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PubMed1989

Inhibition of human hemopoiesis by non-A, non-B hepatitis virus.

All etiologies of acute viral hepatitis are associated with a transient suppression of hemopoiesis and, rarely, with the development of aplastic anemia. Both hepatitis A and hepatitis B viruses directly inhibit the growth and differentiation of human bone marrow progenitor cells in vitro. We now report a similar effect of a non-A, non-B (NANB) hepatitis agent on human bone marrow progenitor cells. Three chimpanzees were inoculated with a putative NANB agent. Coded sera were blindly evaluated for their ability to affect human bone marrow colony formation in vitro. Sera obtained during the acute phase of NANB hepatitis inhibited the in vitro growth of human erythroid (CFU-E, BFU-E) and granulocyte-macrophage (CFU-GM) progenitor cells, compared with sera obtained before inoculation. Sera obtained after remission of both the biochemical and histological hepatitis and sera obtained from a chimpanzee who underwent biochemical but not histological remission did not inhibit the stem cell assays as much as the acute phase sera. These results suggest an approach to identifying the viremic phase of NANB hepatitis. Inhibition of human bone marrow proliferation appears to be a common property of all known hepatitis viruses.

Animals↗
PubMed1996

Influence of oropharyngeal flora and specimen pretreatment on the recovery of Helicobacter pylori.

The recovery of Helicobacter pylori was studied in vitro in the presence of different concentrations of oropharyngeal flora. The effect on the bacterial yield of Helicobacter pylori and oropharyngeal flora of washing biopsies once, twice or thrice before culture was then studied in biopsies taken from 35 patients. A low rate of recovery of low Helicobacter pylori concentrations (< or = 10(3) cfu/ml) was related to the presence of relatively high oropharyngeal flora concentrations (> or = 10(4) cfu/ml). After three washings a decrease in oropharyngeal flora between 10-fold and 100-fold cfu/biopsy was observed in seven patient samples. In another three specimens a more than 100-fold reduction in cfu/biopsy was observed. In the case of Helicobacter pylori, a 60-fold decrease in log cfu/biopsy was observed in only one patient. Pretreatment of the specimens by washing of the biopsies improved the recovery of small amounts of Helicobacter pylori in 10 of 28 patients. For optimal detection of Helicobacter pylori in gastric mucosal biopsies, washing of the specimens in addition to the use of selective media is recommended.

Biopsy↗
PubMed2000

Haemopoiesis in mice genetically lacking granulocyte-macrophage colony stimulating factor during chronic infection with Mycobacterium avium.

In order to test the role of granulocyte-macrophage colony stimulating factor (GM-CSF) in haemopoiesis during chronic infection, mice with a targeted disruption of the gene for GM-CSF were infected intraperitoneally with the facultative intracellular pathogen, Mycobacterium avium. The bacteria spread to lungs, liver and spleen and persisted for more than 10 weeks at levels between 105 and 106 CFU. Bacterial numbers did not differ significantly between infected GM-CSF-/- and wild-type mice, making this an excellent model in which to study the effects of GM-CSF deficiency on haemopoietic cells without complications of interpretation relating to differences in bacterial load. Haemopoietic colony forming cells (CFC) in the bone marrow of GM-CSF-/- mice before infection were not different from wild-type. However, whereas CFC in wild-type mice increased 1.5-fold with infection, GM-CSF-/- mice were unable to increase their CFC and numbers were significantly lower than in infected wild-type mice. Cells attracted to the peritoneal cavity of the GM-CSF-/- mice following i.p. injection of bacteria were notably lacking in the large, granular macrophages of activated appearance, which were a feature in wild-type mice. Nitric oxide production by peritoneal cells from GM-CSF-/- mice was deficient. Thus, GM-CSF is not critical for haemopoiesis during chronic infection, but in its absence the mice are unable to increase their output of haemopoietic cells and there are deficiencies in macrophage activation.

Animals↗
PubMed2001

Culture at high density improves the ability of human macrophages to control mycobacterial growth.

