bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vitro Selection of Resistance in
Haemophilus influenzae
by Amoxicillin-Clavulanate, Cefpodoxime, Cefprozil, Azithromycin, and Clarithromycin
Authors: Clark Catherine , Bozdogan Bülent , Peric Mihaela , Dewasse Bonifacio , Jacobs Michael R. , Appelbaum Peter C.
Published: October 2002

Abilities of amoxicillin-clavulanate, cefpodoxime, cefprozil, azithromycin, and clarithromycin to select resistant mutants of
Haemophilus influenzae
were tested by multistep and single-step methodologies. For multistep studies, 10 random strains were tested: 5 of these were β-lactamase positive. After 50 daily subcultures in amoxicillin-clavulanate, MICs did not increase more than fourfold. However, cefprozil MICs increased eightfold for one strain. Clarithromycin and azithromycin gave a >4-fold increase in 8 and 10 strains after 14 to 46 and 20 to 50 days, respectively. Mutants selected by clarithromycin and azithromycin were associated with mutations in 23S rRNA and ribosomal proteins L4 and L22. Three mutants selected by clarithromycin or azithromycin had alterations in ribosomal protein L4, while five had alterations in ribosomal protein L22. Two mutants selected by azithromycin had mutations in the gene encoding 23S rRNA: one at position 2058 and the other at position 2059 (
Escherichia coli
numbering), with replacement of A by G. One clone selected by clarithromycin became hypersusceptible to macrolides. In single-step studies azithromycin and clarithromycin had the highest mutation rates, while amoxicillin-clavulanate had the lowest. All resistant clones were identical to parents as observed by pulsed-field gel electrophoresis. The MICs of azithromycin for azithromycin-resistant clones were 16 to >128 μg/ml, and those of clarithromycin for clarithromycin-resistant clones were 32 to >128 μg/ml in multistep studies. For strains selected by azithromycin, the MICs of clarithromycin were high and vice versa. After 50 daily subcultures in the presence of drugs, MICs of amoxicillin-clavulanate and cefpodoxime against
H. influenzae
did not rise more than fourfold, in contrast to cefprozil, azithromycin, and clarithromycin, whose MICs rose to variable degrees.
In Vitro Interaction of Flucytosine Combined with Amphotericin B or Fluconazole against Thirty-Five Yeast Isolates Determined by both the Fractional Inhibitory Concentration Index and the Response Surface Approach
Authors: Te Dorsthorst D. T. A. , Verweij P. E. , Meletiadis J. , Bergervoet M. , Punt N. C. , Meis J. F. G. M. , Mouton J. W.
Published: October 2002

Combination therapy could be of benefit for the treatment of invasive yeast infections. However, in vitro interaction studies are relatively scarce and the interpretation of the fractional inhibitory concentration (FIC) index can be contradictory due to various definitions used; not all information on the interaction study is used in the index, and different MIC end points exist for different classes of drugs. Fitting an interaction model to the whole response surface and estimation of an interaction coefficient alpha (ICα) would overcome these objections and has the additional advantage that confidence intervals of the interaction are obtained. The efficacy of flucytosine (5FC) in combination with amphotericin B (AB) and fluconazole (FCZ) was studied against 35 yeast isolates in triplicate (
Candida albicans
[
n
= 9],
Candida glabrata
[
n
= 9],
Candida krusei
[
n
= 9], and
Cryptococcus neoformans
[
n
= 8]) using a broth microdilution checkerboard method and measuring growth after 48 h by a spectrophotometer. The FIC index and ICα were determined, the latter by estimation from the response surface approach described by Greco et al. (W. R. Greco, G. Bravo, and J. C. Parsons, Pharmacol. Rev.
47:
331-385, 1995) by using a computer program developed for that purpose. For the 5FC-FCZ combination, the interactions determined by the ICα generally were in concordance with the interactions determined by the FIC index, but large discrepancies were found between both methods for the 5FC-AB combination. These could mainly be explained by shortcomings in the FIC approach. The in vitro interaction of 5FC-AB demonstrated variable results depending on the tested
Candida
isolate. In general, the 5FC-FCZ combination was antagonistic against
Candida
species, but for some
Candida
isolates synergism was found. For
C. neoformans
the interaction for both combinations was highly dependent on the tested isolate and the method used. Response surface approach is an alternative method for determining the interaction between antifungal agents. By using this approach, some of the problems encountered with the FIC were overcome.
The Antifungal Echinocandin Caspofungin Acetate Kills Growing Cells of
Aspergillus fumigatus
In Vitro
Authors: Bowman J. C. , Hicks P. Scott , Kurtz M. B. , Rosen H. , Schmatz D. M. , Liberator P. A. , Douglas C. M.
Published: October 2002

