Seasonal Retention and Release of Cryptosporidium parvum Oocysts by Environmental Biofilms in the Laboratory

Cryptosporidium is a genus of waterborne protozoan parasites that causes significant gastrointestinal disease in humans. These parasites can accumulate in environmental biofilms and be subsequently released to contaminate water supplies. Natural microbial assemblages were collected each season from an eastern Pennsylvania stream and used to grow biofilms in laboratory microcosms in which influx, efflux, and biofilm retention were determined from daily oocyst counts. For each seasonal biofilm, oocysts attached to the biofilm quickly during oocyst dosing. Upon termination of oocyst dosing, the percentage of oocysts retained within the biofilm decreased to a new steady state within 5 days. Seasonal differences in biofilm retention of oocysts were observed. The spring biofilm retained the greatest percentage of oocysts, followed (in decreasing order) by the winter, summer, and fall biofilms. There was no statistically significant correlation between the percentage of oocysts attached to the biofilm and (i) any measured stream water quality parameter (including temperature, pH, conductivity, and dissolved organic carbon concentration) or (ii) experimental temperature. Seasonal differences in oocyst retention persisted when biofilms were tested with stream water from a different season. These data suggest that seasonal variation in the microbial community and resulting biofilm architecture may be more important to oocyst transport in this stream site than water quality. The biofilm attachment and detachment dynamics of C. parvum oocysts have implications for public health, and the drinking water industry should recognize that the potential exists for pathogen-free water to become contaminated during the distribution process as a result of biofilm dynamics.Cryptosporidium is a genus of waterborne protozoan parasites that cause a gastrointestinal disease in humans (cryptosporidiosis) that can be prolonged and life-threatening for people with compromised immune systems. Recent advances in medical treatment for cryptosporidiosis exist but are not entirely effective for immunocompromised patients (1). In addition, conventional water treatment does not effectively target Cryptosporidium oocysts because the oocysts'' small size (4 to 8 μm) limits the ability of filters to remove them and, more importantly, oocysts are resistant to chlorine (25). Therefore, environmental control of Cryptosporidium is important to protect public health. To determine the risk of human exposure and infection, the fate and transport of Cryptosporidium oocysts in the environment, including biofilms, should be examined.Within the past two decades, biofilms have been recognized as ubiquitous habitats found on most surfaces exposed to water (20, 24). Environmental biofilms can rapidly accumulate pathogens at densities much higher than water column densities, and the potential for gradual or sudden pathogen loss from the biofilm exists long after entrapment (8, 22). Biofilm sloughing events are commonplace, occurring when a biofilm detaches from the substrate to be resuspended as large particles in the water column, and may result in the release of pathogen reservoirs from the biofilm into the water column (8).Biofilms have been identified as a possible contamination source for drinking water supplies, which may lead to infections for which the source cannot be identified (7, 9). An example of the impact of biofilm sloughing events on human health is seen in the cryptosporidiosis outbreak that occurred in Lancashire County, England, in March 2000 (10). After the outbreak, the oocyst source was identified as cattle feces from adjacent farmland that contaminated the drinking water after abnormally heavy rainfall. The water source was subsequently changed to two upland impounding reservoirs containing filtered surface water. However, oocysts persisted in the water distribution system for 19 days, with large peaks associated with major water main disturbance events, including the initial flushing of the system and a burst in the main supply pipe. This persistence of oocysts in the water supply was attributed to the release of oocysts trapped in biofilms on the interior surface of the distribution pipes and may have contributed to additional infections.Several studies have examined pathogen transport dynamics in biofilms using glass or latex beads of various sizes as surrogates for pathogens (5, 8, 16, 17). A few studies examined the attachment of C. parvum oocysts to biofilms but did not use natural microbial assemblages to make the biofilms (3, 23) or quantify how many oocysts attached or sloughed (9, 22). Rogers and Keevil (22) showed that oocysts attached to a biofilm composed of a natural microbial assemblage collected from a reservoir at a concentration of 1,400 oocysts/cm² after the addition of 10⁸ oocysts in 10 ml of sterile water. Dai and Hozalski (3) and Searcy et al. (23) used pure culture biofilms to demonstrate oocyst attachment; however, only Searcy et al. (23) accounted for sloughing, although no oocyst release from the biofilm was seen during the course of their experiments. Helmi et al. (9) noted attachment and detachment of oocysts from a natural biofilm but did not include a quantitative analysis to account for all oocysts in the flow system over time. None of these studies examined pathogen attachment seasonally over the course of a year. Seasonal changes in temperature, precipitation, and water quality (including nutrient availability) may have significant impacts on the microbial composition and functional structure of a biofilm (14). These changes include structural changes in the biofilm thickness and morphology, as well as changes in the water composition and suspended matter. In addition, seasonal changes in stream flow dynamics may alter biofilm composition and morphology, as well as oocyst attachment and release patterns.This study provides novel information about C. parvum oocyst attachment to biofilms grown in the laboratory from natural microbial assemblages collected seasonally (i.e., in January, April, July, and October) from Monocacy Creek in Bethlehem, PA. Previous work (26) showed that (i) a significant fraction of C. parvum oocysts adhered to the surface of experimental biofilms during a 3-day oocyst dosing period, (ii) a portion of the adhered oocysts immediately released from the biofilm, and (iii) a portion of the oocysts remained attached to the biofilm for a period of days after termination of oocyst dosing. Here, we test the hypotheses that (i) oocyst retention by biofilms varies seasonally and (ii) seasonal changes in water quality influence oocyst retention.     

