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PubMed2004

Development of joint application strategies for two microbial gene finders.

MOTIVATION: As a starting point in annotation of bacterial genomes, gene finding programs are used for the prediction of functional elements in the DNA sequence. Due to the faster pace and increasing number of genome projects currently underway, it is becoming especially important to have performant methods for this task. RESULTS: This study describes the development of joint application strategies that combine the strengths of two microbial gene finders to improve the overall gene finding performance. Critica is very specific in the detection of similarity-supported genes as it uses a comparative sequence analysis-based approach. Glimmer employs a very sophisticated model of genomic sequence properties and is sensitive also in the detection of organism-specific genes. Based on a data set of 113 microbial genome sequences, we optimized a combined application approach using different parameters with relevance to the gene finding problem. This results in a significant improvement in specificity while there is similarity in sensitivity to Glimmer. The improvement is especially pronounced for GC rich genomes. The method is currently being applied for the annotation of several microbial genomes. AVAILABILITY: The methods described have been implemented within the gene prediction component of the GenDB genome annotation system.

Algorithms↗
PubMedDate not supplied

[The use of a cholera coagulating diagnostic agent under the cholera epidemic conditions in Dagestan].

Dried cholera diagnosticum for the slide coagglutination test was obtained. The diagnosticum, found to be highly active and specific, permitted the detection of Vibrio cholerae in the analyzed material at a concentration of 10(6)-10(8) microbial cells/ml. The diagnosticum was used during cholera epidemic in Daghestan for the detection and rapid identification of cholera vibrios. In all cases the positive results of the coagglutination test were confirmed by other investigation methods (no cases of hyperdiagnosis was registered). The production of these diagnostica in small batches on the basis of specialized research laboratories was recommended.

Agglutination Tests↗
PubMed1995

The inflammatory Papanicolaou smear: what does it mean?

OBJECTIVE: To determine the correlation between inflammation detected on Papanicolaou smear and specific lower genital tract agents, and, based on these findings, to develop recommendations for follow-up tests and treatment of young women with inflammation on smears. METHODS: A high-risk population of 779 randomly selected women attending a sexually transmitted disease (STD) clinic and a low-risk population of 1050 consecutive women presenting for annual examination at a university student health center underwent a standardized history and gynecologic examination. Univariate and multivariate analyses, focusing on the association between dense inflammation on Papanicolaou smear and specific lower genital tract pathogens or findings on cervical examination, were done for each population. RESULTS: Dense inflammation was present on the Papanicolaou smear of 256 (33%) of the 779 women in the STD clinic and 200 (19%) of 1050 students. Dense inflammation on Papanicolaou smear was independently associated with mucopus, cervical ectopy, cervical infection with Neisseria gonorrhoeae, Chlamydia trachomatis, herpes simplex virus (HSV), and vaginal infection by Trichomonas vaginalis in the STD population; in the student population, it was associated with cervical ectopy, C trachomatis, and mucopus. CONCLUSION: Although dense inflammation on Papanicolaou smear was a common finding in both the high- and low-risk populations, about half of the inflammation detected in the high-risk setting was associated with a specific microbial organism (C trachomatis, N gonorrhoeae, HSV, or T vaginalis), whereas less than 10% of the dense inflammation detected in the low-risk setting was linked with a specific pathogen (C trachomatis). In both settings, a substantial population of sexually active women had dense inflammation associated with cervical ectopy but none of the specific organisms evaluated in this study.

Adult↗
PubMed2000

Sub-micron particle manipulation in an ultrasonic standing wave: applications in detection of clinically important biomolecules.

