Richard Felleisen

Identification of Yersinia enterocolitica at the Species and Subspecies Levels by Fourier Transform Infrared Spectroscopy

ABSTRACT Yersinia enterocolitica and other Yersinia species, such as Y. pseudotuberculosis, Y. bercovieri, and Y. intermedia, were differentiated using Fourier transform infrared spectroscopy (FT-IR) combined with artificial neural network analysis. A set of well defined Yersinia strains from Switzerland and Germany was used to create a method for FT-IR-based differentiation of Yersinia isolates at the species level. The isolates of Y. enterocolitica were also differentiated by FT-IR into the main biotypes (biotypes 1A, 2, and 4) and serotypes (serotypes O:3, O:5, O:9, and “non-O:3, O:5, and O:9”). For external validation of the constructed methods, independently obtained isolates of different Yersinia species were used. A total of 79.9% of Y. enterocolitica sensu stricto isolates were identified correctly at the species level. The FT-IR analysis allowed the separation of all Y. bercovieri, Y. intermedia, and Y. rohdei strains from Y. enterocolitica, which could not be differentiated by the API 20E test system. The probability for correct biotype identification of Y. enterocolitica isolates was 98.3% (41 externally validated strains). For correct serotype identification, the probability was 92.5% (42 externally validated strains). In addition, the presence or absence of the ail gene, one of the main pathogenicity markers, was demonstrated using FT-IR. The probability for correct identification of isolates concerning the ail gene was 98.5% (51 externally validated strains). This indicates that it is possible to obtain information about genus, species, and in the case of Y. enterocolitica also subspecies type with a single measurement. Furthermore, this is the first example of the identification of specific pathogenicity using FT-IR.

Rapid detection of total and viable Legionella pneumophila in tap water by immunomagnetic separation, double fluorescent staining and flow cytometry

We developed a rapid detection method for Legionella pneumophila (Lp) by filtration, immunomagnetic separation, double fluorescent staining, and flow cytometry (IMS‐FCM method). The method requires 120 min and can discriminate ‘viable’ and ‘membrane‐damaged’ cells. The recovery is over 85% of spiked Lp SG 1 cells in 1 l of tap water and detection limits are around 50 and 15 cells per litre for total and viable Lp, respectively. The method was compared using water samples from house installations in a blind study with three environmental laboratories performing the ISO 11731 plating method. In 53% of the water samples from different taps and showers significantly higher concentrations of Lp were detected by flow cytometry. No correlation to the plate culture method was found. Since also ‘viable but not culturable’ (VNBC) cells are detected by our method, this result was expected. The IMS‐FCM method is limited by the specificity of the used antibodies; in the presented case they target Lp serogroups 1–12. This and the fact that no Lp‐containing amoebae are detected may explain why in 21% of all samples higher counts were observed using the plate culture method. Though the IMS‐FCM method is not yet fit to completely displace the established plating method (ISO 11731) for routine Lp monitoring, it has major advantages to plating and can quickly provide important insights into the ecology of this pathogen in water distribution systems.

Novel Bacteroides host strains for detection of human- and animal-specific bacteriophages in water

Bacteriophages active against specific Bacteroides host strains were shown to be suitable for detection of human faecal pollution. However, the practical application of this finding is limited because some specific host strains were restricted to certain geographic regions. In this study, novel Bacteroides host strains were isolated that discriminate human and animal faecal pollution in Switzerland. Two strains specific for bacteriophages present in human faecal contamination and three strains specific for bacteriophages indicating animal faecal contamination were evaluated. Bacteriophages infecting human strains were exclusively found in human wastewater, whereas animal strains detected bacteriophages only in animal waste. The newly isolated host strains could be used to determine the source of surface and spring water faecal contamination in field situations. Applying the newly isolated host Bacteroides thetaiotaomicron ARABA 84 for detection of bacteriophages allowed the detection of human faecal contamination in spring water.

Market Surveillance for Contamination with Thermotolerant Campylobacters on Various Categories of Chicken Meat in Switzerland

From April 2009 to April 2010, 1,132 samples of different types of chicken meat were tested qualitatively and quantitatively for thermotolerant campylobacters. Samples were recovered at retail in shops from the entire territory of Switzerland and comprised imported meat and meat from domestic production. The meat categories covered by the study were refrigerated and frozen meat, meat with and without skin, and meat preparations. Overall, 38.4% of the samples were positive, and in 27.8%, Campylobacter bacteria could be quantified. Counts ranged from ≥10 to <10(4) CFU/g with a maximum value of 8 × 10(3) CFU/g in a sample of refrigerated chicken meat with skin. The contamination frequencies were 45.2% in meat with skin, 40.8% in meat without skin, and 27.4% in meat preparations. Refrigerated meat was contaminated with Campylobacter bacteria more often than frozen meat (53.9 versus 20.0%). The study also showed considerable differences between the contamination rates found for samples from different large retail chains. In 2010, a further study with 120 samples of refrigerated and sliced chicken meat and fresh chicken liver was carried out in order to test a possible seasonal variation of the occurrence of Campylobacter bacteria. The contamination frequency of sliced meat increased from 10.0% in the period from February to March to 36.7% during July to August. In both sampling periods, the counts remained in the range of ≥10 to <100 CFU/g with a maximum value of 30 CFU/g. For chicken liver, a 10.0% contamination rate was observed in the period from December to January, which rose to 100% in the period from August to October. Contrary to the results for sliced meat, not only did the frequency of contamination increase but so did the Campylobacter counts, with the highest recorded value being 2.2 × 10(4) CFU/g.

