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PubMed1980

Production of interferon and other lymphokines during murine tumour growth. I. Lymphokines in cell--free fluid of rat Zajdela ascites hepatoma.

Cell-free ascites of rat Zajdela hepatoma was assayed for the presence of lymphokine-like factors. Macrophage migration inhibitory and microbial growth inhibitory cytokines were detected with peak activities at day 4-8 and 10-11 after tumour inoculation, respectively. Occasionally, skin reactive activity was found, whereas only borderline titres of an interferon-like substance were demonstrated. Preliminary studies indicated that both migration inhibitory and microbial growth inhibitory factors are proteins resembling the corresponding lymphocyte-derived lymphokines. The cellular site of formation of these factors remains to be determined.

Animals↗

PubMed1993

Efficacy of 1% sodium fluoride as a preservative in urine samples containing glucose and Candida albicans.

Whether urine samples used in forensic science DUI testing can be compromised by endogenous ethanol production is a recurrent and yet unresolved issue. This study first assessed unpreserved urine samples that were collected, processed, and analyzed repeatedly over 13 to 41 days using a standard gas chromatographic procedure for ethanol analysis. Despite extensive microbial growth, ethanol was not detected in any test sample. The extent of ethanol production in samples supplemented with glucose, Candida albicans, or both was determined to evaluate the potential for ethanol production in urine samples associated with pathological conditions such as urinary tract yeast infections and diabetes mellitus. Ethanol production under each of the above treatment conditions was assessed in the presence and absence of 1% sodium fluoride as a microbial suppressant. Mean ethanol concentrations were determined for unpreserved samples containing urine only (0.003 +/- 0.005 g%), urine plus yeast (0.006 +/- 0.009 g%) and urine plus glucose (0.067 +/- 0.070 g%). Unpreserved samples supplemented with both yeast and glucose attained mean ethanol concentrations of 0.164 +/- 0.057 g% (P < 0.01). Ethanol could not be detected in any corresponding duplicate samples, which were preserved with 1% sodium fluoride. A lack of ethanol production in any of the unpreserved urine samples indicates that false DUI convictions due to endogenous ethanol production are very unlikely. And while endogenous ethanol production is possible in the presence of both glucose and contaminating C. albicans, 1% sodium fluoride completely eliminated microbial fermentation.

Alcoholic Intoxication↗

PubMed1997

Nuclear magnetic resonance timecourse studies of glyphosate metabolism by microbial soil isolates.

1. Triple Resonance Isotope EDited nmr spectroscopy (TRIED) has been developed to detect and examine minute levels of glyphosate metabolites in microbial soil isolates. Using stable isotopic labelling (13C and 15N), TRIED allows the simultaneous detection of multiple metabolites in crude matrices at submicrogram levels. An improvement over earlier techniques where milligrams are needed, TRIED can detect 500 ng of triply labelled compound in a crude sample (1:14,000 mass ratio) in just hours. 2. TRIED is used here to compare the kinetics and metabolic pathways of glyphosate metabolism by two strains of Ochrobactrum anthropi, LBAA and S5. Both LBAA and S5 appear to metabolize glyphosate primarily via the aminomethylphosphonate (AMPA) pathway, since no detectable levels of glycine or sarcosine are observed in the media or lysates of either microbe. The formation of N-methylAMPA is common to the metabolism of both microorganisms, but N-acetylAMPA is observed only in LBAA. N-methylacetamide is detected predominantly in media and lysates of S5, although some evidence also points to the formation of this metabolite in LBAA. 3. Results are consistent with conventional radioactive tracer studies. TRIED nmr provides more specific structural information complementary to radiolabel methods. Both nmr and radioactivity studies show S5 glyphosate metabolism to be much slower than that of LBAA.

Animals↗

PubMed2001

Application of rRNA-targeted oligonucleotide probes in biotechnology.

