bluuebunny/crossref_metadata_2025 huggingface dataset:
Antiviral Activities of Oral 1-O-Hexadecylpropanediol-3-Phosphoacyclovir and Acyclovir in Woodchucks with Chronic Woodchuck Hepatitis Virus Infection
Authors: Hostetler Karl Y. , Beadle James R. , Hornbuckle William E. , Bellezza Christine A. , Tochkov Ilia A. , Cote Paul J. , Gerin John L. , Korba Brent E. , Tennant Bud C.
Published: July 2002

Acyclovir triphosphate is a potent inhibitor of hepatitis B virus DNA polymerase, but acyclovir treatment provides no benefit in patients with hepatitis B virus infection. This is due in part to the fact that hepatitis B virus, unlike herpes simplex virus, does not code for a viral thymidine kinase which catalyzes the initial phosphorylation of acyclovir. We synthesized 1-O-octadecyl-sn-glycero-3-phospho (3-P)-acyclovir and found that it was highly active in reducing hepatitis B virus replication in 2.2.15 cells, while acyclovir was inactive. The greater antiviral activity of 1-O-octadecyl-sn-glycero-3-P-acyclovir appeared to be due to liver cell metabolism of the compound to acyclovir monophosphate (K. Y. Hostetler et al., Biochem. Pharmacol. 53:1815–1822, 1997). However, a closely related compound without a hydroxyl group at thesn-2 position of glycerol, 1-O-hexadecylpropanediol-3-P-acyclovir, was more active and selective in 2.2.15 cells in vitro. In this study, we treated woodchucks chronically infected with woodchuck hepatitis virus with increasing oral doses of 1-O-hexadecylpropanediol-3-P-acyclovir and assessed the response to therapy versus acyclovir or a placebo. At a dosage of 10 mg/kg of body weight twice a day, the test compound significantly inhibited viral replication in vivo, as indicated by a 95% reduction in serum woodchuck hepatitis virus DNA levels and by a 54% reduction in levels of woodchuck hepatitis virus replicative intermediates in the liver. Higher doses were somewhat less effective. In contrast, 20 mg of acyclovir/kg twice daily, a 5.3-fold-higher molar dosage, had no demonstrable activity against woodchuck hepatitis virus. Oral 1-O-hexadecylpropanediol-3-P-acyclovir appeared to be safe and effective in chronic woodchuck hepatitis virus infection.
Tilmicosin Induces Apoptosis in Bovine Peripheral Neutrophils in the Presence or in the Absence ofPasteurella haemolyticaand Promotes Neutrophil Phagocytosis by Macrophages
Authors: Chin Alex C. , Lee Wilson D. , Murrin Katherine A. , Morck Douglas W. , Merrill John K. , Dick Paul , Buret Andre G.
Published: July 2002

Pathogen virulence factors and inflammation are responsible for tissue injury associated with respiratory failure in bacterial pneumonia, as seen in the bovine lung infected withPasteurella haemolytica. Tilmicosin is a macrolide antibiotic used for the treatment of bovine bacterial pneumonia. Recent evidence suggests that tilmicosin-induced neutrophil apoptosis may have anti-inflammatory effects. Using bovine leukocytes, we sought to define whether liveP. haemolyticaaffected tilmicosin-induced neutrophil apoptosis, assessed the proapoptotic effects of tilmicosin in comparison with other drugs, and characterized its impact on phagocytic uptake of neutrophils by macrophages. Induction of apoptosis in the presence or absence ofP. haemolyticawas assessed by using an enzyme-linked immunosorbent assay for apoptotic nucleosomes. In addition, fluorescent annexin-V staining identified externalized phosphatidylserine in neutrophils treated with tilmicosin, penicillin, ceftiofur, oxytetracycline, or dexamethasone. Neutrophil membrane integrity was assessed by using propidium iodide and trypan blue exclusion. As phagocytic clearance of apoptotic neutrophils by macrophages contributes to the resolution of inflammation, phagocytosis of tilmicosin-treated neutrophils by esterase-positive cultured bovine macrophages was assessed with light microscopy and transmission electron microscopy. Unlike bovine neutrophils treated with penicillin, ceftiofur, oxytetracycline, or dexamethasone, neutrophils exposed to tilmicosin became apoptotic, regardless of the presence or absence ofP. haemolytica. Tilmicosin-treated apoptotic neutrophils were phagocytosed at a significantly greater rate by bovine macrophages than were control neutrophils. In conclusion, tilmicosin-induced neutrophil apoptosis occurs regardless of the presence or absence of liveP. haemolytica, exhibits at least some degree of drug specificity, and promotes phagocytic clearance of the dying inflammatory cells.
ATP Synthase Is Necessary for Microcin H47 Antibiotic Action
Authors: Trujillo Mónica , Rodrı́guez Eliana , Laviña Magela
Published: July 2002

Microcin H47 is a gene-encoded peptide antibiotic produced by a natural
Escherichia coli
strain isolated in Uruguay. In order to identify cellular components necessary for its antibiotic action, microcin H47-resistant mutants isolated in this work, as well as previously described mutants affected in membrane proteins, were analyzed. These studies indicated that (i) receptor outer membrane proteins for ferric-catechol siderophores would be involved in microcin-specific binding to the cell surface, (ii) the TonB pathway is needed for microcin H47 uptake, and (iii) the presence of the ATP synthase complex is necessary for microcin action. The possibility that this last structure contains the antibiotic target is discussed.
Treatment of Vancomycin-Resistant Enterococcal Infections in the Immunocompromised Host: Quinupristin-Dalfopristin in Combination with Minocycline
Authors: Raad Issam , Hachem Ray , Hanna Hend , Girgawy Essam , Rolston Kenneth , Whimbey Estella , Husni Rola , Bodey Gerald
Published: July 2002

