specific plant species, such as Stenotrophomonas, Yersinia, Labrys and Luteibacter. Several endophytes were occasionally observed in the rhizosphere, and vice versa. This is the first survey of culturable endophytic bacteria associated with uncultivated plants in Québec. The culturable bacterial community studied herein are assumed to represent a portion of the entire phytomicrobiome of the evaluated plants. Results confirmed that the crops and uncultivated plants of Québec represent an extremely rich reservoir of diverse rhizobacteria.     

Isolation of uncultivated anaerobic thermophiles from compost by supplementing cell extract of Geobacillus toebii in enrichment culture medium

Several researchers have reported that microorganisms can be cultivated only in the presence of other microorganisms. We suggest that a portion of uncultivated microorganisms might be cultivated in the presence of cellular components released from bacteria in their natural environments. In this study, the cell extract of Geobacillus toebii was used to enrich uncultivated thermophiles from compost. In the process of enrichment cultures, cell extract supplementation apparently changed the community composition. This change was monitored by PCR-DGGE targeting 16S rRNA gene. Five novel groups of microorganisms (similarity of 16S rRNA gene to the closest relative <96%) were specifically isolated from enrichment cultures by using cell extract-supplemented culture media. Their growth was found to be dependent on the addition of extract of G. toebii. Putting these findings together, we suggest that the extracts of bacteria could be one of the growth factors in the thermal ecosystem with a possibility of extending other ecological niches.     

Isolation of uncultivable anaerobic thermophiles of the family Clostridiaceae requiring growth-supporting factors

Novel groups of uncultivable anaerobic thermophiles were isolated from compost by enrichment cultivation in medium with a cell-free extract of Geobacillus toebii. The cell-free extract of G. toebii provided the medium with growth-supporting factors (GSF) needed to cultivate the previously uncultured microorganisms. Twenty-nine GSF-requiring candidates were successfully cultivated, and 16 isolated novel bacterial strains were classified into three different groups of uncultivable bacteria. The similarity among these 16 isolates and a phylogenetic analysis using 16S rRNA gene sequences revealed that these GSF-requiring strains represented novel groups within the family Clostridiaceae.     

人类口腔小生境微生物的多样性

本文论述了人类口腔中微生物的物种多样性,口腔小生境的复杂性与微生物多样性的关系,以及口腔中微生物变化与人类疾病和健康的关系。     

Radioisotopic, culture-based, and oligonucleotide microchip analyses of thermophilic microbial communities in a continental high-temperature petroleum reservoir

Activity measurements by radioisotopic methods and cultural and molecular approaches were used in parallel to investigate the microbial biodiversity and its physiological potential in formation waters of the Samotlor high-temperature oil reservoir (Western Siberia, Russia). Sulfate reduction with rates not exceeding 20 nmol of H(2)S liter(-1) day(-1) occurred at 60 and 80 degrees C. In upper horizons (AB, A, and B), methanogenesis (lithotrophic and/or acetoclastic) was detected only in wells in which sulfate reduction did not occur. In some of the wells from deeper (J) horizons, high-temperature sulfate reduction and methanogenesis occurred simultaneously, the rate of lithotrophic methanogenesis exceeding 80 nmol of CH(4) liter(-1) day(-1). Enrichment cultures indicated the presence of diverse physiological groups representing aerobic and anaerobic thermophiles and hyperthermophiles; fermentative organotrophs were predominant. Phylogenetic analyses of 15 isolates identified representatives of the genera Thermotoga, Thermoanaerobacter, Geobacillus, Petrotoga, Thermosipho, and Thermococcus, the latter four being represented by new species. Except for Thermosipho, the isolates were members of genera recovered earlier from similar habitats. DNA obtained from three samples was hybridized with a set of oligonucleotide probes targeting selected microbial groups encompassing key genera of thermophilic bacteria and archaea. Oligonucleotide microchip analyses confirmed the cultural data but also revealed the presence of several groups of microorganisms that escaped cultivation, among them representatives of the Aquificales/Desulfurobacterium-Thermovibrio cluster and of the genera Desulfurococcus and Thermus, up to now unknown in this habitat. The unexpected presence of these organisms suggests that their distribution may be much wider than suspected.     

