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PubMed2006

Comparison of most probable number-PCR and most probable number-foci detection method for quantifying infectious Cryptosporidium parvum oocysts in environmental samples.

Microbial contamination of public water supplies is of significant concern, as numerous outbreaks, including Cryptosporidium, have been reported worldwide. Detection and enumeration of Cryptosporidium parvum oocysts in water supplies is important for the prevention of future cryptosporidiosis outbreaks. In addition to not identifying the oocyst species, the U.S. EPA Method 1622 does not provide information on oocyst viability or infectivity. As such, current detection strategies have been coupled with in vitro culture methods to assess oocyst infectivity. In this study, a most probable number (MPN) method was coupled with PCR (MPN-PCR) to quantify the number of infectious oocysts recovered from seeded raw water concentrates. The frequency of positive MPN-PCR results decreased as the oocyst numbers decreased. Similar results were observed when MPN was coupled to the foci detection method (MPN-FDM), which was done for comparison. For both methods, infectious oocysts were not detected below 10(3) seeded oocysts and the MPN-PCR and MPN-FDM estimates for each seed dose were generally within one-log unit of directly enumerated foci of infection. MPN-PCR estimates were 0.25, 0.54, 0 and 0.66 log(10) units higher than MPN-FDM estimates for the positive control, 10(5), 10(4) and 10(3) seed doses, respectively. The results show the MPN-PCR was the better method for the detection of infectious C. parvum oocysts in environmental water samples.

Animals↗
PubMed1980

[Blood cultures. Comparative study with 7 known and 2 new nutrient media for the detection of aerobic and anaerobic microorganisms (author's transl)].

The growth promoting properties of seven commercially manufactured and two recently developed culture media are compared according to a standardized method. The study is carried out alternately without and with the addition of 10% fresh human blood to the culture media. The carefully selected test strains include 20 species of bacteria and 2 species of yeasts which represent obligatory aerobic, facultative anaerobic and obligatory anaerobic microorganisms with quite different nutritional and atmospherical requirements. The 9 tested media are good enough for the purpose of culturing nonfastidious bacteria. However, most of the commercially prepared media failed to detect small inocula of very fastidious microbial agents, especially when no blood is added. Collectively, thiol broth is the most noneffective medium. The last but one is thioglycollate medium. Three culture media based on brain heart infusion formula prove to be effective. Those are the commercial brain heart dextrose and the two media recently developed by the authors, namely brain heart dipeptone (BHD) and brain heart dipeptone cysteine (BHDC). BHD is the most suitable medium for the detection of obligatory aerobic and facultative anaerobic fastidious microorganisms. BHDC detects anaerobic fastidious bacteria quite effectively. The other media, namely Columbia, trypticase soy, trypticase soy sucrose, and Rosenow are of limited value with regard to the detection of small inocula of fastidious microorganisms. The causes of the unsatisfactory results with different commercial media are discussed in detail. The authors point out to the possible use of hypertonic media in special cases. Properties that should be fulfilled by blood culture media are proposed.

Anaerobiosis↗
PubMed1968

Bioautographic technique for a rapid survey of microbial populations for the production of antibiotics and other metabolites.

A method is described for the detection of microorganisms capable of producing antibiotics or other metabolites. The microbial population under study was bioautographed against any desired indicator organism. This was easily accomplished by lifting the entire agar layer out of a petri dish on which the microbial population was grown and transferring it to an agar surface seeded with an indicator organism. The use of this technique in the search for metabolites other than antibiotics is pointed out. The advantages of the method, over previously reported methods, are discussed.

Anti-Bacterial Agents↗
PubMed2026

Metagenomic profiling of blood-associated microbial DNA signatures in leukemia-associated febrile neutropenia.

