The effect of prebiotic fortified infant formulas on microbiota composition and dynamics in early life.

Gastrointestinal (GI) microbiota composition differs between breastfed and formula-fed infants. Today's infant formulas are often fortified with prebiotics to better mimic properties of human milk with respect to its effect on GI microbiota composition and function. We used Illumina HiSeq sequencing of PCR-amplified 16S rRNA gene fragments to investigate the composition of faecal microbiota in 2-12 week old infants receiving either breastmilk, infant formulas fortified with prebiotics, or mixed feeding. We compared these results with results from infants fed traditional formulas used in the Netherlands in 2002-2003, which contained no added prebiotics. We showed that today's formulas supplemented with either scGOS (0.24-0.50 g/100 ml) or scGOS and lcFOS (at a 9:1 ratio; total 0.6 g/100 ml) had a strong bifidogenic effect as compared to traditional formulas, and they also resulted in altered patterns of microbial colonisation within the developing infant gastrointestinal tract. We identified three microbial states (or developmental stages) in the first 12 weeks of life, with a gradual transition pattern towards a bifidobacteria dominated state. In infants receiving only fortified formulas, this transition towards the bifidobacteria dominated state was accelerated, whereas in infants receiving mixed feeding the transition was delayed, as compared to exclusively breastfed infants.

The potential role of 'Candidatus Microthrix parvicella' in phosphorus removal during sludge bulking in two full-scale enhanced biological phosphorus removal plants.

We investigated the bacterial community compositions and phosphorus removal performance under sludge bulking and non-bulking conditions in two biological wastewater treatment systems (conventional A²/O (anaerobic/anoxic/aerobic) and inverted A²/O (anoxic/anaerobic/aerobic) processes) receiving the same raw wastewater. Sludge bulking resulted in significant shift in bacterial compositions from Proteobacteria dominance to Actinobacteria dominance, characterized by the significant presence of filamentous 'Candidatus Microthrix parvicella'. Quantitative real-time polymerase chain reaction (PCR) analysis revealed that the relative abundance of 'Candidatus Accumulibacter phosphatis', a key polyphosphate-accumulating organism responsible for phosphorus removal, with respect to 16s rRNA genes of total bacteria was 0.8 and 0.7%, respectively, for the conventional and inverted A²/O systems when sludge bulking occurred, which increased to 8.2 and 12.3% during the non-bulking period. However, the total phosphorus removal performance during the bulking period (2-week average: 97 ± 1 and 96 ± 1%, respectively) was not adversely affected comparable to that during the non-bulking period (2-week average: 96 ± 1 and 96 ± 1%, respectively). Neisser staining revealed the presence of large polyphosphate granules in 'Candidatus Microthrix parvicella', suggesting that this microbial group might have been responsible for phosphorus removal during the sludge bulking period when 'Candidatus Accumulibacter phosphatis' was excluded from the systems.

Microbial community changes during the start-up of an anaerobic/aerobic/anoxic-type sequencing batch reactor.

An anaerobic/aerobic/anoxic-type sequencing batch reactor was started up during a summer rainy season to obtain enhanced biological phosphorus removal (EBPR), and its sludge microbial community was also monitored in the hope of observing the microbial community evolution of polyphosphate-accumulating organisms (PAOs). During the start-up process, a total of 17 bands of highest species richness were detected in the sludge microbial community, including Alpha-, Beta-, and Gamma- Proteobacteria, as well as Actinobacteria and Planctomycetes. Major microbial community structural change was observed in Rhodocyclus-related and Acinetobacter-related PAOs, glycogen-accumulating organisms (GAOs), and Actinobacteria. In contrast to the current belief that enrichment of PAOs is essential for the establishment of EBPR, PAOs were not favourably enriched in this study. Instead, Actinobacteria and GAOs overwhelmingly flourished. The overall conclusion of this study challenges the conventional view that EBPR cannot live without traditional PAOs. However, it suggests an non-negligible role of denitrifying phosphorus-accumulating bacteria in EBPR systems, as well as other uncultured bacteria.

Clonal expansion of Mycobacterium tuberculosis isolates and coexisting drug resistance in patients newly diagnosed with pulmonary tuberculosis in Hanoi, Vietnam.

