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PubMed2006

Comparison of disruption procedures for enumeration of activated sludge floc bacteria by flow cytometry.

BACKGROUND: In a wastewater treatment plant, the degradation process is performed by a variable and mixed community of microorganisms in an aerobic aquatic environment. The activated-sludge process is based on the formation of strong microbial flocs where many bacteria are attached to sludge flocs. METHODS: Cytometric analysis requires an homogeneous cell suspension and so detachment of bacteria from flocs is required. In this study, sonication and homogenization were compared to find the most adequate pretreatment method for bacterial cytometric analysis in activated sludge samples. Bacterial viability was tested with a nucleic acid double-staining (NADS) protocol (Barbesti et al., Cytometry 2000;40:214-218) and on flow cytometry. RESULTS: Each method showed a good efficiency in terms of bacterial detachment; thus finally, the choice of which could be the best treatment method was based on both viability results and analysis rapidity. On the basis of the degree of cell detachment and viability, the maximum value was obtained by sonication (2 x 45''). CONCLUSIONS: The use of flow cytometry in conjunction with fluorescent dyes and an adequate pretreatment represents a useful method to rapidly detect and enumerate bacteria in activated sludge samples.

Bacteria↗
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↗
PubMed1993

Recent applications of flow cytometry in aquatic microbial ecology.

Microorganisms (unicellular algae, bacteria) constitute fundamental compartments of aquatic ecosystems because of their high concentrations and activities. The evaluation and understanding of their behavior and role raise different problems for which traditional methodologies are often inadequate, whether they refer to global or classical microscopic analyses. Flow cytometry (FCM) has been recently used to study microorganisms in aquatic environments. Although this technology is still applied on a limited scale in our field, a large number of works has been done showing that FCM seems to be a promising tool for aquatic microbial ecology. This paper summarizes, from the literature produced during the last decade and with original data obtained in our laboratory, the main questions related to the cell identification, the evaluation of cell viability, biomasses and productions and the measurements of bacterial and phytoplanktonic activities. The representatives of sampling and observation scales is also discussed within the framework of the FCM measurements.

Bacteria↗
PubMed1999

Application of laser scanning for the rapid and automated detection of bacteria in water samples.

It is widely accepted that the heterotrophic plate count method may not support the growth of all viable bacteria which may be present within a water sample and that alternative procedures using 'viability markers' may yield additional information. In this study, ChemChrome B (CB), which is converted to a fluorescent product by esterase activity, was used to stain viable bacteria (captured by membrane filtration) from potable water samples. The labelled bacteria from each sample were subsequently enumerated using a novel laser scanning instrument (ChemScan). Analysis of 107 potable water samples using this procedure demonstrated the presence of a significantly greater number of bacteria than were detected by culture (z-test, P < 0.05). The mean number of bacteria isolated by culture on R2A agar incubated at 22 degrees C for 7 d was only 25.2% of the total number of viable bacteria detected using the CB/ChemScan viability assay. Further analysis of 81 water samples using a 5-cyano-2,3,4-tolyl-tetrazolium chloride (CTC) viability assay also demonstrated the presence of many viable bacteria which were not capable of growth under the culture conditions employed in this study. However, the results indicate that ChemChrome B has the ability to stain a significantly greater number of heterotrophs than CTC (z-test, P < 0.05). In contrast, six potable waters were identified in which the CTC viability assay resulted in counts greater than those obtained using CB. The ChemScan instrument was successfully used for rapid and accurate enumeration of labelled micro-organisms, allowing information on the total viable microbial load of a water sample to be determined within 1 h. Furthermore, the ChemScan system has the potential for use in detecting specific organisms labelled with fluorescently-labelled antibodies or nucleic acid probes.

Bacteria↗
PubMed2001

Menadione-catalyzed luminol chemiluminescent assay for the viability of Escherichia coli ATCC 25922.

Escherichia coli ATCC 25922 produced O2- in the presence of menadione, and O2- -dependent luminol chemiluminescence intensity was proportional to colony-forming unit (CFU) in the exponential phase. CFU was determined by using a 96-well plate at a range of 3 X 10(3) to 8 x 10(7) CFU /well (0.1 ml) after a 10-min incubation with menadione, followed by chemiluminescent assay for 5 s. After a 4-hr incubation of E. coli (10(5) CFU/0.1 ml) with menadione and an antimicrobial agent inhibiting the synthesis of peptidoglycan, protein, and DNA, the inhibitory concentration (IC) of the antimicrobial agent determined by menadione-catalyzed luminol chemiluminescent assay was in good agreement with minimal inhibitory concentration (MIC) of the NCCLS (National Committee for Clinical Laboratory Standard) method requiring 18 hr. Menadione-catalyzed luminol chemiluminescent assay is expected to be useful for the rapid determination of cell viability under the conditions of various cell growths and stresses.