Separation of particles from the suspending phase is of interest, among others, to clinical analysts. A system that enables manipulation of sub-micron sized particles in suspensions of analytical scale volume (10-50 microl) using a non-cavitating ultrasonic standing wave is described. Particle suspensions, contained in glass capillary tubes of 1-2 mm internal dimension, are treated on the axis of a tubular transducer generating a radial standing wave field at 4.5 MHz. Microparticles (of average diameter range 0.3-10 microm) suspended in buffer are concentrated within seconds at preferred regions separated by submillimetre distances. Concentration of suspended latex particles was inhibited in solutions containing protein at levels similar to those occurring in clinical specimens when the suspensions were sonicated in capillaries of circular cross-section. This effect was associated with acoustic streaming of the suspending fluid. Silica microparticles (more dense and less compressible than latex) could be concentrated in the presence of streaming. Latex particles concentrated readily in square cross-section capillaries where no streaming was observed. With sub-micron particles, the geometry of the sample chamber, the suspending phase composition and the size, density and compressibility of the microparticles all influence particle manipulation. The radial standing wave system has been used to enhance agglutination of antibody-coated latex microparticles in the presence of antigen allowing rapid and highly sensitive detection of clinically important biomolecules. The sensitivity of conventional diagnostic tests for microbial antigen has been improved by application of ultrasound and clinical utility has been demonstrated, in particular, for detection of meningitis-causing bacteria.

Antigens, Bacterial↗
PubMed1995

Microbial biotransformation of the angiotensin II antagonist GR117289 by Streptomyces rimosus to identify a mammalian metabolite.

Screening a range of microorganisms incubated with the angiotensin II antagonist GR117289 resulted in the use of Streptomyces rimosus to generate five related biotransformation products. These comprised three compounds hydroxylated on the aliphatic side chain, one further oxidized to a ketone, and one hydroxylated on the phenyl ring. These microbial metabolites were used as standards to identify a human metabolite detected in plasma and urine, but present in insufficient quantities for full structural characterisation. This further demonstrates how the use of microbial biotransformation systems at an early stage of drug metabolism studies can act as a valuable tool in facilitating identification of minor human metabolites.

Angiotensin Receptor Antagonists↗
PubMed1998

Isolation of broad-host-range replicons from marine sediment bacteria.

Naturally occurring plasmids isolated from heterotrophic bacterial isolates originating from coastal California marine sediments were characterized by analyzing their incompatibility and replication properties. Previously, we reported on the lack of DNA homology between plasmids from the culturable bacterial population of marine sediments and the replicon probes specific for a number of well-characterized incompatibility and replication groups (P. A. Sobecky, T. J. Mincer, M. C. Chang, and D. R. Helinski, Appl. Environ. Microbiol. 63:888-895, 1997). In the present study we isolated 1.8- to 2.3-kb fragments that contain functional replication origins from one relatively large (30-kb) and three small (<10-kb) naturally occurring plasmids present in different marine isolates. 16S rRNA sequence analyses indicated that the four plasmid-bearing marine isolates belonged to the alpha and gamma subclasses of the class Proteobacteria. Three of the marine sediment isolates are related to the gamma-3 subclass organisms Vibrio splendidus and Vibrio fischeri, while the fourth isolate may be related to Roseobacter litoralis. Sequence analysis of the plasmid replication regions revealed the presence of features common to replication origins of well-characterized plasmids from clinical bacterial isolates, suggesting that there may be similar mechanisms for plasmid replication initiation in the indigenous plasmids of gram-negative marine sediment bacteria. In addition to replication in Escherichia coli DH5alpha and C2110, the host ranges of the plasmid replicons, designated repSD41, repSD121, repSD164, and repSD172, extended to marine species belonging to the genera Achromobacter, Pseudomonas, Serratia, and Vibrio. While sequence analysis of repSD41 and repSD121 revealed considerable stretches of homology between the two fragments, these regions do not display incompatibility properties against each other. The replication origin repSD41 was detected in 5% of the culturable plasmid-bearing marine sediment bacterial isolates, whereas the replication origins repSD164 and repSD172 were not detected in any plasmid-bearing bacteria other than the parental isolates. Microbial community DNA extracted from samples collected in November 1995 and June 1997 and amplified by PCR yielded positive signals when they were hybridized with probes specific for repSD41 and repSD172 replication sequences. In contrast, replication sequences specific for repSD164 were not detected in the DNA extracted from marine sediment microbial communities.