The mechanisms through which granuloma formation helps control mycobacterial infection are poorly understood, but it is possible that the accumulation of macrophages at high density at sites of infection promotes the differentiation of macrophages into cells with improved mycobactericidal activity. To test this possibility, varying numbers of monocytes were cultured in 96-well plates for 3 days, infected with Mycobacterium bovis bacillus Calmette-Guérin, and mycobacterial number was assessed 7 days after infection based on the measurement of luciferase activity expressed by a mycobacterial reporter strain or by counting CFU. Mycobacterial growth was optimal in cultures containing 5 x 10(4) cells/well, but increasing the number of cells to 2 x 10(5) cells/well resulted in complete inhibition of mycobacterial growth. This effect could not be explained by differences in mycobacterial uptake, multiplicity of infection, acidification of the extracellular medium in high density cultures, enhanced NO production, or paracrine stimulation resulting from secretion of cytokines or other proteins. The morphology of cells cultured at high density was strikingly different from that of monocytes cultured at 5 x 10(4) cells/well, including the appearance of numerous giant cells. The bacteriostatic activity of monocyte-derived macrophages was also dependent on cell number, but fewer of these more mature cells were required to control mycobacterial growth. Thus, the ability of human macrophages to control mycobacterial infection in vitro is influenced by the density of cells present, findings that may help explain why the formation of granulomas in vivo appears to be a key event in the control of mycobacterial infections.

Cell Adhesion↗
PubMed2000

Safe water supply without disinfection in a large city case study: Berlin.

Berlin's water supplies originate exclusively from groundwater. For sustainable water management, river water is treated by flocculation and filtration and used either for artificial groundwater recharge (rivers Spree and Havel) or for bank filtration (Nordgraben and Lake Tegel). Drinking water chlorination was abandoned in Berlin (West) in 1978, and in Berlin (East) in 1992, following German unification. Chlorine consumption for the purpose of weekly performance checks in the chlorination plants of Berlin's 11 waterworks and occasional chlorination within the pipe system following pipe burst events amounts to 2500 kg per year. Based on the annual water demand of 250 million cubic metres, this is equivalent to 0.01 mg of chlorine per litre. Microbiological monitoring at the 11 waterworks and at 383 sampling points within the pipe system shows CFU at less than 10/1 ml-1 and coliforms and E. coli invariably at 0/100 ml-1. In view of the low AOX content, a multiplication of bacteria within the pipe system can be expected to occur not at all or only to a small extent. Resource protection measures, filter backwashing and pipe system maintenance in observance of the relevant technical rules will continue to ensure that the quality of Berlin's drinking water meets stringent hygiene requirements without chlorination.

Berlin↗
PubMed1995

Granulocyte colony-stimulating factor improves myelopoiesis and host defense in fulminant intra-abdominal sepsis in rats.

The therapeutic efficacy of granulocyte colony-stimulating factor (G-CSF) was studied in a model of fulminant sepsis in rats. Polymicrobial peritonitis was induced by a 4 mm cecal perforation and 10 micrograms/kg recombinant human G-CSF was given intravenously every 12 h, with the first dose at sepsis induction or 4 h post-induction. Rats were sacrificed at various intervals throughout sepsis to measure levels of neutrophil progenitors in the bone marrow and neutrophils and bacteria in blood and peritoneal fluid. Sepsis gave a sustained neutropenia and bacteremia, but did not affect numbers of blast- or GM-colonies, and only a delayed and moderate proliferation of G-clones was seen. Treatment with G-CSF at sepsis induction improved myelopoiesis by doubling the numbers of GM- and G-progenitors at 12 and 24 h post-induction. Concentrations of neutrophils increased twofold in blood and 5-fold in peritoneal fluid, while bacteria counts in the same compartments declined logarithmically. Mortality was 92% in untreated sepsis and declined to 46% when G-CSF therapy was started at sepsis induction, and to 42% following 4 h delayed therapy.

Acute Disease↗
PubMed1998

An in vivo comparison of the antimicrobial activities of three mouthrinses.