Caspofungin acetate is an antifungal antibiotic that inhibits synthesis of 1,3-β-
d
-glucan, an essential component of the fungal cell wall. While caspofungin causes cell death in yeasts and dimorphic fungi such as
Candida albicans
, its effect on
Aspergillus fumigatus
is less well understood. We used the fluorescent dyes 5,(6)-carboxyfluorescein diacetate (CFDA) and bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC), which stain live and dead cells, respectively, to further characterize the antifungal activity of caspofungin. For comparison, compounds whose mode of action was either fungistatic (fluconazole, itraconazole) or fungicidal (amphotericin B) were also evaluated. A correlation between caspofungin-induced loss of viability, decreased CFDA staining, and increased DiBAC staining was established first with
C. albicans
. For
A. fumigatus
, caspofungin caused similar dye-staining changes, which were quantified by fluorimetric analysis of stained hyphae grown in a medium that promoted dispersed growth. The minimum concentration of caspofungin required to produce these changes also decreased the level of growth-dependent reduction of the indicator dye Alamar Blue. We observed a differential effect of caspofungin as a function of cell position: 88% of apical cells and 61% of subapical branching cells failed to stain with the viable dye CFDA, but only 24% of subapical cells were unstained. Complementary results were seen with germlings from DiBAC-stained, caspofungin-treated cultures. Extended incubation of
A. fumigatus
with a single dose of caspofungin affected the same proportion of apical and subapical branching cells for up to 72 h. The dye-staining patterns illustrate that the cells at the active centers for new cell wall synthesis within
A. fumigatus
hyphae are killed when they are exposed to caspofungin.
Mutation in 23S rRNA Associated with Macrolide Resistance in
Neisseria gonorrhoeae
Authors: Ng Lai-King , Martin Irene , Liu Gary , Bryden Louis
Published: October 2002

Fifty-six azithromycin-resistant (MICs, 2.0 to 4.0 μg/ml)
Neisseria gonorrhoeae
strains with cross-resistance to erythromycin (MICs, 2.0 to 64.0 μg/ml), isolated in Canada between 1997 and 1999, were characterized, and their mechanisms of azithromycin resistance were determined. Most (58.9%) of them belonged to auxotype-serotype class NR/IB-03, with a 2.6-mDa plasmid. Based on resistance to crystal violet (MICs ≥ 1 μg/ml), 96.4% of these macrolide-resistant strains appeared to have increased efflux. Nine of the eleven strains selected for further characterization were found to have a promoter region
mtrR
mutation, a single-base-pair (A) deletion in the 13-bp inverted repeat, which is believed to cause overexpression of the
mtrCDE
-encoded efflux pump. The two remaining macrolide-resistant strains (erythromycin MIC, 64.0 μg/ml; azithromycin MIC, 4.0 μg/ml), which did not have the mutation in the
mtrR
promoter region, were found to have a C2611T mutation (
Escherichia coli
numbering) in the peptidyltransferase loop in domain V of the 23S rRNA alleles. Although mutations in domain V of 23S rRNA alleles had been reported in other bacteria, including
E. coli
,
Streptococcus pneumoniae
, and
Helicobacter pylori
, this is the first observation of these mutations associated with macrolide resistance in
N. gonorrhoeae
.
In Vitro Interaction of Caspofungin Acetate with Voriconazole against Clinical Isolates of
Aspergillus
spp
Authors: Perea Sofia , Gonzalez Gloria , Fothergill Annette W. , Kirkpatrick William R. , Rinaldi Michael G. , Patterson Thomas F.
Published: October 2002

The interaction between caspofungin acetate and voriconazole was studied in vitro by using 48 clinical
Aspergillus
spp. isolates obtained from patients with invasive aspergillosis. MICs were determined by the NCCLS broth microdilution method. Synergy, defined as a fractional inhibitory concentration (FIC) index of <1, was detected in 87.5% of the interactions; an additive effect, defined as an FIC index of 1.0, was observed in 4.2% of the interactions; and a subadditive effect, defined as an FIC index of 1.0 to 2.0, was found in 8.3% of the interactions. No antagonism was observed. Animal models are required to validate the in vivo significance of these in vitro data presented for the combination of caspofungin and voriconazole.
Incidence of High-Level Evernimicin Resistance in
Enterococcus faecium
among Food Animals and Humans
Authors: Aarestrup Frank Møller , McNicholas Paul M.
Published: October 2002

Six high-level evernimicin-resistant
Enterococcus faecium
isolates were identified among 304 avilamycin-resistant
E
.
faecium
isolates from animals and 404 stool samples from humans with diarrhea. All four animal isolates, and one of the human isolates, were able to transfer resistance to a susceptible
E
.
faecium
strain. The resulting transconjugants all tested positive for the presence of
emtA
, a gene encoding a methyltransferase previously linked with high-level evernimicin resistance. The four transconjugants derived from animal isolates all carried the same plasmid, while a differently sized plasmid was found in the isolate from humans. This study demonstrated a low incidence of high-level evernimicin resistance mediated by the
emtA
gene in different
E
.
faecium
isolates of animal and human origin.
Inhibition of
Listeria innocua
in Cheddar Cheese by Addition of Nisin Z in Liposomes or by In Situ Production in Mixed Culture
Authors: Benech R.-O. , Kheadr E. E. , Laridi R. , Lacroix C. , Fliss I.
Published: October 2002

The effect of addition of purified nisin Z in liposomes to cheese milk and of in situ production of nisin Z by
Lactococcus lactis
subsp.
lactis
biovar diacetylactis UL719 in the mixed starter on the inhibition of
Listeria innocua
in cheddar cheese was evaluated during 6 months of ripening. A cheese mixed starter culture containing
Lactococcus lactis
subsp.
lactis
biovar diacetylactis UL719 was selected for high-level nisin Z and acid production. Experimental cheddar cheeses were produced on a pilot scale, using the selected starter culture, from milk with added
L. innocua
(10
5
to 10
6
CFU/ml). Liposomes with purified nisin Z were prepared from proliposome H and added to cheese milk prior to renneting to give a final concentration of 300 IU/g of cheese. The nisin Z-producing strain and nisin Z-containing liposomes did not significantly affect cheese production and gross chemical composition of the cheeses. Immediately after cheese production, 3- and 1.5-log-unit reductions in viable counts of
L. innocua
were obtained in cheeses with encapsulated nisin and the nisinogenic starter, respectively. After 6 months, cheeses made with encapsulated nisin contained less than 10 CFU of
L. innocua
per g and 90% of the initial nisin activity, compared with 10
4
CFU/g and only 12% of initial activity in cheeses made with the nisinogenic starter. This study showed that encapsulation of nisin Z in liposomes can provide a powerful tool to improve nisin stability and inhibitory action in the cheese matrix while protecting the cheese starter from the detrimental action of nisin during cheese production.
Identification of the Bacterial Microflora in Dairy Products by Temporal Temperature Gradient Gel Electrophoresis
Authors: Ogier Jean-Claude , Son Olivier , Gruss Alexandra , Tailliez Patrick , Delacroix-Buchet Agnes
Published: October 2002