Between February 1994 and November 1998, 56 oncology patients infected with vancomycin-resistant enterococci (VRE) were treated with quinopristin-dalfopristin (Q-D) plus minocycline (MIN). Infections included bacteremia, urinary tract infection, pneumonia, and wound infection. The response rate was 68%, and the most frequent adverse event was arthralgia or myalgia (36%). Q-D–MIN is effective for VRE infection in cancer patients but is associated with a substantial frequency of arthralgia or myalgia.
Activities of a New Fluoroketolide, HMR 3787, and Its (Des)-Fluor Derivative RU 64399 Compared to Those of Telithromycin, Erythromycin A, Azithromycin, Clarithromycin, and Clindamycin against Macrolide-Susceptible or -Resistant
Streptococcus pneumoniae
and
S. pyogenes
Authors: Nagai Kensuke , Davies Todd A. , Ednie Lois M. , Bryskier Andre , Palavecino Elizabeth , Jacobs Michael R. , Appelbaum Peter C.
Published: July 2002

Activities of HMR 3787 and RU 64399 were compared to those of three macrolides, telithromycin, and clindamycin against 175
Streptococcus pneumoniae
isolates and 121
Streptococcus pyogenes
isolates. HMR3787 and telithromycin were the most active compounds tested against pneumococci. Telithromycin and RU 64399 were equally active against macrolide-susceptible (MICs, 0.008 to 0.06 μg/ml) and -resistant
S. pyogenes
isolates, but HMR 3787 had lower MICs for
ermB
strains.
Susceptibilities of
Eikenella corrodens
,
Prevotella intermedia
, and
Prevotella nigrescens
Clinical Isolates to Amoxicillin and Tetracycline
Authors: Luong N. , Tsai J. , Chen C.
Published: July 2002

The AB Biodisk Etest showed that 106 (100%) and 98 (92%) isolates of
Eikenella corrodens
were susceptible to amoxicillin and tetracycline, respectively. Twenty-three (68%)
Prevotella intermedia
isolates and 14 (67%)
Prevotella nigrescens
isolates were susceptible to amoxicillin. Seventy-nine percent of the
P. intermedia
isolates and 67% of the
P. nigrescens
isolates were susceptible to tetracycline. A higher percentage of β-lactamase-producing isolates of
P. intermedia
and
P. nigrescens
were identified with selective agar containing amoxicillin than with nonselective agar.
In Vitro and In Vivo Activities of Aminoadamantane and Aminoalkylcyclohexane Derivatives againstTrypanosoma brucei
Authors: Kelly John M. , Quack Guenter , Miles Michael M.
Published: July 2002

We reported recently that the bloodstream form of the African trypanosome,Trypanosoma brucei, is sensitive to the anti-influenza virus drug rimantadine. In the present report we describe the trypanocidal properties of a further 62 aminoadamantane and aminoalkylcyclohexane derivatives. Seventeen of the compounds were found to be more active than rimantadine, with four inhibiting growth in vitro ofT. bruceiby >90% at concentrations of 1 μM. The most active derivative (1-adamantyl-4-amino-cyclohexane) was about 20 to 25 times more effective than rimantadine. We observed a correlation between structural features of the derivatives and their trypanocidal properties; hydrophobic substitutions to the adamantane or cyclohexane rings generally enhanced activity. As with rimantadine, the activity in vitro varied with the pH.T. bruceiwas more sensitive in an alkaline environment (including a normal bloodstream pH of 7.4) and less sensitive under acidic conditions. Tests for activity in vivo were carried out with a mouse model of infection with a virulent strain ofT. brucei. Although the parasitemia was not eliminated, it could be transiently suppressed by >98% with the most active compounds tested. These results suggest that aminoadamantane derivatives could have potential as a new class of trypanocidal agents.
In Vitro Characterization of A-315675, a Highly Potent Inhibitor of A and B Strain Influenza Virus Neuraminidases and Influenza Virus Replication
Authors: Kati Warren M. , Montgomery Debra , Carrick Robert , Gubareva Larisa , Maring Clarence , McDaniel Keith , Steffy Kevin , Molla Akhteruzzaman , Hayden Frederick , Kempf Dale , Kohlbrenner William
Published: July 2002

A-315675 is a novel, pyrrolidine-based compound that was evaluated in this study for its ability to inhibit A and B strain influenza virus neuraminidases in enzyme assays and influenza virus replication in cell culture. A-315675 effectively inhibited influenza A N1, N2, and N9 and B strain neuraminidases with inhibitor constant (

K
i

) values between 0.024 and 0.31 nM. These values were comparable to or lower than the

K
i

values measured for oseltamivir carboxylate (GS4071), zanamivir, and BCX-1812, except for the N1 enzymes that were found to be the most sensitive to BCX-1812. The time-dependent inhibition of neuraminidase catalytic activity observed with A-315675 is likely due to its very low rate of dissociation from the active site of neuraminidase. The half times for dissociation of A-315675 from B/Memphis/3/89 and A/Tokyo/3/67 (H3N2) influenza virus neuraminidases of 10 to 12 h are significantly slower than the half times measured for oseltamivir carboxylate (33 to 60 min). A-315675 inhibited the replication of several laboratory strains of influenza virus in cell culture with potencies that were comparable or superior to those for oseltamivir carboxylate and BCX-1812, except for the A/H1N1 viruses that were found to be two- to fourfold more susceptible to BCX-1812. A-315675 and oseltamivir carboxylate exhibited comparable potencies against a panel of A/H1N1 and A/H3N2 influenza virus clinical isolates, but A-315675 was found to be significantly more potent than oseltamivir carboxylate against the B strain isolates. The favorable in vitro results relative to other clinically effective agents provide strong support for the further investigation of A-315675 as a potential therapy for influenza virus infections.
Use of IMiD3, a Thalidomide Analog, as an Adjunct to Therapy for Experimental Tuberculous Meningitis
Authors: Tsenova Liana , Mangaliso Bande , Muller George , Chen Yong , Freedman Victoria H. , Stirling David , Kaplan Gilla
Published: July 2002

