Model studies on a membrane filtration method for the enumeration of coagulase-positive staphylococci in swimming-pool water using rabbit plasma-bovine fibrinogen agar

The recovery of Staphylococcus aureus from swimming-pool water by membrane filtration was studied in model experiments. On the nonselective medium tryptone soya agar (TSA) there was no difference in counts of noninjured S. aureus with all membrane filters tested and with pour plates. Chlorine-injured S. aureus was enumerated most efficiently on TSA by Gelman Tuffryn HT-450 and Sartorius SM 13806 filters. Tuffryn filters were also most productive when used in combination with the selective medium rabbit plasma - bovine fibrinogen agar (RPFA). Other filters, particularly Gelman GN-6 and Millipore HAWP, when used on RPFA were shown to have a synergistic inhibitory effect on both noninjured and chlorine-injured S. aureus. This effect was not found on Baird-Parker agar. Using Tuffryn filters, counts on RPFA were equal to those on TSA for noninjured S. aureus and 0.1-2.0 log units less for chlorine-injured S. aureus. Despite this, the possibility for reading the in situ coagulase reaction for individual colonies on RPFA is considered such an advantage of this medium that its general use for enumeration of S. aureus in swimming pools is recommended. Further studies should be carried out to allow better resuscitation.     

A method for the enumeration of male-specific bacteriophages in sewage

Male-specific bacteriophages adsorb to F-pili and thus can only infect male host strains. A method was developed for the selective enumeration of these phages, based on the observation that in sewage there are few phages capable of infecting F- -salmonellas--usually less than 10 pfu/ml. Using a male Salmonella strain, constructed by the introduction of the plasmid F'42 lac::Tn5 into Salmonella typhimurium phage type 3, plaque counts in secondary effluent were found to be in the range of 60-8200 pfu/ml. Practically all the phages detected had a host range restricted to male Salmonella or Escherichia coli strains, were resistant to chloroform and their infectivity was inhibited by RNase. Electron microscopy of lysates revealed phage particles that were morphologically identical to the male-specific single-strand RNA phages. Similar results were obtained with a strain of Salm. indiana carrying F'42 lac. A derivative of the Salm. typhimurium LT2 strain carrying an F-plasmid (F'42 lac fin P301) derepressed for fertility inhibition by the resident plasmid pSLT was equally sensitive to male-specific phages, but from sewage samples many other phages infecting F- E. coli but not F- Salmonella were isolated using this host strain.     

Proficiency testing of water microbiology laboratories in The Netherlands

In a 3-year period, four series of simulated water samples containing selected test strains were distributed to more than 50 laboratories in The Netherlands for bacteriological testing. Participating laboratories examined the samples by enrichment or membrane filtration methods, or both, for total coliform organisms, thermotol-erant coliform organisms, faecal streptococci and standard plate counts (37˙ and 22˙C) according to Dutch standard methods. The results were quantitatively satisfactory: the distribution of positive and negative results with subsamples conformed to stochastic variation; the standard deviation of membrane or plate counts was usually in the range which may be expected from a Poisson distribution, and there was good correspondence between average counts in participating laboratories and those expected from controls in the organizing laboratory. Problems of a qualitative nature were frequently encountered, however. Among them were a false positive response with a strain of Enterobacter cloacae in the thermotolerant coliform test; a false positive result with Clostridium perfringens in enrichment tests for total or thermotolerant coliform organisms and false positive results with Micrococcus varians in the faecal streptococcus test by membrane filtration. It is concluded that quality assessment should be a consistent activity in water microbiology laboratories. For this purpose, stable and well characterized reference materials are needed.     

A Simple and Widely Applicable Method for Preparing Homogeneous and Stable Quality Control Samples in Water Microbiology

Test strains suspended in skim milk, quickly frozen in dry ice-ethanol, and stored at - 70°C can be used as quality control samples that are immediately available by quickly thawing at 37°C. The samples remain homogeneous and stable for at least 1 year, except for Aeromonas hydrophila, which decreases 20 to 30% in 1 year.     

F-specific RNA bacteriophages are adequate model organisms for enteric viruses in fresh water.