Bacteria↗
PubMed1988

Effect of prolonged subclinical infection with Trichostrongylus colubriformis on the performance and nitrogen metabolism of growing lambs.

Ten 5-month-old lambs, reared free of parasites and prepared with abomasal and ileal cannulas, were paired and given rations of pelleted dried grass by means of continuous feeders. From 7 months of age one of each pair was dosed daily with 2500 Trichostrongylus colubriformis larvae for 34 weeks. An untreated animal served as a pair-fed control to each infected lamb. During 5 experimental periods (pre-infection, and Weeks 6-8, 12-14, 18-20 and 29-34 of dosing) measurements were made of the amounts of N leaving the small intestine, the digestibility of 35[S]-labelled microbial protein in the small intestine, and the amounts of plasma protein leaking into the digestive tract. The infection caused a growth check (P less than 0.05) from Week 6 to approximately Week 13 of dosing. This resulted from inappetence, but also from a disruption in the N digestion and metabolism of the lambs. There was a marked eosinophilia at this time. Measurements of ileal flow rates during Weeks 6 and 12 indicated that significantly more N flowed to the terminal ileum of infected lambs during this period (Week 6, 3.1 g N day-1 P less than 0.05; Week 12, 5.0 g N day-1 P less than 0.001). Plasma N leakage into the intestine during Weeks 8 and 14 was higher in infected lambs (1.2 g N day-1 P less than 0.01 and 0.9 g N day-1 P less than 0.05, respectively) but the digestibility of 35[S]-labelled microbial protein was depressed below that of the controls during the later part of this period, i.e. during Week 13. Continued larval challenge beyond this point caused a less severe nutritional penalty. Voluntary feed intake increased and the animals started to gain weight. The amount of extra N flowing to the terminal ileum of infected lambs was less at Week 18 of dosing (3.5 g N day-1 P less than 0.01) than it had been at Week 12. Similarly, extra plasma N leakage into the tract of infected lambs was less during Week 20 (0.26 g N day-1) than during Week 14. There was however, still a detectable reduction in the coefficient of digestibility of 35S microbial protein in infected lambs during Week 19. By Weeks 29-34 the only detectable difference between the infected and control lambs was in their eosinophil counts.

Animals↗
PubMed1988

[Epidemiologic problems of pneumococcal infection].

During the microbiological examination of 314 tentatively healthy donors, residents of Leningrad, Streptococcus pneumoniae was detected in the rhinopharynx of 94 donors (29.9%) in amounts ranging from a few individual microbial cells to 10(6) microbial cells with the mean geometric concentration equal to 3.58 lg. Antipneumococcal antibodies were detected in the blood of 147 donors (46.8%). The occurrence of antipneumococcal antibodies was approximately 2.5 times higher than the average level of carriership, but in most cases the dynamics of immunological reactions changed in the same direction. Considerable monthly and annual fluctuations in the occurrence of pneumococcal carriership and antipneumococcal antibodies, as well as their correlation with the epidemic rises of acute respiratory diseases, influenza and other acute respiratory viral diseases, were established. These data indicate that pneumococcal infection plays a definite role in the epidemic rises of acute respiratory diseases.

Antibodies, Bacterial↗
PubMed2006

Vaginal microflora in postmenopausal women on hormone replacement therapy.