Tuberculous meningitis (TBM), the most severe form ofMycobacterium tuberculosisinfection in humans, is associated with significant morbidity and mortality despite successful treatment with antituberculous drugs. This is due to the irreversible brain damage subsequent to the local inflammatory response of the host toM. tuberculosis.Corticosteroids have been used in conjunction with antituberculous therapy in an attempt to modulate the inflammatory response, but this strategy has been of limited success. Therefore, we examined whether combining antituberculous drugs with the immunomodulatory drug thalidomide or with a new thalidomide analog, immunomodulatory drug 3 (IMiD3), would be effective in reducing morbidity and mortality in an experimental rabbit model of TBM. Intracisternal inoculation of 5 × 104CFU ofMycobacterium bovisRavenel in rabbits induced progressive subacute meningitis characterized by high cerebrospinal fluid (CSF) leukocytosis, protein influx, release of tumor necrosis factor (TNF), substantial meningeal inflammation, and mortality by day 28. Treatment with antituberculous drugs or with antituberculous drugs plus thalidomide improved the clinical course of disease somewhat and increased survival to about 50%. In contrast, treatment with antituberculous drugs in combination with IMiD3 limited pathological neurologic changes and resulted in marked improvement (73%) in survival. IMiD3 treatment was also associated with reduced leukocytosis in the CSF and significantly lower levels of TNF in CSF and plasma. Histologically, the meningeal inflammation in animals treated with antituberculous drugs plus IMiD3 was considerably attenuated compared to that of the other treatment groups. These results suggest a potential role for IMiD3 in the management of TBM in patients.
Characterization, Seasonal Occurrence, and Diel Fluctuation of Poly(hydroxyalkanoate) in Photosynthetic Microbial Mats
Authors: Rothermich Mary M. , Guerrero Ricardo , Lenz Robert W. , Goodwin Steve
Published: July 2002

In situ poly(hydroxyalkanoate) (PHA) levels and repeating-unit compositions were examined in stratified photosynthetic microbial mats from Great Sippewissett Salt Marsh, Mass., and Ebro Delta, Spain. Unlike what has been observed in pure cultures of phototrophic bacteria, the prevalence of hydroxyvalerate (HV) repeating units relative to hydroxybutyrate (HB) repeating units was striking. In the cyanobacteria-dominated green material of Sippewissett mats, the mole percent ratio of repeating units was generally 1HB:1HV. In the purple sulfur bacteria-dominated pink material the relationship was typically 1HB:2HV. In Sippewissett mats, PHA contributed about 0.5 to 1% of the organic carbon in the green layer and up to 6% in the pink layer. In Ebro Delta mats, PHA of approximately 1HB:2HV-repeating-unit distribution contributed about 2% of the organic carbon of the composite photosynthetic layers (the green and pink layers were not separated). Great Sippewissett Salt Marsh mats were utilized for more extensive investigation of seasonal, diel, and exogenous carbon effects. When the total PHA content was normalized to organic carbon, there was little seasonal variation in PHA levels. However, routine daily variation was evident at all sites and seasons. In every case, PHA levels increased during the night and decreased during the day. This phenomenon was conspicuous in the pink layer, where PHA levels doubled overnight. The daytime declines could be inhibited by artificial shading. Addition of exogenous acetate, lactate, and propionate induced two- to fivefold increases in the total PHA levels when applied in the daylight but had no effect when applied at night. The distinct diel pattern of in situ PHA accumulation at night appears to be related, in some phototrophs, to routine dark energy metabolism and is not influenced by the availability of organic nutrients.
Marine Planktonic Archaea Take Up Amino Acids
Authors: Ouverney Cleber C. , Fuhrman Jed A.
Published: July 2002

Archaea are traditionally thought of as “extremophiles,” but recent studies have shown that marine planktonic Archaea make up a surprisingly large percentage of ocean midwater microbial communities, up to 60% of the total prokaryotes. However, the basic physiology and contribution of Archaea to community microbial activity remain unknown. We have studied Archaea from 200-m depths of the northwest Mediterranean Sea and the Pacific Ocean near California, measuring the archaeal activity under simulated natural conditions (8 to 17°C, dark and anaerobic) by means of a method called substrate tracking autoradiography fluorescence in situ hybridization (STARFISH) that simultaneously detects specific cell types by 16S rRNA probe binding and activity by microautoradiography. In the 200-m-deep Mediterranean and Pacific samples, cells binding the archaeal probes made up about 43 and 14% of the total countable cells, respectively. Our results showed that the Archaea are active in the uptake of dissolved amino acids from natural concentrations (nanomolar) with about 60% of the individuals in the archaeal communities showing measurable uptake. Bacteria showed a similar proportion of active cells. We concluded that a portion of these Archaea is heterotrophic and also appears to coexist successfully with Bacteria in the same water.
Relationships between Colony Morphotypes and Oil Tolerance in
Rhodococcus rhodochrous
Authors: Iwabuchi Noriyuki , Sunairi Michio , Anzai Hirosi , Nakajima Mutsuyasu , Harayama Shigeaki
Published: July 2002

A mucoidal strain of
Rhodococcus rhodochrous
was resistant to 10% (vol/vol)
n
-hexadecane, while its rough derivatives were sensitive. When the extracellular polysaccharide (EPS) produced by the mucoidal strain was added to cultures of the rough strains, the rough strains gained resistance to
n
-hexadecane. Thus, EPS confer tolerance to
n
-hexadecane in members of the genus
Rhodococcus.
Enrichment and Molecular Characterization of a Bacterial Culture That Degrades Methoxy-Methyl Urea Herbicides and Their Aniline Derivatives
Authors: El-Fantroussi Said
Published: July 2002