Numerous microorganisms, including bacteria, yeasts, and molds, are present in cheeses, forming a complex ecosystem. Among these organisms, bacteria are responsible for most of the physicochemical and aromatic transformations that are intrinsic to the cheesemaking process. Identification of the bacteria that constitute the cheese ecosystem is essential for understanding their individual contributions to cheese production. We used temporal temperature gradient gel electrophoresis (TTGE) to identify different bacterial species present in several dairy products, including members of the genera
Lactobacillus
,
Lactococcus
,
Leuconostoc
,
Enterococcus
,
Pediococcus
,
Streptococcus
, and
Staphylococcus
. The TTGE technique is based on electrophoretic separation of 16S ribosomal DNA (rDNA) fragments by using a temperature gradient. It was optimized to reveal differences in the 16S rDNA V3 regions of bacteria with low-G+C-content genomes. Using multiple control strains, we first set up a species database in which each species (or group of species) was characterized by a specific TTGE fingerprint. TTGE was then applied to controlled dairy ecosystems with defined compositions, including liquid (starter), semisolid (home-made fermented milk), and solid (miniature cheese models) matrices. Finally, the potential of TTGE to describe the bacterial microflora of unknown ecosystems was tested with various commercial dairy products. Subspecies, species, or groups of species of lactic acid bacteria were distinguished in dairy samples. In conclusion, TTGE was shown to distinguish bacterial species in vitro, as well as in both liquid and solid dairy products.
Comparative Resistance of Phage Isolates of Four Genotypes of F-Specific RNA Bacteriophages to Various Inactivation Processes
Authors: Schaper M. , Durán A. E. , Jofre J.
Published: October 2002

The effect of natural inactivation in freshwater, chlorination, ammonia, extreme pHs, temperature, and salt content on phage inactivation was evaluated on mixtures of F-specific RNA bacteriophage isolates belonging to genotypes I, II, III, and IV. The bacteriophages studied were previously but recently isolated from natural samples, characterized as F-specific RNA bacteriophages and genotyped by plaque hybridization with genotype-specific probes. Natural inactivation in river water was modeled by in situ incubation of bacteriophages inside submerged dialysis tubes. After several days bacteriophages of genotype I showed the highest persistence, which was significantly different from that of bacteriophages of genotype II, IV, or III. The pattern of resistance of phages belonging to the various genotypes to extreme pHs, ammonia, temperature, salt concentration, and chlorination was similar. In all cases, phages of genotype I showed the highest persistence, followed by the phages of genotypes II, III, and IV. The phages of genotypes III and IV were the least resistant to all treatments, and resistance of genotypes III and IV to the treatments was similar. Bacteriophages of genotype II showed intermediate resistance to some of the treatments. The resistance of four phages of genotype I to natural inactivation and chlorination did not differ significantly. These results indicate that genotypes III and IV are much more sensitive to environmental stresses and to treatments than the other genotypes, especially than genotype I. This should be taken into consideration in future studies aimed at using genotypes of F-specific RNA bacteriophages to fingerprint the origin of fecal pollution.
Plasmid-Borne Genes Code for an Angular Dioxygenase Involved in Dibenzofuran Degradation by
Terrabacter
sp. Strain YK3
Authors: Iida Toshiya , Mukouzaka Yuki , Nakamura Kaoru , Kudo Toshiaki
Published: October 2002

The genes responsible for angular dioxygenation of dibenzofuran in actinomycetes were cloned by using a degenerate set of PCR primers designed by using conserved sequences of the dioxygenase alpha subunit genes. One sequence of alpha subunit genes was commonly amplified from four dibenzofuran-utilizing actinomycetes:
Terrabacter
sp. strains YK1 and YK3,
Rhodococcus
sp. strain YK2, and
Microbacterium
sp. strain YK18. A 5.2-kb
Pst
I fragment encoding the alpha and beta subunits of the terminal dioxygenase, ferredoxin, and ferredoxin reductase (designated
dfdA1
to
dfdA4
, respectively) was cloned from the large circular plasmid pYK3 isolated from
Terrabacter
sp. strain YK3. We confirmed that transcription of the
dfdA1
gene was induced by dibenzofuran in
Terrabacter
sp. strain YK3. Southern blot hybridization analysis revealed that this type of dioxygenase gene is distributed among diverse dibenzofuran-utilizing actinomycetes. However, genes homologous to
dfdA1
were not detected in dibenzofuran utilization-deficient mutants of
Terrabacter
,
Rhodococcus
, and
Microbacterium
species. When the
dfdA1
to
dfdA4
genes were introduced into a non-dibenzofuran-degrading mutant of
Rhodococcus
sp. strain YK2, strain YK2-RD2, which had spontaneously lost the gene homologous to
dfdA1
, the ability to degrade dibenzofuran was restored. Analysis of the breakdown products indicated that DfdA has angular dioxygenase activity. This
dfdA
transformant degraded several aromatic compounds, indicating that the novel angular dioxygenase possesses broad substrate specificity.
Biodiversity of Denitrifying and Dinitrogen-Fixing Bacteria in an Acid Forest Soil
Authors: Rösch Christopher , Mergel Alexander , Bothe Hermann
Published: October 2002

