Characteristics of extended-spectrum cephalosporin-resistant Escherichia coli isolated from Swiss and imported poultry meat.

A worrisome phenomenon is the progressive global spread of Enterobacteriaceae in poultry and chicken meat expressing plasmid-mediated enzymes that inactivate ß-lactam antibiotics, suggesting that the food chain might play a role in the epidemiology and the transmission of extended-spectrum cephalosporin-resistant Enterobacteriaceae to humans. The aim of the present study was to further characterize 24 extended-spectrum cephalosporin-resistant Enterobacteriaceae isolated from domestic and imported poultry meat by antibiotic susceptibility testing, identification of the blaESBL/blapAmpC genes, conjugation mating experiments and determination of plasmid incompatibility types, multilocus sequence typing, and analysis of the Escherichia coli phylogenetic groups. On account of their resistance patterns, 21 of the total 24 isolates were classified as multidrug resistant. Eleven isolates carried a blaCMY-2 gene, whereas 13 isolates harbored a blaCTX-M-1 gene. All isolates harbored plasmids that were assigned to 8 of the 18 described plasmid incompatibility groups, the most frequent of which were IncI1, IncFIB, IncB/O, and IncFrepB. The blaESBL/blapAmpC genes were harbored mainly by transferable IncI1 and IncB/O plasmids. Multilocus sequence typing as well as E. coli phylogenetic group typing revealed a high heterogenicity even among different isolates of the same sample.

Detection of adeno- and lentiviral (HIV1) contaminations on laboratory surfaces as a tool for the surveillance of biosafety standards.

AIMS: As a biosafety laboratory, we survey the handling of adenovirus type 5 (Ad5) and HIV1-derived lentivirus in contained-use facilities in Switzerland to identify insufficiencies of the safety precautions taken by the laboratories. METHODS AND RESULTS: In the past 9 years, we took 430 swab samples from various types of surfaces in research laboratories. Samples were examined for Ad5 contaminations by real-time PCR and infectivity assay or for the presence of lentivirus (HIV1) nucleic acids by real-time (RT) PCR. Samples collected from centrifuges did not only contain Ad5 DNA more frequently but also exhibited higher numbers of Ad5 and lentiviral (HIV1) DNA copies than swabs from any other area of sampling. Five of ten samples containing infectious Ad5 particles or lentivirus (HIV1) RNA were found in samples taken from centrifuges. Ad5 contamination rates were higher in the tube holder and lower on the inner wall of the rotor chamber in centrifuges that were fitted with aerosol tight covers compared to centrifuges without covers. CONCLUSIONS: Our results allowed the comparison of hygiene standards of different laboratories and lead to the identification of centrifuges as hotspots for contaminations. SIGNIFICANCE AND IMPACT OF THE STUDY: Based on our results, we propose to use the collected data as a tool for rating future swab results. Furthermore, the amount of Ad5 and HIV1-derived lentivirus DNA could serve as an indicator of the level of good laboratory practice in contained-use laboratories handling these viral vectors.

Contaminations of laboratory surfaces with Staphylococcus aureus are affected by the carrier status of laboratory staff.

AIM: As a biosafety laboratory, we take samples from surfaces in microbiological laboratories to survey the handling of micro-organisms. Whereas contaminations with other micro-organisms were rare, Staphylococcus aureus was found in the working environment of many laboratories. As 20-60% of the healthy population are carriers of S. aureus we wanted to asses the effect of carriers on our sampling results. METHODS AND RESULTS: Nasal swabs of staff members in nonmicrobiological laboratories and offices as well as surface samples from their personal work environment were taken and analysed for S. aureus DNA. In addition S. aureus strains were isolated using S. aureus-specific agar plates and analysed by randomly amplified polymorphic DNA (RAPD)-PCR and multilocus sequence typing (MLST). Our data show that contaminations with S. aureus in nonmicrobiological environments are common with 29% of the surface samples containing S. aureus DNA. In the working environment of carriers, the number of contaminations was significantly increased compared to the environment of noncarriers. CONCLUSION: The carrier status of staff members significantly affects the number of contaminations on laboratory surfaces. Therefore, even in the absence of intentional handling of S. aureus, contaminations can be detected on a substantial amount of surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: Sampling procedures need to be adapted based on these results with respect to the locations where samples are taken and the threshold for significant contaminations. Because of its wide distribution, S. aureus can serve as a marker for hygienic standards in laboratories.