Soil treated with linuron for more than 10 years showed high biodegradation activity towards methoxy-methyl urea herbicides. Untreated control soil samples taken from the same location did not express any linuron degradation activity, even after 40 days of incubation. Hence, the occurrence in the field of a microbiota having the capacity to degrade a specific herbicide was related to the long-term treatment of the soil. The enrichment culture isolated from treated soil showed specific degradation activity towards methoxy-methyl urea herbicides, such as linuron and metobromuron, while dimethyl urea herbicides, such as diuron, chlorotoluron, and isoproturon, were not transformed. The putative metabolic intermediates of linuron and metobromuron, the aniline derivatives 3,4-dichloroaniline and 4-bromoaniline, were also degraded. The temperature of incubation drastically affected degradation of the aniline derivatives. Whereas linuron was transformed at 28 and 37°C, 3,4-dichloroaniline was transformed only at 28°C. Monitoring the enrichment process by reverse transcription-PCR and denaturing gradient gel electrophoresis (DGGE) showed that a mixture of bacterial species under adequate physiological conditions was required to completely transform linuron. This research indicates that for biodegradation of linuron, several years of adaptation have led to selection of a bacterial consortium capable of completely transforming linuron. Moreover, several of the putative species appear to be difficult to culture since they were detectable by DGGE but were not culturable on agar plates.
Leaching of
Escherichia coli
O157:H7 in Diverse Soils under Various Agricultural Management Practices
Authors: Gagliardi Joel V. , Karns Jeffrey S.
Published: July 2002

Application of animal manures to soil as crop fertilizers is an important means for recycling the nitrogen and phosphorus which the manures contain. Animal manures also contain bacteria, including many types of pathogens. Manure pathogen levels depend on the source animal, the animal's state of health, and how the manure was stored or treated before use. Rainfall may result in pathogen spread into soil by runoff from stored or unincorporated manure or by leaching through the soil profile. Steady rainfall consisting of 16.5 mm h
−1
was applied to 100-mm disturbed soil cores that were treated with manure and inoculated with
Escherichia coli
O157:H7 strain B6914. The level of B6914 in leachate was near the inoculum level each hour for 8 h, as was the level of B6914 at several soil depths after 24 h, indicating that there was a high rate of growth. Bacterial movement through three different types of soil was then compared by using disturbed (tilled) and intact (no-till) soil cores and less intense rainfall consisting of 25.4 mm on 4 consecutive days and then four more times over a 17-day period. Total B6914 levels exceeded the inoculum levels for all treatments except intact clay loam cores. B6914 levels in daily leachate samples decreased sharply with time, although the levels were more constant when intact sandy loam cores were used. The presence of manure often increased total B6914 leachate and soil levels in intact cores but had the opposite effect on disturbed soil cores. Ammonia and nitrate levels correlated with B6914 and total coliform levels in leachate. We concluded that tillage practice, soil type, and method of pathogen delivery affect but do not prevent vertical
E. coli
O157:H7 and coliform transport in soil and that soluble nitrogen may enhance transport.
Frequency and Biodiversity of 2,4-Diacetylphloroglucinol-Producing Bacteria Isolated from the Maize Rhizosphere at Different Stages of Plant Growth
Authors: Picard C. , Di Cello F. , Ventura M. , Fani R. , Guckert A.
Published: July 2002

APseudomonas2,4-diacetylphloroglucinol (DAPG)-producing population that occurred naturally on the roots, in rhizosphere soil ofZea maysand in the nonrhizosphere soil was investigated in order to assess the microbial diversity at five stages of plant growth. A total of 1,716 isolates were obtained, and 188 of these isolates were able to produce DAPG. DAPG producers were isolated at each stage of plant growth, indicating that the maize rhizosphere is colonized by natural DAPG producers throughout development. The frequency of DAPG producers was very low in the first stage of plant growth and increased over time. An analysis of the level of biodiversity of the DAPG producers at the species level was performed by comparing theAluI restriction patterns of the 16S ribosomal DNAs (rDNAs) amplified by PCR from 167 isolates. This comparison allowed us to cluster the isolates into four amplified rDNA restriction analysis (ARDRA) groups, and the main group (ARDRA group 1) contained 89.8% of the isolates. The diversity of the 150 isolates belonging to ARDRA group 1 was analyzed by the random amplified polymorphic DNA (RAPD) technique. An analysis of RAPD patterns by a molecular variance method revealed that there was a high level of genetic diversity in this population and that the genetic diversity was related to plant age. Finally, we found that some of the DAPG producers, which originated from all stages of plant growth, had the same genotype. These DAPG producers could be exploited in future screening programs for biocontrol agents.
Cloning, Characterization, Controlled Overexpression, and Inactivation of the Major Tributyrin Esterase Gene of
Lactococcus lactis
Authors: Fernández Leonides , Beerthuyzen Marke M. , Brown Julie , Siezen Roland J. , Coolbear Tim , Holland Ross , Kuipers Oscar P.
Published: July 2002

