bluuebunny/crossref_metadata_2025 huggingface dataset:
Aminoglycoside Resistance in
Mycobacterium kansasii
,
Mycobacterium avium-M. intracellulare
, and
Mycobacterium fortuitum
: Are Aminoglycoside-Modifying Enzymes Responsible?
Authors: Ho I. I. Y. , Chan C. Y. , Cheng A. F. B.
Published: April 2009

Aminoglycoside acetyltransferase was detected in
Mycobacterium kansasii
and
M. fortuitum
but not in
M. avium-M. intracellulare
when they were screened by a radioassay. Aminoglycoside phosphotransferase and nucleotidyltransferase activities were absent from all three species tested. Acetyltransferases from both
M. kansasii
and
M. fortuitum
displayed relatively high

K
m

s, all at the millimolar level, for substrates including tobramycin, neomycin, and kanamycin A. The

K
m

of each substrate was well above the corresponding maximum achievable level in serum. The low affinities of these enzymes for their substrates suggested that drug modification in vivo was very unlikely. Among the various substrates tested, no apparent positive correlation was found between substrate affinity and resistance level. The presence of aminoglycoside-modifying enzymes in these mycobacterial species was therefore not shown to confer resistance to aminoglycosides.
Bacteriologic Efficacies of Oral Azithromycin and Oral Cefaclor in Treatment of Acute Otitis Media in Infants and Young Children
Authors: Dagan Ron , Leibovitz Eugene , Fliss Dan M. , Leiberman Alberto , Jacobs Michael R. , Craig William , Yagupsky Pablo
Published: April 2009

A prospective, open-label, randomized study was conducted in order to determine the bacteriologic efficacies of cefaclor and azithromycin in acute otitis media (AOM). Tympanocentesis was performed on entry into the study and 3 to 4 days after initiation of treatment. Bacteriologic failure after 3 to 4 days of treatment with both drugs occurred in a high proportion of culture-positive patients, especially in those in whom AOM was caused by
Haemophilus influenzae
(16 of 33 [53%] of those treated with azithromycin and 13 of 34 [52%] of those treated with cefaclor). Although a clear correlation of the persistence of the pathogen with increased MICs of the respective drugs could be demonstrated for
Streptococcus pneumoniae
, no such correlation was found for
H. influenzae
. It is proposed that susceptibility breakpoints for
H. influenzae
should be considerably lower than the current ones for both cefaclor and azithromycin for AOM caused by
H. influenzae
.
The CXCR4 Antagonist AMD3100 Efficiently Inhibits Cell-Surface-Expressed Human Immunodeficiency Virus Type 1 Envelope-Induced Apoptosis
Authors: Blanco Julià , Barretina Jordi , Henson Geoffrey , Bridger Gary , De Clercq Erik , Clotet Bonaventura , Esté José A.
Published: April 2009

Infection by human immunodeficiency virus type 1 (HIV-1) has been associated with increased cell death by apoptosis in infected and uninfected cells. The envelope glycoprotein complex ([gp120/gp41]n) of X4 HIV-1 isolates is involved in both infected and uninfected cell death via its interaction with cellular receptors CD4 and CXCR4. We studied the effect of the blockade of CXCR4 receptors by the agonist stromal derived factor (SDF-1α) and the antagonist bicyclam AMD3100 on apoptotic cell death of CD4+cells in different models of HIV infection. In HIV-infected CEM or SUP-T1 cultures, AMD3100 showed antiapoptotic activity even when added 24 h after infection. In contrast, other antiviral agents, such as zidovudine, failed to block apoptosis under these conditions. The antiapoptotic activity of AMD3100 was also studied in coculture of peripheral blood mononuclear cells or CD4+cell lines with chronically infected H9/IIIB cells. AMD3100 was found to inhibit both syncytium formation and apoptosis induction with 50% inhibitory concentrations ranging from 0.009 to 0.24 μg/ml, depending on the cell type. When compared to SDF-1α, AMD3100 showed higher inhibitory potency in all cell lines tested. Our data indicate that the bicyclam AMD3100 not only inhibits HIV replication but also efficiently blocks cell-surface-expressed HIV-1 envelope-induced apoptosis in uninfected cells.
In Vitro Activities of a New Lipopeptide Antifungal Agent, FK463, against a Variety of Clinically Important Fungi
Authors: Tawara Shuichi , Ikeda Fumiaki , Maki Katsuyuki , Morishita Yoshihiko , Otomo Kazumi , Teratani Noriko , Goto Toshio , Tomishima Masaki , Ohki Hidenori , Yamada Akira , Kawabata Koji , Takasugi Hisashi , Sakane Kazuo , Tanaka Hirokazu , Matsumoto Fumio , Kuwahara Shogo
Published: April 2009

The in vitro antifungal activity and spectrum of FK463 were compared with those of amphotericin B, fluconazole, and itraconazole by using a broth microdilution method specified by National Committee for Clinical Laboratory Standards document M27-A (National Committee for Clinical Laboratory Standards, Wayne, Pa., 1997). FK463 exhibited broad-spectrum activity against clinically important pathogens including
Candida
species (MIC range, ≦0.0039 to 2 μg/ml) and
Aspergillus
species (MIC range, ≦0.0039 to 0.0313 μg/ml), and its MICs for such fungi were lower than those of the other antifungal agents tested. FK463 was also potently active against azole-resistant
Candida albicans
as well as azole-susceptible strains, and there was no cross-resistance with azoles. FK463 showed fungicidal activity against
C. albicans
, i.e., a 99% reduction in viability after a 24-h exposure at concentrations above 0.0156 μg/ml. The minimum fungicidal concentration (MFC) assays indicated that FK463 was fungicidal against most isolates of
Candida
species. In contrast, the MFCs of FK463 for
A. fumigatus
isolates were much higher than the MICs, indicating that its action is fungistatic against this species. FK463 had no activity against
Cryptococcus neoformans
,
Trichosporon
species, or
Fusarium solani
. Neither the test medium (kind and pH) nor the inoculum size greatly affected the MICs of FK463, while the addition of 4% human serum albumin increased the MICs for
Candida
species and
A. fumigatus
more than 32 times. Results from preclinical in vitro evaluations performed thus far indicate that FK463 should be a potent parenteral antifungal agent.
The R467K Amino Acid Substitution in
Candida albicans
Sterol 14α-Demethylase Causes Drug Resistance through Reduced Affinity
Authors: Lamb David C. , Kelly Diane E. , White Theodore C. , Kelly Steven L.
Published: April 2009

