Non-cultivable Species Research Articles

Comparing NGS-Based identification of bloodstream infections to traditional culture methods for enhanced ICU care: a comprehensive study.

Accurate identification of infectious diseases using molecular techniques, such as PCR and NGS, is well-established. This study aims to assess the utility of Bactfast and Fungifast in diagnosing bloodstream infections in ICU settings, comparing them against traditional culture methods. The objectives include evaluating sensitivity and specificity and identifying a wide range of pathogens, including non-culturable species. We collected 500 non-duplicate blood samples from ICU patients between January 2023 and December 2023. Specimens underwent traditional culture, MALDI-TOF, VITEK®2 compact system, and NGS-based Bactfast and Fungifast analyses. Out of the 500 samples, 26.8% (n=134) showed bacterial growth via traditional culture methods, while 4.8% (n=24) were positive for fungal growth. MALDI-TOF and VITEK®2 compact system yielded comparable results, identifying 26.4% (n=132) of specimens with bacterial growth. NGS-based Bactfast detected bacterial presence in 38.2% (n=191) of samples, including non-culturable bacteria missed by traditional methods. However, NGS-based Fungifast showed concordant fungal detection rates with culture methods. Among identified pathogens by culture method included Klebsiella pneumoniae 20.89% (n=28), Enterococcus faecalis 18.65% (n=25), Escherichia coli 15.67% (n=21), Pseudomonas aeruginosa 12.68% (n=17), Acinetobacter baumannii 10.44% (n=14), various Streptococcus species 7.46% (n=10), Mycobacterium tuberculosis 6.71% (n=9), Mycobacterium abscessus 4.47% (n=6), and Salmonella spp 2.98% (n=4). Non-culture-based NGS identified additional (n=33) pathogens, including Klebsiella pneumoniae 27.27% (n=9), Bacteroides fragilis 21.21% (n=7), Aerococcus viridans 15.15% (n=5), Elizabethkingia anopheles 12.12% (n=4), Aeromonas salmonicida 9% (n=3), Clostridium 9% (n=3), and Bacteroides vulgatus 6% (n=2). Candida albicans was reported in 5% (n=24) of samples by both methods. NGS-based Bactfast and Fungifast demonstrate high sensitivity in identifying a wide array of bacterial and fungal pathogens in ICU patients, outperforming traditional culture methods in detecting non-culturable organisms. These molecular assays offer rapid and comprehensive diagnostic capabilities, potentially improving clinical outcomes through timely and accurate pathogen identification.

Mammals' sperm microbiome: current knowledge, challenges, and perspectives on metagenomics of seminal samples.

Bacterial growth is highly detrimental to sperm quality and functionality. However, during the last few years, using sequencing techniques with a metagenomic approach, it has been possible to deepen the study of bacteria-sperm relationships and describe non-culturable species and synergistic and antagonistic relationships between the different species in mammalian animals. We compile the recent metagenomics studies performed on mammalian semen samples and provide updated evidence to understand the importance of the microbial communities in the results of sperm quality and sperm functionality of males, looking for future perspectives on how these technologies can collaborate in the development of andrological knowledge.