BACKGROUND: Newly diagnosed patients without anti-tuberculosis (TB) treatment histories have not often undergone drug susceptibility testing (DST), but have received the standard treatment regimen without information about their DST profiles in many countries with inadequate resources. METHODS: We collected 346 clinical isolates from previously untreated patients with smear-positive active TB in Hanoi, the capital of Vietnam. Of these, 339 were tested for susceptibility to four first-line anti-TB drugs, including isoniazid (INH), rifampicin (RMP), streptomycin (SM), and ethambutol (EMB), using the proportion method. A pyrazinamidase (PZase) test was used to assess pyrazinamide (PZA) resistance. Results of the culture-based drug susceptibility tests were confirmed by those from reverse hybridization-based line probe assays (LiPAs) that detected mutations associated with RMP, INH, PZA, and fluoroquinolone (FQ) resistance. To investigate a diversity of these strains, IS6110-probed restriction fragment length polymorphisms (RFLPs) were analyzed. Nucleotide sequences for furA-katG and fabG1-inhA operons, transcription units responsible for INH resistance, were also determined. RESULTS: Of the isolates tested, 127 (37.5%) were resistant to at least one of the four drugs, which included 93 (27.4%) isolates that were resistant to INH. RFLP analysis identified four clusters defined by similarity of the band patterns, which accounted for 46.1% of the tested isolates. Among the clustered isolates, 37.7% were resistant to INH, most of which (85.4%) carried a g944c mutation, which causes an S315T amino acid substitution, in the katG gene. CONCLUSIONS: Our results suggest that drug-resistant strains, particularly those with INH resistance characterized by a single mutation, S315T, are spreading in Hanoi, Vietnam. When RMP resistance is combined with this setting, patients are not easily cured by conventional short-term treatment. We will need to carefully monitor these trends and search for the origins and transmission routes of these strains.

New efficient DNA extraction method to access the microbiome of Ricinus communis seeds.

Ricinus communis (castor bean) seeds are used to produce an alcohol-soluble oil that is used in more than 400 industrial processes. Despite its economic importance, there has been little research on the endophytic microbiota of castor bean seeds. This microbiota is important for plant metabolic processes and may have considerable biotechnological potential, such as production of lipases and plant growth promoter agents. We evaluated several DNA extraction methodologies in order to access the microbial diversity of castor bean through a metagenomic approach. Based on our observations, we developed a new methodology that takes advantage of the low solubility of calcium phosphates and the high affinity of these phosphates for proteins and polysaccharides. The extracted DNA quality was evaluated by PCR, using a selective primer pair for bacterial and mitochondrial 16S rDNA genes (799F and 1492R). We found this methodology quantitatively and qualitatively more efficient than the other approaches. In evaluating this new extraction methodology, we found that the difficulties of DNA extraction from castor bean seeds, such as abundant oil, polysaccharides, phenolic compounds, and plant enzymes, could be overcome. The resulting extracts had high concentration and purity, and they were obtained faster than with previous methods. The samples contained virtually all of the DNA, including the microbial DNA; this was validated by PCR analysis.

Development of a loop-mediated isothermal amplification procedure as a sensitive and rapid method for detection of 'candidatus Liberibacter solanacearum' in potatoes and Psyllids.

This study reports the development of a loop-mediated isothermal amplification procedure (LAMP) for polymerase chain reaction (PCR)-based detection of 'Candidatus Liberibacter solanacearum', the bacterial causal agent of potato zebra chip (ZC) disease. The 16S rDNA gene of 'Ca. Liberibacter solanacearum' was used to design a set of six primers for LAMP PCR detection of the bacterial pathogen in potato plants and the psyllid vector. The advantage of the LAMP method is that it does not require a thermocycler for amplification or agarose gel electrophoresis for resolution. Positive LAMP results can be visualized directly as a precipitate. The LAMP strategy reported here reliably detected 'Ca. Liberibacter solanacearum' and the closely related species 'Ca. Liberibacter asiaticus', the causative agent of huanglongbing disease of citrus, in plant DNA extracts. Although not as sensitive as quantitative real-time PCR, LAMP detection was equivalent to conventional PCR in tests of ZC-infected potato plants from the field. Thus, the LAMP method shows strong promise as a reliable, rapid, and cost-effective method of detecting 'Ca. Liberibacter' pathogens in psyllids and field-grown potato plants and tubers.

Lesion bacterial communities in American lobsters with diet-induced shell disease.