The cytochrome P450 sterol 14α-demethylase (CYP51) of
Candida albicans
is involved in an essential step of ergosterol biosynthesis and is the target for azole antifungal compounds. We have undertaken site-directed mutation of
C. albicans CYP51
to produce a recombinant mutant protein with the amino acid substitution R467K corresponding to a mutation observed clinically. This alteration perturbed the heme environment causing an altered reduced-carbon monoxide difference spectrum with a maximum at 452 nm and reduced the affinity of the enzyme for fluconazole, as shown by ligand binding studies. The specific activity of CYP51(R467K) for the release of formic acid from 3β-[32-
3
H]hydroxylanost-7-en-32-ol was 70 pmol/nmol of P450/min for microsomal protein compared to 240 pmol/nmol of P450/min for microsomal fractions expressing wild-type CYP51. Furthermore, inhibition of activity by fluconazole revealed a 7.5-fold-greater azole resistance of the recombinant protein than that of the wild type. This study demonstrates that resistance observed clinically can result from the altered azole affinity of the fungal CYP51 enzyme.
Zanamivir Susceptibility Monitoring and Characterization of Influenza Virus Clinical Isolates Obtained during Phase II Clinical Efficacy Studies
Authors: Barnett J. M. , Cadman A. , Gor D. , Dempsey M. , Walters M. , Candlin A. , Tisdale M. , Morley P. J. , Owens I. J. , Fenton R. J. , Lewis A. P. , Claas E. C. J. , Rimmelzwaan G. F. , De Groot R. , Osterhaus A. D. M. E.
Published: April 2009

Zanamivir is a highly selective neuraminidase (NA) inhibitor with demonstrated clinical efficacy against influenza A and B virus infections. In phase II clinical efficacy trials (NAIB2005 and NAIB2008), virological substudies showed mean reductions in virus shedding after 24 h of treatment of 1.5 to 2.0 log
10
50% tissue culture infective doses compared to a placebo, with no reemergence of virus after the completion of therapy. Paired isolates (
n
= 41) obtained before and during therapy with zanamivir demonstrated no shifts in susceptibility to zanamivir when measured by NA assays, although for a few isolates NA activity was too low to evaluate. In plaque reduction assays in MDCK cells, the susceptibility of isolates to zanamivir was extremely variable even at baseline and did not correlate with the speed of resolution of virus shedding. Isolates with apparent limited susceptibility to zanamivir by plaque reduction proved highly susceptible in vivo in the ferret model. Further sequence analysis of paired isolates revealed no changes in the hemagglutinin and NA genes in the majority of isolates. The few changes observed were all natural variants. No amino acid changes that had previously been identified in vitro as being involved with reduced susceptibility to zanamivir were observed. These studies highlighted problems associated with monitoring susceptibility to NA inhibitors in the clinic, in that no reliable cell-based assay is available. At present the NA assay is the best available predictor of susceptibility to NA inhibitors in vivo, as measured in the validated ferret model of infection.
In Vitro Activity of Riboflavin against the Human Malaria Parasite
Plasmodium falciparum
Authors: Akompong Thomas , Ghori Nafisa , Haldar Kasturi
Published: April 2009

The human malaria parasite
Plasmodium falciparum
digests hemoglobin and polymerizes the released free heme into hemozoin. This activity occurs in an acidic organelle called the food vacuole and is essential for survival of the parasite in erythrocytes. Since acidic conditions are known to enhance the auto-oxidation of hemoglobin, we investigated whether hemoglobin ingested by the parasite was oxidized and whether the oxidation process could be a target for chemotherapy against malaria. We released parasites from their host cells and separately analyzed hemoglobin ingested by the parasites from that remaining in the erythrocytes. Isolated parasites contained elevated amounts (38.5% ± 3.5%) of oxidized hemoglobin (methemoglobin) compared to levels (0.8% ± 0.2%) found in normal, uninfected erythrocytes. Further, treatment of infected cells with the reducing agent riboflavin for 24 h decreased the parasite methemoglobin level by 55%. It also inhibited hemozoin production by 50% and decreased the average size of the food vacuole by 47%. Administration of riboflavin for 48 h resulted in a 65% decrease in food vacuole size and inhibited asexual parasite growth in cultures. High doses of riboflavin are used clinically to treat congenital methemoglobinemia without any adverse side effects. This activity, in conjunction with its impressive antimalarial activity, makes riboflavin attractive as a safe and inexpensive drug for treating malaria caused by
P. falciparum
.
Persistence of Infectious Herpes Simplex Virus Type 2 in the Nervous System in Mice after Antiviral Chemotherapy
Authors: Thackray Alana M. , Field Hugh J.
Published: April 2009