基于流式细胞仪高通量分选的深海微生物单细胞培养

【目的】建立适用于海洋微生物的流式细胞分选与高通量单细胞培养的方法,通过该方法从印度洋深海样品中分离微生物纯培养菌株。【方法】利用流式细胞仪单细胞分选功能,以前向角(FSC)和侧向角(SSC)散射光信号代替荧光信号作为分选逻辑,对深海水体和沉积物样品中微生物进行单细胞高通量分选和培养。【结果】确定了流式细胞分选的区域和条件,发现所建立方法适于分离海洋水体微生物,而不是沉积物微生物。从印度洋深海水体样品中获得61个潜在新菌株,分属于6个新属种,占分离菌株总数的26.29%,其16S rRNA基因序列与已培养的模式菌株相似性为89.79%–95.37%。【结论】本研究所建立的方法有助于提高发现海洋微生物新物种的效率,获得更多新的海洋微生物资源。     

Exploration and isolation of novel thermophiles in frozen enrichment cultures derived from a terrestrial acidic hot spring

An isolation strategy, exploring novel microorganisms in frozen enrichment cultures (ENFE), which uses a combination of enrichment culture and 16S rRNA gene clone analysis, was evaluated for isolating uncultured thermophiles from a terrestrial acidic hot spring. The procedure comprised (a) multiple enrichment cultures under various conditions, (b) cryostorage of all enrichments, (c) microbial community analyses of the enrichments using 16S rRNA gene sequences, and (d) purification of microorganisms from enrichments containing previously uncultured microorganisms. The enrichments were performed under a total of 36 conditions, and 16 of these enrichments yielded positive microbial growth with the detection of three previously uncultured archaea. Two of the three previously uncultured archaea, strains HS-1 and HS-3, were successfully isolated. Strain HS-1 and HS-3 represented a novel lineage of the order Sulfolobales and novel species of the genus Sulfolobus, respectively. Although innovative isolation methods play strategic roles in isolating previously uncultured microorganisms, the ENFE strategy showed potential for characterizing and isolating such microorganisms using conventional media and techniques.     

Microflora participating in the decomposition of carboxymethyl cellulose continuously added to the soil

The microbial community in the soil was analyzed during four weeks of a continuous enrichment of structural chernozem soil samples with a 0.1% solution of carboxymethyl cellulose (CMC) under aerobic and semianaerobic conditions. During the first 14 d, the total amount of the aerobic and anaerobic, cellulose-degrading microorganisms increased significantly. Various metabolic pathways were u‘ed te decompose the substrate: diverse metabolic systems were activated and different groups of microorganisms preferred in dependence on the presence of oxygen or the source of mineral nitrogen. In the later phases of cultivation, a decrease in the concentration of zymogenous microflora and in the level of substrate mineralization was observed ovon though CM-cellulase activity remained high. During the fourth week of cultivation, a conspicuous increase in the numbers of oligothropic bacteria occurring in the colcnies of the microorganisms degrading cellulose was found. The representatives of prosthecobacteria (Caulobacter, Hyphomicrobium, Prosthecomicrobium spp.) andSeliberia sp. were thus identified. This “microflora of dispersion” attends the zymogenous microbes degrading CMC and indicates later phases of the process of decomposition.     

Accessing previously uncultured marine microbial resources by a combination of alternative cultivation methods

Few microbes can grow under laboratory conditions, highlighting the fact that the majority of microbes in environment are still uncultured and untapped resources. This study used alternative cultivation methods, diffusion chambers (DC), dilution-to-extinction culture (DTE) and modified agar preparation step (PS media) to cultivate previously uncultured marine bacterial species. These methods were applied to samples from a coastal intertidal zone, and the results were compared with those from standard direct plating (SDP) cultivation. Among the strains isolated with DC, DTE and PS media methods, 28%, 48% and 33% were novel species, respectively, while the SDP method resulted in the isolation of only 9% of novel species. Most isolates were unique to the method used for their cultivation. This implies that each method is selective in its own way, which is different from SDP, thus able to access species that are difficult to obtain using conventional approaches. Comparing the diversity showed that 75 genera were recovered by the alternative methods, 2.7 times higher than that of the SDP cultivation, which constituted 45% of total diversity from culture-independent sequencing. We conclude that combining alternative cultivation methods represents a highly promising key for accessing ‘microbial dark matter’.     