Ribosomal RNA-targeted oligonucleotide probes have become valuable tools for the detection of microorganisms involved in important biotechnological processes. Microorganisms which are of major importance for processes such as wastewater treatment, microbial leaching or methane production can be detected and quantified in situ within a complex microbial community. For certain processes, such as nitrification or biological phosphate removal, new microorganisms have become the focus of interest and have led to an improved understanding of these bioremediation techniques. Hybridization techniques have become fast and reliable alternatives to conventional cultivation techniques in the food industry as a control method for starter cultures for fermentation processes or product control. Recent analytical tools such as flow cytometry and digital image processing have improved the efficiency of these techniques. This review is intended to present a summary of methodological aspects of rRNA-based hybridization techniques and their application in biotechnology.

Bioreactors↗

PubMed1994

Variability of polymerase chain reaction-based detection of human papillomavirus DNA is associated with the composition of vaginal microbial flora.

The results of repeated human papillomavirus (HPV) DNA testing were compared to changes in cervical pathology and the composition of vaginal microorganisms. A cohort of 19 women with HPV cervical infections in the absence of cervical intraepithelial neoplasia at enrollment was reexamined on average at 7.3-month intervals over a 2-year period. At each follow-up visit, cytological and colposcopic examinations were done and vaginal microorganisms were assessed quantitatively by Gram staining of secretions, and anaerobic and aerobic culture. HPV genotypes 6, 11, 16, and 18 were detected by polymerase chain reaction analysis using DNA isolated from exfoliated cervical cells. The detection of HPV DNA was significantly associated with carriage of Grade II flora (P < 0.001), isolation of Gardnerella vaginalis (P = 0.03), Ureaplasma urealyticum (P = 0.04), Candida albicans (P = 0.01), Bacteroides species (P = 0.01), and overgrowth by anaerobes (P = 0.004). Normal vaginal flora, characterised by the predominance of Lactobacillus species, was significantly associated (P < 0.001) with a negative HPV test. The detection of HPV DNA is associated with the composition of microorganisms present in the vagina at the time of testing.

Adult↗

PubMed1995

[Detection of enzyme activity in decontaminated spices in industrial use].

A range of decontaminated species of industrial use have been examined for their enzymes (catalase, peroxidase, amylase, lipase activity). The genuine enzymes remain fully active in irradiated spices, whereas the microbial load is clearly reduced. In contrast steam treated spices no longer demonstrate enzyme activities. Steam treatment offers e.g. black pepper without lipase activity, which can no longer cause fat deterioration. Low microbial load in combination with clearly detectable enzyme activity in spices is an indication for irradiation, whereas, reduced microbial contamination combined with enzyme inactivation indicate steam treatment of raw material.

Amylases↗

PubMed1993

Application of flow cytometry to rapid microbial analysis in food and drinks industries.

In food and drinks industries, the time required for conventional tests can lead to substantial delays in product release to the market. Flow cytometry (FCM) has been used in conjunction with viability markers for rapid counting of yeast, mould and bacterial cells in food products. A single-parameter flow cytometer has proved applicable to the rapid detection of low numbers of microbial contaminants in finished products. The excellent correlation between FCM results and product quality shelf-life expiry date has allowed the establishment of realistic quality control criteria for rapid positive release of product. Used for the monitoring of microbial biomass during manufacturing processes, flow cytometry allowed a direct assessment of bacterial growth. The reproducibility of the results and the proven correlation with standard plate count method obtained in industrial conditions make FCM a good predictive method for product and process quality control.

Bacteria↗

PubMed2005

[Improving microbiological diagnostics in septic orthopaedic surgery. Comparative study of patients receiving systemic antibiotic therapy].