METAGENOMICS IN-SIGHT INTO THE MICROBIAL STRUCTURAL DIVERSITY OF ANAEROBIC DIGESTER UTILISING FRUIT WASTE AS SUBSTRATE

Anaerobic digester has emerged as a technology of choice in management of waste and production of biogas. However, the microbial ecology of digesters utilizing various substrates are very poorly understood. The ecology of anaerobic digester utilizing Citrullus lanatus fruit waste was analyzed using metagenomics. Slurry substrate sample was collected from a functional digester aseptically and anaerobically. Metagenomic DNA was extracted using ZYMO DNA extraction Kit (Model D 6001, Zymo Research, USA) following manufacturer’s instruction. Extracted DNA was amplified using the 16S rRNA gene amplicon PCR primers set and sequenced using Illumina MiSeq platform. Taxonomic analysis of the reads was performed using NCBI-BLAST-2.2.24 and CLC bio Genomics workbench v7.5.1. Taxonomic classification of the sequences revealed that bacteria and archae were the top two kingdoms with reads counts of 57,554 and 80, respectively. The top 7 phyla were Unknown, Firmicutes, Proteobacteria, Planctomycetes, Bacteroidetes, Actinobacteria and Euryarchaeota in decreasing order of counts. A total of 30 microbial classes, 43 orders, 79 families and 210 species were further classified. Over half of the 210 species detected were not routinely cultured species in the laboratory, indicating that there is still a relatively wide gap between culturable and non-culturable species in an anaerobic digester. The Acidogens captured in this study were Clostridium, Uncultured rumen bacteria and Bacteroidetes species. Furthermore, we also detected uncultured syntrophic Acetogens such as Syntrophomonas species and the hydrogenotrophic thermophile, Methanothermobacter sp. The Syntrophomonas species is known to breakdown short chain fatty acids, like propionate and butanoate in concert with hydrogenotrophic Methanogens indicating methane generation was via the hydrogenotrophic route. However, the main representative hydrogenotrophic methanogens detected were Methanoculleus bourgensis and Methanoculleus marisnigri, with the former being more abundant. In addition to the aforementioned species, other species captured were largely classified as unknown or uncultured species and they include Uncultured species of Clostridium, Syntrophomonas, Synergistetes, Synergistaceae Anaerobic sp, Ruminococcaceae, Rumen sp, Thermomonas, Thermoanaerobacteriales, Bacterium, Compost, Firmicutes, Bacteroidetes, Chloroflexi, Clostriaceae, Acetobacter. Eubacterium, Alpha and Aacteroides. The results of the study revealed that culture-independent approach is better able to capture the anaerobes including both culturable and unknown that dominate anaerobic digesters and are responsible for the bioconversion of organic waste into biogas.

Investigations on Contamination of Environmental Water Samples by <i>Legionella</i> using Real-Time Quantitative PCR Combined with Amoebic Co-Culturing

We analyzed the contamination of environmental water samples with Legionella spp. using a conventional culture method, real-time quantitative PCR (qPCR), and real-time qPCR combined with an amoebic co-culture method. Samples (n = 110) were collected from 19 cooling towers, 31 amenity water facilities, and 60 river water sources of tap water in Japan. Legionella was detected in only three samples (3/110, 2.7%) using the culture method. The rate of Legionella detection using amoebic co-culture followed by qPCR was 74.5%, while that using qPCR without amoebic co-culture was 75.5%. A higher than 10-fold bacterial count was observed in 19 samples (19/110, 17.3%) using real-time qPCR subsequent to amoebic co-culture, compared with identical samples analyzed without co-culture. Of these 19 samples, 13 were identified as Legionella spp., including L. pneumophila and L. anisa, and the non-culturable species were identified as L. lytica and L. rowbothamii. This study showed that the detection of Legionella spp., even in those samples where they were not detected by the culture method, was possible using real-time qPCR and an amoebic co-culture method. In addition, this analytical test combination is a useful tool to detect viable and virulent Legionella spp..

생물의약품 제조공정에서 마이코플라스마 정량 검출을 위한 TaqMan Probe Real-Time PCR

Mycoplasma is well recognized as one of the most prevalent and serious microbial contaminants of biologic manufacturing processes. Conventional methods for mycoplasma testing, direct culture method and indirect indicator cell culture method, are lengthy, costly and less sensitive to noncultivable species. In this report, we describe a new TaqMan probe-based real-time PCR method for rapid and quantitative detection of mycoplasma contamination during manufacture of biologics. Universal mycoplasma primers were used for mycoplasma PCR and mycoplasma DNA was quantified by use of a specific TaqMan probe. Specificity, sensitivity, and robustness of the real-time PCR method was validated according to the European Pharmacopoeia. The validation results met required criteria to justify its use as a replacement for the culture method. The established real-time PCR assay was successfully applied to the detection of mycoplasma from human keratinocyte and mesenchymal stem cell as well as Vero cell lines artificially infected with mycoplasma. The overall results indicated that this rapid, specific, sensitive, and robust assay can be reliably used for quantitative detection of mycoplasma contamination during manufacture of biologics.

