Generalized estimating equation modeling on correlated microbiome sequencing data with longitudinal measures.

Existing models for assessing microbiome sequencing such as operational taxonomic units (OTUs) can only test predictors' effects on OTUs. There is limited work on how to estimate the correlations between multiple OTUs and incorporate such relationship into models to evaluate longitudinal OTU measures. We propose a novel approach to estimate OTU correlations based on their taxonomic structure, and apply such correlation structure in Generalized Estimating Equations (GEE) models to estimate both predictors' effects and OTU correlations. We develop a two-part Microbiome Taxonomic Longitudinal Correlation (MTLC) model for multivariate zero-inflated OTU outcomes based on the GEE framework. In addition, longitudinal and other types of repeated OTU measures are integrated in the MTLC model. Extensive simulations have been conducted to evaluate the performance of the MTLC method. Compared with the existing methods, the MTLC method shows robust and consistent estimation, and improved statistical power for testing predictors' effects. Lastly we demonstrate our proposed method by implementing it into a real human microbiome study to evaluate the obesity on twins.

PlasClass improves plasmid sequence classification.

Many bacteria contain plasmids, but separating between contigs that originate on the plasmid and those that are part of the bacterial genome can be difficult. This is especially true in metagenomic assembly, which yields many contigs of unknown origin. Existing tools for classifying sequences of plasmid origin give less reliable results for shorter sequences, are trained using a fraction of the known plasmids, and can be difficult to use in practice. We present PlasClass, a new plasmid classifier. It uses a set of standard classifiers trained on the most current set of known plasmid sequences for different sequence lengths. We tested PlasClass sequence classification on held-out data and simulations, as well as publicly available bacterial isolates and plasmidome samples and plasmids assembled from metagenomic samples. PlasClass outperforms the state-of-the-art plasmid classification tool on shorter sequences, which constitute the majority of assembly contigs, allowing it to achieve higher F1 scores in classifying sequences from a wide range of datasets. PlasClass also uses significantly less time and memory. PlasClass can be used to easily classify plasmid and bacterial genome sequences in metagenomic or isolate assemblies. It is available under the MIT license from: https://github.com/Shamir-Lab/PlasClass.

Sequential and Selective Detection of Two Molecules with a Single Solid-Contact Chronopotentiometric Ion-Selective Electrode.

A polymeric membrane ion-selective electrode (ISE) is typically designed for the determination of one specific ion using a conventional method. In this work, we demonstrate a simple, versatile, and sensitive platform for simultaneous detection of two molecules with a single ISE. Under a series of periodic galvanostatic polarization, a solid-contact ISE without ion exchanger properties under zero-current conditions has been successfully used for simultaneous detection of two opposite charged ions with high sensitivity, good selectivity, and fast reversibility. By integration of biorecognition elements with the potentiometric measurement, highly sensitive and selective detection of a broad range of different molecular targets can be predicted. As a proof of concept, a potentiometric genosensor based on magnetic beads-enzyme sandwich assay has been designed for sensitive and selective detection of pathogenic bacteria Escherichia coli O157:H7 and Staphylococcus aureus. Under optimal conditions, two bacteria nucleic acid sequences can be detected simultaneously with high sensitivity and good selectivity by using a single solid-contact potentiometric ISE. The detection limits of Escherichia coli O157:H7 DNA and Staphylococcus aureus DNA are 120 and 54 fM (3σ), respectively. Because of its simplicity, this potentiometric technique based on ISE can be an attractive tool or detector to perform two analyte measurements.

Analysis of fecal microbiota in patients with functional constipation undergoing treatment with synbiotics.

