Short communication: Recombinant bacteriophage endolysin PlyC is nontoxic and does not alter blood neutrophil oxidative response in lactating dairy cows.

Mastitis is the leading cause of antimicrobial use on dairy farms. The potential for antimicrobial resistance has led to the examination of alternative strategies for controlling mastitis. One such alternative is PlyC, a potent peptidoglycan hydrolase derived from the streptococcal C1 bacteriophage that causes targeted lysis of the cell wall of Streptococcus uberis. At a concentration of 1.0 µg/mL, recombinant PlyC can induce lytic activity, suggesting that a low dose may successfully eliminate infection. We evaluated the dose effect of PlyC (1-50 µg/mL) on cytotoxicity and oxidative response on bovine blood polymorphonuclear leukocytes (PMN) obtained from 12 healthy, mid-lactation primiparous dairy cows. Following incubation at 0.5 and 2 h, cytotoxicity was characterized by measuring lactate dehydrogenase release from isolated cells. Oxidative burst response was characterized as the intensity of chemiluminescence produced in the interaction of reactive oxygen species generated in response to 0 or 1.6 µg/mL of phorbol 12-myristate-13-acetate (PMA) with a luminescent substrate with and without addition to PlyC to the incubation matrix. Data were analyzed as a complete randomized block design using mixed model procedures. Cytotoxicity of PlyC was not affected by concentrations up to 50 µg/mL. As expected, PlyC cytotoxicity on PMN varied across incubation time with greater cell toxicity measured at 2 h of incubation as compared with 0.5 h and is primarily attributed to the short life of PMN ex vivo. Concentrations of PlyC up to 50 µg/mL did not affect oxidative response; however, oxidative response was affected by incubation time and PMA concentration. In summary, varying doses of PlyC are nontoxic as estimated by lactate dehydrogenase release from cells and do not appear to alter PMA-stimulated reactive oxygen species production in bovine PMN. These early observations support continued work on the potential for application of this novel agent in combating mastitis.

Effects of environmental factors on microbial induced calcium carbonate precipitation.

AIMS: To gain an understanding of the environmental factors that affect the growth of the bacterium Sporosarcina pasteurii, the metabolism of the bacterium and the calcium carbonate precipitation induced by this bacterium to optimally implement the biological treatment process, microbial induced calcium carbonate precipitation (MICP), in situ. METHODS AND RESULTS: Soil column and batch tests were used to assess the effect of likely subsurface environmental factors on the MICP treatment process. Microbial growth and mineral precipitation were evaluated in freshwater and seawater. Environmental conditions that may influence the ureolytic activity of the bacteria, such as ammonium concentration and oxygen availability, as well as the ureolytic activities of viable and lysed cells were assessed. Treatment formulation and injection rate, as well as soil particle characteristics are other factors that were evaluated for impact on uniform induction of cementation within the soils. CONCLUSIONS: The results of the study presented herein indicate that the biological treatment process is equally robust over a wide range of soil types, concentrations of ammonium chloride and salinities ranging from distilled water to full seawater; on the time scale of an hour, it is not diminished by the absence of oxygen or lysis of cells containing the urease enzyme. SIGNIFICANCE AND IMPACT OF STUDY: This study advances the biological treatment process MICP towards field implementation by addressing key environmental hurdles faced with during the upscaling process.

Keratinocyte transglutaminase expression varies in squamous cell carcinomas.

Type I transglutaminase (TGase I, keratinocyte or particulate transglutaminase) is a 92-kilodalton (kDa) protein expressed in abundance in cultured keratinocytes and in the hyperproliferative skin disorder psoriasis. To determine the expression of TGase I protein and mRNA, we studied tissue and established squamous carcinoma lines derived from different sources. Immunohistochemistry and Western blotting were used to detect TGase I protein with the B.C1 mouse monoclonal antibody. Only well-differentiated, skin-derived squamous carcinomas stained for TGase I. However, a precocious pattern of expression was seen overlying less-differentiated tumors. Compared to cultured human keratinocytes, squamous cell carcinoma (SCC) had many times less to 7.8 times more TGase I protein, greatest in the two most differentiated tumor lines 14-83 and ME-180. TGase I mRNA levels ranged from 0.010 to 0.00004 pg/microgram total RNA by reverse transcriptase-polymerase chain reaction using an internal standard. Protein expression correlated with mRNA levels in most SCC lines. When a human TGase I promoter was isolated and used to study genomic DNA, SCC1-83 was shown to have unique restriction enzyme fragments, including one indicative of methylation differences, also present within DNA from the KB line. These studies suggest that transcriptional control of TGase I gene expression in squamous carcinomas may be influenced both by cis elements in the promoter and by the degree of tumor squamous differentiation.