The gene encoding the major intracellular tributyrin esterase of
Lactococcus lactis
was cloned using degenerate DNA probes based on 19 known N-terminal amino acid residues of the purified enzyme. The gene, named
estA
, was sequenced and found to encode a protein of 258 amino acid residues. The transcription start site was mapped 233 nucleotides upstream of the start codon, and a canonical promoter sequence was identified. The deduced amino acid sequence of the
estA
product contained the typical GXSXG motif found in most lipases and esterases. The protein was overproduced up to 170-fold in
L. lactis
by use of the nisin-controlled expression system recently developed for lactic acid bacteria. The
estA
gene was inactivated by chromosomal integration of a temperature-sensitive integration vector. This resulted in the complete loss of esterase activity, which could then be recovered after complementation of the constructed esterase-deficient strain with the wild-type
estA
gene. This confirms that EstA is the main enzyme responsible for esterase activity in
L. lactis
. Purified recombinant enzyme showed a preference for short-chain acyl esters, surprisingly also including phospholipids. Medium- and long-acyl-chain lipids were also hydrolyzed, albeit less efficiently. Intermediate characteristics between esterases and lipases make intracellular lactococcal EstA difficult to classify in either of these two groups of esterolytic enzymes. We suggest that, in vivo, EstA could be involved in (phospho)lipid metabolism or cellular detoxification or both, as its sequence showed significant similarity to
S
-formylglutathione hydrolase (FGH) of
Paracoccus denitrificans
and human EstD (or FGH), which are part of a universal formaldehyde detoxification pathway.
Naphthalene and Donor Cell Density Influence Field Conjugation of Naphthalene Catabolism Plasmids
Authors: Hohnstock A. M. , Stuart-Keil K. G. , Kull E. E. , Madsen E. L.
Published: July 2002

We examined transfer of naphthalene-catabolic genes from donor microorganisms native to a contaminated site to site-derived, rifampin-resistant recipient bacteria unable to grow on naphthalene. Horizontal gene transfer (HGT) was demonstrated in filter matings using groundwater microorganisms as donors. Two distinct but similar plasmid types, closely related to pDTG1, were retrieved. In laboratory-incubated sediment matings, the addition of naphthalene stimulated HGT. However, recipient bacteria deployed in recoverable vessels in the field site (in situ) did not retrieve plasmids from native donors. Only when plasmid-containing donor cells and naphthalene were added to the in situ mating experiments did HGT occur.
Quantitative Determination of the Biodegradable Polymer Poly(β-hydroxybutyrate) in a Recombinant
Escherichia coli
Strain by Use of Mid-Infrared Spectroscopy and Multivariative Statistics
Authors: Kansiz Mustafa , Billman-Jacobe Helen , McNaughton Don
Published: July 2002

Fourier transform infrared (FTIR) spectroscopy in combination with the partial least squares (PLS) multivariative statistical technique was used for quantitative analysis of the poly(β-hydroxybutyrate) (PHB) contents of bacterial cells. A total of 237 replicate spectra from 34 samples were obtained together with gas chromatography-determined reference PHB contents. Using the PLS regression, we were able to relate the infrared spectra to the reference PHB contents, and the correlation coefficient between the measured and predicted values for the optimal model with a standard error of prediction of 1.49% PHB was 0.988. With this technique, there are no solvent requirements, sample preparation is minimal and simple, and analysis time is greatly reduced; our results demonstrate the potential of FTIR spectroscopy as an alternative to the conventional methods used for analysis of PHB in bacterial cells.
Simultaneous Recovery of RNA and DNA from Soils and Sediments
Authors: Hurt Richard A. , Qiu Xiaoyun , Wu Liyou , Roh Yul , Palumbo A. V. , Tiedje J. M. , Zhou Jizhong
Published: July 2002

Recovery of mRNA from environmental samples for measurement of in situ metabolic activities is a significant challenge. A robust, simple, rapid, and effective method was developed for simultaneous recovery of both RNA and DNA from soils of diverse composition by adapting our previous grinding-based cell lysis method (Zhou et al., Appl. Environ. Microbiol. 62:316–322, 1996) for DNA extraction. One of the key differences is that the samples are ground in a denaturing solution at a temperature below 0°C to inactivate nuclease activity. Two different methods were evaluated for separating RNA from DNA. Among the methods examined for RNA purification, anion exchange resin gave the best results in terms of RNA integrity, yield, and purity. With the optimized protocol, intact RNA and high-molecular-weight DNA were simultaneously recovered from 19 soil and stream sediment samples of diverse composition. The RNA yield from these samples ranged from 1.4 to 56 μg g of soil−1dry weight), whereas the DNA yield ranged from 23 to 435 μg g−1. In addition, studies with the same soil sample showed that the DNA yield was, on average, 40% higher than that in our previous procedure and 68% higher than that in a commercial bead milling method. For the majority of the samples, the DNA and RNA recovered were of sufficient purity for nuclease digestion, microarray hybridization, and PCR or reverse transcription-PCR amplification.
Clostridium beijerinckii
Cells Expressing
Neocallimastix patriciarum
Glycoside Hydrolases Show Enhanced Lichenan Utilization and Solvent Production
Authors: López-Contreras Ana M. , Smidt Hauke , van der Oost John , Claassen Pieternel A. M. , Mooibroek Hans , de Vos Willem M.
Published: July 2002

Growth and the production of acetone, butanol, and ethanol by
Clostridium beijerinckii
NCIMB 8052 on several polysaccharides and sugars were analyzed. On crystalline cellulose, growth and solvent production were observed only when a mixture of fungal cellulases was added to the medium. On lichenan growth and solvent production occurred, but this polymer was only partially utilized. To increase utilization of these polymers and subsequent solvent production, the genes for two new glycoside hydrolases,
celA
and
celD
from the fungus
Neocallimastix patriciarum
, were cloned separately into
C. beijerinckii
. To do this, a secretion vector based on the pMTL500E shuttle vector and containing the promoter and signal sequence coding region of the
Clostridium saccharobutylicum
NCP262
eglA
gene was constructed and fused either to the
celA
gene or the
celD
gene. Stable
C. beijerinckii
transformants were obtained with the resulting plasmids, pWUR3 (
celA
) and pWUR4 (
celD
). The recombinant strains showed clear halos on agar plates containing carboxymethyl cellulose upon staining with Congo red. In addition, their culture supernatants had significant endoglucanase activities (123 U/mg of protein for transformants harboring
celA
and 78 U/mg of protein for transformants harboring
celD
). Although
C. beijerinckii
harboring either
celA
or
celD
was not able to grow, separately or in mixed culture, on carboxymethyl cellulose or microcrystalline cellulose, both transformants showed a significant increase in solvent production during growth on lichenan and more extensive degradation of this polymer than that exhibited by the wild-type strain.
Effects of Combined Water Potential and Temperature Stresses on
Cryptosporidium parvum
Oocysts
Authors: Walker Mark , Leddy Katherine , Hagar Elaine
Published: July 2002