Young adult mice were inoculated with herpes simplex virus type 2 (HSV-2) in the ear pinna. A relatively severe infection resulted, and 45% of the mice died by 11 days postinfection. Therapy at 1 mg/ml by means of the drinking water with either famciclovir for periods of 5 or 10 days or valaciclovir for 5, 10, 15, or 20 days decreased clinical signs and reduced mortality to 15% or less. Throughout a period of 27 days, mice were tested daily for the presence of infectious virus in the ear pinna, brain stem, and ipsilateral trigeminal ganglia. Virus was cleared from these tissues in surviving, untreated animals by 12 days postinfection, and no infectious virus was detected subsequently in any tissue. Furthermore, no infectious virus was detected after day 9 in mice that had been treated with famciclovir. In mice that had received valaciclovir therapy, however, infectious virus was repeatedly detected in the trigeminal ganglia and brain stem tissue samples up to 7 days after treatment was discontinued. To date, no specific mechanism to account for these results has been discovered; however, possible mechanisms for the persistence of potentially infectious virus in neural tissue of treated mice are discussed.
Sequestration of Zinc Oxide by Fimbrial Designer Chelators
Authors: Kjærgaard Kristian , Sørensen Jack K. , Schembri Mark A. , Klemm Per
Published: April 2009

Type 1 fimbriae are surface organelles of
Escherichia coli
. By engineering a structural component of the fimbriae, FimH, to display a random peptide library, we were able to isolate metal-chelating bacteria. A library consisting of 4 × 10
7
independent clones was screened for binding to ZnO. Sequences responsible for ZnO adherence were identified, and distinct binding motifs were characterized. The sequences selected exhibited various degrees of affinity and specificity towards ZnO. Competitive binding experiments revealed that the sequences recognized only the oxide form of Zn. Interestingly, one of the inserts exhibited significant homology to a specific sequence in a putative zinc-containing helicase, which suggests that searches such as this one may aid in identifying binding motifs in nature. The zinc-binding bacteria might have a use in detoxification of metal-polluted water.
The
ntrB
and
ntrC
Genes Are Involved in the Regulation of Poly-3-Hydroxybutyrate Biosynthesis by Ammonia in
Azospirillum brasilense
Sp7
Authors: Sun Jun , Peng Xuan , Van Impe Jan , Vanderleyden Jos
Published: April 2009

Azospirillum brasilense
Sp7 and its
ntrA
(
rpoN
),
ntrBC
, and
ntrC
mutants have been evaluated for their capabilities of poly-3-hydroxybutyrate (PHB) accumulation in media with high and low ammonia concentrations. It was observed that the
ntrBC
and
ntrC
mutants can produce PHB in both low- and high-C/N-ratio media, while no significant PHB production was observed for the wild type or the
ntrA
mutant in low-C/N-ratio media. Further investigation by fermentation analysis indicated that the
ntrBC
and
ntrC
mutants were able to grow and accumulate PHB simultaneously in the presence of a high concentration of ammonia in the medium, while little PHB was produced in the wild type and
ntrA
(
rpoN
) mutant during active growth phase. These results provide the first genetic evidence that the
ntrB
and
ntrC
genes are involved in the regulation of PHB synthesis by ammonia in
A. brasilense
Sp7.
Plasmid Transfer between the
Bacillus thuringiensis
Subspecies
kurstaki
and
tenebrionis
in Laboratory Culture and Soil and in Lepidopteran and Coleopteran Larvae
Authors: Thomas D. John I. , Morgan J. Alun W. , Whipps John M. , Saunders Jon R.
Published: April 2009

Plasmid transfer between
Bacillus thuringiensis
subsp.
kurstaki
HD1 and
B. thuringiensis
subsp.
tenebrionis
donor strains and a streptomycin-resistant
B. thuringiensis
subsp.
kurstaki
recipient was studied under environmentally relevant laboratory conditions in vitro, in soil, and in insects. Plasmid transfer was detected in vitro at temperatures of 5 to 37°C, at pH 5.9 to 9.0, and at water activities of 0.965 to 0.995, and the highest transfer ratios (up to 10
−1
transconjugant/donor) were detected within 4 h. In contrast, no plasmid transfer was detected in nonsterile soil, and rapid formation of spores by the introduced strains probably contributed most to the lack of plasmid transfer observed. When a
B. thuringiensis
subsp.
kurstaki
strain was used as the donor strain, plasmid transfer was detected in killed susceptible lepidopteran insect (
Lacanobia oleracea
) larvae but not in the nonsusceptible coleopteran insect
Phaedon chocleriae
. When a
B. thuringiensis
subsp.
tenerbrionis
strain was used as the donor strain, no plasmid transfer was detected in either of these insects even when they were killed. These results show that in larger susceptible lepidopteran insects there is a greater opportunity for growth of
B. thuringiensis
strains, and this finding, combined with decreased competition due to a low initial background bacterial population, can provide suitable conditions for efficient plasmid transfer in the environment.
Impact of an Urban Effluent on Antibiotic Resistance of Riverine
Enterobacteriaceae
and
Aeromonas
spp
Authors: Goñi-Urriza Marisol , Capdepuy Michèle , Arpin Corinne , Raymond Nathalie , Caumette Pierre , Quentin Claudine
Published: April 2009

