Janine Schwartzbrod

Integrated Analysis of Established and Novel Microbial and Chemical Methods for Microbial Source Tracking

ABSTRACT Several microbes and chemicals have been considered as potential tracers to identify fecal sources in the environment. However, to date, no one approach has been shown to accurately identify the origins of fecal pollution in aquatic environments. In this multilaboratory study, different microbial and chemical indicators were analyzed in order to distinguish human fecal sources from nonhuman fecal sources using wastewaters and slurries from diverse geographical areas within Europe. Twenty-six parameters, which were later combined to form derived variables for statistical analyses, were obtained by performing methods that were achievable in all the participant laboratories: enumeration of fecal coliform bacteria, enterococci, clostridia, somatic coliphages, F-specific RNA phages, bacteriophages infecting Bacteroides fragilis RYC2056 and Bacteroides thetaiotaomicron GA17, and total and sorbitol-fermenting bifidobacteria; genotyping of F-specific RNA phages; biochemical phenotyping of fecal coliform bacteria and enterococci using miniaturized tests; specific detection of Bifidobacterium adolescentis and Bifidobacterium dentium; and measurement of four fecal sterols. A number of potentially useful source indicators were detected (bacteriophages infecting B. thetaiotaomicron, certain genotypes of F-specific bacteriophages, sorbitol-fermenting bifidobacteria, 24-ethylcoprostanol, and epycoprostanol), although no one source identifier alone provided 100% correct classification of the fecal source. Subsequently, 38 variables (both single and derived) were defined from the measured microbial and chemical parameters in order to find the best subset of variables to develop predictive models using the lowest possible number of measured parameters. To this end, several statistical or machine learning methods were evaluated and provided two successful predictive models based on just two variables, giving 100% correct classification: the ratio of the densities of somatic coliphages and phages infecting Bacteroides thetaiotaomicron to the density of somatic coliphages and the ratio of the densities of fecal coliform bacteria and phages infecting Bacteroides thetaiotaomicron to the density of fecal coliform bacteria. Other models with high rates of correct classification were developed, but in these cases, higher numbers of variables were required.

Comparison of Two Target Genes for Detection and Genotyping of Giardia lamblia in Human Feces by PCR and PCR-Restriction Fragment Length Polymorphism

ABSTRACT A PCR assay targeting the tpi gene was developed to detect and to genotype Giardia lamblia in human feces. Our assay was specific and discriminated between G. lamblia assemblages A and B. G. lamblia cysts isolated from human feces were also analyzed with two previously described PCR-restriction fragment length polymorphism (RFLP) assays, which are based on the detection of tpi or gdh genes. These RFLP analyses distinguished groups I and II within assemblage A or groups III and IV within assemblage B. Among 26 fecal samples from patients with sporadic giardiasis diagnosed by hospital laboratories, the tpi gene was amplified from 25 (96%) with our PCR assay, whereas only 21 (81%) samples were positive when the gdh gene was targeted. Of the 25 positive samples, nine (36%) contained assemblage A and 16 (64%) contained assemblage B. Thus, RFLP analysis classified eight samples (32%) in assemblage A group II, eight (32%) in assemblage B group III, and five (20%) in assemblage B group IV. The group could not be specified for four samples. The tpi and gdh genes of G. lamblia assemblage B were amplified from 14 (93%) of 15 samples collected only from French soldiers coming back from the Ivory Coast. All of these contained assemblage B group III. The PCR method developed is sensitive, simple, and specific and shows that the tpi gene is well adapted for G. lamblia genotyping.

Irradiation of Ascaris ova in sludge using an electron beam accelerator.

The efficiency of sludge disinfection by irradiation was investigated using an electron beam accelerator, with the Ascaris ovum as a model. Ova suspensions prepared by worm dissection, immediately after preparation and after storage at 4 degrees C for 2 months were tested. Suspensions of ova extracted from slaughterhouse sludge were also tested. The ova were irradiated in sludge to determine, by probit analysis, the dose that inactivated 90% of viable ova. The D10 values obtained for irradiation of residual sludge contaminated with ova depended on the source of the ova, the D10 values were 788 +/- 172 Gy for suspensions of ova extracted from slaughterhouse sludge and 1125 +/- 145 Gy for suspensions freshly prepared by dissection. Ova suspensions freshly prepared by dissection were more proof against irradiation. Similarly, the D10 value was affected by storage: 1125 +/- 145 Gy for freshly produced ova suspensions and 661 +/- 45 Gy for suspensions of ova stored for 2 months at 4 degrees C in deionized water. The medium in which the ova were irradiated (deionized water or sludge) also affected D10 values, the indirect effects were smaller in samples of contaminated sludge, which were rich in organic matter, with the action of the radiation being mostly direct.

Optimised immunofluorescence procedure for enumeration of Cryptosporidium parvum oocyst suspensions.

The aim of this study was to evaluate an optimised immunofluorescence assay in terms of the variability of sets of counts for Cryptosporidium parvum oocyst suspensions and data recovery and the reliability of the procedure. A coefficient of variation (CV) of 10% was determined to be the maximum value acceptable for count variability. It was found that the optimised IFA tested provided a high precision for the sets of enumerations for suspensions containing 800-20,000 oocysts/mL. The procedure was found to be robust and providing high recovery level (96.3%). In terms of counting precision, the technique described here approaches the performance of flow cytometry and surpasses other manual techniques with a CV of 10% for a concentration close to 800 oocysts/mL. The procedure described is particularly suitable for the production of seed doses and for other applications requiring the titration of oocyst suspensions with a high degree of precision and accuracy.