Febrile neutropenia (FN) is a life-threatening complication of chemotherapy, but the low microbial biomass of blood makes shotgun metagenomic profiles highly sensitive to technical background. We reanalyzed 47 publicly available patient sequencing runs representing 43 unique patient-timepoint samples from 19 SRA-labeled patients, together with 23 no-template-control (NTC) runs spanning 21 sequencing batches. To distinguish reference-catalogue content from progressively stronger evidence of patient-associated signal, we applied batch-matched NTC correction together with nested abundance thresholds and a feature-specific global NTC envelope. CheckM2 evaluated 1,013 bins; 13 met completeness &#x2265;50% and contamination <10%, and dereplication yielded 11 draft MAG representatives. Ten representatives showed positive patient-to-control abundance excess, but only four showed recurrent support above both threefold matched-control abundance and the global NTC envelope. Functional annotations were therefore interpreted as reference-genome homologs rather than evidence of expression, phenotype, viability or bloodstream origin. Matched-control correction retained 19 read-level ARG types, but only seven subjects contributed complete longitudinal ARG-profile contrasts, limiting reliable temporal inference. The resulting run-resolved, nested evidence framework identified a subset of microbial DNA and ARG signals that remained detectable under increasingly stringent control criteria while distinguishing them from catalogue-level or background-sensitive signals. These findings support cautious reporting of patient-enriched microbial DNA and ARG signals rather than inference of a resident blood microbiome or clinical resistance phenotype.

antimicrobial resistance genes↗
PubMed2000

Long-term treatment with subantimicrobial dose doxycycline exerts no antibacterial effect on the subgingival microflora associated with adult periodontitis.

BACKGROUND: The purpose of this study was to determine whether treatment with subantimicrobial dose doxycycline (SDD), 20 mg bid, exerted an antimicrobial effect on the microflora associated with adult periodontitis. METHODS: Following the approval of the protocol and informed consent forms by the respective IRBs at the University of Florida and West Virginia University, 76 subjects with adult periodontitis were entered and randomly assigned to receive SDD or placebo. A split-mouth design was utilized, with each subject receiving subgingival scaling and root planing (SRP) in two quadrants immediately following baseline data collection, while the remaining two quadrants were left unscaled (non-SRP). Microbial samples were collected prior to treatment, after 3, 6, and 9 months of treatment, and after 3 months of no treatment. The samples were examined by microscopy and by enumeration on selective and non-selective media. RESULTS: All treatments resulted in statistically significant decreases in the proportions of spirochetes and motile rods (P <0.05) and in an increase in the proportion of coccoid forms (P <0.0001) relative to baseline. No between-treatment differences were detected between the SDD and placebo treatments in either the SRP or non-SRP design, with the exception of the small and large spirochetal groups. The spirochetal proportions present in the SDD group were significantly lower (P<0.05) than the paired placebo group during the 9-month treatment and was preceded by a significant decrease (P<0.01) in the proportion of microbiologic sample sites that bled on probing. No between- treatment differences were detected in any of the other microbial parameters. CONCLUSION: The microbial differences observed were attributed to the anticollagenase and anti-inflammatory properties of SDD and not to an antimicrobial effect.

Adult↗
PubMed1990

Analysis of positive cultures from endodontically treated teeth: a retrospective study.

A study of 12,150 endodontically treated teeth from 11,797 patients in a private patient setup revealed an incidence of 10.2% positive cultures obtained just before obturation of the root canal. Streptococcal predominance was reflected by a presence of nearly 60% in the pure culture and nearly 98% in the mixed cultures. No significant changes in microbial predominance or bacterial sensitivities were detected over the 7-year period of study. Ampicillin, cephalothin, erythromycin, and penicillin were the most effective antibiotics against the viridans streptococci, the most prevalent organism in the infected teeth.

Adolescent↗
PubMed1998

Biochemical and pathogenic properties of Shewanella alga and Shewanella putrefaciens.

We characterized 49 strains of Shewanella spp. from clinical (n = 31) and nonhuman (n = 18) sources. Most Shewanella alga organisms (Gilardi biovar 2; Centers for Disease Control and Prevention [CDC] biotype 2) originated from clinical material (92%), failed to produce acid from carbohydrates other than D-ribose, and were biochemically and enzymatically fairly homogeneous. In contrast, Shewanella putrefaciens organisms (Gilardi biovars 1 and 3; CDC biotype 1) were more often associated with nonhuman sources (70%), were able to utilize a number of sugars (sucrose, L-arabinose, and maltose), and were found to exhibit wider variations in biochemical characteristics; three biotypes within S. putrefaciens were detected. Notable differences between the two species in enzymatic activity, determined with the API-ZYM system (bioMérieux, Hazelwood, Mo.), and cellular fatty acid profiles, determined by the MIDI system (Microbial ID Inc., Newark, Del.), were also detected. Pathogenicity studies of mice indicate that S. alga appears to be the more virulent species, possibly due to the production of a hemolytic substance.

