Evaluation of swabs and transport media for the recovery of Yersinia pestis.

The Government Accountability Office report investigating the surface sampling methods used during the 2001 mail contamination with Bacillus anthracis brought to light certain knowledge gaps that existed regarding environmental sampling with biothreat agents. Should a contamination event occur that involves non-spore forming biological select agents, such as Yersinia pestis, surface sample collection and processing protocols specific for these organisms will be needed. Two Y. pestis strains (virulent and avirulent), four swab types (polyester, macrofoam, rayon, and cotton), two pre-moistening solutions, six transport media, three temperatures, two levels of organic load, and four processing methods (vortexing, sonicating, combined sonicating and vortexing, no agitation) were evaluated to determine the conditions that would yield the highest percent of cultivable Y. pestis cells after storage. The optimum pre-moistening agent/transport media combination varied with the Y. pestis strain and swab type. Directly inoculated macrofoam swabs released the highest percent of cells into solution (93.9% recovered by culture) and rayon swabs were considered the second best swab option (77.0% recovered by culture). Storage at 4°C was found to be optimum for all storage times and transport media. In a worst case scenario, where the Y. pestis strain is not known and sample processing and analyses could not occur until 72h after sampling, macrofoam swabs pre-moistened with PBS supplemented with 0.05% Triton X-100 (PBSTX), stored at 4°C in neutralizing buffer (NB) as a transport medium (PBSTX/NB) or pre-moistened with NB and stored in PBSTX as a transport medium (NB/PBSTX), then vortexed 3min in the transport medium, performed significantly better than all other conditions for macrofoam swabs, regardless of strain tested (mean 12 - 72h recovery of 85.9-105.1%, p<0.001). In the same scenario, two combinations of pre-moistening medium/transport medium were found to be optimal for rayon swabs stored at 4°C (p<0.001), then sonicated 3min in the transport medium; PBSTX/PBSTX and NB/PBSTX (mean 12-72h recovery of 83.7-110.1%).

Variability of Burkholderia pseudomallei strain sensitivities to chlorine disinfection.

Burkholderia pseudomallei is a select agent and the causative agent of melioidosis. Variations in previously reported chlorine and monochloramine concentration time (Ct) values for disinfection of this organism make decisions regarding the appropriate levels of chlorine in water treatment systems difficult. This study identified the variation in Ct values for 2-, 3-, and 4-log(10) reductions of eight environmental and clinical isolates of B. pseudomallei in phosphate-buffered water. The greatest calculated Ct values for a 4-log(10) inactivation were 7.8 mg.min/liter for free available chlorine (FAC) at pH 8 and 5 degrees C and 550 mg.min/liter for monochloramine at pH 8 and 5 degrees C. Ionic strength of test solutions, culture hold times in water, and cell washing were ruled out as sources of the differences in prior observations. Tolerance to FAC was correlated with the relative amount of extracellular material produced by each isolate. Solid-phase cytometry analysis using an esterase-cleaved fluorochrome assay detected a 2-log(10)-higher level of organisms based upon metabolic activity than did culture, which in some cases increased Ct values by fivefold. Despite strain-to-strain variations in Ct values of 17-fold for FAC and 2.5-fold for monochloramine, standard FAC disinfection practices utilized in the United States should disinfect planktonic populations of these B. pseudomallei strains by 4 orders of magnitude in less than 10 min at the tested temperatures and pH levels.

Effects of Fis on Escherichia coli gene expression during different growth stages.

Fis is a nucleoid-associated protein in Escherichia coli that is abundant during early exponential growth in rich medium but is in short supply during stationary phase. Its role as a transcriptional regulator has been demonstrated for an increasing number of genes. In order to gain insight into the global effects of Fis on E. coli gene expression during different stages of growth in rich medium, DNA microarray analyses were conducted in fis and wild-type strains during early, mid-, late-exponential and stationary growth phases. The results uncovered 231 significantly regulated genes that were distributed over 15 functional categories. Regulatory effects were observed at all growth stages examined. Coordinate upregulation was observed for a number of genes involved in translation, flagellar biosynthesis and motility, nutrient transport, carbon compound metabolism, and energy metabolism at different growth stages. Coordinate down-regulation was also observed for genes involved in stress response, amino acid and nucleotide biosynthesis, energy and intermediary metabolism, and nutrient transport. As cells transitioned from the early to the late-exponential growth phase, different functional categories of genes were regulated, and a gradual shift occurred towards mostly down-regulation. The results demonstrate that the growth phase-dependent Fis expression triggers coordinate regulation of 15 categories of functionally related genes during specific stages of growth of an E. coli culture.

Growth phase-dependent regulation and stringent control of fis are conserved processes in enteric bacteria and involve a single promoter (fis P) in Escherichia coli.

The intracellular concentration of the Escherichia coli factor for inversion stimulation (Fis), a global regulator of transcription and a facilitator of certain site-specific DNA recombination events, varies substantially in response to changes in the nutritional environment and growth phase. Under conditions of nutritional upshift, fis is transiently expressed at very high levels, whereas under induced starvation conditions, fis is repressed by stringent control. We show that both of these regulatory processes operate on the chromosomal fis genes of the enterobacteria Klebsiella pneumoniae, Serratia marcescens, Erwinia carotovora, and Proteus vulgaris, strongly suggesting that the physiological role of Fis is closely tied to its transcriptional regulation in response to the nutritional environment. These transcriptional regulatory processes were previously shown to involve a single promoter (fis P) preceding the fis operon in E. coli. Recent work challenged this notion by presenting evidence from primer extension assays which appeared to indicate that there are multiple promoters upstream of fis P that contribute significantly to the expression and regulation of fis in E. coli. Thus, a rigorous analysis of the fis promoter region was conducted to assess the contribution of such additional promoters. However, our data from primer extension analysis, S1 nuclease mapping, beta-galactosidase assays, and in vitro transcription analysis all indicate that fis P is the sole E. coli fis promoter in vivo and in vitro. We further show how certain conditions used in the primer extension reactions can generate artifacts resulting from secondary annealing events that are the likely source of incorrect assignment of additional fis promoters.