The effect of open-back vehicles on casualty rates: the case of Papua New Guinea.

Vehicle characteristics and vehicle use are frequently cited in the literature as potentially important factors contributing to the high motor-vehicle-related fatality rates reported by developing countries. Vehicles in developing countries are frequently overloaded, and improper vehicles are used to transport passengers. This paper seeks to estimate the importance of occupancy and vehicle type on the high motor-vehicle-related fatality rates in developing nations, using data for Papua New Guinea, a small South Pacific developing nation. This is achieved by establishing patterns of crash involvement and morbidity rates for vehicle types and ownership. Relative risk of crash involvement and relative risk of casualty for drivers and passengers are examined for open and closed vehicles. Finally, an estimate of the importance of vehicle type on the fatality rate is developed. The results demonstrate the need to disaggregate factors contributing to severity and occurrence in the study of road safety in developing countries.

Outcomes measurement and management with a large Medicaid population: a public/private collaboration.

Recipients, consumer advocates and purchasers expect managed care companies, in partnership with their provider networks, to measure and to continuously improve quality of care. This task is especially challenging when the beneficiaries of that care are covered by a publicly funded program. Yet this will be an increasingly common occurrence as more states contract with managed care companies for the care management of their Medicaid populations. In this article, the authors describe the outcomes measurement and management program of the first statewide managed Medicaid behavioral health carve-out program. Much of the foundation of that program has been built through collaborative efforts between the Massachusetts Division of Medical Assistance as purchaser, MHMA as managed care vendor, recipients, providers and other stakeholders. The authors report on the FMH/MHMA experience and what they learned. The principles derived from this outcomes program may be helpful to other states and to managed care companies undertaking similar public/private partnerships.

DNA-DNA Solution Hybridization Studies of the Bacterial Symbionts of Hydrothermal Vent Tube Worms (Riftia pachyptila and Tevnia jerichonana).

The giant tube worm, Riftia pachyptila (phylum Vestimentifera), is known only from four widely separated sulfide-rich deep-sea hydrothermal vent systems. This invertebrate is nourished by intracellular, chemoautotrophic bacterial symbionts which reside in a specialized trophosome tissue. The symbiont has not been cultured independently and is believed to be acquired de novo by host larvae of each generation. In the current study, R. pachyptila symbiont DNA was purified from the two most distant sites on the basis of its difference in density versus host DNA. These two standards were hybridized against trophosome DNAs of 13 individuals from the Guaymas Basin, Galapagos Rift, and 13 degrees N vents. This indicated that all R. pachyptila symbionts are conspecific and that the variability in DNA-DNA hybridization (relative binding ratio [RBR]) was comparable within or between widely separated vents. The symbiont of another tube worm, Tevnia jerichonana, was found to be the same as that of R. pachyptila, the first case in which distinct hosts possess the same sulfur bacterial symbiont. By contrast, Lamellibrachia sp. (same class as T. jerichonana) showed insignificant RBR with the R. pachyptila symbiont. DNA derived from solely eucaryotic tissue of R. pachyptila showed a surprisingly high RBR (20 to 50) with density-separated DNA standards. With DNAs obtained from physically separated symbionts, independent solution hybridization experiments confirmed the above-described conclusions. Possible explanations for this host-symbiont homology are discussed.

Bacterial zonation, photosynthesis, and spectral light distribution in hot spring microbial mats of Iceland.