Mitochondrial Disease: A Historical,Biochemical, and London Perspective

Roland Luft is credited with describing the first truly mitochondrial disorder in the late 1950s and early 1960s. Cases such as his have proven to be exceptionally rare. Some years later, methods of mitochondrial analysis--enzymatic, polarographic, and spectroscopic, which had been developed primarily by groups in Philadelphia--were applied to the study of mitochondria isolated from skeletal muscle biopsies of patients thought to have defects of oxidative phosphorylation. In the vanguard of these investigations were groups in New York and London. John Clark led the latter group. Application of biochemical studies, more recently supplemented by molecular mtDNA and nuclear DNA studies, have revealed that mitochondrial disorders are among the most common of all metabolic disorders.     

Effect of epinephrine on muscle glycogenolysis during exercise in trained men

Febbraio, M. A., D. L. Lambert, R. L. Starkie, J. Proietto,and M. Hargreaves. Effect of epinephrine on muscle glycogenolysis during exercise in trained men. J. Appl.Physiol. 84(2): 465-470, 1998.To test thehypothesis that an elevation in circulating epinephrine increasesintramuscular glycogen utilization, six endurance-trained men performedtwo 40-min cycling trials at 71 ± 2% of peak oxygen uptake in20-22°C conditions. On the first occasion, subjects wereinfused with saline throughout exercise (Con). One week later, afterdetermination of plasma epinephrine levels in Con, subjects performedthe second trial (Epi) with an epinephrine infusion, which resulted ina twofold higher (P < 0.01) plasmaepinephrine concentration in Epi compared with Con. Although oxygenuptake was not different when the two trials were compared, respiratoryexchange ratio was higher throughout exercise in Epi compared with Con(0.93 ± 0.01 vs. 0.89 ± 0.01; P < 0.05). Muscle glycogenconcentration was not different when the trials were comparedpreexercise, but the postexercise value was lower(P < 0.01) in Epi compared with Con.Thus net muscle glycogen utilization was greater during exercise withepinephrine infusion (224 ± 37 vs. 303 ± 30 mmol/kg for Con andEpi, respectively; P < 0.01). Inaddition, both muscle and plasma lactate and plasma glucoseconcentrations were higher (P < 0.05) in Epi compared with Con. These data indicate that intramuscularglycogen utilization, glycolysis, and carbohydrate oxidation areaugmented by elevated epinephrine during submaximal exercise in trainedmen.