Isolated soil DNA from an oak-hornbeam forest close to Cologne, Germany, was suitable for PCR amplification of gene segments coding for the 16S rRNA and nitrogenase reductase (NifH), nitrous oxide reductase (NosZ), cytochrome
cd
1
-containing nitrite reductase (NirS), and Cu-containing nitrite reductase (NirK) of denitrification. For each gene segment, diverse PCR products were characterized by cloning and sequencing. None of the 16S rRNA gene sequences was identical to any deposited in the data banks, and therefore each of them belonged to a noncharacterized bacterium. In contrast, the analyzed clones of
nifH
gave only a few different sequences, which occurred many times, indicating a low level of species richness in the N
2
-fixing bacterial population in this soil. Identical
nifH
sequences were also detected in PCR amplification products of DNA of a soil approximately 600 km distant from the Cologne area. Whereas biodiversity was high in the case of
nosZ
, only a few different sequences were obtained with
nirK
. With respect to
nirS
, cloning and sequencing of the PCR products revealed that many false gene segments had been amplified with DNA from soil but not from cultured bacteria. With the 16S rRNA gene data, many sequences of uncultured bacteria belonging to the
Acidobacterium
phylum and actinomycetes showed up in the PCR products when isolated DNA was used as the template, whereas sequences obtained for
nifH
and for the denitrification genes were closely related to those of the proteobacteria. Although in such an experimental approach one has to cope with the enormous biodiversity in soils and only a few PCR products can be selected at random, the data suggest that denitrification and N
2
fixation are not genetic traits of most of the uncultured bacteria.
Oxalobacter formigenes
and Its Potential Role in Human Health
Authors: Duncan Sylvia H. , Richardson Anthony J. , Kaul Poonam , Holmes Ross P. , Allison Milton J. , Stewart Colin S.
Published: October 2002

Oxalate degradation by the anaerobic bacterium
Oxalobacter formigenes
is important for human health, helping to prevent hyperoxaluria and disorders such as the development of kidney stones. Oxalate-degrading activity cannot be detected in the gut flora of some individuals, possibly because
Oxalobacter
is susceptible to commonly used antimicrobials. Here, clarithromycin, doxycycline, and some other antibiotics inhibited oxalate degradation by two human strains of
O. formigenes
. These strains varied in their response to gut environmental factors, including exposure to gastric acidity and bile salts.
O. formigenes
strains established oxalate breakdown in fermentors which were preinoculated with fecal bacteria from individuals lacking oxalate-degrading activity. Reducing the concentration of oxalate in the medium reduced the numbers of
O. formigenes
bacteria. Oxalate degradation was established and maintained at dilution rates comparable to colonic transit times in healthy individuals. A single oral ingestion of
O. formigenes
by adult volunteers was, for the first time, shown to result in (i) reduced urinary oxalate excretion following administration of an oxalate load, (ii) the recovery of oxalate-degrading activity in feces, and (iii) prolonged retention of colonization.
Occurrence of Toxigenic
Aspergillus versicolor
Isolates and Sterigmatocystin in Carpet Dust from Damp Indoor Environments
Authors: Engelhart Steffen , Loock Annette , Skutlarek Dirk , Sagunski Helmut , Lommel Annette , Färber Harald , Exner Martin
Published: October 2002

Over the past decade, there has been growing concern regarding the role of toxigenic fungi in damp indoor environments; however, there is still a lack of field investigations on exposure to mycotoxins. The goal of our pilot study was to quantify the proportion of toxigenic
Aspergillus versicolor
isolates in native carpet dust from damp dwellings with mold problems and to determine whether sterigmatocystin can be detected in this matrix. Carpet dust samples (
n
= 11) contained from <2.5 × 10
1
to 3.6 × 10
5
(median, 3.1 × 10
4
)
A. versicolor
CFU/g of dust, and the median proportion of
A. versicolor
from total culturable fungi was 18%. Based on thin-layer chromatography detection of sterigmatocystin, 49 of 50
A. versicolor
isolates (98%) were found to be toxigenic in vitro. By using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry, sterigmatocystin could be detected in low concentrations (2 to 4 ng/g of dust) in 2 of 11 native carpet dust samples. From this preliminary study, we conclude that most strains of
A. versicolor
isolated from carpet dust are able to produce sterigmatocystin in vitro and that sterigmatocystin may occasionally occur in carpet dust from damp indoor environments. Further research and systematic field investigation are needed to confirm our results and to provide an understanding of the health implications of mycotoxins in indoor environments.
Diversity, Abundance, and Activity of Archaeal Populations in Oil-Contaminated Groundwater Accumulated at the Bottom of an Underground Crude Oil Storage Cavity
Authors: Watanabe Kazuya , Kodama Yumiko , Hamamura Natsuko , Kaku Nobuo
Published: October 2002