The zonation and structure of phototrophic microbial mats were studied along two thermal gradients in sulfide-rich hot springs of southwest Iceland. The green, filamentous bacteriumChloroflexus and the unicellular, "high-temperature form" (HTF) ofMastigocladus formed mats growing up to a temperature limit of 62-66°C. The dominant phototrophs wereChloroflexus sp.,Mastigocladus laminosus, andPhormidium laminosum, respectively, at the three temperature intervals: >60°C, 60°C to 55-50°C, and <55-50°C. AChloroflexus mat growing at 60°C under 60µM H2S was anoxic in the light with the exception of a 0.5 mm thick band of HTFMastigocladus which produced oxygen. The oxygenic photosynthesis of these H2S-sensitive cyanobacteria was probably dependent on a preceding sulfide depletion by the anoxygenicChloroflexus. Measurements of spectral radiance gradients with a fiberoptic microprobe showed maximum light attenuation by carotenoids and bacteriochlorophyllC. AM. laminosus mat growing at 52°C was oxic throughout and showed maximum light attenuation by carotenoids, chlorophyllA, and phycocyanin, but no detectable phycoerythrocyanin absorption.

Characterization of the gene encoding the autotrophic ATP sulfurylase from the bacterial endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila.

ATP sulfurylase is a key enzyme in the energy-generating sulfur oxidation pathways of many chemoautotrophic bacteria. The utilization of reduced sulfur compounds to fuel CO2 fixation by the still-uncultured bacterial endosymbionts provides the basis of nutrition in invertebrates, such as the tubeworm Riftia pachyptila, found at deep-sea hydrothermal vents. The symbiont-containing trophosome tissue contains high levels of ATP sulfurylase activity, facilitating the recent purification of the enzyme. The gene encoding the ATP sulfurylase from the Riftia symbiont (sopT) has now been cloned and sequenced by using the partial amino acid sequence of the purified protein. Characterization of the sopT gene has unequivocally shown its bacterial origin. This is the first ATP sulfurylase gene to be cloned and sequenced from a sulfur-oxidizing bacterium. The deduced amino acid sequence was compared to those of ATP sulfurylases reported from organisms which assimilate sulfate, resulting in the discovery that there is substantial homology with the Saccharomyces cerevisiae MET3 gene product but none with the products of the cysDN genes from Escherichia coli nor with the nodP and nodQ genes from Rhizobium meliloti. This and emerging evidence from other sources suggests that E. coli may be atypical, even among prokaryotic sulfate assimilators, in the enzyme it employs for adenosine 5'-phosphosulfate formation. The sopT gene probe also was shown to specifically identify chemoautotrophic bacteria which utilize ATP sulfurylase to oxidize sulfur compounds.

Organic carbon utilization by obligately and facultatively autotrophic beggiatoa strains in homogeneous and gradient cultures.

Marine Beggiatoa strains MS-81-6 and MS-81-1c are filamentous gliding bacteria that use hydrogen sulfide and thiosulfate as electron donors for chemolithotrophic energy generation. They are known to be capable of chemolithoautotrophic growth in sulfide gradient media; here we report the first successful bulk cultivation of these strains in a defined liquid medium. To investigate their nutritional versatilities, strains MS-81-6 and MS-81-1c were grown in sulfide-oxygen gradient media supplemented with single organic compounds. Respiration rates and biomass production relative to those of controls grown in unsupplemented sulfide-limited media were monitored to determine whether organic compounds were utilized as sources of energy and/or cell carbon. With cells grown in sulfide gradient and liquid media, we showed that strain MS-81-6 strongly regulates two enzymes, the tricarboxylic acid cycle enzyme 2-oxoglutarate dehydrogenase and the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, in response to the presence of organic carbon (acetate) in the growth medium. In contrast, strain MS-81-1c lacked 2-oxoglutarate dehydrogenase activity and regulated ribulose-1,5-bisphosphate carboxylase/oxygenase activity only slightly in response to organic substrates. Tracer experiments with radiolabeled acetate showed that strain MS-81-1c did not oxidize acetate to CO(inf2) but could synthesize approximately 20% of its cell carbon from acetate. On the basis of these results, we conclude that Beggiatoa strain MS-81-1c is an obligate chemolithoautotroph, while strain MS-81-6 is a versatile facultative chemolithoautotroph.

Sulfur-oxidizing symbionts have not co-evolved with their hydrothermal vent tube worm hosts: an RFLP analysis.