MATERIAL AND METHODS: This prospective study included 63 patients with confirmed infections of soft tissue, joints, bones or implants. During 110 surgical interventions, 124 swab and deep tissue sample pairs were taken and analysed microbiologically using standard procedures. RESULTS: In 40 patients who had not received prior antibiotic treatment, 57 sample pairs (swabs/tissue) were taken. In 70%, growth of microorganisms could be observed in both swabs and tissue samples. Growth in tissue sample only was observed in 14% and in 14% no growth could be detected. In 67 sample pairs taken from 23 patients who had received systemic antimicrobial treatment prior to surgery, microbial growth in both specimens was detected in 40%. Growth in tissue sample only was observed in 22% and 34% of the samples remained without detectable growth. The overall sensitivity of the tissue samples (70%) was significantly higher than in swab samples (44%) for the pretreated group. CONCLUSION: The use of intraoperative tissue samples for microbiological diagnostics in septic orthopaedic surgery must be considered a "gold standard". The higher sensitivity of intraoperative tissue samples is particularly important in patients receiving systemic antibiotic therapy prior to surgical interventions.

Anti-Bacterial Agents↗

PubMed2003

The evolutionary genomics of pathogen recombination.

A pressing problem in studying the evolution of microbial pathogens is to determine the extent to which these genomes recombine. This information is essential for locating pathogenicity loci by using association studies or population genetic approaches. Recombination also complicates the use of phylogenetic approaches to estimate evolutionary parameters such as selection pressures. Reliable methods that detect and estimate the rate of recombination are, therefore, vital. This article reviews the approaches that are available for detecting and estimating recombination in microbial pathogens and how they can be used to understand pathogen evolution and to identify medically relevant loci.

Animals↗

PubMed1988

A medium for the detection of yeasts using a conductimetric method.

A new medium is described for the detection of yeasts using a Malthus microbial growth analyser which measures conductivity. This medium is superior to previous media devised for use in the Malthus, and allows the detection of a wide range of yeasts including those important in food spoilage. The new medium was inferior to existing media used to detect yeasts in the Bactometer 32 instrument which measures impedance.

Colony Count, Microbial↗

PubMed2004

Microbial community dynamics based on 16S rRNA gene profiles in a Pacific Northwest estuary and its tributaries.

We analyzed bacterioplankton community structure in Tillamook Bay, Oregon and its tributaries to evaluate phylogenetic variability and its relation to changes in environmental conditions along an estuarine gradient. Using eubacterial primers, we amplified 16S rRNA genes from environmental DNA and analyzed the PCR products by length heterogeneity polymerase chain reaction (LH-PCR), which discriminates products based on naturally occurring length differences. Analysis of LH-PCR profiles by multivariate ordination methods revealed differences in community composition along the estuarine gradient that were correlated with changes in environmental variables. Microbial community differences were also detected among different rivers. Using partial 16S rRNA sequences, we identified members of dominant or unique gene fragment size classes distributed along the estuarine gradient. Gammaproteobacteria and Betaproteobacteria and members of the Bacteroidetes dominated in freshwater samples, while Alphaproteobacteria, Cyanobacteria and chloroplast genes dominated in marine samples. Changes in the microbial communities correlated most strongly with salinity and dissolved silicon, but were also strongly correlated with precipitation. We also identified specific gene fragments that were correlated with inorganic nutrients. Our data suggest that there is a significant and predictable change in microbial species composition along an estuarine gradient, shifting from a more complex community structure in freshwater habitats to a community more typical of open ocean samples in the marine-influenced sites. We also demonstrate the resolution and power of LH-PCR and multivariate analyses to provide a rapid assessment of major community shifts, and show how these shifts correlate with environmental variables.

Bacteria↗

PubMed2005

Role of the microbiology laboratory in infectious disease surveillance, alert and response.