The purpose of this in vivo study was to determine and compare the antimicrobial effectiveness of three commercial mouthrinses and a water control. The antimicrobial efficacy of the products was determined against aerobic, micro-aerophilic, and anaerobic bacteria. Twenty human subjects participated in this study. At each experimental session for a given subject, a pre-test saliva sample was taken. This sample was divided and used to grow three bacteria cultures under the different incubation environments. After giving the pre-test sample, the subject rinsed with one of the mouthrinses or the water control for 30 seconds, then waited one hour, at which time a post-test saliva sample was collected. Again, the sample was divided and used to culture the different types of bacteria. Following a 48-hour incubation period, the numbers of microbial colonies on each plate were counted and compared. The results indicated that all of the mouthrinses tested performed significantly better than the water control. Herbal Mouth and Gum Therapy and Peridex did not demonstrate a statistically significant difference in inhibiting aerobic, microaerophilic, and anaerobic bacteria. Both Herbal Mouth and Gum Therapy and Peridex were significantly more effective than Listerine in inhibiting the three different types of bacteria.

Adult↗
PubMed1994

An in vivo comparison of antimicrobial activities of four commercial mouthwashes.

The purpose of this in vivo double-blind study was to determine and compare the antimicrobial effectiveness of four commercial over-the-counter mouthwashes and a water control. Two of the products tested contained alcohol (Listerine and Scope) and two products were alcohol-free (Rembrandt Mouth Refreshing Rincs and Clear Choice). The antimicrobial efficacy of the products was determined against aerobic, microaerophilic, and anaerobic bacteria in saliva. Thirty human subjects participated in the study. At each experimental session for a given subject, a pretest saliva sample was taken. This sample was divided and used to grow three bacteria cultures under the different incubation environments. After giving the pretest sample, the subject rinsed with one of the commercial mouthwashes or the water control for 30 seconds, then waited one hour at which time a posttest saliva sample was collected. Again, the sample was divided and used to culture the different type of bacteria. Following a 48-hour incubation period, the numbers of microbial colonies on each plate were counted and compared. The results indicated that all of the mouthwashes tested performed significantly better than the water control. Both of the non-alcohol-containing products showed efficacy in inhibiting aerobic, microaerophilic, and anaerobic bacteria. Rembrandt Mouth Refreshing Rinse and Listerine inhibited all three types of bacteria, and were not significantly different from each other in efficacy. The Rembrandt mouth wash inhibited all three types of bacteria more effectively than Clear Choice mouthwash, and inhibited two of the three types of bacteria more effectively than Scope mouthwash.

Analysis of Variance↗
PubMed1994

Effect of chlorhexidine-containing varnish (Cervitec) on microbial vitality and accumulation of supragingival dental plaque in humans.

The effect on local plaque formation of a varnish containing 1% chlorhexidine and 1% thymol (Cervitec) was evaluated. Ten volunteers with clinically healthy oral conditions were asked to refrain from any kind of oral hygiene measures for three periods of three days. Undisturbed plaque formation was recorded during the first experimental period. At the beginning of the second period, the varnish was applied to six vestibular enamel surfaces and removed after 1 h. The third experimental period was initiated 12 weeks after varnish application to assess a potential long-term effect. During each period of plaque formation, samples were collected from the vestibular surfaces after 24 h (from teeth 15/25), after 48 h (from teeth 14/24) and after 72 h (from teeth 13/23), respectively, and evaluated for total microscopic bacterial counts (BC) and colony forming units (CFU). Microbial vitality was assessed by the plating efficiency [PE = (CFU/BC) x 100] and directly by a vital fluorescence (VF) technique. VF of 48- and 72-hour plaque was significantly reduced after Cervitec application. An inhibitory effect by Cervitec could not be discerned 12 weeks after varnish treatment.

Adult↗
PubMed1990

Total count of microorganisms contaminating water supply system of Ismailia City, Egypt.

The efficiency of water purification in the French and the British systems of Ismailia city was evaluated concerning the contaminating microorganisms. Counting of microbial contaminants was based on the phenotypic characters of the colony type. Colony-types of eubacteria, eumycetes, Micromonospores and Streptomyces enumerated in the crude water and the different steps of purification. Total counts of microflora in both systems were higher than the counts in canal water. Meanwhile, microbial isolates were higher in the French system than that of the British one. The increased counts in tap water, compared with canal water, were recorded in winter for bacteria and in spring for fungi. These data showed variations in the microbial total counts according to: plant system, steps of purification, chlorination season and type of microbes.

Bacteria↗
PubMed2001

Treatment of broiler litter with organic acids.