Animals↗
PubMed2002

Development and application of new nucleic acid-based technologies for microbial community analyses in foods.

Several challenges still persist in the analysis of microorganisms in foods, particularly in studies of complex communities. Nucleic acid-based methods are promising tools in addressing new questions concerning microbial communities. We have developed several new methods in the field of nucleic acid-based microbial community analyses. These methods cover both sample preparation and detection approaches. The sample preparation method involves simplified DNA purification using paramagnetic beads. As an extension of this method, the same paramagnetic beads are used for both cell separation and DNA purification. This enables full automation. The separate detection of viable and dead bacteria is a major issue in nucleic acid-based diagnostics. We have applied a living/dead dye that binds covalently to DNA and inhibits the PCR from dead cells. In addition, a DNA array-based detection assay has been developed. The assay combines the specificity obtained by enzymatic labeling of DNA probes with the possibility of detecting several targets simultaneously by DNA array hybridization. In combination with 16S rDNA amplification, this is a promising tool for community analyses. Also, we have developed a novel approach for multiplex quantitative PCR. The multiplex PCR has been combined with our DNA array-based detection method. Finally, we are now in the process of adapting a system for monitoring microbial growth and death in real-time through the tagging of bacteria with green fluorescent protein (GFP) combined with fluorescence detection using a high-resolution confocal laser scanner.

Bacteria↗
PubMed2005

[Molecular analysis of the microbial communities of the Dagang Kongdian flooding bed oilfield].

Both PCR-TGGE (temperature gradient gel electrophoresis, TGGE) and 16S rRNA gene clone library construction were used to comparatively analyze the microbial communities of a water injection well (WW) and an oil well (OW) in Dagang oilfield. TGGE analysis of the PCR amplified 16S rDNA V3 region products showed great difference between these two microbial communities. Six major bands were detected in the TGGE profile of the WW sample, while only one predominant band in the OW sample was found. Two 16S rRNA gene clone libraries were also constructed, and 108 and 50 clones were selected from the WW and OW library respectively for amplified ribosomal DNA restriction analysis (ARDRA). 33 taxanomic operational units (OTUs) were found in the WW library with 6 major OTUs, while only 8 OTUs were found in the OW library with one OTU predominant. The results of TGGE and clone library profiling analysis both indicated that microbial community of the WW had higher diversity than the OW. Sequence analysis of the representative clone of each OTU showed that most bacteria of the WW were affiliated with alpha, beta, and gamma Proteobacteria and Actinobacteria, especially Rhodobacter (47%). Most bacteria of the OW were affiliated with alpha, beta, and gamma Proteobacteria, especially Pseudomonas (62%). Molecular analysis of the microbial diversity in oilfield provides foundation for better application of MEOR (Microbial Enhanced Oil Recovery).

Actinobacteria↗
PubMed2005

Diagnostic system for rapid and sensitive differential detection of pathogens.

Naturally emerging and deliberately released pathogens demand new detection strategies to allow early recognition and containment. We describe a diagnostic system for rapid, sensitive, multiplex discrimination of microbial gene sequences and report its application for detecting 22 respiratory pathogens in clinical samples.

Bacterial Infections↗
PubMed2004

Use of RAPD-PCR and TTGE for the evaluation of biodiversity of whey cultures for Grana Padano cheese.

AIMS: This work was carried out in order to evaluate the microbial diversity of whey cultures collected from different Grana Padano cheese plants in Veneto region (north-east Italy) by means of RAPD-PCR and Temporal Temperature Gradient Gel Electrophoresis (TTGE) analysis. METHODS AND RESULTS: Lactobacillus helveticus was the dominant species among isolated thermophilic lactobacilli. RAPD-PCR with primers M13 and D8635 resulted a suitable method for typing Lact. helveticus at strain level. Thirteen different Lact. helveticus biotypes were detected in the seven whey cultures studied with one biotype present in all the whey cultures. Besides Lact. helveticus, Lact. delbrueckii subsp. lactis was the main microbial species detected by TTGE. CONCLUSIONS: RAPD-PCR resulted very useful in studying Lact. helveticus biodiversity; furthermore, TTGE analysis allowed to detect the dominant thermophilic microflora characteristic of Grana Padano cheese whey cultures. IMPACT OF THE STUDY: By the combined used of RAPD-PCR and TTGE it could be possible to follow the behaviour in strain or species composition of whey cultures during time.