A fine-scale phylogenetic comparison was made among the symbionts of different genera of hydrothermal vent tube worms. These included Riftia pachyptila and Tevnia jerichonona, which inhabit sites along the east Pacific Rise, and Ridgeia piscesae from the Juan de Fuca Ridge. An analysis of restriction fragment length polymorphism (RFLP) was employed using three symbiont-specific gene probes: eubacterial 16S rRNA, RuBPC/O Form II, and ATP sulfurylase (recently cloned from the Riftia symbiont). Results indicated that all of the symbionts from the three different hosts were conspecific and the Riftia and Tevnia symbionts were indistinguishable over and 1800-km range. Significantly, this indicates that the symbionts have not co-evolved with their respective hosts, which are known to belong to separate families. This study strongly supports the conclusion that the symbionts are acquired de novo by each generation of juvenile tube worms from a common source in the surrounding sea water.

Use of Reduced Sulfur Compounds by Beggiatoa spp.: Enzymology and Physiology of Marine and Freshwater Strains in Homogeneous and Gradient Cultures.

The marine Beggiatoa strains MS-81-6 and MS-81-1c are filamentous, gliding, colorless sulfur bacteria. They have traditionally been cultured in very limited quantities in sulfide gradient media, where they grow as chemolithoautotrophs, forming a thin horizontal plate well below the air-agar interface. There, the facultatively chemolithoautotrophic strain MS-81-6 quantitatively harvests the flux of sulfide diffusing from below and oxidizes it to sulfate by using oxygen as the electron acceptor. Only recently have these strains been cultivated in bulk in defined liquid media (K. D. Hagen and D. C. Nelson, Appl. Environ. Microbiol. 62:947-953, 1996). In the current study, the obligately chemolithoautotrophic strain MS-81-1c was shown to have, despite much greater storage of elemental sulfur, an apparent Y(infH)(inf(inf2))(infS) twice that of MS-81-6 when the two strains were grown in identical sulfide-limited gradient media. While the basis of this difference in energy conservation has not been established, differences in sulfur oxidation enzymes were noted. Strain MS-81-1c appeared to be able to oxidize sulfite by using either the adenosine phosphosulfate (APS) pathway or a sulfite:acceptor oxidoreductase. APS pathway enzymes (ATP sulfurylase and APS reductase) were present at relatively high and constant levels regardless of growth conditions, while the sulfite:acceptor oxidoreductase activity varied at least eightfold, with the highest activity produced in sulfide gradient medium. By contrast, strain MS-81-6 showed no detectable activity of the APS pathway enzymes and possessed a sulfite:acceptor oxidoreductase activity just sufficient to account for its observed rate of growth in sulfide gradient medium. Freshwater strain OH-75-2a showed activity and regulation of sulfite:acceptor oxidoreductase consistent with lithotrophic energy conservation, a feature not yet proven for any freshwater Beggiatoa strain.

A direct-reading comparison spectroradiometer.

A modification of the Cary Model 14 for radiometry is described which allows recording in microwatts per square mlllimeter per nanometer on the ordinate and linear wavelength on the abscissa through the range 250-2500 nm. A 5.08-cm diam rotating integrating sphere is used to compare standard to unknown sources at a 30 c/s rate. The undispersed radiation from the sphere is directed through the entrance slit of the monochromator. After dispersion by the monochromator the energy is received by a single phototube or PbS detector as alternate pulses of standard and unknown energy. This design results in common optical elements throughout the instrument except for the standard and unknown sources themselves. An electrical calibration system permits an ordinate scale reading irradiance directly in microwatts per square centimeter per nanometer with simple provisions for range change. The instrument provides a dynamic range of 2 x 10(6) to 1. A large open area is provided for mounting unknown sources. The entrance optical system is designed so that remote sources can be viewed conveniently.

Use of reduced sulfur compounds by Beggiatoa sp.