This study was performed to identify changes to microbial composition after treatment with synbiotics in patients with functional constipation and to define the key microbiota in the pathogenesis of functional constipation. Fecal samples from 53 patients diagnosed with chronic functional constipation according to the Rome III criteria were analyzed using 16S rRNA sequencing. After treatment with synbiotics for 1 month, fecal samples were collected from 36 patients; after a total of 3 months, fecal samples were collected from 15 patients. The outcomes were compared with the intestinal microbiota profiles of 53 healthy community volunteers. The microbiota in the constipation group differed from that in the treatment group and healthy group. After synbiotic treatment for 1 and 3 months, the abundance of Escherichia/Shigella decreased, whereas that of Prevotella_9 and Lactococcus increased. Comparison of the microbiota among the three groups showed that Prevotella_9 was the characteristic bacteria that decreased in the constipation group and increased in the treatment group. Synbiotic treatment can improve the microbiota in patients with constipation. Identification of the key bacterial genus is important to reveal the mechanism and provide a reliable theoretical basis of synbiotic treatment. It will also promote relevant research of microbiota treatment and individualized treatments.

Variable-order sequence modeling improves bacterial strain discrimination for Ion Torrent DNA reads.

BACKGROUND: Genome sequencing provides a powerful tool for pathogen detection and can help resolve outbreaks that pose public safety and health risks. Mapping of DNA reads to genomes plays a fundamental role in this approach, where accurate alignment and classification of sequencing data is crucial. Standard mapping methods crudely treat bases as independent from their neighbors. Accuracy might be improved by using higher order paired hidden Markov models (HMMs), which model neighbor effects, but introduce design and implementation issues that have typically made them impractical for read mapping applications. We present a variable-order paired HMM that we term VarHMM, which addresses central issues involved with higher order modeling for sequence alignment. RESULTS: Compared with existing alignment methods, VarHMM is able to model higher order distributions and quantify alignment probabilities with greater detail and accuracy. In a series of comparison tests, in which Ion Torrent sequenced DNA was mapped to similar bacterial strains, VarHMM consistently provided better strain discrimination than any of the other alignment methods that we compared with. CONCLUSIONS: Our results demonstrate the advantages of higher ordered probability distribution modeling and also suggest that further development of such models would benefit read mapping in a range of other applications as well.

Bacterial cell cycle classification. Application to DNA synthesis and DNA content at any cell age.

A proposal is presented for classifying bacterial cell cycles into twelve discrete groups. This classification translates the three temporal parameters that define a cell cycle into numbers that facilitate an algorithmic approach to analyse the replication state of a single bacterium and of a bacterial population during steady-state of exponential growth. The classification and its implementation offer easy ways to obtain the rate of DNA synthesis and the amount of DNA per cell at any age in batch cultures.

The skin microbiome of cow-nose rays (Rhinoptera bonasus) in an aquarium touch-tank exhibit.

Public aquaria offer numerous educational opportunities for visitors while touch-tank exhibits offer guests the ability to directly interact with marine life via physical contact. Despite the popularity of touch-tanks, there is a paucity of research about animal health in these exhibits and, in particular, there is little research on the microbial communities in these highly interactive exhibits. Microbial community structure can have implications for both host health and habitat function. To better understand the microbiome of a touch-tank we used high-throughput sequencing of the 16S rRNA gene to analyze the microbial community on the dorsal and ventral surfaces of cow-nose rays (Rhinoptera bonasus) as well as their environment in a frequently visited touch-tank exhibit at the New England Aquarium. Our analyses revealed a distinct microbial community associated with the skin of the ray that had lower diversity than the surrounding habitat. The ray skin was dominated by three orders: Burkholderiales (∼55%), Flavobacteriales (∼19%), and Pseudomonadales (∼12%), taxonomic groups commonly associated with other fish species. Our results provide a survey of ray-associated bacterial communities in a touch-tank environment, thereby laying the foundation for future studies examining the role of potential challenges to ray microbiota and their associated health.

Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods.