In order to evaluate the impact of an urban effluent on antibiotic resistance of freshwater bacterial populations, water samples were collected from the Arga river (Spain), upstream and downstream from the wastewater discharge of the city of Pamplona. Strains of
Enterobacteriaceae
(representative of the human and animal commensal flora) (110 isolates) and
Aeromonas
(typically waterborne bacteria) (118 isolates) were selected for antibiotic susceptibility testing. Most of the
Aeromonas
strains (72%) and many of the
Enterobacteriaceae
(20%) were resistant to nalidixic acid. Singly nalidixic acid-resistant strains were frequent regardless of the sampling site for
Aeromonas
, whereas they were more common upstream from the discharge for enterobacteria. The most common resistances to antibiotics other than quinolones were to tetracycline (24.3%) and beta-lactams (20.5%) for
Enterobacteriaceae
and to tetracycline (27.5%) and co-trimoxazole (26.6%) for
Aeromonas
. The rates of these antibiotic resistances increased downstream from the discharge at similar degrees for the two bacterial groups; it remained at high levels for enterobacteria but decreased along the 30-km study zone for
Aeromonas
. Genetic analysis of representative strains demonstrated that these resistances were mostly (enterobacteria) or exclusively (
Aeromonas
) chromosomally mediated. Moreover, a reference strain of
Aeromonas caviae
(CIP 7616) could not be transformed with conjugative R plasmids of enterobacteria. Thus, the urban effluent resulted in an increase of the rates of resistance to antibiotics other than quinolones in the riverine bacterial populations, despite limited genetic exchanges between enterobacteria and
Aeromonas
. Quinolone resistance probably was selected by heavy antibiotic discharges of unknown origin upstream from the urban effluent.
Genetic Diversity of
Vibrio cholerae
in Chesapeake Bay Determined by Amplified Fragment Length Polymorphism Fingerprinting
Authors: Jiang Sunny C. , Louis Valerie , Choopun Nipa , Sharma Anjana , Huq Anwar , Colwell Rita R.
Published: April 2009

Vibrio cholerae
is indigenous to the aquatic environment, and serotype non-O1 strains are readily isolated from coastal waters. However, in comparison with intensive studies of the O1 group, relatively little effort has been made to analyze the population structure and molecular evolution of non-O1
V. cholerae
. In this study, high-resolution genomic DNA fingerprinting, amplified fragment length polymorphism (AFLP), was used to characterize the temporal and spatial genetic diversity of 67
V. cholerae
strains isolated from Chesapeake Bay during April through July 1998, at four different sampling sites. Isolation of
V. cholerae
during the winter months (January through March) was unsuccessful, as observed in earlier studies (J. H. L. Kaper, R. R. Colwell, and S. W. Joseph, Appl. Environ. Microbiol. 37:91–103, 1979). AFLP fingerprints subjected to similarity analysis yielded a grouping of isolates into three large clusters, reflecting time of the year when the strains were isolated. April and May isolates were closely related, while July isolates were genetically diverse and did not cluster with the isolates obtained earlier in the year. The results suggest that the population structure of
V. cholerae
undergoes a shift in genotype that is linked to changes in environmental conditions. From January to July, the water temperature increased from 3°C to 27.5°C, bacterial direct counts increased nearly an order of magnitude, and the chlorophyll
a
concentration tripled (or even quadrupled at some sites). No correlation was observed between genetic similarity among isolates and geographical source of isolation, since isolates found at a single sampling site were genetically diverse and genetically identical isolates were found at several of the sampling sites. Thus,
V. cholerae
populations may be transported by surface currents throughout the entire Bay, or, more likely, similar environmental conditions may be selected for a specific genotype. The dynamic nature of the population structure of this bacterial species in Chesapeake Bay provides new insight into the ecology and molecular evolution of
V. cholerae
in the natural environment.
Generation of Novel Bacterial Regulatory Proteins That Detect Priority Pollutant Phenols
Authors: Wise Arlene A. , Kuske Cheryl R.
Published: April 2009

The genetic systems of bacteria that have the ability to use organic pollutants as carbon and energy sources can be adapted to create bacterial biosensors for the detection of industrial pollution. The creation of bacterial biosensors is hampered by a lack of information about the genetic systems that control production of bacterial enzymes that metabolize pollutants. We have attempted to overcome this problem through modification of DmpR, a regulatory protein for the phenol degradation pathway of
Pseudomonas
sp. strain CF600. The phenol detection capacity of DmpR was altered by using mutagenic PCR targeted to the DmpR sensor domain. DmpR mutants were identified that both increased sensitivity to the phenolic effectors of wild-type DmpR and increased the range of molecules detected. The phenol detection characteristics of seven DmpR mutants were demonstrated through their ability to activate transcription of a
lacZ
reporter gene. Effectors of the DmpR derivatives included phenol, 2-chlorophenol, 2,4-dichlorophenol, 4-chloro-3-methylphenol, 2,4-dimethylphenol, 2-nitrophenol, and 4-nitrophenol.
Microcystin Production byMicrocystis aeruginosain a Phosphorus-Limited Chemostat
Authors: Oh Hee-Mock , Lee Seog June , Jang Min-Ho , Yoon Byung-Dae
Published: April 2009

The production of microcystins (MC) fromMicrocystis aeruginosaUTEX 2388 was investigated in a P-limited continuous culture. MC (MC-LR, MC-RR, and MC-YR) from lyophilizedM. aeruginosawere extracted with 5% acetic acid, purified by a Sep-Pak C18cartridge, and then analyzed by high-performance liquid chromatography with a UV detector and Nucleosil C18reverse-phase column. The specific growth rate (μ) ofM. aeruginosawas within the range of 0.1 to 0.8/day and was a function of the cellular P content under a P limitation. The N/P atomic ratio of steady-state cells in a P-limited medium varied from 24 to 15 with an increasing μ. The MC-LR and MC-RR contents on a dry weight basis were highest at μ of 0.1/day at 339 and 774 μg g−1, respectively, while MC-YR was not detected. The MC content ofM. aeruginosawas higher at a lower μ, whereas the MC-producing rate was linearly proportional to μ. The C fixation rate at an ambient irradiance (160 microeinsteins m−2s−1) increased with μ. The ratios of the MC-producing rate to the C fixation rate were higher at a lower μ. Accordingly, the growth ofM. aeruginosawas reduced under a P limitation due to a low C fixation rate, whereas the MC content was higher. Consequently, increases in the MC content per dry weight along with the production of the more toxic form, MC-LR, were observed under more P-limited conditions.
Autumnal Biomass and Potential Productivity of Salt Marsh Fungi from 29° to 43° North Latitude along the United States Atlantic Coast
Authors: Newell Steven Y. , Blum Linda K. , Crawford Richard E. , Dai Ting , Dionne Michele
Published: April 2009