Fluorescence in situ hybridization has shown that cells labeled with an
Archaea
-specific probe (ARCH915) accounted for approximately 10% of the total cell count in oil-contaminated groundwater accumulated at the bottom of an underground crude oil storage cavity. Although chemical analyses have revealed vigorous consumption of nitrate in cavity groundwater, the present study found that the methane production rate was higher than the nitrate consumption rate. To characterize the likely archaeal populations responsible for methane production in this system, fragments of 16S ribosomal DNA (rDNA) were amplified by PCR using eight different combinations of universal and
Archaea
-specific primers. Sequence analysis of 324 clones produced 23 different archaeal sequence types, all of which were affiliated with the kingdom
Euryarchaeota
. Among them, five sequence types (KuA1, KuA6, KuA12, KuA16, and KuA22) were obtained in abundance. KuA1 and KuA6 were closely related to the known methanogens
Methanosaeta concilii
(99% identical) and
Methanomethylovorans hollandica
(98%), respectively. Although no closely related organism was found for KuA12, it could be affiliated with the family
Methanomicrobiaceae
. KuA16 and KuA22 showed substantial homology only to some environmental clones. Both of these branched deeply in the
Euryarchaeota
, and may represent novel orders. Quantitative competitive PCR showed that KuA12 was the most abundant, accounting for ∼50% of the total archaeal rDNA copies detected. KuA1 and KuA16 also constituted significant proportions of the total archaeal rDNA copies (7 and 17%, respectively). These results suggest that limited species of novel archaea were enriched in the oil storage cavity. An estimate of specific methane production rates suggests that they were active methanogens.
The Peptidyl-Prolyl Isomerase Motif Is Lacking in PmpA, the PrsA-Like Protein Involved in the Secretion Machinery of
Lactococcus lactis
Authors: Drouault Sophie , Anba Jamila , Bonneau Sophie , Bolotin Alexander , Ehrlich S. Dusko , Renault Pierre
Published: October 2002

The
prsA
-like gene from
Lactococcus lactis
encoding its single homologue to PrsA, an essential protein triggering the folding of secreted proteins in
Bacillus subtilis
, was characterized. This gene, annotated
pmpA
, encodes a lipoprotein of 309 residues whose expression is increased 7- to 10-fold when the source of nitrogen is limited. A slight increase in the expression of the PrsA-like protein (PLP) in
L. lactis
removed the degradation products previously observed with the
Staphylococcus hyicus
lipase used as a model secreted protein. This shows that PmpA either triggers the folding of the secreted lipase or activates its degradation by the cell surface protease HtrA. Unlike the case for
B. subtilis
, the inactivation of the gene encoding PmpA reduced only slightly the growth rate of
L. lactis
in standard conditions. However, it almost stopped its growth when the lipase was overexpressed in the presence of salt in the medium. Like PrsA of
B. subtilis
and PrtM of
L. lactis
, the
L. lactis
PmpA protein could thus have a foldase activity that facilitates protein secretion. These proteins belong to the third family of peptidyl-prolyl
cis
/
trans
-isomerases (PPIases) for which parvulin is the prototype. Almost all PLP from gram-positive bacteria contain a domain with the PPIase signature. An exception to this situation was found only in
Streptococcaceae
, the family to which
L. lactis
belongs. PLP from
Streptococcus pneumoniae
and
Enterococcus faecalis
possess this signature, but those of
L. lactis
,
Streptococcus pyogenes
, and
Streptococcus mutans
do not. However, secondary structure predictions suggest that the folding of PLP is conserved over the entire length of the proteins, including the unconserved signature region. The activity associated with the expression of PmpA in
L. lactis
and these genomic data show that either the PPIase motif is not necessary for PPIase activity or, more likely, PmpA foldase activity does not necessarily require PPIase activity.
Endotoxin Activity of
Moraxella osloensis
against the Grey Garden Slug,
Deroceras reticulatum
Authors: Tan Li , Grewal Parwinder S.
Published: October 2002

Moraxella osloensis
is a gram-negative bacterium associated with
Phasmarhabditis hermaphrodita
, a slug-parasitic nematode that has prospects for biological control of mollusk pests, especially the grey garden slug,
Deroceras reticulatum
. This bacterium-feeding nematode acts as a vector that transports
M. osloensis
into the shell cavity of the slug, and the bacterium is the killing agent in the nematode-bacterium complex. We discovered that
M. osloensis
produces an endotoxin(s), which is tolerant to heat and protease treatments and kills the slug after injection into the shell cavity. Washed or broken cells treated with penicillin and streptomycin from 3-day
M. osloensis
cultures were more pathogenic than similar cells from 2-day
M. osloensis
cultures. However, heat and protease treatments and 2 days of storage at 22°C increased the endotoxin activity of the young broken cells but not the endotoxin activity of the young washed cells treated with the antibiotics. This suggests that there may be a proteinaceous substance(s) that is structurally associated with the endotoxin(s) and masks its toxicity in the young bacterial cells. Moreover, 2 days of storage of the young washed bacterial cells at 22°C enhanced their endotoxin activity if they were not treated with the antibiotics. Furthermore, purified lipopolysaccharide (LPS) from the 3-day
M. osloensis
cultures was toxic to slugs, with an estimated 50% lethal dose of 48 μg per slug, thus demonstrating that the LPS of
M. osloensis
is an endotoxin that is active against
D. reticulatum
. This appears to be the first report of a biological toxin that is active against mollusks.
Molecular Cloning and Characterization of the Gene Coding for the Aerobic Azoreductase from
Xenophilus azovorans
KF46F
Authors: Blümel Silke , Knackmuss Hans-Joachim , Stolz Andreas
Published: October 2002