There is growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality management. The transition from a research tool to a standardized protocol requires a high degree of confidence in data quality across laboratories. Data quality is typically determined through a series of specifications that ensure good experimental practice and the absence of bias in the results due to DNA isolation and amplification interferences. However, there is currently a lack of consensus on how best to evaluate and interpret human fecal source identification qPCR experiments. This is, in part, due to the lack of standardized protocols and information on interlaboratory variability under conditions for data acceptance. The aim of this study is to provide users and reviewers with a complete series of conditions for data acceptance derived from a multiple laboratory data set using standardized procedures. To establish these benchmarks, data from HF183/BacR287 and HumM2 human-associated qPCR methods were generated across 14 laboratories. Each laboratory followed a standardized protocol utilizing the same lot of reference DNA materials, DNA isolation kits, amplification reagents, and test samples to generate comparable data. After removal of outliers, a nested analysis of variance (ANOVA) was used to establish proficiency metrics that include lab-to-lab, replicate testing within a lab, and random error for amplification inhibition and sample processing controls. Other data acceptance measurements included extraneous DNA contamination assessments (no-template and extraction blank controls) and calibration model performance (correlation coefficient, amplification efficiency, and lower limit of quantification). To demonstrate the implementation of the proposed standardized protocols and data acceptance criteria, comparable data from two additional laboratories were reviewed. The data acceptance criteria proposed in this study should help scientists, managers, reviewers, and the public evaluate the technical quality of future findings against an established benchmark.

[A NOVEL APPROACH TO GENOTYPING OF HOSPITAL ISOLATES OF CLOSTRIDIUM DIFFICILE].

AIM: Development of a novel approach in genotyping of Clostridium difficile and its testing on the example of 140 hospital isolates. MATERIALS AND METHODS: The approach is based on an idea of double digest and selective label (DDSL), used previously during genotyping of other bacterial pathogens. Selection of optimal enzymes for restriction of MluI and Mph1103I was carried out, condition of DDSL reaction execution were optimized. RESULTS: Genotyping of C. difficile hospital isolates was carried out, index of strain discrimination was calculated, conclusions regarding possibilities of the method in elucidation of spread pathways and identification of infection sources were made. CONCLUSION: The developed method of genotyping has a number of advantages over the existing method and can be used to'address issues in epidemiology of infections caused by C. difficile.

Variability in gene cassette patterns of class 1 and 2 integrons associated with multi drug resistance patterns in Staphylococcus aureus clinical isolates in Tehran-Iran.

BACKGROUND: To investigate antibiotic resistance, the occurrence and distribution of class 1 and 2 integrons in multidrug- resistant Staphylococcus aureus isolates from hospitals in Tehran, Iran. The isolates were examined for susceptibility to antimicrobial agents. The mecA gene, class 1 and 2 integrons were detected by PCR. Integrase positive strains were further analysed for the presence of resistance gene cassettes using specific primers and were sequenced. RESULTS: Among 139 S.aureus isolates, 109 (78.4 %) and 112 (80.5 %) strains were considered as multidrug resistant and mecA positive, respectively. Class 1 integrons and internal variable regions were found in 72.6 % (101/139) and 97 % (98/101) and class 2 integrons and variable regions also in 35.2 % (49/139) and 65.3 % (32/49) of S.aureus clinical isolates, respectively. Twelve distinct cassette arrays were found, containing genes encoding resistance to ß-lactams, aminoglycosides, streptothricin, trimethoprim, chloramphenicol,a putative glucose dehydrogenase precursor and a protein with unknown function. Gene cassette arrays aadB, aadA2 and dhfrA1-sat2-aadA1 were common in S.aureus isolates. We detected a completely new gene cassettes which contained aadB, oxa2, aacA4, orfD-aacA4-catB8, aadB-catB3, orfD-aacA4 and aadB-aadA1-cmlA6 of class 1 and dhfrA1-sat2-aadA1, dhfrA11, dhfrA1-sat2 of class 2 integrons. CONCLUSIONS: This is the first study to report carriage of class 1 and 2 integrons and associated gene cassettes among in S.aureus isolates from Iran.

Optimized Protocol for PFGE Analysis of Anginosus (milleri) Streptococci.