Experiments for treatment of contaminated broiler litter with citric, tartaric and salicylic acids were performed. At days 2 and 6 after the treatment, pH values (using a pH-meter), the ammonia concentrations (titration with 0.1 N HCl) and the microbial cells counts were determined in both experimental and control specimens of litter. The cost of acidification of litter was also determined. Our studies showed that the treatment of the contaminated litter with 5 per cent citric acid, 4 per cent tartaric acid and 1.5 per cent salicylic acid created an acid medium with pH under 5.0 and thus reduced the microbial counts to 2.2 x 10(3)colony forming units per gram manure litter. The treatment reduced the content of ammonia in the litter and in the air under the hygienic limits, i.e. 25-50 ppm. The cost of acidification of litter with these organic acids amounted to 0.1 $ per bird and 1.5 $ per 15 birds on one square metre in a growth period of 50 days.

Ammonia↗
PubMed1998

Essential roles for granulocyte-macrophage colony-stimulating factor (GM-CSF) and G-CSF in the sustained hematopoietic response of Listeria monocytogenes-infected mice.

The in vivo roles of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte (G)-CSF were studied in factor-deficient gene-targeted knockout mice infected with the facultative intracellular bacterium Listeria monocytogenes. Previous results showed that G-CSF-/- mice had an underlying selective deficiency in granulopoiesis, but GM-CSF-/- mice had little disturbance in resting hematopoiesis. Nevertheless, in this study it is revealed that 3 days after intraperitoneal infection with 2 x 10(5) Listeria, GM-CSF-/- mice harbored 50-fold more organisms in their spleen and liver than similarly infected wild-type mice. This was accompanied by a severe depletion of bone marrow hematopoietic cells and a deficient inflammatory response in their peritoneal cavity. Thus, GM-CSF is essential for emergency, but not resting, hematopoiesis. In contrast, G-CSF-/- mice were markedly susceptible to low doses (2 x 10(4)) of Listeria intraperitoneally. After infection, the acute (1 day) granulocyte infiltration to the peritoneal cavity was normal compared with wild type, but the more prolonged monocyte response was deficient, reflecting a continued decrease in bone marrow cellularity and hematopoiesis over 3 days, which was not observed in infected wild-type mice. It is thus apparent that G-CSF deficiency affects monocytopoiesis as well as granulopoiesis during infection.

Animals↗
PubMed1998

COVASIAM: an image analysis method that allows detection of confluent microbial colonies and colonies of various sizes for automated counting.

In this work we introduce the confluent and various sizes image analysis method (COVASIAM), an automated colony count technique that uses digital imaging technology for detection and separation of confluent microbial colonies and colonies of various sizes growing on petri dishes. The proposed method takes advantage of the optical properties of the surfaces of most microbial colonies. Colonies in the petri dish are epi-illuminated in order to direct the reflection of concentrated light coming from a halogen lamp towards an image-sensing device. In conjunction, a multilevel threshold algorithm is proposed for colony separation and counting. These procedures improved the quantification of colonies showing confluence or differences in size. We tested COVASIAM with a sample set of microorganisms that form colonies with contrasting physical properties: Saccharomyces cerevisiae, Aspergillus nidulans, Escherichia coli, Azotobacter vinelandii, Pseudomonas aeruginosa, and Rhizobium etli. These physical properties range from smooth to hairy, from bright to opaque, and from high to low convexities. COVASIAM estimated an average of 95.47% (sigma = 8.55%) of the manually counted colonies, while an automated method based on a single-threshold segmentation procedure estimated an average of 76% (sigma = 16.27) of the manually counted colonies. This method can be easily transposed to almost every image-processing analyzer since the procedures to compile it are generically standard.

Aspergillus nidulans↗
PubMed1991

Mechanism of protective effect of recombinant human granulocyte colony-stimulating factor (rG-CSF) on Pseudomonas infection.