Surveillance is usually defined as the ongoing and systematic collection, analysis and interpretation of health data essential to the planning, implementation and evaluation of public health practice. During recent years, most of these programmes have been developed in the field of antimicrobial resistance and nosocomial infections, but efforts have also been made in other areas. Recent experiences of emerging microbial threats, including severe acute respiratory syndrome and new influenza variants affecting humans, the re-emergence of infectious disease problems and the possibility of bioterrorism have evidenced the need for implementation of infectious disease surveillance programmes. clinical microbiology laboratories play a pivotal role in these programmes. They have the first opportunity to detect these problems and should participate in the design of reporting strategies and dissemination of this information. Policies for the flow of data to national and international authorities should be established using passive surveillance strategies. However, active surveillance programmes taking advantage of new methodologies, including virtual tools and mathematical programs, should be the goal for early detection of unusual patterns of microbial pathogens, outbreaks and healthcare-associated infections. In addition, early implementation of response strategies should be designed and performed with the cooperation of microbiology laboratories, and intervention and response protocols should be defined with the participation of clinical microbiologists.

Communicable Disease Control↗

PubMed1987

Simple method of monitoring colonising microbial load in chronic bronchial sepsis: pilot comparison of reduction in colonising microbial load with antibiotics given intermittently and continuously.

Aerobic and anaerobic culture of sputum on selective bacteriological media, combined with a new method of plating and plate reading, permitted rapid identification and quantitation of three genera of bacteria commonly associated with chronic bronchial sepsis (Haemophilus spp, Pseudomonas aeruginosa, and Staphylococcus aureus) and avoided time consuming serial dilution of sputum and subculture of organisms. The accuracy of this new technique was assessed in patients with chronic bronchial sepsis and was used to detect changes in the colonising microbial load of Haemophilus spp and Ps aeruginosa in patients with bronchiectasis receiving one of three different antibiotic regimens: intermittent seven day courses of amoxycillin for exacerbations; or a six month course of continuous oral or nebulised amoxycillin. The colonising microbial load of Haemophilus spp was reduced only temporarily (+++ to ++) after each intermittent course of antibiotic, but a sustained and greater reduction in the colonising microbial load of both Haemophilus spp (+++ to +) and antibiotic resistant P aeruginosa (+++ to +) was seen during both continuous treatments. Sputum purulence decreased in parallel with colonising microbial load, reflecting a reduction in host inflammatory response to the colonising microbial load.

Amoxicillin↗

PubMed2003

Culture-independent molecular analysis of microbial constituents of the healthy human outer ear.

Molecular-phylogenetic sequence analyses have provided a new perspective on microbial communities by allowing the detection and identification of constituent microorganisms in the absence of cultivation. In this study we used broad-specificity amplification of ribosomal DNA (rDNA) genes to survey organisms present in the human outer ear canal. Samples were obtained from 24 individuals, including members of three extended families, in order to survey the resident microbiota and to examine microbial population structures in individuals related by familial or household associations. To examine the stability of the microbial populations, one individual was sampled four times and another twice over a 14-month period. We found that a distinct set of microbial types was present in the majority of the subjects sampled. The two most prevalent rDNA sequence types that were identified in multiple individuals corresponded closely to those of Alloiococcus otitis and Corynebacterium otitidis, commonly thought to be associated exclusively with infections of the middle ear. Our results suggest, therefore, that the outer ear canal may serve as a reservoir for normally commensal microbes that can contribute to pathogenesis upon introduction into the middle ear. Alternatively, culture analyses of diseases of the middle ear may have been confounded by these contaminating commensal organisms.

Bacteria↗

PubMed2002

Subgingival microbial profiles in refractory periodontal disease.