Bacterial Typing Techniques↗
PubMed1995

Qualitative and quantitative characterization of spoilage bacteria from packed fish.

The large cells recently suggested to be responsible for spoilage of packed cod, have been identified as Photobacterium phosphoreum. The spoilage activity of these cells, of Shewanella putrefaciens and of other microorganisms isolated form spoiled packed cod has been studied. Both qualitative and quantitative tests were used for characterization of the microbial spoilage activity. The importance of the different groups of microorganisms was evaluated by comparison of microbial spoilage activity determined in model substrates and in product experiments. The yield factor for production of trimethylamine (YTMA/CFU) and the cell concentration determined at the time of off-odour detection were used as quantitative measurements of microbial spoilage activity. On average cells of P. phosphoreum produced 30 times more TMA than cells of S. putrefaciens, YTMA/CFU of the two organisms were 10(-8.0) mg-N TMA/cfu and 10(-9.5) mg-N TMA/cfu, respectively. With these yield factors the level of TMA found in spoiled packed cod (30 mg-N TMA/100g) corresponds to about 10(7) cfu/g of P. phosphoreum and to 10(8)-10(9) cfu/g of S. putrefaciens. 10(7) cfu/g of P. phosphoreum were actually found in spoiled packed cod suggesting this organism could be responsible for spoilage. High cell concentrations of more than 10(8) cfu/g of S. putrefaciens were required for production of detectable off-odours and is was concluded that this organism is without importance for spoilage of packed cod.

Animals↗
PubMed2006

Matrix-suppressed laser desorption/ionisation mass spectrometry and its suitability for metabolome analyses.

Matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry was investigated for the simultaneous detection of several metabolites, as applicable to global metabolite analysis (metabolomics). The commonly employed organic matrices alpha-cyano-4-hydroxycinnamic acid and 3,5-dihydroxybenzoic acid, in both the crystalline and ionic liquid forms, were investigated. The employment of a low matrix-to-analyte molar ratio suppressed matrix peaks and was effective in detecting all the metabolites with a unique mass in a 30-metabolite synthetic cocktail, albeit to varying degrees. These matrix-suppressed laser desorption/ionisation (MSLDI) analyses were performed in the positive ion mode, and metabolites were detected as the protonated [M+H]+, sodiated [M+Na]+ or potassiated [M+K]+ species. The spectral signals were dominated by basic metabolites. It was possible to detect components of a synthetic cocktail when it was spiked quantitatively into a microbial extract, demonstrating the feasibility of using the technique for detecting metabolite signals in a complex biological matrix. However, analyte suppression effects were noted when the relative proportion of one analyte was allowed to increasingly dominate the others in a mixture. The implications of the findings with respect to applications in metabolomic investigations are discussed.

Amino Acids↗
PubMed2000

Survival and filamentation of Salmonella enterica serovar enteritidis PT4 and Salmonella enterica serovar typhimurium DT104 at low water activity.

In this study we investigated the long-term survival of and morphological changes in Salmonella strains at low water activity (a(w)). Salmonella enterica serovar Enteritidis PT4 and Salmonella enterica serovar Typhimurium DT104 survived at low a(w) for long periods, but minimum humectant concentrations of 8% NaCl (a(w), 0. 95), 96% sucrose (a(w), 0.94), and 32% glycerol (a(w), 0.92) were bactericidal under most conditions. Salmonella rpoS mutants were usually more sensitive to bactericidal levels of NaCl, sucrose, and glycerol. At a lethal a(w), incubation at 37 degrees C resulted in more rapid loss of viability than incubation at 21 degrees C. At a(w) values of 0.93 to 0.98, strains of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium formed filaments, some of which were at least 200 microm long. Filamentation was independent of rpoS expression. When the preparations were returned to high-a(w) conditions, the filaments formed septa, and division was complete within approximately 2 to 3 h. The variable survival of Salmonella strains at low a(w) highlights the importance of strain choice when researchers produce modelling data to simulate worst-case scenarios or conduct risk assessments based on laboratory data. The continued increase in Salmonella biomass at low a(w) (without a concomitant increase in microbial count) would not have been detected by traditional microbiological enumeration tests if the tests had been performed immediately after low-a(w) storage. If Salmonella strains form filaments in food products that have low a(w) values (0.92 to 0.98), there are significant implications for public health and for designing methods for microbiological monitoring.