It has been established that substantial amounts of fungal mass accumulate in standing decaying smooth cordgrass (
Spartina alterniflora
) marshes in the southeastern United States (e.g., in standing decaying leaf blades with a total fungal organic mass that accounts for about 20% of the decay system organic mass), but it has been hypothesized that in marshes farther north this is not true. We obtained samples of autumnal standing decaying smooth cordgrass from sites in Florida to Maine over a 3-year period. The variation in latitude could not explain any of the variation in the living fungal standing crop (as determined by ergosterol content) or in the instantaneous rates of fungal growth (as determined by acetate incorporation into ergosterol at a standard temperature, 20°C), which led to the conclusion that the potential levels of fungal production per unit of naturally decaying grass are not different in northern and southern marshes. Twenty-one percent of the variation in the size of the living fungal standing crop could be explained by variation in the C/N ratio (the higher the C/N ratio the smaller the fungal crop), but the C/P ratio was not related to the size of the fungal crop. Instantaneous rates of fungal growth were negatively related to the size of the living fungal crop (
r
= −0.35), but these rates were not correlated with C/nutrient ratios. The same two predominant species of ascomycetes (one
Phaeosphaeria
species and one
Mycosphaerella
species) were found ejecting ascospores from standing decaying smooth cordgrass blades at all of the sites examined from Florida to Maine.
Kinetic and Nuclear Magnetic Resonance Studies of Xylose Metabolism by Recombinant
Zymomonas mobilis
ZM4(pZB5)
Authors: Kim In Seop , Barrow Kevin D. , Rogers Peter L.
Published: April 2009

The specific rates of growth, substrate utilization, and ethanol production as well as yields of biomass and ethanol production on xylose for the recombinant
Zymomonas mobilis
ZM4(pZB5) were shown to be much less than those on glucose or glucose-xylose mixtures. Typical fermentations with ZM4(pZB5) growing on glucose-xylose mixtures followed two-phase growth kinetics with the initial uptakes of glucose and xylose being followed by slower growth and metabolic uncoupling on xylose after glucose depletion. The reductions in rates and yields from xylose metabolism were considered in the present investigation and may be due to a number of factors, including the following: (i) the increased metabolic burden from maintenance of plasmid-related functions, (ii) the production of by-products identified as xylitol, acetate, lactate, acetoin, and dihydroxyacetone by
13
C-nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography, (iii) growth inhibition due to xylitol by the putative inhibitory compound xylitol phosphate, and (iv) the less energized state of ZM4(pZB5). In vivo
31
P-NMR studies have established that the levels of NTP and UDP sugars on xylose were less than those on glucose, and this energy limitation is likely to restrict the growth of the recombinant strain on xylose media.
A Simple Methodological Approach for Counting and Identifying Culturable Viruses Adsorbed to Cellulose Nitrate Membrane Filters
Authors: Papageorgiou Georgios T. , Mocé-Llivina Laura , Christodoulou Christina G. , Lucena Francisco , Akkelidou Dina , Ioannou Eleni , Jofre Juan
Published: April 2009

We identified conditions under which Buffalo green monkey cells grew on the surfaces of cellulose nitrate membrane filters in such a way that they covered the entire surface of each filter and penetrated through the pores. When such conditions were used, poliovirus that had previously been adsorbed on the membranes infected the cells and replicated. A plaque assay method and a quantal method (most probable number of cytopathic units) were used to detect and count the viruses adsorbed on the membrane filters. Polioviruses in aqueous suspensions were then concentrated by adsorption to cellulose membrane filters and were subsequently counted without elution, a step which is necessary when the commonly used methods are employed. The pore size of the membrane filter, the sample contents, and the sample volume were optimized for tap water, seawater, and a 0.25 M glycine buffer solution. The numbers of viruses recovered under the optimized conditions were more than 50% greater than the numbers counted by the standard plaque assay. When ceftazidime was added to the assay medium in addition to the antibiotics which are typically used, the method could be used to study natural samples with low and intermediate levels of microbial pollution without decontamination of the samples. This methodological approach also allowed plaque hybridization either directly on cellulose nitrate membranes or on Hybond N+ membranes after the preparations were transferred.
Development of Methods To Detect “Norwalk-Like Viruses” (NLVs) and Hepatitis A Virus in Delicatessen Foods: Application to a Food-Borne NLV Outbreak
Authors: Schwab Kellogg J. , Neill Frederick H. , Fankhauser Rebecca L. , Daniels Nicholas A. , Monroe Stephan S. , Bergmire-Sweat David A. , Estes Mary K. , Atmar Robert L.
Published: April 2009

