Extended-spectrum beta-lactamases (ESBL) of the TEM, SHV, or CTX-M type confer resistance to betalactam antibiotics in Gram-negative bacteria. The activity of these enzymes against beta-lactam antibiotics and their resistance against inhibitors can be influenced by genetic variation at the single-nucleotide level. Here, we describe the development and validation of an oligonucleotide microarray for the rapid identification of ESBLs in Gram-negative bacteria by simultaneously genotyping bla TEM , bla SHV , and bla CTX-M . The array consists of 618 probes that cover mutations responsible for 156 amino acid substitutions. As this comprises unprecedented genotyping coverage, the ESBL array has a high potential for epidemiological studies and infection control. With an assay time of 5 h, the ESBL microarray also could be an attractive option for the development of rapid antimicrobial resistance tests in the future. The validity of the DNA microarray was demonstrated with 60 blinded clinical isolates, which were collected during clinical routines. Fifty-eight of them were characterized phenotypically as ESBL producers. The chip was characterized with regard to its resolution, phenotype-genotype correlation, and ability to resolve mixed genotypes. ESBL phenotypes could be correctly ascribed to ESBL variants of bla CTX-M (76%), bla SHV (22%), or both (2%), whereas no ESBL variant of bla TEM was found. The most prevalent ESBLs identified were CTX-M-15 (57%) and SHV-12 (18%).

BackgroundBifidobacteria belong to one of the predominant bacterial groups in the intestinal microbiota of infants and adults. Several beneficial effects on the health status of their human hosts have been demonstrated making bifidobacteria interesting candidates for probiotic applications. Adhesion of probiotics to the intestinal epithelium is discussed as a prerequisite for colonisation of and persistence in the gastrointestinal tract.ResultsIn the present study, 15 different strains of bifidobacteria were tested for adhesion. B. bifidum was identified as the species showing highest adhesion to all tested intestinal epithelial cell (IEC) lines. Adhesion of B. bifidum S17 to IECs was strongly reduced after treatment of bacteria with pronase. These results strongly indicate that a proteinaceous cell surface component mediates adhesion of B. bifidum S17 to IECs. In silico analysis of the currently accessible Bifidobacterium genomes identified bopA encoding a lipoprotein as a B. bifidum-specific gene previously shown to function as an adhesin of B. bifidum MIMBb75. The in silico results were confirmed by Southern Blot analysis. Furthermore, Northern Blot analysis demonstrated that bopA is expressed in all B. bifidum strains tested under conditions used to cultivate bacteria for adhesion assays. The BopA gene was successfully expressed in E. coli and purified by Ni-NTA affinity chromatography as a C-terminal His6-fusion. Purified BopA had an inhibitory effect on adhesion of B. bifidum S17 to IECs. Moreover, bopA was successfully expressed in B. bifidum S17 and B. longum/infantis E18. Strains overexpressing bopA showed enhanced adhesion to IECs, clearly demonstrating a role of BopA in adhesion of B. bifidum strains.ConclusionsBopA was identified as a B. bifidum-specific protein involved in adhesion to IECs. Bifidobacterium strains expressing bopA show enhanced adhesion. Our results represent the first report on recombinant bifidobacteria with improved adhesive properties.

Bifidobacteria are an important component of the human gastrointestinal microbiota and are frequently used as probiotics. The genetic inaccessibility and lack of molecular tools commonly used in other bacteria have hampered a detailed analysis of the genetic determinants of bifidobacteria involved in their adaptation to, colonization of, and interaction with the host. In the present study, a range of molecular tools were developed that will allow the closing of some of the gaps in functional analysis of bifidobacteria. A number of promoters were tested for transcriptional activity in Bifidobacterium bifidum S17 using pMDY23, a previously published promoter probe vector. The promoter of the gap gene (P gap ) of B. bifidum S17 yielded the highest promoter activity among the promoters tested. Thus, this promoter and the pMDY23 backbone were used to construct a range of vectors for expression of different fluorescent proteins (FPs). Successful expression of cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and mCherry could be shown for three strains representing three different Bifidobacterium spp. The red fluorescent B. bifidum S17/pVG-mCherry was further used to demonstrate application of fluorescent bifidobacteria for adhesion assays and detection in primary human macrophages cultured in vitro. Furthermore, pMGCmCherry was cloned by combining a chloramphenicol resistance marker and expression of the FP mCherry under the control of P gap . The chloramphenicol resistance marker of pMGC-mCherry was successfully used to determine gastrointestinal transit time of B. bifidum S17. Moreover, B. bifidum S17/pMGC-mCherry could be detected in fecal samples of mice after oral administration.

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