Status of nutrition education in Canadian dental and medical schools.

To investigate the present status of nutrition education for dentists and physicians in Canada, we conducted a survey of the nutrition education programs in 10 Canadian dental and 16 medical schools in the academic year 1982-83. Seven of the dental schools and seven of the medical schools had a separate course in nutrition. The average duration of these courses was 22 hours for the dental schools and 26 hours for the medical schools. Nutrition education was integrated with another discipline in 4 of the dental schools and 11 of the medical schools. The average duration of this type of instruction was 14 hours for the dental schools and 18 hours for the medical schools. Six of the dental schools and eight of the medical schools employed a nutritionist/dietitian to provide instruction in nutrition. We recommend that courses in basic and applied clinical nutrition be incorporated throughout the curricula of Canadian dental and medical schools, and that personnel trained in clinical nutrition be employed to provide instruction in this area.     

Management of chronic hepatitis C: clinical audit of biopsy based management algorithm.

OBJECTIVE: To assess the attendance, outcome, compliance with treatment, and response to interferon alfa in patients with chronic hepatitis C who attended during 1995 and were treated according to a biopsy based algorithm. DESIGN: Retrospective audit of all patients with chronic hepatitis C attending outpatient clinics over one year. SETTING: The liver unit at a London teaching hospital. SUBJECTS: 255 patients with chronic hepatitis C. MAIN OUTCOME MEASURES: Patient survival, attendance, and compliance with diagnostic and therapeutic regimens. Response to interferon alfa treatment, based on loss of viraemia three months after cessation of treatment. RESULTS: A large proportion of patients (39%) with newly diagnosed chronic hepatitis C infection do not want to undergo further investigation. Of those patients who do attend for further treatment, a large proportion with severe hepatic fibrosis (42%) do not want to undergo currently available treatment. The response rate to interferon (21%) in treated patients was similar to that previously reported in a trial setting. There was no significant difference in response rates in patients with or without severe fibrosis not amounting to cirrhosis. In patients with cirrhosis there was a high incidence of hepatocellular carcinoma (18%) over a follow up period of 20 months. CONCLUSION: Current strategies aimed at investigating and treating patients with chronic hepatitis C are not acceptable to a large proportion of patients. Many patients with cirrhosis related to hepatitis C infection develop hepatic neoplasms, and management strategies to deal with this problem are urgently required.     

Prophage Carriage and Diversity within Clinically Relevant Strains of Clostridium difficile

Prophages are encoded in most genomes of sequenced Clostridium difficile strains. They are key components of the mobile genetic elements and, as such, are likely to influence the biology of their host strains. The majority of these phages are not amenable to propagation, and therefore the development of a molecular marker is a useful tool with which to establish the extent and diversity of C. difficile prophage carriage within clinical strains. To design markers, several candidate genes were analyzed including structural and holin genes. The holin gene is the only gene present in all sequenced phage genomes, conserved at both terminals, with a variable mid-section. This allowed us to design two sets of degenerate PCR primers specific to C. difficile myoviruses and siphoviruses. Subsequent PCR analysis of 16 clinical C. difficile ribotypes showed that 15 of them are myovirus positive, and 2 of them are also siphovirus positive. Antibiotic induction and transmission electron microscope analysis confirmed the molecular prediction of myoviruses and/or siphovirus presence. Phylogenetic analysis of the holin sequences identified three groups of C. difficile phages, two within the myoviruses and a divergent siphovirus group. The marker also produced tight groups within temperate phages that infect other taxa, including Clostridium perfringens, Clostridium botulinum, and Bacillus spp., which suggests the potential application of the holin gene to study prophage carriage in other bacteria. This study reveals the high incidence of prophage carriage in clinically relevant strains of C. difficile and correlates the molecular data to the morphological observation.     

Antibiotic resistance and virulence traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates

This study compared virulence and antibiotic resistance traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates. E. faecalis isolates harboured a broader spectrum of virulence determinants compared to E. faecium isolates. The virulence traits Cyl-A, Cyl-B, Cyl-M, gel-E, esp and acm were tested and environmental isolates predominantly harboured gel-E (80% of E. faecalis and 31.9% of E. faecium) whereas esp was more prevalent in clinical isolates (67.8% of E. faecalis and 70.4% of E. faecium). E. faecalis and E. faecium isolated from water had different antibiotic resistance patterns compared to those isolated from clinical samples. Linezolid resistance was not observed in any isolates tested and vancomycin resistance was observed only in clinical isolates. Resistance to other antibiotics (tetracycline, gentamicin, ciprofloxacin and ampicillin) was detected in both clinical and water isolates. Clinical isolates were more resistant to all the antibiotics tested compared to water isolates. Multi-drug resistance was more prevalent in clinical isolates (71.2% of E. faecalis and 70.3% of E. faecium) compared to water isolates (only 5.7% E. faecium). tet L and tet M genes were predominantly identified in tetracycline-resistant isolates. All water and clinical isolates resistant to ciprofloxacin and ampicillin contained mutations in the gyrA, parC and pbp5 genes. A significant correlation was found between the presence of virulence determinants and antibiotic resistance in all the isolates tested in this study (p<0.05). The presence of antibiotic resistant enterococci, together with associated virulence traits, in surface recreational water could be a public health risk.     