Streptococcus anginosus (milleri) is a diverse group of gram positive bacteria. Molecular methods to establish relationship between strains are poorly developed. Therefore, main tool to study genetic variability is restriction fragment length polymorphism combined with pulsed field gel electrophoresis (RFLP-PFGE). In this communication, we present optimized protocol for S. anginosus PFGE analysis.

Micronized fibres affect in vitro fermentation under normal buffered and osmotic stress conditions using porcine inocula.

In this in vitro study, the modified Hohenheim gas test was used to determine fermentation activity and bacterial composition of pig's faecal microbial inoculum, when fermenting a standard pig diet with varying levels of crude protein (CP; 20, 24 and 28% CP), and supplemented with one of three fibre sources manufactured by micronization treatment. These were wheat envelopes (MWE), pea fibre (MPF) and lupine fibre (MLF). For comparison, inulin was used. As intestinal bacteria have to cope with varying osmotic conditions in their ecosystem, fermentation was performed under normal buffered and osmotic stress conditions. After 24 h of fermentation, total gas production and ammonia production were measured. In addition, the effect of MWE and inulin on short-chain fatty acid (SCFA) production and numbers of total eubacteria, Lactobacillus spp., Bifidobacterium spp., Enterobacteriaceae, Enterococcus spp., Clostridium cluster XIVa and Clostridium cluster IV, were determined using quantitative real-time PCR. Under normal buffered conditions, supplementation of MWE resulted in increased (p < 0.05) SCFA, acetic, propionic and valerianic acid production at CP levels of 20 and 28%. There was an increase (p < 0.05) in ammonia production for the micronized supplements, and for MWE an increased (p < 0.05) branched-chain proportion was observed, possibly due to higher availability of protein for fermentation which was released during the micronization process. Osmotic stress conditions reduced (p < 0.05) total gas as well as total SCFA, acetic and propionic acid production for all treatments, while cell counts were increased (p < 0.05) for Bifidobacterium spp., Enterococcus spp. and Lactobacillus spp. Under normal buffered conditions in combination with 24 and 28% CP levels, lactobacilli were increased for MWE, compared to inulin (p < 0.05). In conclusion, micronized supplements such as MWE may beneficially modulate pigs' intestinal microbiota by increasing SCFA production in addition to a selective proliferation of lactobacilli.

Melioidosis caused by Burkholderia pseudomallei in drinking water, Thailand, 2012.

We identified 10 patients in Thailand with culture-confirmed melioidosis who had Burkholderia pseudomallei isolated from their drinking water. The multilocus sequence type of B. pseudomallei from clinical specimens and water samples were identical for 2 patients. This finding suggests that drinking water is a preventable source of B. pseudomallei infection.

Phylogeography of cylindrospermopsin and paralytic shellfish toxin-producing nostocales cyanobacteria from mediterranean europe (Spain).

Planktonic Nostocales cyanobacteria represent a challenge for microbiological research because of the wide range of cyanotoxins that they synthesize and their invasive behavior, which is presumably enhanced by global warming. To gain insight into the phylogeography of potentially toxic Nostocales from Mediterranean Europe, 31 strains of Anabaena (Anabaena crassa, A. lemmermannii, A. mendotae, and A. planctonica), Aphanizomenon (Aphanizomenon gracile, A. ovalisporum), and Cylindrospermopsis raciborskii were isolated from 14 freshwater bodies in Spain and polyphasically analyzed for their phylogeography, cyanotoxin production, and the presence of cyanotoxin biosynthesis genes. The potent cytotoxin cylindrospermopsin (CYN) was produced by all 6 Aphanizomenon ovalisporum strains at high levels (5.7 to 9.1 µg CYN mg(-1) [dry weight]) with low variation between strains (1.5 to 3.9-fold) and a marked extracellular release (19 to 41% dissolved CYN) during exponential growth. Paralytic shellfish poisoning (PSP) neurotoxins (saxitoxin, neosaxitoxin, and decarbamoylsaxitoxin) were detected in 2 Aphanizomenon gracile strains, both containing the sxtA gene. This gene was also amplified in non-PSP toxin-producing Aphanizomenon gracile and Aphanizomenon ovalisporum. Phylogenetic analyses supported the species identification and confirmed the high similarity of Spanish Anabaena and Aphanizomenon strains with other European strains. In contrast, Cylindrospermopsis raciborskii from Spain grouped together with American strains and was clearly separate from the rest of the European strains, raising questions about the current assumptions of the phylogeography and spreading routes of C. raciborskii. The present study confirms that the nostocalean genus Aphanizomenon is a major source of CYN and PSP toxins in Europe and demonstrates the presence of the sxtA gene in CYN-producing Aphanizomenon ovalisporum.