Proteomics meets blue biotechnology: A wealth of novelties and opportunities

Blue biotechnology, in which aquatic environments provide the inspiration for various products such as food additives, aquaculture, biosensors, green chemistry, bioenergy, and pharmaceuticals, holds enormous promise. Large-scale efforts to sequence aquatic genomes and metagenomes, as well as campaigns to isolate new organisms and culture-based screenings, are helping to push the boundaries of known organisms. Mass spectrometry-based proteomics can complement 16S gene sequencing in the effort to discover new organisms of potential relevance to blue biotechnology by facilitating the rapid screening of microbial isolates and by providing in depth profiles of the proteomes and metaproteomes of marine organisms, both model cultivable isolates and, more recently, exotic non-cultivable species and communities. Proteomics has already contributed to blue biotechnology by identifying aquatic proteins with potential applications to food fermentation, the textile industry, and biomedical drug development. In this review, we discuss historical developments in blue biotechnology, the current limitations to the known marine biosphere, and the ways in which mass spectrometry can expand that knowledge. We further speculate about directions that research in blue biotechnology will take given current and near-future technological advancements in mass spectrometry.

Selective digestive decontamination and bacterial resistance

1 van der Meer JW, Vandenbroucke-Grauls CM, Vincent JL. Resistance to selective decontamination: the jury is still out. Lancet Infect Dis 2013; 13: 282–83. 2 Daneman N, Sarwar S, Fowler RA, Cuthbertson BH; for SuDDICU Canadian Study Group. Eff ect of selective decontamination on antimicrobial resistance in intensive care units: a systematic review and meta-analysis. Lancet Infect Dis 2013; 13: 328–41. 3 Vincent JL. Prevention of nosocomial bacterial pneumonia. Thorax 1999; 54: 544–549. 4 van Saene HKF, Stoutenbeek CP. Selective decontamination. J Antimicrob Chemother 1987; 20: 462–65. 5 Silvestri L, de la Cal MA, van Saene HKF. Selective decontamination of the digestive tract: the mechanism of action is control of gut overgrowth. Intensive Care Med 2012; 38: 1738–50. 6 Stoutenbeek CP, Miranda DR, van Saene HKF. A new technique for infection prevention in the intensive care unit. Proceedings of the 6th European Congress of Anaesthesiology. Anaesthesia 1982; 56: 98 (abstr). patients treated in an intensivecare unit for longer than 5 days. A 2% chlorhexidine rinse was used for throat decontamination. This regimen was given to 55 patients: 18 had abnormal fl ora in the throat and gut, particularly Pseudomonas spp and Acinetobacter spp. These isolates were sensitive to colistin, but resistant to co-trimoxazole. 12 patients developed pneumonia. Co-trimoxazole is absorbable and caused side-eff ects including thrombo cytopenia and allergy, so was replaced by parenteral cefotaxime and enteral, non-absorbable tobramycin. This combination, used in 35 RCTs, was analysed by the authors of the meta-analysis. van der Meer JW and VandenbrouckeGrauls make two statements: that benefi t with SDD is controversial and that the long-term eff ects of SDD on resistance are unknown. They say that decades are required to assess the eff ect of SDD on the development of resistance, particularly on the noncultivable species in the digestive tract. We strongly disagree with these statements. SDD reduces morbidity and mortality, and the Surviving Sepsis Campaign has recommended SDD as an infection control measure. van der Meer JW and Vandenbroucke-Grauls’ demands for further research into SDD and resistance, in particular in non-cultivable species, do not make sense because these organisms have no clinical impact. We ask whether the contributors have applied similar requirements for their traditional use of parenteral antibiotics with regards to the emergence of resistance?