The gene coding for an aerobic azoreductase was cloned from
Xenophilus azovorans
KF46F (formerly
Pseudomonas
sp. strain KF46F), which was previously shown to grow with the carboxylated azo compound 1-(4′-carboxyphenylazo)-2-naphthol (carboxy-Orange II) as the sole source of carbon and energy. The deduced amino acid sequence encoded a protein with a molecular weight of 30,278 and showed no significant homology to amino acid sequences currently deposited at the relevant data bases. A presumed NAD(P)H-binding site was identified in the amino-terminal region of the azoreductase. The enzyme was heterologously expressed in
Escherichia coli
and the azoreductase activities of resting cells and cell extracts were compared. The results suggested that whole cells of the recombinant
E. coli
strains were unable to take up sulfonated azo dyes and therefore did not show in vivo azoreductase activity. The turnover of several industrially relevant azo dyes by cell extracts from the recombinant
E. coli
strain was demonstrated.
Life at the Energetic Edge: Kinetics of Circumneutral Iron Oxidation by Lithotrophic Iron-Oxidizing Bacteria Isolated from the Wetland-Plant Rhizosphere
Authors: Neubauer Scott C. , Emerson David , Megonigal J. Patrick
Published: October 2002

Batch cultures of a lithotrophic Fe(II)-oxidizing bacterium, strain BrT, isolated from the rhizosphere of a wetland plant, were grown in bioreactors and used to determine the significance of microbial Fe(II) oxidation at circumneutral pH and to identify abiotic variables that affect the partitioning between microbial oxidation and chemical oxidation. Strain BrT grew only in the presence of an Fe(II) source, with an average doubling time of 25 h. In one set of experiments, Fe(II) oxidation rates were measured before and after the cells were poisoned with sodium azide. These experiments indicated that strain BrT accounted for 18 to 53% of the total iron oxidation, and the average cellular growth yield was 0.70 g of CH
2
O per mol of Fe(II) oxidized. In a second set of experiments, Fe(II) was constantly added to bioreactors inoculated with live cells, killed cells, or no cells. A statistical model fitted to the experimental data demonstrated that metabolic Fe(II) oxidation accounted for up to 62% of the total oxidation. The total Fe(II) oxidation rates in these experiments were strongly limited by the rate of Fe(II) delivery to the system and were also influenced by O
2
and total iron concentrations. Additionally, the model suggested that the microbes inhibited rates of abiotic Fe(II) oxidation, perhaps by binding Fe(II) to bacterial exopolymers. The net effect of strain BrT was to accelerate total oxidation rates by up to 18% compared to rates obtained with cell-free treatments. The results suggest that neutrophilic Fe(II)-oxidizing bacteria may compete for limited O
2
in the rhizosphere and therefore influence other wetland biogeochemical cycles.
Mechanisms and Rates of Bacterial Colonization of Sinking Aggregates
Authors: Kiørboe Thomas , Grossart Hans-Peter , Ploug Helle , Tang Kam
Published: October 2002

Quantifying the rate at which bacteria colonize aggregates is a key to understanding microbial turnover of aggregates. We used encounter models based on random walk and advection-diffusion considerations to predict colonization rates from the bacteria's motility patterns (swimming speed, tumbling frequency, and turn angles) and the hydrodynamic environment (stationary versus sinking aggregates). We then experimentally tested the models with 10 strains of bacteria isolated from marine particles: two strains were nonmotile; the rest were swimming at 20 to 60 μm s
−1
with different tumbling frequency (0 to 2 s
−1
). The rates at which these bacteria colonized artificial aggregates (stationary and sinking) largely agreed with model predictions. We report several findings. (i) Motile bacteria rapidly colonize aggregates, whereas nonmotile bacteria do not. (ii) Flow enhances colonization rates. (iii) Tumbling strains colonize aggregates enriched with organic substrates faster than unenriched aggregates, while a nontumbling strain did not. (iv) Once on the aggregates, the bacteria may detach and typical residence time is about 3 h. Thus, there is a rapid exchange between attached and free bacteria. (v) With the motility patterns observed, freely swimming bacteria will encounter an aggregate in <1 day at typical upper-ocean aggregate concentrations. This is faster than even starving bacteria burn up their reserves, and bacteria may therefore rely solely on aggregates for food. (vi) The net result of colonization and detachment leads to a predicted equilibrium abundance of attached bacteria as a function of aggregate size, which is markedly different from field observations. This discrepancy suggests that inter- and intraspecific interactions among bacteria and between bacteria and their predators may be more important than colonization in governing the population dynamics of bacteria on natural aggregates.
Cloning and Inactivation of a Branched-Chain-Amino-Acid Aminotransferase Gene from
Staphylococcus carnosus
and Characterization of the Enzyme
Authors: Madsen Søren M. , Beck Hans Christian , Ravn Peter , Vrang Astrid , Hansen Anne Maria , Israelsen Hans
Published: October 2002