To study the spectrum of vaginal microflora in postmenopausal women on hormone replacement therapy (HRT) and to compare the efficacy of Papanicolaou (Pap) smears with other methods for their detection. Eighty postmenopausal women were recruited for the study. These included 40 women who had attained spontaneous and were on HRT (User 1); 20 hysterectomised women on only estrogen therapy (User 2) and 20 controls (Non users). Their clinical data was recorded and specimens were collected for vaginal cultures (for aerobic bacteria and fungi), vaginal pH, Gram stain and Pap stain on cervical-vaginal smears and toluidine blue on wet smears. Vaginal pH was significantly lower in Users as compared to Non users. Lactobacilli and Gardnerella were more frequently isolated from Users while Bacteroides and E. coli were more common in Non users. Cultures were significantly more sensitive than Gram stained direct vaginal smears in detection of aerobic bacteria; however, Candida could be detected on Gram stain alone in all the cases. Frequency of detection of organisms significantly improved by application of Gram stain to the cervico-vaginal smears. However, clinically relevant organisms like Candida, Gardnerella and Mobiluncus could be identified on Pap smears alone in >50% cases. Lactobacilli could be readily identified in Pap smears in 98% cases. Wet mounts could detect cocci more easily as compared to Pap smears. Altered vaginal microbial profile in post menopausal women receiving HRT may cause bacterial and fungal vaginitis. Although culture studies remain the gold standard to detect these microorganisms, Pap and Gram stains and wet smears provide useful supplements and may be used as alternative procedures especially in resource limited settings lacking adequate culture facilities.

Bacteria↗
PubMed2003

Development of a biosensor for on-line detection of tributyltin with a recombinant bioluminescent Escherichia coli strain.

A biosensor was developed for the detection of tributyltin (TBT), using a bioluminescent recombinant Escherichia coli:: luxAB strain. Dedicated devices allowed the on-line measurement of bioluminescence, pH and dissolved oxygen values and the feed-back regulation of temperature. Bacterial physiology was monitored by the measurement of the cellular density, respiratory activity and the intracellular level of ATP, glucose and acetate levels. Our results showed that a synthetic glucose medium gave a better TBT detection limit than LB medium (respectively 0.02 micro M and 1.5 micro M TBT). High growth and dilution rates ( D=0.9 h(-1)) allowed maximum light emission from the bacterium. Moreover, simple atmospheric air bubbling was sufficient to provide oxygen for growth and the bioluminescence reaction. Real-time monitoring of bioluminescence after TBT induction occurred with continuous addition of decanal up to 300 micro M, which was not toxic throughout a 7-day experiment. The design of our biosensor and the optimization of the main parameters that influence microbial activity led to the capacity for the detection of TBT.

Biosensing Techniques↗
PubMed2003

Acclimation of mesophilic anaerobic sludge to thermophilic conditions: PCR genera detection methodology.

In Mexico, sludge from wastewater treatment plants is a public health problem, especially for the infant population due to the presence of pathogens and parasite eggs. Therefore, it is of great interest to apply proper treatment methods for those wastes. An option proposed for the removal of possible infectious sources is thermophilic anaerobic digestion, even though a limitation for its acceptance is the slow start-up of the reactors. The transformation of a mesophilic anaerobic sludge for its utilization as a thermophilic inoculum, by means of direct temperature increase up to 55 degrees C and feeding of the inoculum with synthetic medium was possible in two months. As a main part of the work and with the objective of evaluating the changes in the methanogenic microbial population during the sludge treatment, a specific detection technique for these bacteria has been developed and validated, based on the use of the polymerase chain reaction (PCR).

Bacteria↗
PubMed2026

Identification and Classification of Expressed Orphan Genes, Spurious Orphan Genes, and Conserved Genes in the Human Gut Microbiome.