Ulcerated and nonulcerated nontuberculous cutaneous mycobacterial granulomas in cats and dogs

Mycobacterial granulomas of the skin and subcutis can be caused by one of a number of pathogens. This review concentrates on noncultivable species that cause diseases characterized by focal granuloma(s), namely leproid granuloma (in dogs) and feline leprosy (in cats). Clinically indistinguishable lesions can be caused by tuberculous organisms (Mycobacterium bovis and Mycobacterium microti) and members of the Mycobacterium avium complex. Rapidly growing mycobacterial species that cause infection of the subcutaneous panniculus associated with draining tracts are not discussed. Disease caused by Mycobacterium ulcerans is an important emerging differential diagnosis for ulcerated cutaneous nodules in certain localized regions. CLINICAL LESIONS: Lesions comprise one or multiple nodules in the skin/subcutis. These are generally firm and well circumscribed, and typically become denuded of hair. They may or may not ulcerate, depending on the virulence of the causal organisms and the immune response of the host. The most inexpensive, noninvasive means of diagnosis is by submission of methanol-fixed, Romanowsky-stained smears to a Mycobacterium Reference Laboratory after detecting negatively stained or acid-fast bacilli on cytological smears. Scrapings of material from slides usually provide sufficient mycobacterial DNA to enable identification of the causal organism using sequence analysis of amplicons after PCR using specific mycobacterial primers. Therapy relies upon a combination of marginal resection of easily accessible lesions and treatment using two or three drugs effective against slowly growing mycobacteria, choosing amongst rifampicin, clarithromycin, clofazimine and pradofloxacin/moxifloxacin.

Defining bacterial species in the genomic era: insights from the genus Acinetobacter.

BackgroundMicrobial taxonomy remains a conservative discipline, relying on phenotypic information derived from growth in pure culture and techniques that are time-consuming and difficult to standardize, particularly when compared to the ease of modern high-throughput genome sequencing. Here, drawing on the genus Acinetobacter as a test case, we examine whether bacterial taxonomy could abandon phenotypic approaches and DNA-DNA hybridization and, instead, rely exclusively on analyses of genome sequence data.ResultsIn pursuit of this goal, we generated a set of thirteen new draft genome sequences, representing ten species, combined them with other publically available genome sequences and analyzed these 38 strains belonging to the genus. We found that analyses based on 16S rRNA gene sequences were not capable of delineating accepted species. However, a core genome phylogenetic tree proved consistent with the currently accepted taxonomy of the genus, while also identifying three misclassifications of strains in collections or databases. Among rapid distance-based methods, we found average-nucleotide identity (ANI) analyses delivered results consistent with traditional and phylogenetic classifications, whereas gene content based approaches appear to be too strongly influenced by the effects of horizontal gene transfer to agree with previously accepted species.ConclusionWe believe a combination of core genome phylogenetic analysis and ANI provides an appropriate method for bacterial species delineation, whereby bacterial species are defined as monophyletic groups of isolates with genomes that exhibit at least 95% pair-wise ANI. The proposed method is backwards compatible; it provides a scalable and uniform approach that works for both culturable and non-culturable species; is faster and cheaper than traditional taxonomic methods; is easily replicable and transferable among research institutions; and lastly, falls in line with Darwin’s vision of classification becoming, as far as is possible, genealogical.

Improvement of Detection of Bacterial Pathogens in Normally Sterile Body Sites with a Focus on Orthopedic Samples by Use of a Commercial 16S rRNA Broad-Range PCR and Sequence Analysis

A new commercially available universal 16S and 18S rRNA gene PCR test, which is followed by sequence analysis of amplicons (SepsiTest), was evaluated for rapid identification of pathogens in the diagnosis of bone and joint infections. Eighty-three orthopedic samples and 21 specimens from other normally sterile body sites collected from 84 patients were analyzed in parallel by culture and PCR for detection of bacteria and fungi. Compared to culture, the diagnostic sensitivity and specificity of PCR were 88.5% and 83.5%, respectively. The detection rate of PCR (34.6%) was higher than that of bacterial culture (25.0%) as a consequence of the presence of fastidious and noncultivable species in samples and antibiotic treatment of patients. Thirteen culture-negative infections were identified by PCR, and PCR was able to detect culture-proven polymicrobial infections. On the other hand, three samples were culture positive but PCR negative. SepsiTest was demonstrated to be a valuable supplemental tool in the rapid detection of bacteria, especially for fastidious and noncultivable organisms, allowing earlier initiation of pathogen-adapted therapy in patients with bone and joint infections.