“Norwalk-like viruses” (NLVs) and hepatitis A virus (HAV) are the most common causes of virus-mediated food-borne illness. Epidemiological investigations of outbreaks associated with these viruses have been hindered by the lack of available methods for the detection of NLVs and HAV in foodstuffs. Although reverse transcription (RT)-PCR methods have been useful in detecting NLVs and HAV in bivalve mollusks implicated in outbreaks, to date such methods have not been available for other foods. To address this need, we developed a method to detect NLVs and HAV recovered from food samples. The method involves washing of food samples with a guanidinium-phenol-based reagent, extraction with chloroform, and precipitation in isopropanol. Recovered viral RNA is amplified with HAV- or NLV-specific primers in RT-PCRs, using a viral RNA internal standard control to identify potential sample inhibition. By this method, 10 to 100 PCR units (estimated to be equivalent to 10
2
to 10
3
viral genome copies) of HAV and Norwalk virus seeded onto ham, turkey, and roast beef were detected. The method was applied to food samples implicated in an NLV-associated outbreak at a university cafeteria. Sliced deli ham was positive for a genogroup II NLV as determined by using both polymerase- and capsid-specific primers and probes. Sequence analysis of the PCR-amplified capsid region of the genome indicated that the sequence was identical to the sequence from virus detected in the stools of ill students. The developed method is rapid, simple, and efficient.
Safety Evaluation of Sous Vide-Processed Products with Respect to Nonproteolytic
Clostridium botulinum
by Use of Challenge Studies and Predictive Microbiological Models
Authors: Hyytiä-Trees Eija , Skyttä Eija , Mokkila Mirja , Kinnunen Arvo , Lindström Miia , Lähteenmäki Liisa , Ahvenainen Raija , Korkeala Hannu
Published: April 2009

Sixteen different types of sous vide-processed products were evaluated for safety with respect to nonproteolytic group II
Clostridium botulinum
by using challenge tests with low (2.0-log-CFU/kg) and high (5.3-log-CFU/kg) inocula and two currently available predictive microbiological models, Food MicroModel (FMM) and Pathogen Modeling Program (PMP). After thermal processing, the products were stored at 4 and 8°C and examined for the presence of botulinal spores and neurotoxin on the sell-by date and 7 days after the sell-by date. Most of the thermal processes were found to be inadequate for eliminating spores, even in low-inoculum samples. Only 2 of the 16 products were found to be negative for botulinal spores and neurotoxin at both sampling times. Two products at the high inoculum level showed toxigenesis during storage at 8°C, one of them at the sell-by date. The predictions generated by both the FMM thermal death model and the FMM and PMP growth models were found to be inconsistent with the observed results in a majority of the challenges. The inaccurate predictions were caused by the limited number and range of the controlling factors in the models. Based on this study, it was concluded that the safety of sous vide products needs to be carefully evaluated product by product. Time-temperature combinations used in thermal treatments should be reevaluated to increase the efficiency of processing, and the use of additional antibotulinal hurdles, such as biopreservatives, should be assessed.
Elucidation of Enzymes in Fermentation Pathways Used by
Clostridium thermosuccinogenes
Growing on Inulin
Authors: Sridhar Jayanth , Eiteman Mark A. , Wiegel Juergen W.
Published: April 2009

Based on the presence and absence of enzyme activities, the biochemical pathways for the fermentation of inulin by
Clostridium thermosuccinogenes
DSM 5809 are proposed. Activities of nine enzymes (lactate dehydrogenase, phosphoenolpyruvate carboxylase, malate dehydrogenase, fumarase, fumarate reductase, phosphotransacetylase, acetate kinase, pyruvate kinase, and alcohol dehydrogenase) were measured at four temperatures (37, 47, 58, and 70°C). Each of the enzymes increased 1.5 to 2.0-fold in activity between 37 and 58°C, but only lactate dehydrogenase, fumarate reductase, malate dehydrogenase, and fumarase increased at a similar rate between 58 and 70°C. No acetate kinase activity was observed at 70°C. Arrhenius energies were calculated for each of these nine enzymes and were in the range of 9.8 to 25.6 kcal/mol. To determine if a relationship existed between product formation and enzyme activity, serum bottle fermentations were completed at the four temperatures. Maximum yields (in moles per mole hexose unit) for succinate (0.23) and acetate (0.79) and for biomass (29.5 g/mol hexose unit) occurred at 58°C, whereas the maximum yields for lactate (0.19) and hydrogen (0.25) and the lowest yields for acetate (0.03) and biomass (19.2 g/mol hexose unit) were observed at 70°C. The ratio of oxidized products to reduced products changed significantly, from 0.52 to 0.65, with an increase in temperature from 58 to 70°C, and there was an unexplained detection of increased reduced products (ethanol, lactate, and hydrogen) with a concomitant decrease in oxidized-product formation at the higher temperature.
Increased Production of Hydrogen Peroxide by
Lactobacillus delbrueckii
subsp.
bulgaricus
upon Aeration: Involvement of an NADH Oxidase in Oxidative Stress
Authors: Marty-Teysset C. , de la Torre F. , Garel J.-R.
Published: April 2009

The growth of
Lactobacillus delbrueckii
subsp.
bulgaricus
(
L. delbrueckii
subsp.
bulgaricus
) on lactose was altered upon aerating the cultures by agitation. Aeration caused the bacteria to enter early into stationary phase, thus reducing markedly the biomass production but without modifying the maximum growth rate. The early entry into stationary phase of aerated cultures was probably related to the accumulation of hydrogen peroxide in the medium. Indeed, the concentration of hydrogen peroxide in aerated cultures was two to three times higher than in unaerated ones. Also, a similar shift from exponential to stationary phase could be induced in unaerated cultures by adding increasing concentrations of hydrogen peroxide. A significant fraction of the hydrogen peroxide produced by
L. delbrueckii
subsp.
bulgaricus
originated from the reduction of molecular oxygen by NADH catalyzed by an NADH:H
2
O
2
oxidase. The specific activity of this NADH oxidase was the same in aerated and unaerated cultures, suggesting that the amount of this enzyme was not directly regulated by oxygen. Aeration did not change the homolactic character of lactose fermentation by
L. delbrueckii
subsp.
bulgaricus
and most of the NADH was reoxidized by lactate dehydrogenase with pyruvate. This indicated that NADH oxidase had no (or a very small) energetic role and could be involved in eliminating oxygen.
Detection and Identification of Gastrointestinal
Lactobacillus
Species by Using Denaturing Gradient Gel Electrophoresis and Species-Specific PCR Primers
Authors: Walter J. , Tannock G. W. , Tilsala-Timisjarvi A. , Rodtong S. , Loach D. M. , Munro K. , Alatossava T.
Published: April 2009