Orphan genes (OGs)-genes lacking detectable homologs outside a species-are widespread in microbial genomes and are thought to contribute to their adaptation and molecular innovation. However, not all predicted OGs may represent novel functional coding sequences. False positive OGs, also called spurious OGs, can arise from gene prediction errors. We reason that OGs lacking detectable expression are more likely to be spurious. To test this, we combined large-scale metatranscriptomic profiling of the human gut microbiome with machine learning to distinguish expressed OGs from spurious ones and compare them with conserved genes (CGs) found in multiple species. Using nearly 5,000 metatranscriptome libraries, we identified &#x223c;218,000 OGs supported by expression evidence, while &#x223c;330,000 predicted OGs lacked detectable expression and were classified as spurious. We extracted 154 features for sequence, structural, and evolutionary properties for each gene and trained XGBoost classifiers while accounting for genomic representation. The models achieved an area under the receiver operating characteristic curve (AUC) of 0.82 in distinguishing expressed OGs from spurious OGs and an AUC of 0.93 in distinguishing expressed OGs from CGs. Interpretation based on SHAP (SHapley Additive exPlanations) revealed clear biological signals. Particularly, expressed orphans were present in more genomes than spurious ones, and expressed OGs were shorter than CGs. This work improves OG discovery and suggests that expressed OGs differ systematically from CGs and spurious OGs in sequence composition, structural constraints, and evolutionary signals.

Humans↗
PubMed2025

Large Quantities of Bacterial DNA and Protein in Common Dietary Protein Source Used in Microbiome Studies.

Diet has been shown to greatly impact the intestinal microbiota. To understand the role of individual dietary components, defined diets with purified components are frequently used in diet-microbiota studies. Defined diets frequently use purified casein as the protein source. Previous work indicated that casein contains microbial DNA potentially impacting results of microbiome studies. Other diet-based microbially derived molecules that may impact microbiome measurements, such as proteins detected by metaproteomics, have not been determined for casein. Additionally, other protein sources used in microbiome studies have not been characterized for their microbial content. We used metagenomics and metaproteomics to identify and quantify microbial DNA and protein in a casein-based defined diet to better understand potential impacts on metagenomic and metaproteomic microbiome studies. We further tested six additional defined diets with purified protein sources with an integrated metagenomic-metaproteomic approach and found that contaminating microbial protein is unique to casein within the tested set as microbial protein was not identified in diets with other protein sources. We also illustrate the contribution of diet-derived microbial protein in diet-microbiota studies by metaproteomic analysis of stool samples from germ-free mice (GF) and mice with a conventional microbiota (CV) following consumption of diets with casein and non-casein protein. This study highlights a potentially confounding factor in diet-microbiota studies that must be considered through evaluation of the diet itself within a given study.

Animals↗
PubMedDate not supplied

[Treatment of chronic rhinitis in children].

This paper discusses involvement of intestinal dysbacteriosis in nasal inflammation. Altogether 42 children of preschool age with chronic rhinitis were examined. It was found that microbial changes in the nasal cavity were related to the degree of intestinal dysbacteriosis, which was detected in most patients. The drugs that were used in combined therapy to treat microbial changes in the nasal mucosa and intestine produced good clinical and bacteriological effects. The 4 month follow-up showed that the children on the combined therapeutic protocol remained in the remission stage throughout this time period, whereas 85 +/- 10% of the control children exhibited exacerbation of the inflammatory process in the nasal cavity within 2-3 weeks after onset of standard treatment.

Bacterial Infections↗
PubMed2006

Greyhound meningoencephalitis: PCR-based detection methods highlight an absence of the most likely primary inducing agents.