Staphylococcus carnosus
and
Staphylococcus xylosus
are widely used as aroma producers in the manufacture of dried fermented sausages. Catabolism of branched-chain amino acids (BCAAs) by these strains contributes to aroma formation by production of methyl-branched aldehydes and carboxy acids. The first step in the catabolism is most likely a transamination reaction catalyzed by BCAA aminotransferases (IlvE proteins). In this study, we cloned the
ilvE
gene from
S. carnosus
by using degenerate oligonucleotides and PCR. We found that the deduced amino acid sequence was 80% identical to that of the corresponding enzyme in
Staphylococcus aureus
and that the
ilvE
gene was constitutively expressed as a monocistronic transcript. To study the influence of
ilvE
on BCAA catabolism, we constructed an
ilvE
deletion mutant by gene replacement. The IlvE protein from
S. carnosus
was shown mainly to catalyze the transamination of isoleucine, valine, leucine, and, to some extent, methionine using pyridoxal 5′-phosphate as a coenzyme. The
ilvE
mutant degraded less than 5% of the BCAAs, while the wild-type strain degraded 75 to 95%. Furthermore, the mutant strain produced approximately 100-fold less of the methyl-branched carboxy acids, 2-methylpropanoic acid, 2-methylbutanoic acid, and 3-methylbutanoic acid, which derived from the BCAA catabolism, clearly emphasizing the role of IlvE in aroma formation. In contrast to previous reports, we found that IlvE was the only enzyme that catalyzed the deamination of BCAAs in
S. carnosus
. The
ilvE
mutant strain showed remarkably lower growth rate and biomass yield compared to those of the wild-type strain when grown in rich medium. Normal growth rate and biomass yield were restored by addition of the three BCAA-derived α-keto acids, showing that degradation products of BCAAs were essential for optimal cell growth.
Transfer of Microorganisms, Including
Listeria monocytogenes
, from Various Materials to Beef
Authors: Midelet Graziella , Carpentier Brigitte
Published: October 2002

The quantity of microorganisms that may be transferred to a food that comes into contact with a contaminated surface depends on the density of microorganisms on the surface and on the attachment strengths of the microorganisms on the materials. We made repeated contacts between pieces of meat and various surfaces (stainless steel and conveyor belt materials [polyvinyl chloride and polyurethane]), which were conditioned with meat exudate and then were contaminated with
Listeria monocytogenes
,
Staphylococcus sciuri
,
Pseudomonas putida
, or
Comamonas
sp. Attachment strengths were assessed by the slopes of the two-phase curves obtained by plotting the logarithm of the number of microorganisms transferred against the order number of the contact. These curves were also used to estimate the microbial population on the surface by using the equation of A. Veulemans, E. Jacqmain, and D. Jacqmain (Rev. Ferment. Ind. Aliment. 25:58-65, 1970). The biofilms were characterized according to their physicochemical surface properties and structures. Their exopolysaccharide-producing capacities were assessed from biofilms grown on polystyrene. The
L. monocytogenes
biofilms attached more strongly to polymers than did the other strains, and attachment strength proved to be weaker on stainless steel than on the two polymers. However, in most cases, it was the population of the biofilms that had the strongest influence on the total number of CFU detached. Although attachment strengths were weaker on stainless steel, this material, carrying a smaller population of bacteria, had a weaker contaminating capacity. In most cases the equation of Veulemans et al. revealed more bacteria than did swabbing the biofilms, and it provided a better assessment of the contaminating potential of the polymeric materials studied here.
Molecular Breeding of Transgenic Rice Plants Expressing a Bacterial Chlorocatechol Dioxygenase Gene
Authors: Shimizu Masami , Kimura Tetsuya , Koyama Takayoshi , Suzuki Katsuhisa , Ogawa Naoto , Miyashita Kiyotaka , Sakka Kazuo , Ohmiya Kunio
Published: October 2002

The
cbnA
gene encoding the chlorocatechol dioxygenase gene from
Ralstonia eutropha
NH9 was introduced into rice plants. The
cbnA
gene was expressed in transgenic rice plants under the control of a modified cauliflower mosaic virus 35S promoter. Western blot analysis using anti-CbnA protein indicated that the
cbnA
gene was expressed in leaf tissue, roots, culms, and seeds. Transgenic rice calluses expressing the
cbnA
gene converted 3-chlorocatechol to 2-chloromucote efficiently. Growth and morphology of the transgenic rice plants expressing the
cbnA
gene were not distinguished from those of control rice plants harboring only a Ti binary vector. It is thus possible to breed transgenic plants that degrade chloroaromatic compounds in soil and surface water.
Chlorxanthomycin, a Fluorescent, Chlorinated, Pentacyclic Pyrene from a
Bacillus
sp
Authors: Magyarosy Andrew , Ho Jonathan Z. , Rapoport Henry , Dawson Scott , Hancock Joe , Keasling Jay D.
Published: October 2002

A gram-positive
Bacillus
sp. that fluoresces yellow under long-wavelength UV light on several common culture media was isolated from soil samples. On the basis of carbon source utilization studies, fatty acid methyl ester analysis, and 16S ribosomal DNA analysis, this bacterium was most similar to
Bacillus megaterium
. Chemical extraction yielded a yellow-orange fluorescent pigment, which was characterized by X-ray crystallography, mass spectrometry, and nuclear magnetic resonance spectroscopy. The fluorescent compound, chlorxanthomycin, is a pentacyclic, chlorinated molecule with the molecular formula C
22
H
15
O
6
Cl and a molecular weight of 409.7865. Chlorxanthomycin appears to be located in the cytoplasm, does not diffuse out of the cells into the culture medium, and has selective antibiotic activity.
Possible Quorum Sensing in Marine Snow Bacteria: Production of Acylated Homoserine Lactones by
Roseobacter
Strains Isolated from Marine Snow
Authors: Gram Lone , Grossart Hans-Peter , Schlingloff Andrea , Kiørboe Thomas
Published: October 2002

