The Luminex Gastrointestinal Pathogen Panel (xTAG(®) GPP) detects in one assay the most common gastroenteritis-causing pathogens and toxins, namely adenovirus 40/41, norovirus genogroup (NG) I/II, rotavirus A, Clostridium difficile toxin A/B, Campylobacter sp., Escherichia coli O157, Enterotoxigenic E. coli heat-labile enterotoxin/heat-stable enterotoxin, Salmonella sp., Shiga-toxin producing E. coli, Shiga-like toxin (Stx)1/2, Shigella sp., Vibrio cholerae, Yersinia enterocolitica, Cryptosporidium sp., Entamoeba histolytica and Giardia sp. In this study, we compared the results that were obtained by testing 393 faecal samples, collected during November and December 2011 at our laboratory, using the xTAG(®) GPP assay with the results of the routine diagnostic procedure. This procedure includes culture for bacteria and real-time PCR for viruses and parasites, but only if the test was requested by the clinician. If the clinician did not request the test for an xTAG(®) GPP-positive target, real-time PCR assays were used to confirm xTAG(®) GPP positivity. Discrepant results were also tested with real-time PCR assays. A total of 83 targets were detected in 76 samples using xTAG(®) GPP. The xTAG(®) GPP assay detected 43 additional positives compared with the routine diagnostic procedure, of which 11 targets could not be confirmed by real-time PCR. The non-confirmed targets were Campylobacter (one sample), Salmonella (four samples), Shigella (one sample) and E. histolytica (five samples). The xTAG(®) GPP was shown to be a convenient and sensitive assay for detection of 15 major gastrointestinal pathogens in a single molecular test, but for detection of E. histolytica and Salmonella, a confirmatory assay is indicated.

A chromosomal gene of Enterobacter cloacae encoding an outer membrane protein (OmpX) has been cloned. Overproduction of the OmpX protein decreased the quantity of porins in the outer membrane of the parental strain and of Escherichia coli HB101. The The outer membranes of gram-negative bacteria obtain their nonspecific permeability for hydrophilic solutes from the presence of porins, a class of proteins that form waterfilled diffusion channels (22). Lack of these proteins leads to resistance to most beta-lactam antibiotics and quinolones (1, 21). In a previous report (28), we described the cloning of a restriction fragment of Enterobacter cloacae chromosomal DNA that was associated with resistance to beta-lactam antibiotics in transformants both of Escherichia coli and of the parental E. cloacae strain. In these transformants, the amount of the porin proteins OmpF and OmpC present decreased, and an outer membrane protein of approximately 18 kDa was overproduced. In vitro transcription and translation of the recombinant plasmid showed that this protein was encoded by a gene on the cloned fragment. The function of the protein is still unknown, but its overproduction clearly interferes with the presence of OmpF and OmpC in the outer membrane. We propose the name OmpX for this protein, as long as its proper function is obscure. In the accompanying paper (29), the biological characterization of OmpX is presented. In this report, we present the nucleotide sequence of the ompX gene and the deduced amino acid sequence. From these data, some physical properties of OmpX are derived. MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains, plasmids, and bacteriophages used are listed in Table 1.Growth conditions. Bacteria were grown at 37°C in brain heart infusion broth (Oxoid Ltd.) supplemented when required with chloramphenicol (34 jig/ml Insertions of the transposable element -yb (11) in plasmid pJS04 were obtained as follows. DNA of plasmid pJS04 was introduced into the F' lac+-containing E. coli CE1304 by transformation. Transformants were selected on Iso-Sensitest agar (Oxoid CM471) containing chloramphenicol (34 ,ug/ml). Several transformants were subsequently mated with E. coli W3110 by filter mating on agar plates for 18 h at 37°C. Cells were collected from the filter, and the mixture was plated on Iso-Sensitest agar plates containing chloramphenicol and nalidixic acid (80 ,ug/ml). Transconjugants were screened subsequently for growth on Iso-Sensitest agar plates containing cephalothin (10 ,g/ml). The positions of the -yb sequence insertions were localized by restriction enzyme digestions.Analysis of plasmid-encoded proteins. Plasmid-encoded proteins were synthesized in vitro in a procaryotic DNAdirected transcription-translation system (Amersham, U.K.

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The NucliSens Extractor is an automated nucleic acid isolation system based on guanidinium thiocyanate (GuSCN)-silica extraction technology. The system has been validated for the isolation of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) RNAs from human samples in combination with nucleic acid sequence-based amplification-and reverse transcription-PCR-based methods. We evaluated the extractor for hepatitis B virus (HBV) DNA extraction from human samples using a noncommercial HBV DNA PCR. Several sample pretreatment procedures in combination with the extractor were compared with the Qiagen extraction method, and the impact of the sample volume used in the extraction on the sensitivity was investigated. Heating of the lysed sample prior to extractor isolation and the use of a large sample volume resulted in highly sensitive detection of HBV DNA. Incubation of a 1-ml sample in GuSCN at 80°C (10 min) and at 37°C (30 min) allowed detection of 4 and 40 HBV genome equivalents/ml, respectively, in standard dilution panels. Sample lysis in GuSCN at room temperature and proteinase K treatment prior to use of the extractor were less efficient procedures. All clinical samples that were PCR positive after Qiagen extraction and/or that were HBsAg positive were also PCR positive after extractor isolation. HBV DNA, HCV RNA, and HIV type 1 RNA were efficiently coextracted from a single sample, allowing reliable detection of viral genomes.