BACKGROUND/AIM: The purpose of the present investigation was to examine subgingival microbial profiles associated with refractory periodontitis and to seek such profiles in periodontally healthy, periodontally well-maintained elder and untreated periodontitis subjects. METHODS: 36 subjects were defined as refractory on the basis of further attachment loss after scaling and root planing, surgery and systemically administered antibiotics. A total of 890 subgingival plaque samples (mean/subject=24.7) were taken from the mesial aspect of each tooth in each subject at baseline and individually processed for their content of 40 subgingival taxa using checkerboard DNA-DNA hybridization. Cluster analysis was performed on mean within subject species counts using the chord coefficient and an average unweighted linkage sort. Significant differences among clusters for individual and complexes of species were sought using the Kruskal Wallis test. The microbial profiles of the refractory subjects were compared with those of 27 periodontally healthy subjects (n plaque samples=708), 35 periodontally well-maintained elder subjects (n plaque samples=801) and 115 untreated adult periodontitis subjects (n plaque samples=2871). RESULTS: 28 of 36 refractory subjects fell into 4 clusters with >29% similarity. 10 of 40 species and 4 of 7 complexes differed significantly among clusters. Profile (Cluster) I (n=4) was characterized by high proportions of "yellow" and "green" complex species, profile II (n=3) by low total counts and high proportions of "orange" and "purple" complex species, profile III (n=9) by high total counts and counts of Actinomyces and "purple" complex species, profile IV (n=12) by high proportions of "red" and "orange" complex species. The mean profiles of each cluster were subjected to cluster analysis with microbial data from 4380 (mean 24.7) baseline subgingival plaque samples from 27 periodontally healthy, 35 treated, well-maintained elders and 115 untreated adult periodontitis subjects. 12 clusters were formed with >41% similarity. 3 of the refractory profiles were detected in 3 cluster groups. Profile II in a cluster of 1 healthy, 1 elder and 4 untreated periodontitis subjects; profile III in a cluster of 1 healthy, 2 elder and 12 periodontitis subjects; Profile IV, with 1 healthy and 5 untreated periodontitis subjects. The profile not detected in non refractory subjects was dominated by Streptococcus species. 9 clusters did not harbor refractory profiles. 11.1% of healthy, 8.6% of elder and 18.3% of periodontitis subjects were in clusters exhibiting refractory microbial profiles. CONCLUSIONS: 4 subgingival microbial profiles were detected among refractory subjects. "Refractory microbial profiles" could be detected in subjects who had not yet exhibited refractory disease.

Actinobacillus↗

PubMed1998

[Analysis of variance of bacterial counts in milk. 1. Characterization of total variance and the components of variance random sampling error, methodologic error and variation between parallel errors during storage].

In contrast to the prevailing automatized chemical analytical methods, classical microbiological techniques are linked with considerable material- and human-dependent sources of errors. These effects must be objectively considered for assessing the reliability and representativeness of a test result. As an example for error analysis, the deviation of bacterial counts and the influence of the time of testing, bacterial species involved (total bacterial count, coliform count) and the detection method used (pour-/spread-plate) were determined in a repeated testing of parallel samples of pasteurized (stored for 8 days at 10 degrees C) and raw (stored for 3 days at 6 degrees C) milk. Separate characterization of deviation components, namely, unavoidable random sampling error as well as methodical error and variation between parallel samples, was made possible by means of a test design where variance analysis was applied. Based on the results of the study, the following conclusions can be drawn: 1. Immediately after filling, the total count deviation in milk mainly followed the POISSON-distribution model and allowed a reliable hygiene evaluation of lots even with few samples. Subsequently, regardless of the examination procedure used, the setting up of parallel dilution series can be disregarded. 2. With increasing storage period, bacterial multiplication especially of psychrotrophs leads to unpredictable changes in the bacterial profile and density. With the increase in errors between samples, it is common to find packages which have acceptable microbiological quality but are already spoiled by the time of the expiry date labeled. As a consequence, a uniform acceptance or rejection of the batch is seldom possible. 3. Because the contamination level of coliforms in certified raw milk mostly lies near the detection limit, coliform counts with high relative deviation are expected to be found in milk directly after filling. Since no bacterial multiplication takes place during storage, then error between samples always predominates the total variation. 4. The present results cannot be simply applied to other selective enumerations of microorganisms. Yet, a non-homogenous distribution should always be expected at microbial counts close to the detection limit. Technical errors arising from clustering as well as eugonic growth can additionally hamper the counting of colonies of such microorganisms. Effects of these observations in the decision-making process will be dealt with in the second communication.