Identification of Fecal Input Sites in Spring Water by Selection and Genotyping of Multiresistant Escherichia coli

ABSTRACT The localization of fecal input sites is important for water quality management. For this purpose, we have developed a new approach based on a three-step procedure, including a preparatory phase, the screening of multiresistant bacteria using selective agar plates, and a typing phase where selected Escherichia coli isolates are characterized by antibiotic resistance profiles and molecular fingerprinting techniques (pulsed-field gel electrophoresis [PFGE]). These two well-known source tracking methods were combined in order to reduce cost and effort. This approach was successfully applied under field conditions in a study area located in the north-western part of Switzerland. E. coli isolates from spring water and surface water samples collected in this area were screened with selective agar plates. In this way, 21 different groups, each consisting of strains with the same pattern of antibiotic resistance, were found. Of these, four groups were further analyzed using PFGE. Strains with identical PFGE profiles were detected repeatedly, demonstrating the suitability of this method for the localization of fecal input sites over an extended period of time. Identical PFGE patterns of strains detected in water from two different springs were also found in the stream flowing through the study area. These results demonstrated the applicability of the new approach for the examination of incidents of fecal contamination in drinking water. The advantages of the described approach over genotyping methods currently being used to identify sources of fecal contaminants are a reduction in time, costs, and the effort required. Identical isolates could be identified without the construction of large libraries.

Improved detection of Rhodococcus coprophilus with a new quantitative PCR assay

Agricultural practices, such as spreading liquid manure or the utilisation of land as animal pastures, can result in faecal contamination of water resources. Rhodococcus coprophilus is used in microbial source tracking to indicate animal faecal contamination in water. Methods previously described for detecting of R. coprophilus in water were neither sensitive nor specific. Therefore, the aim of this study was to design and validate a new quantitative polymerase chain reaction (qPCR) to improve the detection of R. coprophilus in water. The new PCR assay was based on the R. coprophilus 16S rRNA gene. The validation showed that the new approach was specific and sensitive for deoxyribunucleic acid from target host species. Compared with other PCR assays tested in this study, the detection limit of the new qPCR was between 1 and 3 log lower. The method, including a filtration step, was further validated and successfully used in a field investigation in Switzerland. Our work demonstrated that the new detection method is sensitive and robust to detect R. coprophilus in surface and spring water. Compared with PCR assays that are available in the literature or to the culture-dependent method, the new molecular approach improves the detection of R. coprophilus.

Assessment of source tracking methods for application in spring water

For discriminating between human and animal faecal contamination in water, microbial source tracking (MST) approaches using different indicators have been employed. In the current study, a range of 10 such MST indicators described in the scientific literature were comparatively assessed. Bacteriophages infecting host strains of Bacteroides (GA-17, GB-124 and ARABA 84) as well as sorbitol-fermenting bifidobacteria proved useful for indicating human faecal contamination while Rhodococcus coprophilus was associated with animal-derived faecal contamination. These potential source indicators were present in samples of faecal origin, i.e. either in human wastewater or animal waste, from many different regions in Switzerland and therefore showed a geographic stability. In addition, the MST indicators were abundant in surface water and were even sensitive enough to detect faecal contamination in spring water from two study areas in Switzerland. This is the first study that has compared and successfully applied MST methods in spring water.

Microbial source tracking in highly vulnerable karst drinking water resources

Water resources situated in areas with underlying karst geology are particularly vulnerable to fecal pollution. In such vulnerable systems, microbial source tracking (MST) methods are useful tools to elucidate the pathways of both animal and human fecal pollution, leading to more accurate water use risk assessments. Here, we describe the application of a MST toolbox using both culture-dependent bacteriophage and molecular-dependent 16S rRNA assays at spring and well sites in the karstic St Imier Valley, Switzerland. Culture-dependent and molecular-dependent marker performance varied significantly, with the 16S rRNA assays displaying greater sensitivity than their phage counterpart; HF183 was the best performing human wastewater-associated marker while Rum2Bac was the best performing ruminant marker. Differences were observed in pollution regimes between the well and spring sampling sites, with the spring water being more degraded than the well site. Our results inform the choice of marker selection for MST studies and highlight differences in microbial water quality between well and spring karst sites.

Real-time PCR method for the detection of figwort mosaic virus (FMV) to complement the FMV 34S promoter-specific PCR assay used for screening of genetically modified plants

In this study a new real-time PCR assay for the detection of figwort mosaic virus (FMV) DNA is described. This assay targets a 113-bp-long sequence of the FMV open reading frame Vll, a non-conserved coding region among the caulimoviruses. Detection of FMV DNA is useful to complement screening for the FMV 34S promoter (P-FMV), a genetic element present in several genetically modified (GM) plants. The specificity of the assay was assessed against closely related plant viruses, plant species of agronomic importance or susceptible to infection by FMV, and various GM plants containing the P-FMV sequence. No cross-reactivity of the assay against the tested nontarget organisms was observed. The limit of detection of the FMV real-time PCR assay was determined at approximately 5 copies per reaction. The robustness of the method was tested using different real-time PCR instruments and PCR master mixes with no negative effects on the PCR efficiency and linearity being observed. When amplifying the FMV target DNA at a concentration level close to the detection limit, no negative influence on the PCR performance was observed in the presence of background genomic DNA from various plant species. The precision data of the in-house validation were in line with generally accepted performance requirements. In summary, the method appears fit for the purpose of a specific identification test for the presence of genomic FMV DNA, while preventing false-positive results during P-FMV screening.