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments obtained by PCR amplification of the V2-V3 region of the 16S rRNA gene was used to detect the presence of
Lactobacillus
species in the stomach contents of mice.
Lactobacillus
isolates cultured from human and porcine gastrointestinal samples were identified to the species level by using a combination of DGGE and species-specific PCR primers that targeted 16S-23S rRNA intergenic spacer region or 16S rRNA gene sequences. The identifications obtained by this approach were confirmed by sequencing the V2-V3 region of the 16S rRNA gene and by a BLAST search of the GenBank database.
Efficient Production of Artificially Designed Gelatins with a
Bacillus brevis
System
Authors: Kajino Tsutomu , Takahashi Haruo , Hirai Masana , Yamada Yukio
Published: April 2009

Artificially designed gelatins comprising tandemly repeated 30-amino-acid peptide units derived from human αI collagen were successfully produced with a
Bacillus brevis
system. The DNA encoding the peptide unit was synthesized by taking into consideration the codon usage of the host cells, but no clones having a tandemly repeated gene were obtained through the above-mentioned strategy. Minirepeat genes could be selected in vivo from a mixture of every possible sequence encoding an artificial gelatin by randomly ligating the mixed sequence unit and transforming it into
Escherichia coli
. Larger repeat genes constructed by connecting minirepeat genes obtained by in vivo selection were also stable in the expression host cells. Gelatins derived from the eight-unit and six-unit repeat genes were extracellularly produced at the level of 0.5 g/liter and easily purified by ammonium sulfate fractionation and anion-exchange chromatography. The purified artificial gelatins had the predicted N-terminal sequences and amino acid compositions and a solgel property similar to that of the native gelatin. These results suggest that the selection of a repeat unit sequence stable in an expression host is a shortcut for the efficient production of repetitive proteins and that it can conveniently be achieved by the in vivo selection method. This study revealed the possible industrial application of artificially designed repetitive proteins.
Molecular Characterization of the Toxic Cyanobacterium
Cylindrospermopsis raciborskii
and Design of a Species-Specific PCR
Authors: Wilson Kim M. , Schembri Mark A. , Baker Peter D. , Saint Christopher P.
Published: April 2009

Cylindrospermopsis raciborskii
is a toxic-bloom-forming cyanobacterium that is commonly found in tropical to subtropical climatic regions worldwide, but it is also recognized as a common component of cyanobacterial communities in temperate climates. Genetic profiles of
C. raciborskii
were examined in 19 cultured isolates originating from geographically diverse regions of Australia and represented by two distinct morphotypes. A 609-bp region of
rpoC1
, a DNA-dependent RNA polymerase gene, was amplified by PCR from these isolates with cyanobacterium-specific primers. Sequence analysis revealed that all isolates belonged to the same species, including morphotypes with straight or coiled trichomes. Additional
rpoC1
gene sequences obtained for a range of cyanobacteria highlighted clustering of
C. raciborskii
with other heterocyst-producing cyanobacteria (orders
Nostocales
and
Stigonematales
). In contrast, randomly amplified polymorphic DNA and short tandemly repeated repetitive sequence profiles revealed a greater level of genetic heterogeneity among
C. raciborskii
isolates than did
rpoC1
gene analysis, and unique band profiles were also found among each of the cyanobacterial genera examined. A PCR test targeting a region of the
rpoC1
gene unique to
C. raciborskii
was developed for the specific identification of
C. raciborskii
from both purified genomic DNA and environmental samples. The PCR was evaluated with a number of cyanobacterial isolates, but a PCR-positive result was only achieved with
C. raciborskii
. This method provides an accurate alternative to traditional morphological identification of
C. raciborskii
.
A Theoretical Study on the Metabolic Requirements Resulting from α-Ketoglutarate-Dependent Cleavage of Phenoxyalkanoates
Authors: Müller R. H. , Babel W.
Published: April 2009

The etherolytic cleavage of phenoxyalkanoic acids in various bacteria is catalyzed by an α-ketoglutarate-dependent dioxygenase. In this reaction, the electron acceptor is oxidatively decarboxylated to succinate, whereas the proper substrate is cleaved by forming the oxidized alkanoic acid and the phenolic intermediate. The necessity of regenerating α-ketoglutarate and the consequences for the overall metabolism were investigated in a theoretical study. It was found that the dioxygenase mechanism is accompanied by a significant loss of carbon amounting to up to 62.5% in the assimilatory branch, thus defining the upper limit of carbon conversion efficiency. This loss in carbon is almost compensated for in comparison to a monooxygenase-catalyzed initial step when the dissimilatory efforts of the entire metabolism are included: the yield coefficients become similar. The α-ketoglutarate-dependent dioxygenase mechanism has more drastic consequences for microorganisms which are restricted in their metabolism to the first step of phenoxyalkanoate degradation by excreting the phenolic intermediate as a dead-end product. In the case of phenoxyacetate derivatives, the cleavage reaction would quickly cease due to the exhaustion of α-ketoglutarate and no growth would be possible. With the cleavage products of phenoxypropionate and phenoxybutyrate herbicides, i.e., pyruvate and succinate(semialdehyde), respectively, as the possible products, the regeneration of α-ketoglutarate will be guaranteed for stoichiometric reasons. However, the maintenance of the cleavage reaction ought to be restricted due to physiological factors owing to the involvement of other metabolic reactions in the pool of metabolites. These effects are discussed in terms of a putative recalcitrance of these compounds.
Requirement of Monooxygenase-Mediated Steps for Sterigmatocystin Biosynthesis by
Aspergillus nidulans
Authors: Keller Nancy P. , Watanabe Coran M. H. , Kelkar Hemant S. , Adams Thomas H. , Townsend Craig A.
Published: April 2009