     

The recovery of Arcobacter butzleri NCTC 12481 from various temperature treatments

AIMS: The aim of this study was to determine the growth and survival characteristics for Arcobacter butzleri NCTC 12481. METHODS AND RESULTS: The temperature and pH growth ranges were 15-39 degrees C and pH 6.0-8.0, as determined using impedance microbiology. The maximum specific growth rate was 00.57 h(-1) at 30 degrees C, pH 7.0. Arcobacter butzleri harvested from the exponential phase was more resistant to heat treatment than stationary phase cells (D55 1.1 and 0.4 min, respectively). Fluorescent dye uptake, and the release of UV-absorbing material, increased in heat-treated cells. After 21 d storage at 4 and -20 degrees C, A. butzleri was recovered on blood agar, but not on the isolation media CAT or CCDA. CONCLUSION: Arcobacter butzleri cells from the exponential phase were less heat sensitive than those from the stationary phase. The organism was able to survive cold storage for at least 3 weeks. SIGNIFICANCE AND IMPACT OF THE STUDY: The growth and survival characteristics have been quantified thus providing a greater understanding of this newly emerging pathogen.     

Effect of pH on tolerance of Hormidium rivulare to zinc and copper

Summary The toxicity of zinc to a population of Hormidium rivulare isolated from an acid mine drainage was shown to be least at the optimum pH range for the growth of the alga, pH 3.5–4.0; toxicity increases markedly at higher pH values. Calcium clearly antagonizes the toxicity of zinc. Populations of H. rivulare isolated from higher pH values and which are resistant to zinc, are also especially resistant to low pH values, although they are unlikely ever to encounter such values naturally. Nevertheless raised levels of calcium bring about only a slight improvement of growth at very low pH values in the absence of zinc, so the mechanisms of pH and zinc tolerance are not the same. Although the acid stream population grows in the field in an environment with rather similar levels of zinc and copper, copper is less toxic than zinc at pH 3.5, but much more toxic than zinc at pH 6.0.     

Chlorimipramine: a novel anticancer agent with a mitochondrial target

Mitochondria have been suggested to be a potential intracellular target for cancer chemotherapy. In this report, we demonstrate the ability of the tricyclic antidepressant chlorimipramine to kill human glioma cells in vitro by a molecular mechanism resulting in an increase in caspase 3 activity following inhibition of glioma oxygen consumption. Studies with isolated rat mitochondria showed that chlorimipramine specifically inhibited mitochondrial complex III activity, which causes decreased mitochondrial membrane potential as well as mitochondrial swelling and vacuolation. The use of chlorimipramine in human as an effective, non-toxic cancer therapeutic having a strong selectivity between cancer cells and normal cells on the basis of their mitochondrial function is discussed.     

Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine on differentiating mouse N2a neuroblastoma cells

The effect of the neurotoxin 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) was investigated in mouse N2a neuroblastoma cells, induced to differentiate by serum withdrawal and addition of dibutyryl cyclic AMP, over a 24-h period. Addition of MPTP (10 microM) during differentiation caused a change in cell morphology characterised by an inhibition of axon outgrowth, in the absence of cell death. Biochemical characterisation by western blotting revealed that MPTP had no significant effects on the levels of actin, alpha-tubulin, or total heavy-chain neurofilament (NF-H). However, NF-H phosphorylation appeared to increase following MPTP treatment when blots were probed with the phosphorylation state-specific antibodies RMd09 and Ta51. In addition, indirect immunofluorescence analysis revealed an accumulation of phosphorylated NF-H in the cell perikaryon, suggesting that altered NF-H distribution was associated with the observed effects of MPTP on cell morphology. These changes may represent a useful in vitro marker of MPTP neurotoxicity within a simple differentiating neuronal cell model system.     

CRISPR Diversity in E. coli Isolates from Australian Animals,Humans and Environmental Waters

Seventy four SNP genotypes and 54 E. coli genomes from kangaroo, Tasmanian devil, reptile, cattle, dog, horse, duck, bird, fish, rodent, human and environmental water sources were screened for the presence of the CRISPR 2.1 loci flanked by cas2 and iap genes. CRISPR 2.1 regions were found in 49% of the strains analysed. The majority of human E. coli isolates lacked the CRISPR 2.1 locus. We described 76 CRISPR 2.1 positive isolates originating from Australian animals and humans, which contained a total of 764 spacer sequences. CRISPR arrays demonstrated a long history of phage attacks especially in isolates from birds (up to 40 spacers). The most prevalent spacer (1.6%) was an ancient spacer found mainly in human, horse, duck, rodent, reptile and environmental water sources. The sequence of this spacer matched the intestinal P7 phage and the pO111 plasmid of E. coli.