Hosts infected with the parasite
Cryptosporidium parvum
may excrete oocysts on soils in watersheds that supply public drinking water. Environmental stresses decrease the numbers of oocysts after deposition on soils. However, the rates and effects of combined stresses have not been well characterized, especially for the purposes of estimating decrease in numbers. We subjected oocysts to combined stresses of water potential (−4, −12, and −33 bars), above-freezing temperatures (4 and 30°C), and a subfreezing temperature (−14°C) for 1, 14, and 29 days and one to six freeze-thaw cycles (−14 to 10°C) to estimate coefficients to characterize population degradation using multiplicative error and exponential decay models. The experiments were carried out in NaCl solutions with water potentials of −4, −12, and −33 bars, in combination with temperature stresses at levels that could be expected in natural soils. Increased water potential increased the rate of population degradation for all temperature conditions investigated. Enhanced degradation leads to estimated rates of population degradation that are greater than those that have been reported and used in previous studies conducted to assess risk of water supply contamination from sources of
C. parvum
.
Multiplex PCR Assay for Detection and Identification ofClostridium botulinumTypes A, B, E, and F in Food and Fecal Material
Authors: Lindström Miia , Keto Riikka , Markkula Annukka , Nevas Mari , Hielm Sebastian , Korkeala Hannu
Published: July 2002

Botulism is diagnosed by detecting botulinum neurotoxin andClostridium botulinumcells in the patient and in suspected food samples. In this study, a multiplex PCR assay for the detection ofClostridium botulinumtypes A, B, E, and F in food and fecal material was developed. The method employs four new primer pairs with equal melting temperatures, each being specific to botulinum neurotoxin gene type A, B, E, or F, and enables a simultaneous detection of the four serotypes. A total of 43C. botulinumstrains and 18 strains of other bacterial species were tested. DNA amplification fragments of 782 bp forC. botulinumtype A alone, 205 bp for type B alone, 389 bp for type E alone, and 543 bp for type F alone were obtained. Other bacterial species, includingC. sporogenesand the nontoxigenic nonproteolyticC. botulinum-like organisms, did not yield a PCR product. Sensitivity of the PCR for types A, E, and F was 102cells and for type B was 10 cells per reaction mixture. With a two-step enrichment, the detection limit in food and fecal samples varied from 10−2spore/g for types A, B, and F to 10−1spore/g of sample material for type E. Of 72 natural food samples investigated, two were shown to containC. botulinumtype A, two contained type B, and one contained type E. The assay is sensitive and specific and provides a marked improvement in the PCR diagnostics ofC. botulinum.
Effects of Nondigestible Oligosaccharides onSalmonella entericaSerovar Typhimurium and NonpathogenicEscherichia coliin the Pig Small Intestine In Vitro
Authors: Naughton Patrick J. , Mikkelsen Lene Lind , Jensen Bent Borg
Published: July 2002

An in vitro intestinal tissue model was developed for the investigation of bacterial association in the pig small intestine under different dietary regimes. In preliminary experiments, jejunal and ileal tissue was taken from Danish Landrace pigs fed standard diet and inoculated with eitherSalmonellaor nonpathogenicEscherichia colistrains. Higher numbers of salmonellae associated with the ileal tissues, but the numbers did not reach significance. Hence, jejunal sections were inoculated with nonpathogenicE. coliand ileal sections were inoculated with salmonellae in the presence of mannose or commercial nondigestible oligosaccharides (NDO) at 2.5%. There was a significant decrease inE. coliassociated with the jejunum in the presence of mannose (P< 0.05). Furthermore, in pigs fed a diet supplemented with commercial NDO at 4% there was a significant reduction in the numbers ofE. coliin jejunal organ cultures of pigs fed the FOS diet (P< 0.05). There was a reduction, though not a significant one, in the association ofSalmonellasp. to the ileal sections of pigs fed the commercial FOS diet. The feeding of commercial GOS or its addition to organ cultures did not affectE. coliorSalmonellanumbers.
Fate ofMycobacterium aviumsubsp.paratuberculosisin Swiss Hard and Semihard Cheese Manufactured from Raw Milk
Authors: Spahr U. , Schafroth K.
Published: July 2002

Raw milk was artificially contaminated with declumped cells ofMycobacterium aviumsubsp.paratuberculosisat a concentration of 104to 105CFU/ml and was used to manufacture model hard (Swiss Emmentaler) and semihard (Swiss Tisliter) cheese. Two different strains ofM. aviumsubsp.paratuberculosiswere tested, and for each strain, two model hard and semihard cheeses were produced. The survival ofM. aviumsubsp.paratuberculosiscells was monitored over a ripening period of 120 days by plating out homogenized cheese samples onto 7H10-PANTA agar. In both the hard and the semihard cheeses, counts decreased steadily but slowly during cheese ripening. Nevertheless, viable cells could still be detected in 120-day cheese.Dvalues were calculated at 27.8 days for hard and 45.5 days for semihard cheese. The most important factors responsible for the death ofM. aviumsubsp.paratuberculosisin cheese were the temperatures applied during cheese manufacture and the low pH at the early stages of cheese ripening. Since the ripening period for these raw milk cheeses lasts at least 90 to 120 days, theDvalues found indicate that 103to 104cells ofM. aviumsubsp.paratuberculosisper g will be inactivated.
Diversity and Ubiquity of Bacteria Capable of Utilizing Humic Substances as Electron Donors for Anaerobic Respiration
Authors: Coates John D. , Cole Kimberly A. , Chakraborty Romy , O'Connor Susan M. , Achenbach Laurie A.
Published: July 2002