A strain of Beggiatoa cf. leptomitiformis (OH-75-B, clone 2a) was isolated which is unique among reported strains in its ability to deposit internal sulfur granules from thiosulfate. It also deposited these characteristic granules (as all BEggiatoa species do) from sulfide. In cultures where growth was limited by exhaustion of organic substrates, these granules generally comprised about 20% of the total cell weight. With medium containing acetate and thiosulfate, no measurable utilization of thiosulfate or deposition of elemental sulfur (S0) took place until after the exponential growth phase. Neither sulfide nor thiosulfate added an increment to heterotrophic growth yield except for the weight of the deposited S0. The deposition of S0 from thiosulfate was probably a disproportionation in which S0 and sulfate were produced in a 1:1 ratio. Some of the S0 was further oxidized to sulfate. No autotrophic or mixotrophic growth was demonstrated for this strain. When inoculated in small, well-dispersed quantities into yeast extract medium, this strain grew only after long lags. Addition of the enzyme catalase eliminated initial lags and increased growth rates slightly. In contrast, catalase had no influence on growth rate when added to mineral medium containing acetate. In yeast extract medium, the inhibition of growth rate was presumably because of peroxides. Addition of thiosulfate was almost as effective as catalase in eliminating this inhibition. The S0 granules which, in this case, were deposited during the exponential growth phase, appeared to be partly responsible for this relief. This strain of Beggiatoa sp. remained active for at least 5 days under strictly anaerobic conditions, and under those conditions, it increased its dry weight by about 2.5-fold. Anaerobic "growth" and maintenance required the presence of an energy source, such as acetate. When cells containing much internal S0 were transferred to an organic anaerobic medium, a substantial portion of the internal S0 was eventually converted to sulfide.

Organic nutrition of Beggiatoa sp.

Culture OH-75-B of Beggiatoa sp. differed significantly from any described previously in its utilization of organic carbon and reduced sulfur compounds. It deposited internal sulfur granules characteristic of Beggiatoa sp. with either sulfide or thiosulfate in the medium. This strain (OH-75-B, clone 2a) could be grown in agitated liquid cultures on mineral medium with acetate as the only source of organic carbon. The resultant growth yields and rates were comparable to those for typical heterotrophs. Of the other simple organic compounds tested, only pyruvate, lactate, or ethanol could singly support the growth of this strain. Single sugars or amino acids neither supported growth nor enhanced it when added to acetate-containing medium. In contrast, compounds of the tricarboxylic acid cycle enhanced growth yields when tested in concert with acetate. These and fluoroacetate inhibition results indicate that Beggiatoa sp. possesses a functional tricarboxylic acid cycle. Poor yields characterized the growth of this strain on dilute yeast extract medium, and higher concentrations of yeast extract proved inhibitory. The enzyme catalase, contrary to the findings of others, had no synergistic influence on growth yields when added to medium containing yeast extract or acetate or both.

Renal vein thrombosis associated with nephrotic syndrome and gold therapy in rheumatoid arthritis.

In this case of renal vein thrombosis secondary to a nephrotic syndrome, we postulate renal disease resulted from gold therapy. We know of no previous report relating gold toxicity and renal vein thrombosis. It should be emphasized that with increasing use of gold, proteinuria and nephrotic syndrome may be more common than once suspected and, when present, predispose to the development of renal vein thrombosis.

Microoxic-Anoxic Niche of Beggiatoa spp.: Microelectrode Survey of Marine and Freshwater Strains.

Beggiatoa spp. grow optimally in media containing opposed gradients of oxygen and soluble sulfide, although some strains also require an organic substrate. By using microelectrodes, we characterized oxygen and sulfide gradients during their initial development in uninoculated media and in cultures of marine and freshwater strains. In gradient media, Beggiatoa strains always grew some distance below the air/agar interface as a dense "plate" of constantly gliding filaments with sharply demarcated upper and lower boundaries. Within established plates, the maximum oxygen partial pressure was 0.6 to 6.0% of air saturation and not significantly lower if filaments were fixing nitrogen. Oxygen penetrated only 100 to 300 mum into the plate, and the anoxic fraction increased from less than 10% to approximately 90% during later stages of growth. For lithoautotrophically grown marine strains, the linearity of the oxygen profile above the plate plus its drop to zero therein indicated that oxygen uptake for the entire tube occurred only within the Beggiatoa plate. Consequently, oxygen consumption could be predicted solely from the distance between the air/agar interface and the top of a plate, given the diffusion coefficient for oxygen. By contrast, for freshwater strains grown heterotrophically (with sulfide also in the medium), oxygen profiles were frequently nonlinear because of nonbiological reaction with sulfide which had diffused past the aggregated filaments. For all strains tested, microoxic aggregation also occurred in the absence of sulfide, apparently reflecting a step-up phobic response to oxygen.

Growth Pattern and Yield of a Chemoautotrophic Beggiatoa sp. in Oxygen-Sulfide Microgradients.