Retention and Release of Cryptosporidium parvum Oocysts by Experimental Biofilms Composed of a Natural Stream Microbial Community

Cryptosporidium parvum oocysts accumulate on biofilm surfaces. The percentage of oocysts attached to biofilms remained nearly constant while oocysts were supplied to the system but decreased to a new steady-state level once oocysts were removed from the feed. More oocysts attached to summer biofilm cultures than winter biofilm cultures.Cryptosporidium causes a potentially life-threatening gastrointestinal disease. Because conventional water treatment may not effectively target Cryptosporidium, source water monitoring and protection are important to avoid infection outbreaks.Biofilms can accumulate pathogens at densities that are much higher than water column densities, with the potential for pathogen release long after entrapment (5, 13, 15, 19). Biofilms have been identified as a drinking water contamination source (7), causing infections for which the source cannot be identified (4, 6).Several previous studies examined pathogen transport in biofilms using Cryptosporidium parvum oocysts (2, 6, 15, 16) or beads as pathogen surrogates (3, 5, 11, 12). The former studies did not use natural microbial assemblages (2, 16) or quantify oocyst attachment or sloughing (6, 15). The current study provides novel information about C. parvum oocyst attachment to environmental biofilms, including a mass balance analysis to identify the daily number of oocysts that (i) remained in the flowing water or were sloughed from the biofilm and (ii) were attached to the biofilm. We imaged biofilms using scanning confocal laser microscopy, as used in other studies (9, 17, 20, 21), to identify spatial patterns of oocyst attachment.Biofilms were scraped from rocks found in Monocacy Creek (Bethlehem, PA) into 1 liter of creek water in January 2007 (winter biofilm culture) and July 2008 (summer biofilm culture). The biofilm suspension was vacuum filtered through a 6-μm cellulose filter. The filtrate was centrifuged (1,754 × g for 15 min), and the resulting biofilm pellet was resuspended in 1 ml of raw creek water. The cell concentration was quantified by DAPI (4′,6-diamidino-2-phenylindole) staining (14). Cells were split into aliquots (5 × 10⁶ cells each) and stored at −80°C in cryovials containing 30% glycerol.Single-channel flow chambers (length by width by height, 24 mm by 8 mm by 4 mm) with glass coverslips (Stovall Life Science, Inc., Greensboro, NC) were inoculated with 5 × 10⁶ biofilm cells for 24 h before the flow was started. Filter-sterilized creek water was used as the flow medium. A 12-channel peristaltic pump (Ismatec, Glattbrugg, Switzerland) maintained a constant flow of 0.2 mm/s (1).For biofilm imaging, the following two setups were used: (i) 1 × 10⁴ C. parvum oocysts (Iowa isolate; Waterborne, Inc., New Orleans, LA) (all oocysts were used within 3 weeks of shedding) in the influent each day for 3 days and (ii) 3 × 10⁴ C. parvum oocysts added to the influent for the last 24 h of a 3-day flow experiment. Biofilms were imaged with a Zeiss LSM 510 META laser scanning microscope, using an argon laser (458-nm, 477-nm, 488-nm, and 514-nm excitation wavelengths) and a HeNe1 laser (543-nm excitation wavelength). Biofilms were fixed with methanol, blocked using a 1:10 dilution of fetal bovine serum, and stained with 20 μM SYTO 9 (Invitrogen, Molecular Probes, Eugene, OR) (16). C. parvum oocysts in the biofilm were stained with a Cy3-conjugated monoclonal antibody solution specific for Cryptosporidium (Waterborne, Inc.) (16).For the mass balance analysis, C. parvum oocysts (1 × 10⁴ per day for 3 days) were added to 500 ml constantly stirred influent water to keep oocysts in suspension. Influent water was replaced each day. Experiments to quantify sloughing included 2 or 5 additional days with oocyst-free feed water, for a total of 5 or 8 days. Biofilms used for the 3- and 5-day experiments were grown with the winter biofilm culture; biofilms used for the 8-day experiments were grown with the summer biofilm culture.After each 24-hour period, the remaining influent and effluent waters were processed by membrane filtration (MF) and immunomagnetic separation (IMS) to recover the oocysts. On the last day of each experiment, biofilms were scraped from the flow chambers, resuspended in sterile creek water, and also processed by MF and IMS. MF was performed according to the method of Oda et al. (10), using the 3-μm filter only. IMS was performed on the filtrate using the Aureon IMS kit (ImmTech, Inc., New Windsor, MD), and oocysts were dissociated from the magnetic beads with 0.05 M HCl. IMS products were counted by hemocytometry and corrected for MF and IMS processing losses. An average IMS recovery of 65% ± 4.2% standard error (SE) (determined by four trials using 1 × 10⁴ oocysts in deionized water) was used. MF recoveries were consistent within each day but varied between days. Therefore, an MF recovery control was performed each day using 1 × 10⁴ oocysts in 1 liter deionized water to obtain a daily MF correction factor.The mass balance analysis demonstrated that these methods were effective for tracking oocysts throughout the flow system for the experiment''s duration, accounting for all the oocysts within 8% (Table ​(Table1).1). In a control flow chamber with no biofilm growth (i.e., a clean glass surface), oocyst loss within the system was 1% or less, indicating that very few oocysts attached to any abiotic surface within the flow system. Laboratory biofilms composed of natural microbial assemblages were successfully created, although grazing impacts that would affect biofilm dynamics in the environment were eliminated. The thicknesses of laboratory biofilms (average thickness, 39.6 μm; SD, 4.7 μm; n = 16) were not statistically different (P of 0.17 by independent t test) than those of natural biofilms in Monocacy Creek (average thickness, 35.8 μm; SD, 10.2 μm; n = 36).

TABLE 1.

Mass balance analysis of biofilms grown for 3, 5, and 8 days, with 3-day oocyst dosing^a