Previous studies have demonstrated that reduced humic substances (HS) can be reoxidized by anaerobic bacteria such as
Geobacter
,
Geothrix
, and
Wolinella
species with a suitable electron acceptor; however, little is known of the importance of this metabolism in the environment. Recently we investigated this metabolism in a diversity of environments including marine and aquatic sediments, forest soils, and drainage ditch soils. Most-probable-number enumeration studies were performed using 2,6-anthrahydroquinone disulfonate (AHDS), an analog for reduced HS, as the electron donor with nitrate as the electron acceptor. Anaerobic organisms capable of utilizing reduced HS as an electron donor were found in all environments tested and ranged from a low of 2.31 × 10
1
in aquifer sediments to a high of 9.33 × 10
6
in lake sediments. As part of this study we isolated six novel organisms capable of anaerobic AHDS oxidation. All of the isolates coupled the oxidation of AHDS to the reduction of nitrate with acetate (0.1 mM) as the carbon source. In the absence of cells, no AHDS oxidation was apparent, and in the absence of AHDS, no cell density increase was observed. Generally, nitrate was reduced to N
2
. Analysis of the AHDS and its oxidized form, 2,6-anthraquinone disulfonate (AQDS), in the medium during growth revealed that the anthraquinone was not being biodegraded as a carbon source and was simply being oxidized as an energy source. Determination of the AHDS oxidized and nitrate reduced accounted for 109% of the theoretical electron transfer. In addition to AHDS, all of these isolates could also couple the oxidation of reduced humic substances to the reduction of nitrate. No HS oxidation occurred in the absence of cells and in the absence of a suitable electron acceptor, demonstrating that these organisms were capable of utilizing natural HS as an energy source and that AHDS serves as a suitable analog for studying this metabolism. Alternative electron donors included simple volatile fatty acids such as propionate, butyrate, and valerate as well as simple organic acids such as lactate and pyruvate. Analysis of the complete sequences of the 16S rRNA genes revealed that the isolates were not closely related to each other and were phylogenetically diverse, with members in the alpha, beta, gamma, and delta subdivisions of the
Proteobacteria
. Most of the isolates were closely related to known genera not previously recognized for their ability to couple growth to HS oxidation, while one of the isolates represented a new genus in the delta subclass of the
Proteobacteria
. The results presented here demonstrate that microbial oxidation of HS is a ubiquitous metabolism in the environment. This study represents the first description of HS-oxidizing isolates and demonstrates that microorganisms capable of HS oxidation are phylogenetically diverse.
Isolation and Metabolic Characteristics of Previously Uncultured Members of the Order
Aquificales
in a Subsurface Gold Mine
Authors: Takai Ken , Hirayama Hisako , Sakihama Yuri , Inagaki Fumio , Yamato Yu , Horikoshi Koki
Published: July 2002

Culture-dependent and -independent techniques were combined to characterize the physiological properties and the ecological impacts of culture-resistant phylotypes of thermophiles within the order
Aquificales
from a subsurface hot aquifer of a Japanese gold mine. Thermophilic bacteria phylogenetically associated with previously uncultured phylotypes of
Aquificales
were successfully isolated. 16S ribosomal DNA clone analysis of the entire microbial DNA assemblage and fluorescence in situ whole-cell hybridization analysis indicated that the isolates dominated the microbial population in the subsurface aquifer. The isolates were facultatively anaerobic, hydrogen- or sulfur/thiosulfate-oxidizing, thermophilic chemolithoautotrophs utilizing molecular oxygen, nitrate, ferric iron, arsenate, selenate, and selenite as electron acceptors. Their versatile energy-generating systems may reflect the geochemical conditions of their habitat in the geothermally active subsurface gold mine.
Specificity and Prevalence of Natural Bovine Anti-Alpha Galactosyl (Galα1-6Glc or Galα1-6Gal) Antibodies
Authors: Ni Yawei , Powell Robert , Turner Debra D. , Tizard Ian
Published: July 2002

Immunity against the carbohydrate components of microorganisms mediated by antibodies is an important part of host defenses. Humans and closely related primates, but not other mammals, possess natural anti-Galα1-3Gal antibodies which also, although less avidly, react with melibiose (Galα1-6Glc). Using an enzyme-linked immunosorbent assay (ELISA) with melibiose-bovine serum albumin as an antigen, we analyzed bovine anti-alpha galactosyl antibodies with respect to specificity and distribution in individual animals. Inhibition assays showed that melibiose was the strongest inhibitor, followed equally by stachyose (Galα1-6Galα1-6Glcβ1-2Fru) and raffinose (Galα1-6Glcβ1-2Fru) and then by Galβ1-6Gal, Gal, and Galα1-2Gal. Others, including Galα1-3Gal and Galα1-4Gal, only exhibited minor inhibition. Thus, these bovine anti-alpha galactosyl antibodies appeared to preferentially react with Galα1-6Glc or Galα1-6Gal. The distinction of this specificity from that (Galα1-3Gal) of human antibodies was further demonstrated by the poor reaction of bovine serum to the Galα1-3Gal antigen in comparison to human serum. All 27 healthy bovine serum samples of the three age groups (newborn, calf, and adult) tested contained such antibodies with titers increasing with age. The antibodies purified by affinity chromatography using a melibiose-agarose column were mainly of the immunoglobulin G (IgG) isotype with a concentration of >23 μg/ml in most samples. IgG1 was found to be the primary antimelibiose IgG isotype in all age groups by isotype-specific ELISA, but a significant increase in IgG2, an isotype more related to innate immunity, was observed in calves and adults, compared to newborns. The purified antibodies reacted with the type II bovine strain ofStreptococcus agalactiae, a common pathogen of bovine mastitis. Thus, these anti-Galα1-6Glc or Galα1-6Gal antibodies in cattle might be involved in defense against microbes bearing this or the related epitopes.
Lack of Utility of Specific Immunoglobulin G Antibody Avidity for Serodiagnosis of Reactivated Toxoplasmosis in Immunocompromised Patients
Authors: Mechain Bénédicte , Garin Yves Jean-François , Robert-Gangneux Florence , Dupouy-Camet Jean , Derouin Francis
Published: July 2002