Recently developed techniques involving opposed, gel-stabilized gradients of O(2) and H(2)S permit cultivation of a marine Beggiatoa strain as a chemolithoautotroph which uses gliding motility to precisely track the interface between H(2)S and O(2). In the current study with microelectrodes, vertical profiles of H(2), O(2), and pH were measured in replicate cultures grown for various intervals. After an initial period of exponential biomass increase (doubling time, 11 h), linear growth prevailed throughout much of the time course. This H(2)S-limited growth was followed by a transition to stationary phase when the declining H(2)S flux was sufficient only to supply maintenance energy. During late-exponential and linear growth phases, the Beggiatoa sp. consumed a constant 0.6 mol of H(2)S for each 1.0 mol of O(2), the ratio anticipated for balanced lithoautotrophic growth at the expense of complete oxidation of H(2)S to SO(4). Over the entire range of conditions studied, this consumption ratio varied by approximately twofold. By measuring the extent to which the presence of the bacterial plate diminished the overlap of O(2) and H(2)S, we demonstrated that oxidation of H(2)S by Beggiatoa sp. is approximately 3 orders of magnitude faster than spontaneous chemical oxidation. By integrating sulfide profiles and comparing sulfide consumed with biomass produced, a growth yield of 8.4 g (dry weight) mol of H(2)S was computed. This is higher than that found for sulfide-grown thiobacilli, indicating very efficient growth of Beggiatoa sp. as a chemoautotroph. The methods used here offer a unique opportunity to determine the yield of H(2)S-oxidizing chemolithoautotrophs while avoiding several problems inherent in the use of homogeneous liquid culture. Finally, by monitoring time-dependent formation of H(2)S profiles under anoxic conditions, we demonstrate a method for calculating the molecular diffusion coefficient of soluble substrates in gel-stabilized media.

Characterization of Large, Autotrophic Beggiatoa spp. Abundant at Hydrothermal Vents of the Guaymas Basin.

Filamentous bacteria, identified as members of the genus Beggiatoa by gliding motility and internal globules of elemental sulfur, occur in massive aggregations at the deep-sea hydrothermal vents of the Guaymas Basin, Gulf of California. Cell aggregates covering the surface of sulfide-emanating sediments and rock chimneys were collected by DS R/V Alvin and subjected to shipboard and laboratory experiments. Each sample collected contained one to three discrete width classes of this organism usually accompanied by a small number of "flexibacteria" (width, 1.5 to 4 mum). The average widths of the Beggiatoa classes were 24 to 32, 40 to 42, and 116 to 122 mum. As indicated by electron microscopy and cell volume/protein ratios, the dominant bacteria are hollow cells, i.e., a thin layer of cytoplasm surrounding a large central liquid vacuole. Activities of Calvin-cycle enzymes indicated that at least two of the classes collected possess autotrophic potential. Judging from temperature dependence of enzyme activities and whole-cell CO(2) incorporation, the widest cells were mesophiles. The narrowest Beggiatoa sp. was either moderately thermophilic or mesophilic with unusually thermotolerant enzymes. This was consistent with its occurrence on the flanks of hot smoker chimneys with highly variable exit temperatures. In situ CO(2) fixation rates, sulfide stimulation of incorporation, and autoradiographic studies suggest that these Beggiatoa spp. contribute significantly as lithoautrophic primary producers to the Guaymas Basin vent ecosystems.

Hypernatremia and lactulose therapy.

Lactulose is used in the treatment of portal-systemic encephalopathy. Although it is usually safe, a case of hypernatremia has been reported. To evaluate further the association of lactulose therapy and hypernatremia, records of patients with hepatic failure treated with lactulose for encephalopathy were analyzed. In 20 of 75 courses of lactulose treatment, serum sodium level exceeded 145 mEq/L. Mortality was 41% in patients in whom hypernatremia developed as compared with 14% in those who remained normonatremic. In persons who died, hypernatremia persisted; in patients who eventually recovered, the serum sodium level returned to normal. The precise mechanism of hypernatremia in these cases is not clearly defined. As an osmotic cathartic, however, lactulose may cause fecal water loss in excess of sodium, resulting in contraction of extracellular fluid volume and, therefore, hypernatremia.