Short-Term Fluctuations of Sugar Beet Damping-Off by Pythium ultimum in Relation to Changes in Bacterial Communities After Organic Amendments to Two Soils

Previously, oscillations in beet seedling damping-off by Pythium ultimum, measured as area under the disease progress curve (AUDPC), were demonstrated after incorporation of organic materials into organic and conventional soils. These periodic fluctuations of P. ultimum infections were cross-correlated with oscillations of copiotrophic CFU at lags of 2 to 4 days. For this article, we investigated whether bacterial communities and microbial activities fluctuated after a disturbance from incorporation of organic materials, and whether these fluctuations were linked to the short-term oscillations in AUDPC of beet seedling damping-off and bacterial populations (CFU) in soil. Soil microbial communities studied by polymerase chain reaction-DGGE analysis of 16S DNA after isolation of total DNA from soil and microbial activities measured as CO(2) emission rates were monitored daily for 14 days after addition of grass-clover (GC) or composted manure (CM) into organic versus conventional soils. Similar to our previous findings, AUDPC and density of copiotrophic bacteria oscillated with time. Fluctuations in species richness (S), Shannon diversity index (H), and individual amplicons on DGGE gels were also detected. Oscillations in AUDPC were positively cross-correlated with copiotrophic CFU in all soils. Oscillations in AUDPC were also positively cross-correlated with 19 to 35% of the high-intensity DNA fragments in soils amended with GC but only 2 to 3% of these fragments in CM-amended soils. AUDPC values were negatively cross-correlated with 13 to 17% of the amplicons with low average intensities in CM-amended soils, which were not correlated with densities of copiotrophic CFU. CO(2) emission rates had remarkable variations in the initial 7 days after either of the soil amendments but were not associated with daily changes in AUDPC. The results suggest that infection by P. ultimum is hampered by competition from culturable copiotrophic bacteria and some high-intensity DGGE amplicons, because AUDPC is cross-correlated with these variables at lags of 1 to 4 days. However, negative cross-correlations with low-intensity DNA fragments indicate that P. ultimum infection could also be suppressed by antagonistic bacteria with low densities that may be nonculturable species, especially in CM amended soil. The organic soil generally had lower AUDPC values, higher bacterial diversity, and negative cross-correlations between AUDPC and low-intensity DNA fragments (after CM amendment), indicating that specific bacteria that do not attain high densities may contribute to P. ultimum suppression in organic soils.

Exploration type-specific standard values of extramatrical mycelium – a step towards quantifying ectomycorrhizal space occupation and biomass in natural soil