Greyhound meningoencephalitis is currently classified as a breed-associated idiopathic central nervous system inflammatory disorder. The non-suppurative inflammatory response can be distinguished from the other breed-associated disorders based on histopathology and lesion topography, however the nature of the response primarily suggests a viral infection. In the present study PCR and RT-PCR technologies were employed on frozen cerebral tissue from confirmed cases of meningoencephalitis to target specific viruses and protozoa likely to be implicated and to exclude the presence of bacterial 16SrRNA. Secondly, degenerate primers were used to detect viruses of the herpesvirus and flavivirus families. In addition cerebral tissues were probed for West Nile Virus. Viral nucleic acid sequences to Borna disease virus, to louping ill, tick borne encephalitis, West Nile and other flaviviruses were not detected. Canine distemper virus was detected in one animal with 97% homology to strain A75/15. Degenerate PCR for herpesviruses detected viral amplification products in one animal with 90% homology to canine herpesvirus DNA polymerase gene. Protozoal amplification products were only detected in a single dog with pathological confirmation of a combination of lesions of greyhound meningoencephalitis and a protozoal encephalomyelitis. Neospora was confirmed with sequence homology to Austrian strain 1. Bacterial 16SrRNA was not detected. The present study supports previous observations that many of the known microbial causes of canine meningoencephalitis are not involved. Findings could reflect that the causal agent was not specifically targeted for detection, or that the agent is at undetectable levels or has been eliminated from brain tissue. The potential roles of genetics and of molecular mimicry also cannot be discounted.

Animals↗
PubMed2003

Emerging food pathogens and bacterial toxins.

Many different foodborne diseases have been described. For example, Shigella bacteria, hepatitis A virus and Norwalk virus were shown as a unwashed hands microorganisms, but pathogen Campylobacter and Escherichia coli were named as raw and undercooked meat and poultry or raw milk and untreated water born bacteria. However, two of them: Listeria monocytogenes and Yersinia enterocolitica are known as growing at refrigerator temperatures. Essential virulence determinants of Listeria monocytogenes pathogenicity are well known as a bacterial toxins. Basic molecular mechanisms of pathogenicity depending from these toxins were presented. It was shown that other bacterial toxins may act as very danger food poisoning substances. This is why elimination of pathogenic microorganisms from foods is an obvious solution in some food processes, however this approach is not practical or even desirable in many processes. Thus, risk assessment and microbial monitoring will continue to play important roles in ensuring food safety. Some technological advances have the capability of delivering detection systems that can not only monitor pathogenic microorganisms, but also entire microbial populations in the food matrix.

Bacterial Toxins↗
PubMed1999

Immunochemical detection and isolation of DNA from metabolically active bacteria.

Most techniques used to assay the growth of microbes in natural communities provide no information on the relationship between microbial productivity and community structure. To identify actively growing bacteria, we adapted a technique from immunocytochemistry to detect and selectively isolate DNA from bacteria incorporating bromodeoxyuridine (BrdU), a thymidine analog. In addition, we developed an immunocytochemical protocol to visualize BrdU-labeled microbial cells. Cultured bacteria and natural populations of aquatic bacterioplankton were pulse-labeled with exogenously supplied BrdU. Incorporation of BrdU into microbial DNA was demonstrated in DNA dot blots probed with anti-BrdU monoclonal antibodies and either peroxidase- or Texas red-conjugated secondary antibodies. BrdU-containing DNA was physically separated from unlabeled DNA by using antibody-coated paramagnetic beads, and the identities of bacteria contributing to both purified, BrdU-containing fractions and unfractionated, starting-material DNAs were determined by length heterogeneity PCR (LH-PCR) analysis. BrdU-containing DNA purified from a mixture of DNAs from labeled and unlabeled cultures showed >90-fold enrichment for the labeled bacterial taxon. The LH-PCR profile for BrdU-containing DNA from a labeled, natural microbial community differed from the profile for the community as a whole, demonstrating that BrdU was incorporated by a taxonomic subset of the community. Immunocytochemical detection of cells with BrdU-labeled DNA was accomplished by in situ probing with anti-BrdU monoclonal antibodies and Texas red-labeled secondary antibodies. Using this suite of techniques, microbial cells incorporating BrdU into their newly synthesized DNA can be quantified and the identities of these actively growing cells can be compared to the composition of the microbial community as a whole. Since not all strains tested could incorporate BrdU, these methods may be most useful when used to gain an understanding of the activities of specific species in the context of their microbial community.

Antibodies, Monoclonal↗