Animals↗
PubMed1996

Application of 16S rRNA gene PCR to study bowel flora of preterm infants with and without necrotizing enterocolitis.

The purpose of the present study was to determine the extent to which bacteria not detected by culture contribute to the microbial flora of the bowel of preterm infants with and without neonatal necrotizing enterocolitis (NEC). Fecal samples from 32 preterm infants in special care baby units including samples from 10 infants with NEC were examined by culture and PCR amplification of the 16S rRNA gene (rDNA). The 16S rDNA V3 region was amplified with eubacterial primers, and the amplification products derived from the fecal sample DNA were compared with the products from individual cultured isolates by PCR and denaturing gradient gel electrophoresis (PCR-DGGE), allowing the DNA from uncultured bacteria to be identified. For the 22 infants without NEC weekly samples were examined for a mean of 5.3 postnatal weeks. The total number of types detected by culture combined with PCR-DGGE was 10.1 per infant, of which PCR-DGGE contributed 10.4% of the types identified. Additional types detected by PCR-DGGE were found in 14 (63.6%) of the 22 infants. The majority of the sequences associated with uncultured bacteria showed > 90% 16S rDNA sequence identity with sequences from culturable human enteric flora, and all were found in single infants with the exception of sequences indistinguishable by DGGE from seven infants. These sequences showed > 90% sequence identity with the 16S rDNA of Streptococcus salivarius and may have been derived from upper gastrointestinal or respiratory tract flora. In the present study uncultured bacteria detected by PCR-DGGE were no more frequent in fecal samples from infants with NEC than in samples from infants without NEC, although these findings do not exclude the possibility of unrecognized bacteria associated with the mucosa of the small intestine of infants with NEC.

Bacteria↗
PubMed1996

Microbial removal of chlorinated phenols during aerobic treatment of effluents from radiata pine kraft pulps bleached with chlorine-based chemicals, with or without hemicellulases.

The removal of chlorophenolic compounds from kraft mill effluents bleached with chlorine (cBKME) or chlorine plus hemicellulases (bBKME) was studied in reactors of aerobic treatment lagoons. In these laboratory models, a stable microbial population removed biochemical oxygen demand at similar rates of the mill lagoon. Complete removal of nine chlorophenols and chloroguaiacols during microbial treatment of these effluents was detected by gas chromatography. Abiotic removal was only observed with 2,4-dichlorophenol and 2,4,5-trichlorophenol. There were no significant differences in degradative ability between microorganisms acclimated to grow in reactors fed with cBKME or bBKME. The latter had a lower content of adsorbable organic halogen and chlorophenols than cBKME. Microorganisms acclimated to cBKME or bBKME were only able to grow on phenol or guaiacol as sole carbon source. However, these microorganisms removed (0.1-0.5 mM) 4-chlorophenol, 2,4-dichlorophenol and 2,4-dichlorophenoxyacetate with BKME as primary carbon source. Under these conditions, 2,4,6- and 2,4,5-trichlorophenol, 4,5-dichloroguaiacol, 4,5,6-trichloroguaiacol and tetrachloroguaiacol were not removed. These results suggest that the microbial removal of bleaching chlorophenols and chloroguaiacols during aerobic treatment, probably takes place only because of their very low concentration (1-200 ppb) in BKME.

Bacteria, Aerobic↗
PubMed2005

Construction of Saccharomyces cerevisiae strain FAV20 useful in detection of immunosuppressants produced by soil actinomycetes.

The screening of microbial natural products continues to represent an important route to the discovery of novel chemicals for development of new therapeutic agents. The aim of this work was to develop an efficient method for the detection of immunosuppressive compounds produced by soil actinomycetes. Mutant strain of Saccharomyces cerevisiae, named FAV20, sensitive to FK506 was constructed by disrupting VMA22 gene using the selectable marker kanMX4 which allowed detection of integration events. Actinomycetes were isolated from different soil samples and in a newly developed test with S. cerevisiae FAV20, six strains have been identified that produce bioactive compounds with the same mechanism of action as FK506. S. cerevisiae FAV20 can be easily used as a test strain in drug screening programs based on inhibition of the calcineurin phosphatase dependent signaling pathway in the cell.

Actinobacteria↗