The present study aims at providing standard values for the exploration type (ET)-specific quantification of extramatrical mycelium (EMM) of ectomycorrhizal fungi applicable to ecological field studies. These values were established from mycelial systems of ectomycorrhizae (ECM) synthesized in rhizotrons with near-to-natural peat substrate. Based on image analysis, the “Specific Potential Mycelial Space Occupation” (sPMSO), i.e. the ET-specific complete area that is covered by the EMM systems (mm2 cm−1 ECM−1), and the “Specific Actual Mycelial Space Occupation” (sAMSO), i.e. the projection area of mycelial systems (mm2 cm−1 ECM−1), were analyzed as an extension of a previously described approach. The “Specific Extramatrical Mycelial Length” (sEML) [m cm−1 ECM−1] and the “Specific Extramatrical Mycelial Biomass” (sEMB) (μg cm−1 ECM−1) were calculated for each of the ET via the proportion of hyphal projected area, hyphal length and biomass, the latter two being derived from previous measurements on Piloderma croceum, a “Medium-Distance” (MD)-ET. Both sPMSO and sAMSO were highest for the “Long-Distance” (LD)-ET, whereas those of the “Short-Distance” (SD)-ET and MD-ET were similar, although showing high variation. In contrast, mycelial density per occupied area of the MD-ET was twice as high as that of the LD-ET. Proportional to the sAMSO, the EMM length and biomass differed considerably between the three ET with values of the MD-ET being 1.9 times higher than those of SD-ET, and those of the LD-ET being 15 times higher than those of the SD-ET. These standards in relation to ECM length may ease quantification of mycelial space occupation and biomass in a relatively simple way. Thereby, the ET-specific contribution of EMM can be distinguished—also of non-cultivable species—and up-scaling to large-scale estimation of cost/benefit relations is possible.

Development of soil microbiology methods: from respirometry to molecular approaches

This review deals with techniques and methods used in the study of the function and development of microorganisms occurring in soil with emphasis on the contributions of Czech Academician Ivan Málek and his coworkers or fellows (Jiří Macura, František Kunc) to the development of basic techniques used in soil microbiology. Early studies, including batch cultivation and respirometric techniques, as well as later developments of percolation and continuous-flow methods of cultivation of soil microorganisms are discussed. Recent developments in the application of analytical chemistry (HPLC or GC) and of molecular biological techniques to ecological questions that have revolutionized concepts in soil microbiology and microbial ecology are also briefly mentioned, including denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), phospholipid fatty acid analysis (PLFA) and others. The shift of soil microbiology from the study of individual microorganisms to entire microbial communities, including nonculturable species, is briefly discussed.

Development of gut microbiota in infants not exposed to medical interventions

Knowledge of the composition of a normal healthy gut microbiota during infancy is important for understanding the role of gut microbiota in disease. A limitation of previous studies is that they are based on infants who have been subject to factors, which can have a profound disruptive effect on the natural colonization process. We describe the colonization process, during the first 4 months after birth, in 85 infants who have experienced no major medical or dietary interventions. They were all vaginally delivered, healthy, term infants, who were not exposed to antibiotics, exclusively breastfed during their first month of life and at least partially breastfed up to 4 months. Selected microbial groups were identified by targeting small subunit microbial ribosomal RNA genes. In contrast to more recent studies, but in agreement with older studies, almost all our infants harbored γ-Proteobacteria and Bifidobacterium. Yet undefined non-cultivable species belonging to Bacteroides, as well as microbes identified as Lachnospiraceae 2, were common. Strong associations were observed between some specific constituents of microbiota at day 4 and the concentration of specific microbial groups at day 120, indicating that early gut microbiota may influence later microbiota. Novel information of the undisturbed composition of early gut microbiota in babies is presented.

ClassII peroxidase-encoding genes are present in a phylogenetically wide range of ectomycorrhizal fungi

Fungal peroxidases (ClassII) have a key role in degrading recalcitrant polyphenolic compounds in boreal forest wood, litter and humus. To date, their occurrence and activity have mainly been studied in a small number of white-rot wood decomposers. However, peroxidase activity is commonly measured in boreal forest humus and mineral soils, in which ectomycorrhizal fungi predominate. Here, we used degenerate PCR primers to investigate whether peroxidase-encoding genes are present in the genomes of a wide phylogenetic range of ectomycorrhizal taxa. Cloning and sequencing of PCR products showed that ectomycorrhizal fungi from several different genera possess peroxidase genes. The new sequences represent four major homobasidiomycete lineages, but the majority is derived from Cortinarius, Russula and Lactarius. These genera are ecologically important, but consist mainly of non-culturable species from which little ecophysiological information is available. The amplified sequences contain conserved active sites, both for folding and substrate oxidation. In some Cortinarius spp., there is evidence for gene duplications during the evolution of the genus. ClassII peroxidases seem to be an ancient and a common feature of most homobasidiomycetes, including ectomycorrhizal fungi. Production of extracellular peroxidases may provide ectomycorrhizal fungi with access to nitrogen sequestered in complex polyphenolic sources.