The avidities ofToxoplasma-specific immunoglobulin G serum antibodies were measured in immunocompromised patients presenting with cerebral or extracerebral toxoplasmosis and/or serological reactivation. Since avidity remained high and stable in 39 of 40 patients with toxoplasmosis and 27 of 28 patients with serological reactivation, we conclude that this test cannot help diagnose toxoplasmosis in these patients.
The AIDA Autotransporter System Is Associated with F18 and Stx2e inEscherichia coliIsolates from Pigs Diagnosed with Edema Disease and Postweaning Diarrhea
Authors: Niewerth Ulla , Frey Andreas , Voss Thomas , Le Bouguénec Chantal , Baljer Georg , Franke Sylvia , Schmidt M. Alexander
Published: July 2002

PathogenicEscherichia colistrains are known to cause edema disease (ED) and postweaning diarrhea (PWD) in piglets. Although the exact mechanisms of pathogenicity that lead to ED-PWD remain to be elucidated,E. coli-borne Shiga-like toxin and adhesion-mediating virulence factors such as F18 adhesin or F4 fimbriae are believed to play a central role in ED-PWD. In light of these observations we investigated whether anotherE. coliadhesin, the plasmid-encoded AIDA (adhesin involved in diffuse adherence) might also be present in ED-PWD-causingE. coliisolates. For rapid screening for the AIDA system in large numbers of isolates, a multiplex PCR method along with a duplex Western blot procedure was developed. When screening 104 strains obtained from pigs with or without ED-PWD, we observed a high prevalence of the AIDA operon in porcineE. coliisolates, with over 25% of all strains being AIDA positive, and we could demonstrate a significant association of the intact AIDA gene (orfB) with ED-PWD, while defects inorfBwere associated with the absence of disease. Although our data hint toward a contribution of AIDA to ED-PWD, further studies will be necessary since the presence of the AIDA genes was also associated with the presence of the Shiga-like toxin and F18 adhesin genes, two reported virulence factors for ED-PWD.
Adherence ofGiardia lambliaTrophozoites to Int-407 Human Intestinal Cells
Authors: Céu Sousa M. , Gonçalves C. A. , Bairos V. A. , Poiares-da-Silva J.
Published: July 2002

Attachment ofGiardia lambliatrophozoites to enterocytes is essential for colonization of the small intestine and is considered a prerequisite for parasite-induced enterocyte dysfunction and clinical disease. In this work, coincubation ofGiardiawith Int-407 cells, was used as an in vitro model to study the role of cytoskeleton and surface lectins involved in the attachment of the parasite. This interaction was also studied by scanning and transmission electron microscopy. Adherence was dependent on temperature and was maximal at 37°C. It was reduced by 2.5 mM colchicine (57%), mebendazole (10 μg/ml) (59%), 100 mM glucose (26%), 100 mM mannose (22%), 40 mM mannose-6-phosphate (18%), and concanavalin A (100 μg/ml) (21%). No significant modification was observed whenGiardiawas pretreated with cytochalasins B and D and with EDTA.Giardiaattachment was also diminished by preincubating Int-407 cells with cytochalasin B and D (5 μg/ml) (16%) and by glutaraldehyde fixation of intestinal cells and ofG. lambliatrophozoites (72 and 100%, respectively). Ultrastructural studies showed thatGiardiaattaches to the Int-407 monolayer predominantly by its ventral surface. Int-407 cells contact trophozoites with elongated microvilli, and both trophozoite imprints and interactions ofGiardiaflagella with intestinal cells were also observed. Transmission electron microscopy showed thatGiardialateral crest and ventrolateral flange were important structures in the adherence process. Our results suggest a combination of mechanical and hydrodynamic forces in trophozoite attachment; surface lectins also seem to mediate binding and may be involved in specific recognition of host cells.
Use of Hoechst 33342 Staining To Detect Apoptotic Changes in Bovine Mononuclear Phagocytes Infected withMycobacterium aviumsubsp.paratuberculosis
Authors: Allen S. , Sotos J. , Sylte M. J. , Czuprynski C. J.
Published: July 2002

Mycobacterium aviumsubsp.paratuberculosisis an intracellular pathogen of macrophages that causes a chronic enteritis (Johne's disease) in ruminants. The purpose of this study was to determine whetherM. aviumsubsp.paratuberculosisinfection causes apoptosis in bovine monocytes. Using Hoechst 33342 staining, we observed increased numbers of apoptotic monocytes within 6 h of infection withM. aviumsubsp.paratuberculosis, and these numbers increased further at 24 and 48 h. This effect appeared to require viable bacilli, because monocytes infected with heat-killedM. aviumsubsp.paratuberculosisdid not exhibit a significant increase in apoptosis. Preincubation of monocytes with bovine growth hormone prior to infection withM. aviumsubsp.paratuberculosisdid not significantly alter the number of apoptotic cells.