In southern New England, USA, shell disease affects the profitability of the American lobster Homarus americanus fishery. In laboratory trials using juvenile lobsters, exclusive feeding of herring Clupea harengus induces shell disease typified initially by small melanized spots that progress into distinct lesions. Amongst a cohabitated, but segregated, cohort of 11 juvenile lobsters fed exclusively herring, bacterial communities colonizing spots and lesions were investigated by denaturing gradient gel electrophoresis of 16S rDNA amplified using 1 group-specific and 2 universal primer sets. The Bacteroidetes and Proteobacteria predominated in both spots and lesions and included members of the orders Flavobacteriales (Bacteriodetes), Rhodobacterales, Rhodospirillales and Rhizobiales (Alphaproteobacteria), Xanthomonadales (Gammaproteobacteria) and unclassified Gammaproteobacteria. Bacterial communities in spot lesions displayed more diversity than communities with larger (older) lesions, indicating that the lesion communities stabilize over time. At least 8 bacterial types persisted as lesions developed from spots. Aquimarina 'homaria', a species commonly cultured from lesions present on wild lobsters with epizootic shell disease, was found ubiquitously in spots and lesions, as was the 'Candidatus Kopriimonas aquarianus', implicating putative roles of these species in diet-induced shell disease of captive lobsters.

Large-scale comparative phenotypic and genomic analyses reveal ecological preferences of shewanella species and identify metabolic pathways conserved at the genus level.

The use of comparative genomics for the study of different microbiological species has increased substantially as sequence technologies become more affordable. However, efforts to fully link a genotype to its phenotype remain limited to the development of one mutant at a time. In this study, we provided a high-throughput alternative to this limiting step by coupling comparative genomics to the use of phenotype arrays for five sequenced Shewanella strains. Positive phenotypes were obtained for 441 nutrients (C, N, P, and S sources), with N-based compounds being the most utilized for all strains. Many genes and pathways predicted by genome analyses were confirmed with the comparative phenotype assay, and three degradation pathways believed to be missing in Shewanella were confirmed as missing. A number of previously unknown gene products were predicted to be parts of pathways or to have a function, expanding the number of gene targets for future genetic analyses. Ecologically, the comparative high-throughput phenotype analysis provided insights into niche specialization among the five different strains. For example, Shewanella amazonensis strain SB2B, isolated from the Amazon River delta, was capable of utilizing 60 C compounds, whereas Shewanella sp. strain W3-18-1, isolated from deep marine sediment, utilized only 25 of them. In spite of the large number of nutrient sources yielding positive results, our study indicated that except for the N sources, they were not sufficiently informative to predict growth phenotypes from increasing evolutionary distances. Our results indicate the importance of phenotypic evaluation for confirming genome predictions. This strategy will accelerate the functional discovery of genes and provide an ecological framework for microbial genome sequencing projects.

Occurrence of class 1 and 2 integrons in resistant Enterobacteriaceae collected from a urban wastewater treatment plant: first report from central Italy.

The purpose of this study was to investigate the presence and distribution of integron-carrying isolates among Enterobacteriaceae resistant to ß-lactam antibiotics collected from a wastewater effluent of the city of L'Aquila (Italy). A total of 471 Enterobacteriaceae were collected during a period of 2 years (2005-2006). The presence and distribution of class 1 and 2 integrons was investigated by colony blot hybridization using specific probes labelled with dUTP-fluoresceine kit. The variable region of class 1 and 2 integrons was analyzed by polymerase chain reaction and DNA sequencing in 24 isolates with different random amplified polymorphic DNA profile. The characterization of class 1 and 2 integrons gene cassettes of 24 nonrelated strains showed the presence of four different arrays: dfr17-aadA5; aadA10; dfr1-sat1; dfr1-sat1-aadA1. This is the first report from Italy in which the authors confirm the presence of Enterobacteriaceae carrying class 1 and 2 integrons in a wastewater treatment plant that collects the urban and hospital discharges.

Detection of Clavibacter michiganensis subsp. sepedonicus by AmpliDet RNA, a new technology based on real time monitoring of NASBA amplicons with a molecular beacon.

AIMS: To develop a procedure for direct detection of viable cells of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, based on AmpliDet RNA, in which amplicons generated by nucleic acid sequence based amplification (NASBA) are monitored in real time with a molecular beacon. METHODS AND RESULTS: Five methods were evaluated and fine-tuned for extraction of RNA from Cms. The most efficient non-commercial RNA extraction method included an enzymatic breakdown of the cell wall followed by a phenol extraction. AmpliDet RNA enabled detection of 10,000 molecules of purified rRNA per reaction and 100 cfu of Cms per reaction in more complex samples. Two primer pairs were tested with DNA and RNA purified from Cms. One primer pair was able to distinguish live from dead cells. CONCLUSIONS: An AmpliDet RNA was developed which enabled fast and specific detection of viable cells of Cms in complex substrates at a detection limit of 100 cfu per reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel AmpliDet RNA is carried out in sealed tubes, thus reducing the risk of carry-over contamination. The method will be particularly suitable for studies on the epidemiology of Cms in which viable cells should be exclusively detected.