Identification of oral bacterial species associated with halitosis

The authors examined the tongue bacteria associated with oral halitosis (bad breath originating from the oral cavity), focusing on noncultivable bacteria-bacteria that cannot be identified by bacterial culture techniques. The authors took samples from the dorsal tongue surface of eight adult subjects with halitosis and five control subjects who did not have halitosis. They identified the bacteria in these samples by using both anaerobic culture and direct amplification of 16S ribosomal DNA, a method that can identify both cultivable and noncultivable microorganisms. They analyzed the resulting microbiological data using chi(2) and correlation coefficient tests. Clinical measures of halitosis were correlated highly with each other and with tongue coating scores. Of 4,088 isolates and phylotypes identified from the 13 subjects, 32 species including 13 noncultivable species were found only in subjects with halitosis. Solobacterium moorei was present in all subjects with halitosis but not in any control subjects. Subjects with halitosis harbor some bacterial species on their dorsal tongue surfaces that are distinct from bacterial species found in control subjects. This finding is consistent with the hypothesis that halitosis has a microbial etiology. Like other oral diseases with microbial etiology, halitosis may be amenable to specific and nonspecific antimicrobial therapy targeted toward the bacteria associated with it.

Establishment and Early Succession of a Multispecies Biofilm Composed of Soil Bacteria

Most soil bacteria are likely to be organized in biofilms on roots, litter, or soil particles. Studies of such biofilms are complicated by the many nonculturable species present in soil, as well as the interspecific bacterial interactions affecting biofilm biology. We in this study describe the development of a biofilm flow model and use this system to establish an early (days 1-7) flow biofilm of soil bacteria from agricultural soil. It was possible to follow the succession in the early flow biofilm by denaturing gradient gel electrophoresis (DGGE) analysis, and it was demonstrated that the majority of strains present in the biofilm were culturable. We isolated and identified nine strains, all associated with unique DGGE profiles, and related their intrinsic phenotypes regarding monospecies biofilm formation in microtiter plates and planktonic growth characteristics to the appearance of the strains in the flow biofilm. The ability of the strains to attach to and establish biofilm in microtiter plates was reflected in their flow biofilm appearance, whereas no such reflection of the planktonic growth characteristics in the flow biofilm appearance was observed. One strain-specific synergistic interaction, strongly promoting biofilm formation of two strains when cultured together in a dual-species biofilm, was observed, indicating that some strains promote biofilm formation of others. Thus, the biofilm flow model proved useful for investigations of how intrinsic phenotypic traits of individual species affect the succession in an early soil biofilm consortium.

Development of a PCR method for mycoplasma testing of Chinese hamster ovary cell cultures used in the manufacture of recombinant therapeutic proteins