Enrichment of previously uncultured bacteria from natural complex communities by adhesion to solid surfaces

The adhesion to inert solid surfaces was explored as a novel approach for the enrichment of previously uncultured bacteria from natural microbial communities. Enrichments on solid steel, glass and synthetic polymeric surfaces were established using samples from five freshwater lakes, a marine microbial mat and an alpine soil, and were subsequently analysed by molecular fingerprinting and sequencing of their 16S rRNA gene fragments. The majority of the enriched phylotypes grouped with the Alphaproteobacteria, Betaproteobacteria or Bacteroidetes and in several cases were related to typical biofilm‐forming species and genera. Most enrichments were most closely related to previously uncultured phylotypes and none had previously been cultivated from the original environments even when applying improved high throughput liquid cultivation techniques. Of the 13 phylotypes enriched from freshwater samples, seven were previously unknown, three matched so‐far uncultured environmental clones, and three were identical to previously cultivated bacteria. Of the 17 phylotypes recovered from soil, 12 were previously unknown with five of these phylotypes representing novel genera, whereas five phylotypes were identical to previously cultured soil bacteria. The feasibility of the biofilm‐enrichment approach was exemplified by the successful isolation of a not‐yet cultured Betaproteobacterium that constituted a discernible component of the alpine soil microbial community in situ and exhibited only 93% similarity to its closest cultured relative. Based on these results, cultivation on solid surfaces represents a promising approach to recover isolates that have so far escaped cultivation as suspended cultures in liquid media.     

Analysis of a microbial community oxidizing inorganic sulfide and mercaptans

Successful treatment of refinery spent-sulfidic caustic (which results from the addition of sodium hydroxide solutions to petroleum refinery waste streams) was achieved in a bioreactor containing an enrichment culture immobilized in organic polymer beads with embedded powdered activated carbon (Bio-Sep). The aerobic enrichment culture had previously been selected using a gas mixture of hydrogen sulfide and methyl mercaptan (MeSH) as the sole carbon and energy sources. The starting cultures for the enrichment consisted of several different Thiobacilli spp. (T. thioparus, T. denitrificans, T. thiooxidans, and T. neopolitanus), as well as activated sludge from a refinery aerobic wastewater treatment system and sludge from an industrial anaerobic digester. Microscopic examination (light and SEM) of the beads and of microbial growth on the walls of the bioreactor revealed a great diversity of microorganisms. Further characterization was undertaken starting with culturable aerobic heterotrophic microorganisms (sequencing of PCR-amplified DNA coding for 16S rRNA, Gram staining) and by PCR amplification of DNA coding for 16S rRNA extracted directly from the cell mass, followed by the separation of the PCR products by DGGE (denaturing gradient gel electrophoresis). Eight prominent bands from the DGGE gel were sequenced and found to be closest to sequences of uncultured Cytophagales (3 bands), Gram-positive cocci (Micrococcineae), alpha proteobacteria (3 bands), and an unidentified beta proteobacterium. Culturable microbes included several genera of fungi as well as various Gram-positive and Gram-negative heterotrophic bacteria not seen in techniques using direct DNA extraction.     

Microbial dynamics in anaerobic enrichment cultures degrading di-n-butyl phthalic acid ester

Although anaerobic biodegradation of di-n-butyl phthalic acid ester (DBP) has been studied over the past decade, only little is known about the microorganisms involved in the biological anaerobic degradation pathways. The aim of this work is to characterize the microbial community dynamics in enrichment cultures degrading phthalic acid esters under methanogenic conditions. A selection pressure was applied by adding DBP at 10 and 200 mg L(-1) in semi-continuous anaerobic reactors. The microbial dynamics were monitored using single strand conformation polymorphism (SSCP). While only limited abiotic losses were observed in the sterile controls (20-22%), substantial DBP biodegradation was found in the enrichment cultures (90-99%). In addition, significant population changes were observed. The dominant bacterial species in the DBP-degrading cultures was affiliated to Soehngenia saccharolytica, a microorganism described previously as an anaerobic benzaldehyde degrader. Within the archaeal community, there was a shift between two different species of the genus Methanosaeta sp., indicating a highly specific impact of DBP or degradation products on archaeal species. RNA-directed probes were designed from SSCP sequences, and FISH observations confirmed the dominance of S. saccharolytica, and indicated floccular microstructures, likely providing favourable conditions for DBP degradation.     

A procedure for the enrichment and isolation of Halobacterium

A procedure for the specific enrichment and isolation of species of the genus Halobacterium was designed, based on the ability of Halobacterium cells to grow anaerobically by fermentation of l-arginine. None of the other genera of neutrophilic halophilic Archaea tested grew fermentatively on arginine. Using anaerobic enrichments in the presence of arginine, representatives of the genus Halobacterium were consistently isolated from saltern crystallizer ponds in Eilat (Israel) and San Francisco Bay (California), environments in which Halobacterium represents only a very small fraction of the halophilic archaeal community.