Sterigmatocystin (ST) and aflatoxin B
1
(AFB
1
) are two polyketide-derived
Aspergillus
mycotoxins synthesized by functionally identical sets of enzymes. ST, the compound produced by
Aspergillus nidulans
, is a late intermediate in the AFB
1
pathway of
A. parasiticus
and
A. flavus
. Previous biochemical studies predicted that five oxygenase steps are required for the formation of ST. A 60-kb ST gene cluster in
A. nidulans
contains five genes,
stcB
,
stcF
,
stcL
,
stcS
, and
stcW
, encoding putative monooxygenase activities. Prior research showed that
stcL
and
stcS
mutants accumulated versicolorins B and A, respectively. We now show that strains disrupted at
stcF
, encoding a P-450 monooxygenase similar to
A. parasiticus avnA
, accumulate averantin. Disruption of either StcB (a putative P-450 monooxygenase) or StcW (a putative flavin-requiring monooxygenase) led to the accumulation of averufin as determined by radiolabeled feeding and extraction studies.
Characterization of the Kexin-Like Maturase of
Aspergillus niger
Authors: Jalving Ruud , van de Vondervoort Peter J. I. , Visser Jaap , Schaap Peter J.
Published: April 2009

Secreted yields of foreign proteins may be enhanced in filamentous fungi through the use of translational fusions in which the target protein is fused to an endogenous secreted carrier protein. The fused proteins are usually separated in vivo by cleavage of an engineered Kex2 endoprotease recognition site at the fusion junction. We have cloned the kexin-encoding gene of
Aspergillus niger
(
kexB
). We constructed strains that either overexpressed KexB or lacked a functional
kexB
gene. Kexin-specific activity doubled in membrane-protein fractions of the strain overexpressing KexB. In contrast, no kexin-specific activity was detected in the similar protein fractions of the
kexB
disruptant. Expression in this loss-of-function strain of a glucoamylase human interleukin-6 fusion protein with an engineered Kex2 dibasic cleavage site at the fusion junction resulted in secretion of unprocessed fusion protein. The results show that KexB is the endoproteolytic proprotein processing enzyme responsible for the processing of (engineered) dibasic cleavage sites in target proteins that are transported through the secretion pathway of
A. niger
.
Role of Leaf Surface Sugars in Colonization of Plants by Bacterial Epiphytes
Authors: Mercier Julien , Lindow S. E.
Published: April 2009

The relationship between nutrients leached onto the leaf surface and the colonization of plants by bacteria was studied by measuring both the abundance of simple sugars and the growth of
Pseudomonas fluorescens
on individual bean leaves. Data obtained in this study indicate that the population size of epiphytic bacteria on plants under environmentally favorable conditions is limited by the abundance of carbon sources on the leaf surface. Sugars were depleted during the course of bacterial colonization of the leaf surface. However, about 20% of readily utilizable sugar, such as glucose, present initially remained on fully colonized leaves. The amounts of sugars on a population of apparently identical individual bean leaves before and after microbial colonization exhibited a similar right-hand-skewed distribution and varied by about 25-fold from leaf to leaf. Total bacterial population sizes on inoculated leaves under conditions favorable for bacterial growth also varied by about 29-fold and exhibited a right-hand-skewed distribution. The amounts of sugars on leaves of different plant species were directly correlated with the maximum bacterial population sizes that could be attained on those species. The capacity of bacteria to deplete leaf surface sugars varied greatly among plant species. Plants capable of supporting high bacterial population sizes were proportionally more depleted of leaf surface nutrients than plants with low epiphytic populations. Even in species with a high epiphytic bacterial population, a substantial amount of sugar remained after bacterial colonization. It is hypothesized that residual sugars on colonized leaves may not be physically accessible to the bacteria due to limitations in wettability and/or diffusion of nutrients across the leaf surface.
Quantification of Different
Eubacterium
spp. in Human Fecal Samples with Species-Specific 16S rRNA-Targeted Oligonucleotide Probes
Authors: Schwiertz Andreas , Le Blay Gwenaelle , Blaut Michael
Published: April 2009

Species-specific 16S rRNA-targeted, Cy3 (indocarbocyanine)-labeled oligonucleotide probes were designed and validated to quantify different
Eubacterium
species in human fecal samples. Probes were directed at
Eubacterium barkeri
,
E. biforme
,
E. contortum
,
E. cylindroides
(two probes),
E. dolichum
,
E. hadrum
,
E. lentum
,
E. limosum
,
E. moniliforme
, and
E. ventriosum
. The specificity of the probes was tested with the type strains and a range of common intestinal bacteria. With one exception, none of the probes showed cross-hybridization under stringent conditions. The species-specific probes were applied to fecal samples obtained from 12 healthy volunteers.
E. biforme
,
E. cylindroides
,
E. hadrum
,
E. lentum
, and
E. ventriosum
could be determined. All other
Eubacterium
species for which probes had been designed were under the detection limit of 10
7
cells g (dry weight) of feces
−1
. The cell counts obtained are essentially in accordance with the literature data, which are based on colony counts. This shows that whole-cell in situ hybridization with species-specific probes is a valuable tool for the enumeration of
Eubacterium
species in feces.