Amikacin↗
PubMed1987

The long-term survival of Escherichia coli in river water.

Escherichia coli introduced into autoclaved filtered river water survived for up to 260 d at temperatures from 4 degrees to 25 degrees C with no loss of viability. Survival times were less in water which was only filtered through either a Whatman filter paper or a 0.45 micron Millipore filter or in untreated water, suggesting that competition with the natural microbial flora of the water was the primary factor in the disappearance of the introduced bacteria. Survival was also dependent upon temperature with survival at 4 degrees C greater than 15 degrees C greater than 25 degrees C greater than 37 degrees C for any water sample. Direct counts showed that bacterial cells did not disappear as the viable count decreased. The possession of the antibiotic resistance plasmids, R1drd-19 or R144-3, did not enhance survival nor cause a faster rate of decay, indicating that the metabolic burden imposed by a plasmid was not a factor in survival under starvation conditions. There was no evidence of transfer of either plasmid at 15 degrees C or of loss of plasmid function during starvation.

Escherichia coli↗
PubMed1993

Interactions of dirithromycin with human polymorphonuclear leukocytes.

Dirithromycin, a new macrolide antibiotic, achieves prolonged, high levels in tissue. We previously demonstrated that certain macrolides are highly concentrated within phagocytic cells. This background information prompted us to evaluate the interactions of dirithromycin and human polymorphonuclear leukocytes (PMNs). After incubation with radiolabeled dirithromycin, antibiotic uptake by PMNs was determined by a velocity-gradient centrifugation technique and was expressed as the ratio of the cellular to the extracellular drug concentration (C/E). Dirithromycin was avidly accumulated by PMNs (C/E, 5 at 15 min, 10 at 30 min, 19 at 1 h, and 35 at 2 h). Uptake was dependent on cell viability, physiologic environmental temperature, and pH (optimum 8.6), but was not influenced by potential competitive inhibitors of membrane transport. Incubation with sodium cyanide caused an increase in dirithromycin accumulation by PMNs. Ingestion of microbial particles (mimicking in vivo infection) modestly inhibited the entry of dirithromycin into PMNs. After removal of extracellular drug, the efflux (release) of dirithromycin from PMNs was slow; only 10% was released within the first 30 min. This prolonged retention of dirithromycin within phagocytic cells might allow delivery and release of accumulated drug at sites of infection. The impact of intraphagocytic dirithromycin on cellular function was also evaluated. In a manner similar to that of other highly concentrated, weakly basic antibiotics, dirithromycin inhibited the respiratory burst response (superoxide production) in stimulated PMNs. The presence of dirithromycin slightly increased the intraphagocytic killing of Staphylococcus aureus in human PMNs. These interactions of dirithromycin with phagocytic cells may promote the extraphagocytic, and possibly the intraphagocytic, killing of infecting organisms.

Anti-Bacterial Agents↗
PubMed1996

Growth inhibition of metronidazole-susceptible and metronidazole-resistant strains of Gardnerella vaginalis by Lactobacilli in vitro.

Metronidazole resistance was produced in susceptible Gardnerella vaginalis after subculture in the presence of metronidazole. Metronidazole-resistant gardnerellae were less susceptible to growth inhibition by Lactobacillus culture filtrates. A low pH (+/- 4) and lactic acid accounted for 60 to 95% of inhibitory activity, and H2O2 accounted for only 0 to 30%. However, in the presence of myeloperoxidase, H2O2-producing lactobacilli decreased the viability of metronidazole-susceptible gardnerellae 2,000-fold.

Anti-Bacterial Agents↗
PubMed1971

Involvement of recombination genes in growth and viability of Escherichia coli K-12.