Validation of PCR methods for quantitation of genetically modified plants in food.

For enforcement of the recently introduced labeling threshold for genetically modified organisms (GMOs) in food ingredients, quantitative detection methods such as quantitative competitive (QC-PCR) and real-time PCR are applied by official food control laboratories. The experiences of 3 European food control laboratories in validating such methods were compared to describe realistic performance characteristics of quantitative PCR detection methods. The limit of quantitation (LOQ) of GMO-specific, real-time PCR was experimentally determined to reach 30-50 target molecules, which is close to theoretical prediction. Starting PCR with 200 ng genomic plant DNA, the LOQ depends primarily on the genome size of the target plant and ranges from 0.02% for rice to 0.7% for wheat. The precision of quantitative PCR detection methods, expressed as relative standard deviation (RSD), varied from 10 to 30%. Using Bt176 corn containing test samples and applying Bt176 specific QC-PCR, mean values deviated from true values by -7to 18%, with an average of 2+/-10%. Ruggedness of real-time PCR detection methods was assessed in an interlaboratory study analyzing commercial, homogeneous food samples. Roundup Ready soybean DNA contents were determined in the range of 0.3 to 36%, relative to soybean DNA, with RSDs of about 25%. Taking the precision of quantitative PCR detection methods into account, suitable sample plans and sample sizes for GMO analysis are suggested. Because quantitative GMO detection methods measure GMO contents of samples in relation to reference material (calibrants), high priority must be given to international agreements and standardization on certified reference materials.

IUPAC collaborative trial study of a method to detect genetically modified soy beans and maize in dried powder.

This paper presents results of a collaborative trial study (IUPAC project No. 650/93/97) involving 29 laboratories in 13 countries applying a method for detecting genetically modified organisms (GMOs) in food. The method is based on using the polymerase chain reaction to determine the 35S promotor and the NOS terminator for detection of GMOs. reference materials were produced that were derived from genetically modified soy beans and maize. Correct identification of samples containing 2% GMOs is achievable for both soy beans and maize. For samples containing 0.5% genetically modified soy beans, analysis of the 35S promotor resulted also in a 100% correct classification. However, 3 false-negative results (out of 105 samples analyzed) were reported for analysis of the NOS terminator, which is due to the lower sensitivity of this method. Because of the bigger genomic DNA of maize, the probability of encountering false-negative results for samples containing 0.5% GMOs is greater for maize than for soy beans. For blank samples (0% GMO), only 2 false-positive results for soy beans and one for maize were reported. These results appeared as very weak signals and were most probably due to contamination of laboratory equipment.

A dissection of the cauliflower mosaic virus polyadenylation signal.

Mutagenesis analysis of the polyadenylation [poly(A)] signal from the cauliflower mosaic virus (CaMV), a plant pararetrovirus, revealed striking differences to known vertebrate poly(A) signals. Our results show that (1) the AATAAA sequence is necessary for efficient cleavage at the poly(A) site, although the requirement for an authentic AATAAA might be less stringent in plant than in vertebrate cells; (2) surprisingly and in contrast to the majority of vertebrate poly(A) signals, the sequences downstream of the CaMV poly(A) site do not influence processing efficiency drastically although they affect the precision of cleavage; and (3) deletion of sequences upstream of the CaMV AATAAA sequence decreased processing at the CaMV site dramatically, suggesting the presence of one or several positively acting upstream elements. An oligonucleotide consisting of CaMV upstream sequences could induce the recognition of a normally silent exogenous poly(A) signal when inserted upstream of its AATAAA motif.