Isolation and detection of extended spectrum ß-lactamase (ESBL)-producing enterobacteriaceae from meat using chromogenic agars and isothermal loop-mediated amplification (LAMP) assays.

The aim of this work was to develop a molecular method using loop-mediated isothermal amplification (LAMP) for detection of extended spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae from meat, and to compare it with different isolation agars and microarrays. LAMP assays were developed for CTX-M groups 1, 2, and 9 and OXA-10-like genes. Chicken, lamb, beef, pork, and turkey samples were spiked with 10, 100, and 1,000 cfu/gram using 8 strains of ESBL-producing Enterobacteriaceae (CTX-M sequence types 1, 2, 3, 14, 15, OXA-11, SHV-2, TEM-52) +/- a mix of competitor organisms. Samples were enriched overnight in buffered peptone water (BPW) +/- antibacterials before plating to CHROMagar CTX, OXOID ESBL Brilliance agar, and MacConkey agar with 1 mg/L cefotaxime. Selected BPW broths were also tested using LAMP assays, microarrays and using cefpodoxime discs on agar. For isolation/detection of ESBL producers from beef, pork, lamb, and turkey spiked with 10 or 100 cfu/gram ESBL (natural flora only), all agars and the LAMP assays showed 100% sensitivity and specificity for ESBL spike strains. For chicken samples, both LAMP and chromogenic agars showed improved sensitivity and specificity for isolation of ESBLs compared with MacConkey agar, particularly with competitor bacteria added. In comparison, the cefpodoxime disc method and microarray showed reduced sensitivity.

Plasmid-mediated catabolism of dicamba by Pseudomonas species strain PXM.

Pseudomonas species strain PXM, which is able to use dicamba (3,6-dichloro-2-methoxybenzoic acid; CAS 1918-00-9, Banvel) as its sole carbon source for growth, has been isolated. The catabolism of dicamba and some of its putative metabolic descendants correlates with the presence of a large and unstable plasmid.

Detection, isolation, and stability of megaplasmid-encoded chloroaromatic herbicide-degrading genes within Pseudomonas species.

Dicamba is used as a model system for microbial degradation of chloroaromatic benzoic acids. The detection, isolation, and stability of a megaplasmid within a Pseudomonas sp. is described as the first step in optimizing the growth of this microorganism and other microorganisms similar to it. A large plasmid, pDK1, consisting of approximately 250 kb, was purified from dicamba-degrading Pseudomonas sp. PXM. This plasmid was purified by the method of Allen (personal communication, 1994), which is a modified version of several that have been attempted for the isolation of large plasmids (Lee and Rasheed, 1990). The restriction analysis of this plasmid (pDK1) from PXM. revealed many distinctive bands on agarose gel electrophoresis. Based on the preliminary restriction enzyme analysis, the estimated size of this plasmid is 250 kb, which could make it one of the largest procaryotic plasmids encoding for chloroaromatic degrading enzymes. Allen's methodology results in very high purity and reproducibility compared to the other methods used in this study. As described in this work, the method of Kado and Liu (1981) is easier to perform and results in a more reproducible plasmid preparation than the method of Casse et al. (1979). Casse's protocol requires the use of a highly alkaline SDS solution (pH 12.45) in order to eliminate the chromosomal DNA. However, only incomplete removal of the chromosomal DNA results. Compared to the Casse et al. protocol, the Kado and Liu protocol requires the use of a highly alkaline solution (pH 12.6) and a high temperature (55-65 degrees C) to eliminate the chromosomal DNA. This results in a nearly complete removal of the chromosomal DNA. The high temperature treatment also quickly eliminates the RNA. Another advantage of the protocol of Kado and Liu over the protocol of Casse et al. is that the former uses phenol-chloroform extraction while the latter uses only phenol extraction. The phenol-chloroform extraction step denatures the DNA along with the proteins. In addition to this, the phenol-chloroform mixture minimizes the formation of a brown oxidation pigment that usually occurs with phenol extraction alone. Finally, the time needed to complete the Kado and Liu protocol is much shorter (2 hr) than the time needed to complete the Casse protocol (8 hr). As described previously, a highly purified plasmid preparation with minimal chromosomal DNA was prepared by following the suggestions of L. Allen.(ABSTRACT TRUNCATED AT 400 WORDS)

Chlorobium limicola forma thiosulfatophilum: Biocatalyst in the Production of Sulfur and Organic Carbon from a Gas Stream Containing H(2)S and CO(2).

Chlorobium limicola forma thiosulfatophilum (ATCC 17092) was grown in a 1-liter continuously stirred tank reactor (800-ml liquid volume) at pH 6.8, 30 degrees C, saturated light intensity, and a gas flow rate of 23.6 ml/min from a gas cylinder blend consisting of 3.9 mol% H(2)S, 9.2 mol% CO(2), 86.4 mol% N(2), and 0.5 mol% H(2). This is the first demonstration of photoautotrophic growth of a Chlorobium sp. on a continuous inorganic gas feed. A significant potential exists for applying this photoautotrophic process to desulfurization and CO(2) fixation of gases containing acidic components (H(2)S and CO(2)).