Niche differentiation in nitrogen metabolism among methanotrophs within an operational taxonomic unit.

BACKGROUND: The currently accepted thesis on nitrogenous fertilizer additions on methane oxidation activity assumes niche partitioning among methanotrophic species, with activity responses to changes in nitrogen content being dependent on the in situ methanotrophic community structure Unfortunately, widely applied tools for microbial community assessment only have a limited phylogenetic resolution mostly restricted to genus level diversity, and not to species level as often mistakenly assumed. As a consequence, intragenus or intraspecies metabolic versatility in nitrogen metabolism was never evaluated nor considered among methanotrophic bacteria as a source of differential responses of methane oxidation to nitrogen amendments. RESULTS: We demonstrated that fourteen genotypically different Methylomonas strains, thus distinct below the level at which most techniques assign operational taxonomic units (OTU), show a versatile physiology in their nitrogen metabolism. Differential responses, even among strains with identical 16S rRNA or pmoA gene sequences, were observed for production of nitrite and nitrous oxide from nitrate or ammonium, nitrogen fixation and tolerance to high levels of ammonium, nitrate, and hydroxylamine. Overall, reduction of nitrate to nitrite, nitrogen fixation, higher tolerance to ammonium than nitrate and tolerance and assimilation of nitrite were general features. CONCLUSIONS: Differential responses among closely related methanotrophic strains to overcome inhibition and toxicity from high nitrogen loads and assimilation of various nitrogen sources yield competitive fitness advantages to individual methane-oxidizing bacteria. Our observations proved that community structure at the deepest phylogenetic resolution potentially influences in situ functioning.

A combination of MLST and CRISPR typing reveals dominant Campylobacter jejuni types in organically farmed laying hens.

AIM: To elucidate the Campylobacter jejuni population in organically farmed laying hens in Finland, multilocus sequence typing (MLST) was combined with characterization of clustered regularly interspaced short palindromic repeat (CRISPR) sequences. METHODS AND RESULTS: A total of 147 Camp. jejuni isolates, collected from organically farmed laying hens from 18 farms in 2003-2004, were previously analysed by pulsed-field gel electrophoresis. In the present study, subsets of the isolates were further analysed by MLST and CRISPR sequences. Fourteen STs were found by MLST. ST-50 (27%, 7/18 farms), ST-3272 (20%, 8/18 farms), ST-45 (12%, 7/18 farms) and ST-356 (12%, 5/18 farms) were the most common STs. CRISPR types were identical among all isolates of ST-50 (ST-21 clonal complex (CC)) and the most variable among ST-45 (ST-45 CC). CONCLUSIONS: ST-3272 (UA), a common ST in this study, has been infrequently detected in other hosts. Other major STs (ST-50 and ST-45) have been common in several hosts such as conventional poultry and bovines. CRISPR typing provided additional discrimination between isolates of certain dominant STs and could be useful in further epidemiological studies. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives new information about MLST and CRISPR types of Camp. jejuni among organically farmed laying hens.

MicroSEQ Salmonella spp. Detection Kit using the Pathatrix 10-Pooling Salmonella spp. Kit linked protocol method modification. Performance Tested Method 031001.

