ABSTRACT
Central venous catheter needleless connectors (NCs) have been shown to develop microbial contamination. A protocol was developed for the collection, processing, and examination of NCs to detect and measure biofilms on these devices. Sixty-three percent of 24 NCs collected from a bone marrow transplant center contained biofilms comprised primarily of coagulase-negative staphylococci.
Subject(s)
Biofilms , Catheterization, Central Venous/instrumentation , Catheters, Indwelling , Equipment Contamination , Disinfection/methods , Enterobacter cloacae/isolation & purification , Enterobacter cloacae/physiology , Equipment Design , Geobacillus stearothermophilus/isolation & purification , Geobacillus stearothermophilus/physiology , Humans , Microscopy, Electron, Scanning , Reproducibility of Results , Spores, Bacterial , Staphylococcus/isolation & purification , Staphylococcus/physiology , Staphylococcus/ultrastructureABSTRACT
Enterohemorrhagic Escherichia coli (EHEC) and specifically serotype O157:H7 are a significant cause of hemorrhagic gastrointestinal disease and the hemolytic uremic syndrome. Methods currently used in clinical microbiology labs, such as sorbitol-MacConkey (SMAC) agar, reliably detect only O157:H7. We have evaluated a two-step method that has the potential to identify and isolate all EHEC serotypes, including serotype O157:H7. This method utilizes a chromogenic selective-differential medium for the isolation of E. coli together with an enzyme-linked immunosorbent assay (ELISA) that detects the Shiga-like toxins Stx1 and Stx2. Both are commercially available and usable in a wide range of clinical microbiology laboratories. Compared to a Vero cell cytotoxic assay, SMAC had sensitivities of 23.5% for the identification of all EHEC serotypes and of 50.0% for the identification of O157:H7 alone. The two-step method had sensitivities of 76.5 and 100%, respectively. The ELISA alone had a sensitivity of 82.4% in the detection of Stx1 and Stx2. The specificity was 100% in all cases. Overall, 14 EHEC isolates were obtained: 8 (58%) O157:H7, 2 (14%) O26, 2 (14%) O111:NM, 1 (7%) O103:H2, and 1 (7%) O121:H19. All but one were isolated during the months of May to September. The two-step method was found to be considerably more expensive than SMAC for both positive and negative samples.
Subject(s)
Bacterial Toxins/metabolism , Culture Media , Enzyme-Linked Immunosorbent Assay/methods , Escherichia coli Infections/microbiology , Escherichia coli/classification , Escherichia coli/isolation & purification , Agar , Animals , Bacterial Toxins/immunology , Bacterial Typing Techniques , Chlorocebus aethiops , Chromogenic Compounds/metabolism , Culture Media/economics , Cytotoxicity Tests, Immunologic , Enzyme-Linked Immunosorbent Assay/economics , Escherichia coli/metabolism , Escherichia coli Infections/diagnosis , Escherichia coli Infections/epidemiology , Humans , Serotyping , Shiga Toxins , Vero CellsABSTRACT
Many bacterial genera, including Bacteroides spp., harbor mobilizable transposons, a class of transfer factors that carry genes for conjugal DNA transfer and, in some cases, antibiotic resistance. Mobilizable transposons are capable of inserting into and mobilizing other, nontransferable plasmids and are implicated in the dissemination of antibiotic resistance. This paper presents the isolation and characterization of Tn5520, a new mobilizable transposon from Bacteroides fragilis LV23. At 4,692 bp, it is the smallest mobilizable transposon reported from any bacterial genus. Tn5520 was captured from B. fragilis LV23 by using the transfer-deficient shuttle vector pGAT400DeltaBglII. The termini of Tn5520 contain a 22-bp imperfect inverted repeat, and transposition does not result in a target site repeat. Tn5520 also demonstrates insertion site sequence preferences characterized by A-T-rich nucleotide sequences. Tn5520 has been sequenced in its entirety, and two large open reading frames whose predicted protein products exhibit strong sequence similarity to recombinase-integrase enzymes and mobilization proteins, respectively, have been identified. The transfer, mobilization, and transposition properties of Tn5520 have been studied, revealing that Tn5520 mobilizes plasmids in both B. fragilis and Escherichia coli at high frequency and also transposes in E. coli.
Subject(s)
Bacteroides fragilis/genetics , DNA Transposable Elements , Genes, Bacterial , Integrases/genetics , Bacteroides fragilis/enzymology , Base Sequence , Drug Resistance, Microbial/genetics , Escherichia coli/genetics , Gene Transfer Techniques , Molecular Sequence Data , Open Reading Frames , Repetitive Sequences, Nucleic Acid , Sequence Analysis, DNAABSTRACT
A 4.2-kb plasmid (pLV22a) native to Bacteroides fragilis LV22 became fused to a transfer-deficient Bacteroides spp.-Escherichia coli shuttle vector by an inverse transposition event, resulting in a transferrable phenotype. The transfer phenotype was attributable to pLV22a, which was also capable of mobilization within E. coli when coresident with the IncP beta R751 plasmid. Transposon mutagenesis with Tn1000 localized the mobilization region to a 1.5-kb DNA segment in pLV22a. The mobilization region has been sequenced, and five open reading frames have been identified. Mutants carrying disruptions in any of the three genes designated mbpA, mbpB, and mbpC and coding for deduced products of 11.3, 30.4, and 17.1 kDa, respectively, cannot be mobilized when coresident with R751. Mutations in all three genes can be complemented in the presence of the respective wild-type genes, indicating that the products of mbpA, mbpB, and mbpC have roles in the mobilization process and function in trans. The deduced 30.4-kDa MbpB protein contains a 14-amino-acid conserved motif that is also found in the DNA relaxases of a variety of conjugal and mobilizable plasmids and the conjugative transposon Tn4399. Deletion analysis and complementation experiments have localized a cis-acting region of pLV22a within mbpA.