Culturable enteroviruses were detected by applying concentration techniques and by inoculating the concentrates on the BGM cell line. Samples were obtained from a wide variety of environments, including raw sewage, secondary effluent, coagulated effluent, chlorinated and UV-irradiated effluents, river water, coagulated river water, and lake water. The virus concentrations varied widely between 0.001 and 570/liter. The same cell line also supported growth of reoviruses, which were abundant in winter (up to 95% of the viruses detected) and scarce in summer (less than 15%). The concentrations of three groups of model organisms in relation to virus concentrations were also studied. The concentrations of bacteria (thermotolerant coliforms and fecal streptococci) were significantly correlated with virus concentrations in river water and coagulated secondary effluent, but were relatively low in disinfected effluents and relatively high in surface water open to nonhuman fecal pollution. The concentrations of F-specific RNA bacteriophages (FRNA phages) were highly correlated with virus concentrations in all environments studied except raw and biologically treated sewage. Numerical relationships were consistent over the whole range of environments; the regression equations for FRNA phages on viruses in river water and lake water were statistically equivalent. These relationships support the possibility that enteric virus concentrations can be predicted from FRNA phage data.     

A role for the transient increase of cytoplasmic free calcium in cell rescue after photodynamic treatment.

Chinese hamster ovary (CHO) cells and T24 human bladder transitional carcinoma cells were treated with the photosensitizers aluminum phthalocyanine (AlPc) and hematoporphyrin derivative (HPD), respectively. Exposure of both sensitized cell lines to red light caused an immediate increase of cytoplasmic free calcium, [Ca2+]i, reaching a peak within 5-15 min after exposure and then returning to basal level (approximately 200 nM). The level of the peak [Ca2+]i depended on the light fluence, reaching a maximum of 800-1000 nM at light doses that kill about 90% of the cells. Loading the cells with the intracellular calcium chelators quin2 or BAPTA prior to light exposure enhanced cell killing. This indicates that increased [Ca2+]i after photodynamic therapy (PDT) contributed to survivability of the treated cells by triggering a cellular rescue response. The results of experiments with calcium-free buffer and calcium chelators indicate that both in CHO cells treated with AlPc and with HPD-PDT of T24 cells extracellular Ca2+ influx is mainly responsible for elevated [Ca2+]i. PDT is unique in triggering a cell rescue process via elevated [Ca2+]i. Other cytotoxic agents, e.g., H2O2, produce sustained increase of [Ca2+]i that is involved in the pathological processes leading to cell death.     

Affinity of Ionic Species of Nucleoside Transport Protein Inhibitors

Abstract

A class of very potent nucleoside transport inhibitors is present in two molecular forms around physiological pH. We investigated whether the monoprotonated or the unionized species of these molecules binds to this camer protein with higher affinity.     

A Model for the Early Identification of Sources of Airborne Pathogens in an Outdoor Environment

Background

Source identification in areas with outbreaks of airborne pathogens is often time-consuming and expensive. We developed a model to identify the most likely location of sources of airborne pathogens.

Methods

As a case study, we retrospectively analyzed three Q fever outbreaks in the Netherlands in 2009, each with suspected exposure from a single large dairy goat farm. Model input consisted only of case residential addresses, day of first clinical symptoms, and human population density data. We defined a spatial grid and fitted an exponentially declining function to the incidence-distance data of each grid point. For any grid point with a fit significant at the 95% confidence level, we calculated a measure of risk. For validation, we used results from abortion notifications, voluntary (2008) and mandatory (2009) bulk tank milk sampling at large (i.e. >50 goats and/or sheep) dairy farms, and non-systematic vaginal swab sampling at large and small dairy and non-dairy goat/sheep farms. In addition, we performed a two-source simulation study.

Results

Hotspots – areas most likely to contain the actual source – were identified at early outbreak stages, based on the earliest 2–10% of the case notifications. Distances between the hotspots and suspected goat farms varied from 300–1500 m. In regional likelihood rankings including all large dairy farms, the suspected goat farms consistently ranked first. The two-source simulation study showed that detection of sources is most clear if the distance between the sources is either relatively small or relatively large.

Conclusions

Our model identifies the most likely location of sources in an airborne pathogen outbreak area, even at early stages. It can help to reduce the number of potential sources to be investigated by microbial testing and to allow rapid implementation of interventions to limit the number of human infections and to reduce the risk of source-to-source transmission.