We have studied the growth properties of 17 isogenic strains of Escherichia coli K-12 differing only in the recA, recB, recC, and sbcA alleles. We have observed the following. (i) All recombination deficient strains have decreased growth rates and decreased viabilities compared with recombination proficient strains. The large populations of nonviable cells in Rec(-) cultures may arise by spontaneous lethal sectoring (9). (ii) A recA mutant strain which is entirely recombination deficient and which shows high ultraviolet sensitivity and "reckless" deoxyribonucleic acid (DNA) breakdown has approximately the same growth rate and twice the viability as recB and recC mutant strains which have residual recombination proficiency, moderate ultraviolet sensitivity, and "cautious" DNA breakdown. (iii) Indirectly suppressed (sbcA(-)) recombination proficient (Rec(+)) revertants of recB and recC mutant strains have approximately normal growth rates and are three times as viable as their Rec(-) ancestors (but not as viable as rec(+) cells). We suggest the following hypothesis to account for the low viability of Rec(-)E. coli. Single-strand breaks in the DNA duplex, necessary for normal bacterial growth, may be repaired in a Rec(+) cell. Failure of Rec(-) cells to repair this normal DNA damage may lead to the observed loss of viability.

Bacterial Proteins↗
PubMed1987

[The role of R plasmids in the resistance of bacteria in air].

The study of Escherichia coli J 53, used as a model, has revealed that some R plasmids isolated from Serratia marcescens and Klebsiella pneumoniae, found to be the cause of the outbreak of hospital infection, ensure, besides multiple drug resistance, also their viability in the air.

Air Microbiology↗
PubMed1980

[Influence of the autolytic system on the bactericidal effect induced by beta-lactamic antibiotics (author's transl)].

New experimental conditions showing the phenomena of "antibiotic tolerance" are described. In Streptococcus pneumoniae, a strong protection against the loss of viability induced by penicillin was obtained when TRIS (hydroxymethyl aminomethane), instead of potassium phosphate, was used as buffer of the growth medium. The main pneumococcal autolysin (N-acetyl-muramil amidase) present in S. pneumoniae was found to be, at least partially, responsible for the loss of viability induced by beta-lactamic molecules.

Anti-Bacterial Agents↗
PubMed1996

Effect of nitric oxide on staphylococcal killing and interactive effect with superoxide.

The role of reactive nitrogen intermediates (RNI) such as nitric oxide (.NO) in host defense against pyogenic microorganisms is unclear, and the actual interactive effect of RNI and reactive oxidative intermediates (ROI) for microbial killing has not been determined. Since, in nature, ROI and RNI might be generated together within any local infection, we evaluated the separate and interactive effects of .NO and O2- on staphylococcal survival by using a simplified system devoid of eukaryotic cells. These studies showed that prolonged exposure of staphylococci to .NO does not result in early loss of viability but instead is associated with a dose-related delayed loss of viability. This effect is abrogated by the presence of hemoglobin, providing further evidence that the effect is RNI associated. Superoxide-mediated killing also is dose related, but in contrast to RNI-mediated killing, it is rapid and occurs within 2 h of exposure. We further show that the interaction of .NO and O(2)- results in decreased O(2)--mediated staphylococcal killing at early time points. .NO, however, appears to enhance or stabilize microbial killing over prolonged periods of incubation. This study did not produce evidence of early synergism of ROI and RNI, but it does suggest that .NO may contribute to host defense, especially when ROI-mediated killing is compromised.

Dose-Response Relationship, Drug↗
PubMed2006

Antimicrobial activities of silver used as a polymerization catalyst for a wound-healing matrix.

Wound healing is a complex and orchestrated process that re-establishes the barrier and other functions of the skin. While wound healing proceeds apace in healthy individual, bacterial overgrowth and infection disrupts this process with significant morbidity and mortality. As such, any artificial matrix to promote wound healing must also control infecting microbes. We had earlier developed a two-part space-conforming gel backbone based on polyethyleneglycol (PEG) or lactose, which used ionic silver as the catalyst for gelation. As silver is widely used as an in vitro antimicrobial, use of silver as a catalyst for gelation provided the opportunity to assess its function as an anti-microbial agent in the gels. We found that these gels show bacteriostatic and bactericidal activity for a range of Gram-negative and Gram-positive organisms, including aerobic as well as anaerobic bacteria. This activity lasted for days, as silver leached out of the formed gels over a day in the manner of second-order decay. Importantly the gels did not limit either cell growth or viability, though cell migration was affected. Adding collagen I fragments to the gels corrected this effect on cell migration. We also found that the PEG gel did not interfere with hemostasis. These observations provide the basis for use of the gel backbones for incorporation of anesthetic agents and factors that promote wound repair. In conclusion, silver ions can serve dual functions of catalyzing gelation and providing anti-microbial properties to a biocompatible polymer.

Anti-Bacterial Agents↗