Decrease in resistance to systemic Pseudomonas infection in cyclophosphamide (CPA)-induced neutropenic mice was prevented by injections of recombinant human granulocyte colony-stimulating factor (rG-CSF). In order to explore mechanism of the prevention of CPA-induced decrease in the anti-infectious resistance by rG-CSF, CPA-treated and then rG-CSF-injected mice were inoculated i.p. with P. aeruginosa, and growth of the infecting bacteria and infiltration of leukocytes in the peritoneal cavity were determined. In the mice who had received 4 daily s.c. injections of rG-CSF from the day after CPA-injection, a large number of neutrophils were mobilized into the peritoneal cavity in response to the bacterial inoculation and growth of the infecting Pseudomonas in the cavity was markedly inhibited, whereas in CPA-induced neutropenic mice few neutrophils were mobilized and the infecting bacteria proliferated vigorously in the peritoneal cavity. These results suggest that administration of rG-CSF prevents CPA-induced neutropenia and neutrophils circulating at normal level in the number are normally mobilized into the peritoneal cavity in response to Pseudomonas inoculation, and that the mobilized neutrophils inhibit proliferation of the infecting Pseudomonas.

Animals↗
PubMed1993

Vaginal flora in health and disease.

This chapter has portrayed the normal vaginal flora as a range of microbial species that associates in a stable way with human vaginal epithelium. The human vagina provides these microorganisms with the physical and chemical environment that allows them to use this tissue as a normal habitat. Because the relationship of the normal flora to the underlying tissue is that of an ecosystem, the components of this system are in dynamic equilibrium. Consequently, a change in the environmental conditions provided by the vaginal epithelium will result in changes in the population density or the species composition of the normal flora. Although the microbial flora appears to provide a benefit to the host by reducing the probability of colonization by exogenous microorganisms, it may participate in various undesirable symptoms, including various types of vaginitis or postoperative, postabortion, and post-Caesarean section infections, and upper tract invasion after lower tract infection. All future attempts to understand vaginitis in all its manifestations necessarily must include recognition of the importance of the normal flora because all types of vaginitis are superimposed on an existing endogenous microbial flora.

Animals↗
PubMed2005

Mixed plaques: statistical evidence how plaque assays may underestimate virus concentrations.

Even at very low concentrations human pathogenic viruses may result in infection and possibly subsequent disease. Ideally, viruses are quantified by use of cell culture assays to determine their infectivity. Plaque assays are common tools for enumeration of viruses in inocula and this process is straightforward when a plaque results from the offspring of a single infectious virus particle. In the course of a study on the usefulness of sewage monitoring for surveillance of polio-virus transmission, sewage samples containing a mixture of two live polio vaccine strains (type 1 and type 3) were analyzed. The total poliovirus concentration in plaque forming units (pfu) was estimated by means of a monolayer plaque assay on L20B cells. Subsequent typing of virus directly by neutralisation of virus from excised plaques revealed the occurrence of plaques containing both type 1 and type 3 virus. This means that there must be plaques that originate from more than one initial infectious virus particle. As a consequence, the estimated virus concentration is incorrect. We present statistical methods that utilize these mixed plaque counts to estimate the concentrations of either virus type in our sewage samples. We can also calculate a correction factor for the error in virus concentration, which would result from equating a pfu to a single infectious particle. Since many quantitative methods in microbiology are based on colony counts, we conclude that such counts should be interpreted with caution, especially when data are used in quantitative microbial risk assessment to estimate the public health impact.

Colony Count, Microbial↗
PubMed2001

Disruption of nuclear factor-interleukin-6, a transcription factor, results in severe mycobacterial infection.

Nuclear factor-interleukin-6 (NF-IL-6) is one of several nuclear transcription factors (NF-IL-6, NF-kappaB, PU.1, interferon-regulatory factor 1, Egr-1, and Stat-1). NF-IL-6 and NF-kappaB are expressed in macrophages and is induced by bacterial lipopolysaccharides. To evaluate whether NF-IL-6 is required for the inflammatory immune response to mycobacterial infection, in which epithelioid macrophages comprise the leading cell population, we generated NF-IL-6 knockout (KO) mutant mice. Airborne infection of these mice with Mycobacterium tuberculosis strains induced disseminated tuberculosis lacking granuloma formation, although interferon-gamma, tumor necrosis factor-alpha, and interleukin-12 mRNA expression levels were within the normal range compared with those of wild-type mice. Generation of O2- and mycobacterial killing by neutrophils from these mice were impaired severely compared with wild-type mice. We conclude that NF-IL-6 is a critical transcription factor in mycobacterial control as well as in granulocyte-colony stimulating factor induction resulting in neutrophil activation.

Animals↗