Real-time PCR methods for detecting foodborne pathogens offer the advantages of simplicity and quick time to results compared to traditional culture methods. The addition of a recirculating pooled immunomagnetic separation method prior to real-time PCR analysis increases processing output while reducing both cost and labor. This AOAC Research Institute method modification study validates the MicroSEQ Salmonella spp. Detection Kit [AOAC Performance Tested Method (PTM) 031001] linked with the Pathatrix 10-Pooling Salmonella spp. Kit (AOAC PTM 090203C) in diced tomatoes, chocolate, and deli ham. The Pathatrix 10-Pooling protocol represents a method modification of the enrichment portion of the MicroSEQ Salmonella spp. protocol. The results of the method modification were compared to standard cultural reference methods for diced tomatoes, chocolate, and deli ham. All three matrixes were analyzed in a paired study design. An additional set of chocolate test portions was analyzed using an alternative enrichment medium in an unpaired study design. For all matrixes tested, there were no statistically significant differences in the number of positive test portions detected by the modified candidate method compared to the appropriate reference method. The MicroSEQ Salmonella spp. protocol linked with the Pathatrix individual or 10-Pooling procedure demonstrated reliability as a rapid, simplified, method for the preparation of samples and subsequent detection of Salmonella in diced tomatoes, chocolate, and deli ham.

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 functional structure in response to micro-aerobic conditions in sulfate-reducing sulfur-producing bioreactor.

Limited oxygen supply to anaerobic wastewater treatment systems had been demonstrated as an effective strategy to improve elemental sulfur (S(0)) recovery, coupling sulfate reduction and sulfide oxidation. However, little is known about the impact of dissolved oxygen (DO) on the microbial functional structures in these systems. We used a high throughput tool (GeoChip) to evaluate the microbial community structures in a biological desulfurization reactor under micro-aerobic conditions (DO: 0.02-0.33 mg/L). The results indicated that the microbial community functional compositions and structures were dramatically altered with elevated DO levels. The abundances of dsrA/B genes involved in sulfate reduction processes significantly decreased (p < 0.05, LSD test) at relatively high DO concentration (DO: 0.33 mg/L). The abundances of sox and fccA/B genes involved in sulfur/sulfide oxidation processes significantly increased (p < 0.05, LSD test) in low DO concentration conditions (DO: 0.09 mg/L) and then gradually decreased with continuously elevated DO levels. Their abundances coincided with the change of sulfate removal efficiencies and elemental sulfur (S(0)) conversion efficiencies in the bioreactor. In addition, the abundance of carbon degradation genes increased with the raising of DO levels, showing that the heterotrophic microorganisms (e.g., fermentative microorganisms) were thriving under micro-aerobic condition. This study provides new insights into the impacts of micro-aerobic conditions on the microbial functional structure of sulfate-reducing sulfur-producing bioreactors, and revealed the potential linkage between functional microbial communities and reactor performance.

[Biodiversity of sulfate-reducing bacteria growing on objects of heating systems].

It was shown that sulfate-reducing bacteria developed on the sections of Kyiv municipal heating systems, which are exploited in conditions of different temperatures. The bacteria were different as to their morphological and physiological properties. The bacteria of Desulfovibrio genus were revealed on the sections, which were exploited at a temperature of 35-40 degrees C and bacteria of Desulfomicrobium and Desulfotomaculum genera were revealed on the sections with a higher temperature such as 60 degrees C. Based on of the 16S rRNA gene analysis data, it was demonstrated that sequences of TC2, TC3 and TC4 clones related to Desulfovibrio sp. DSM 12803 (100% sequence similarity), Desulfotomaculum sp. ECP-C-5 (92% sequence similarity) and Desulfomicrobium baculatum strain DSM 2555 (99% sequence similarity), respectively. The identified bacteria are potentially dangerous for heating systems and can be the agents of microbial corrosion.