Analysis of Variance↗

PubMed1996

Comparison of Isolator 1.5 and BACTEC NR660 aerobic 6A blood culture systems for detection of fungemia in children.

The Isolator 1.5 microbial system (ISO 1.5) (Wampole Laboratories, Cranbury, N.J.) was compared with the BACTEC NR660 aerobic NR6A bottle (NR6A) (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) for the detection of fungemia in hospitalized pediatric patients. For 4,825 paired blood cultures evaluated retrospectively from April 1992 to December 1994, at least one blood culture system was positive for 89 clinically important fungal isolates involved in 36 episodes of fungemia in 34 patients. Sixty isolates (44 Candida albicans, 12 Candida parapsilosis, and 4 Candida tropicalis isolates) were recovered from both systems, 13 were recovered from NR6A bottles only (10 C. albicans, 1 C. parapsilosis, and 2 Cryptococcus neoformans isolates), and 16 were recovered from ISO 1.5 tubes only (8 C. albicans and 5 C. parapsilosis isolates and 1 C. tropicalis, 1 Candida lusitaniae, and 1 Rhodotorula glutinis isolate) (P > 0.05). For the 60 Candida isolates from both systems, the mean time to detection was the same in each system. Thirty-seven isolates were detected by both systems on the same day, 9 isolates were detected earlier by NR6A, and 14 isolates were detected earlier by ISO 1.5 (P > 0.05). Of the 36 clinically important episodes of fungemia, 22 were detected by both systems (13 C. albicans isolates and 9 other Candida isolates), 4 were detected by NR6A only (3 C. albicans isolates and 1 C. neoformans isolate), and 10 were detected by ISO 1.5 only (3 C. albicans isolates, 6 other Candida isolates, and 1 R. glutinis isolate) (P > 0.05). Of the 22 episodes in which cultures from both systems were positive at some point during the episode, 12 were detected on the same day by both systems, 8 were detected earlier by NR6A, and 2 were detected earlier by ISO 1.5. Thus, for our pediatric population, NR6A is comparable to ISO 1.5 in both yield and time to detection of yeasts in fungemic patients.

Aerobiosis↗

PubMed2002

Influence of the pattern of peptide supply on microbial activity in the rumen simulating fermenter (RUSITEC).

The source and pattern of N supply was varied in the rumen simulation technique (RUSITEC) in order to determine if continuous, rather than transient, availability of peptides was required for optimum ruminal fermentation. The energy source was fibre prepared from sugar-beet pulp. N was added as NH3 continuously infused (AC) or peptides (Bacto(R) Casitone, a pancreatic hydrolysate of casein; Difco Laboratories, Detroit, MI, USA) continuously infused (PC) or added as a single dose at the time of feeding (PS). Free peptides were detected in the fermenter liquid for 4 h after feeding in the AC treatment, for 10 h in the PS treatment, and at all times with the PC treatment. Treatments had no effect on DM degradation. Approximately 40 % of the degradation occurred during the time no peptides were detected in the PS treatment. Microbial N flow tended to be higher with the peptide additions (P<0.061), with no significant difference between the two peptides treatments. The production of liquid-associated micro-organisms (LAM) was higher in the PC treatment (P<0.05) and the proportion of LAM derived from NH3 lower (P<0.05). However, LAM only accounted for 20-30 % total microbial population. Our main conclusion was that peptides had a small stimulatory effect on the fermentation, but there was no indication that synchrony of supply of energy and amino acid-N in the fermenter promoted a more efficient fermentation than non-synchronous supply. This conclusion must be qualified, however, because some N remained in the fibre and may have become available progressively as the fibre was digested by the micro-organisms.

Animal Nutritional Physiological Phenomena↗