Chinese hamster ovary (CHO) cell cultures used to produce biopharmaceuticals are tested for mycoplasma contamination as part of the ensurance of a safe and pure product. The current U.S. Food and Drug Administration (FDA) regulatory guideline recommends using two procedures: broth/agar cultures and DNA staining of indicator cell cultures. Although these culture methods are relatively sensitive to most species, theoretically capable of detecting as few as 1–10 cfu/ml of most species, the overall procedure is lengthy (28 d), costly and less sensitive to noncultivable species. The detection of mycoplasma using the polymerase chain reaction (PCR) method has been considered an alternative method because it is relatively fast (1–2 d), inexpensive, and independent of culture conditions, however, limitations in sensitivity (limit of detection ≥ 1000 cfu/ml) and the risk of false positive and false negative results have prevented PCR from replacing the traditional culture methods in the industrial setting. In this report, we describe a new PCR assay for mycoplasma detection that appears to resolve these issues while being sufficiently simple and inexpensive for routine use. This assay applies readily available techniques in DNA extraction together with a modified single-step PCR using a previously characterized primer pair that is homologous to a broad spectrum of mycoplasma species known to infect mammalian cell cultures. Analysis is made easy by the detection of only a single amplification product within a narrow size range, 438–470 bp. A high sensitivity and specificity for mycoplasma detection in CHO cell production cultures is made possible through the combination of three key techniques: 8-methoxypsoralen and UV light treatment to decontaminate PCR reagents of DNA; hot-start Taq DNA polymerase to reduce nonspecific priming events; and touchdown- (TD-) PCR to increase sensitivity while also reducing nonspecific priming events. In extracts of mycoplasma DNA, the limit of detection for eight different mycoplasma species is 10 genomic copies. In CHO cell production cultures containing gentamicin, the limit of detection for a model organism, gentamicin-resistant M. hyorhinis, is 1 cfu/ml. The sensitivity and specificity of this PCR assay for mycoplasma detection in CHO cell production cultures appear similar to the currently used culture methods and thus should be considered as an alternative method by the biopharmaceutical industry.

Bacteria and Archaea: Molecular techniques reveal astonishing diversity

The last decade has produced a significant advance in our appreciation of the diversity of prokaryotic organisms (commonly given the generic term “bacteria”). The need for this improvement was clear as the current list of approximately 5,000 accredited species has long been known to be a major underestimate of living prokaryotic species. The primary reasons for this poor census were 1) the inability to cultivate the vast majority of prokaryotic species in the laboratory and 2) a classification system that inherently required laboratory culture. Fortunately, the impact of DNA-based methods has remarkably improved our knowledge by providing both a new alternative classification system (i.e. phylogenetic classification), and critically, new experimental strategies to identify non-culturable species. The resulting data have not only highlighted the true breath of prokaryotic diversity but have also changed some of our previous views of biological evolution. Phylogenetic analysis of gene sequences retrieved from both cultured and uncultured bacteria has shown that all cellular life can be ordered into three taxa (termed Domains)—Bacteria, Archaea, and Eucarya. Intriguingly, this has resulted in a major taxonomic promotion for the Archaea, which were previously thought to be a series of unusual bacterial species. In addition, the use of DNA-based methods to identify and catalogue non-culturable species has radically improved our knowledge of the diversity found within living prokaryotes. This paper describes our current view of prokaryotic diversity describing the impact over the last decade of DNA-based methods. It is a popular adaptation of a previously published paper (2001).

Bacterial community associated with Pfiesteria-like dinoflagellate cultures.

Dinoflagellates (Eukaryota; Alveolata; Dinophyceae) are single-cell eukaryotic microorganisms implicated in many toxic outbreaks in the marine and estuarine environment. Co-existing with dinoflagellate communities are bacterial assemblages that undergo changes in species composition, compete for nutrients and produce bioactive compounds, including toxins. As part of an investigation to understand the role of the bacteria in dinoflagellate physiology and toxigenesis, we have characterized the bacterial community associated with laboratory cultures of four 'Pfiesteria-like' dinoflagellates isolated from 1997 fish killing events in Chesapeake Bay. A polymerase chain reaction with oligonucleotide primers specific to prokaryotic 16S rDNA gene sequences was used to characterize the total bacterial population, including culturable and non-culturable species, as well as possible endosymbiotic bacteria. The results indicate a diverse group of over 30 bacteria species co-existing in the dinoflagellate cultures. The broad phylogenetic types of dinoflagellate-associated bacteria were generally similar, although not identical, to those bacterial types found in association with other harmful algal species. Dinoflagellates were made axenic, and the culturable bacteria were added back to determine the contribution of the bacteria to dinoflagellate growth. Confocal scanning laser fluorescence microscopy with 16S rDNA probes was used to demonstrate a physical association of a subset of the bacteria and the dinoflagellate cells. These data point to a key component in the bacterial community being species in the marine alpha-proteobacteria group, most closely associated with the alpha-3 or SAR83 cluster.