We report here, for the first time, that bacteria associated with marine snow produce communication signals involved in quorum sensing in gram-negative bacteria. Four of 43 marine microorganisms isolated from marine snow were found to produce acylated homoserine lactones (AHLs) in well diffusion and thin-layer chromatographic assays based on the
Agrobacterium tumefaciens
reporter system. Three of the AHL-producing strains were identified by 16S ribosomal DNA gene sequence analysis as
Roseobacter
spp., and this is the first report of AHL production by these α-
Proteobacteria
. It is likely that AHLs in
Roseobacter
species and other marine snow bacteria govern phenotypic traits (biofilm formation, exoenzyme production, and antibiotic production) which are required mainly when the population reaches high densities, e.g., in the marine snow community.
Horizontal Transmission of Shiga Toxin-ProducingEscherichia coliwithin Groups of Dairy Calves
Authors: Cobbold R. , Desmarchelier P.
Published: October 2002

To examine the dissemination of Shiga-toxigenicEscherichia coli(STEC) within cattle groups, dairy calves on two farms utilizing different calf-rearing practices were exposed to a traceable STEC strain. Test strain dissemination differed significantly between farms, with a higher prevalence being associated with group penning. Pen floors and calf hides may be the main environmental mechanisms of transmission. Dairy calf husbandry represents a control point for reducing on-farm STEC prevalence.
Role of the
htrA
Gene in
Klebsiella pneumoniae
Virulence
Authors: Cortés Guadalupe , de Astorza Beatriz , Benedí Vicente J. , Albertí Sebastián
Published: October 2002

We recently described the use of mini-Tn
5
to generate complement-sensitive mutants derived from a complement-resistant
Klebsiella pneumoniae
clinical isolate deficient in the lipopolysaccharide O side chain. One mutant with a reduced capacity to survive in nonimmune human sera carried the transposon inserted in the
htrA
gene. We cloned and sequenced the gene and predicted from the deduced amino acid sequence that the putative HtrA homolog contains structural features similar to those of previously described HtrA proteins. To investigate the biological functions and the role of the
htrA
gene in the virulence of
K. pneumoniae
, we constructed an isogenic mutant by insertion-duplication mutagenesis. Characterization of the mutant showed that it had greater sensitivity to temperature (50°C) and oxidative stress (H
2
O
2
) than the parent strain. Furthermore, the
htrA
mutant produced less capsule, bound more molecules of complement component C3, and was more sensitive to complement and whole-blood killing than was the parent strain. Finally, disruption of the
htrA
gene in a virulent
K. pneumoniae
strain caused a reduction of its virulence in a mice model. Our results indicate that the
htrA
gene plays an important role in the virulence of
K. pneumoniae
.
Immune Cell-Mediated Protection against Vaginal Candidiasis: Evidence for a Major Role of Vaginal CD4+T Cells and Possible Participation of Other Local Lymphocyte Effectors
Authors: Santoni Giorgio , Boccanera Maria , Adriani Daniela , Lucciarini Roberta , Amantini Consuelo , Morrone Stefania , Cassone Antonio , De Bernardis Flavia
Published: October 2002

The protective roles of different lymphocyte subsets were investigated in a rat vaginal candidiasis model by adoptive transfer of vaginal lymphocytes (VL) or sorted, purified CD3+T cells, CD4+or CD8+T cells, or CD3−CD5+B cells from the vaginas of naïve or immune rats following three rounds ofCandida albicansinfection. The adoptive transfer of total VL from nonimmune animals did not alter the course of vaginal candidiasis of the recipient rats. In contrast, the animals receiving total VL or CD3+T cells from immune rats showed a highly significant acceleration of fungus clearance compared with animals which received nonimmune VL. The animals with vaginal CD3−CD5+B cells transferred from immune rats also had fewerCandidaCFU than the controls, but fungal clearance was significantly retarded with respect to the animals administered immune T cells. Sorted, purified CD4+and CD8+vaginal T cells from immune rats were also adoptively transferred to naïve animals. Although both populations were seen to accelerate the clearance of the fungus from the vagina, CD4+T cells were much more effective than CD8+T cells. Overall, there was no difference between the antifungal effects of immune vaginal CD4+T cells and those achievable with the transfer of whole, immune VL. Histological observations of the vaginal tissues of rats with adoptively transferred immune T cells demonstrated a remarkable accumulation of lymphocytes in the subepithelial lamina propria and also infiltrating the mucosal epithelium. These results strongly suggest that distinct vaginal lymphocyte subsets participate in the adaptive anti-Candidaimmunity at the vaginal level, with the vaginal CD4+T cells probably playing a major role.
Immunization with the
Chlamydia trachomatis
Mouse Pneumonitis Major Outer Membrane Protein by Use of CpG Oligodeoxynucleotides as an Adjuvant Induces a Protective Immune Response against an Intranasal Chlamydial Challenge
Authors: Pal Sukumar , Davis Heather L. , Peterson Ellena M. , de la Maza Luis M.
Published: October 2002

Recently, we have shown that a vaccine consisting of a purified preparation of the
Chlamydia trachomatis
mouse pneumonitis (MoPn) major outer membrane protein (MOMP) and Freund's adjuvant can protect mice against a genital challenge. Here, we wanted to determine if CpG motifs could be used as an immune modulator to the MOMP to induce protection in mice against an intranasal (i.n.) challenge. One-week-old BALB/c mice were immunized intramuscularly and subcutaneously either once or three times at 2-week intervals with MOMP and CpG suspended in aluminum hydroxide (alum). Negative controls received ovalbumin, CpG, and alum. Positive controls were immunized i.n. with
C. trachomatis
MoPn elementary bodies (EB). Six weeks after the last immunization, mice were challenged i.n. with 10
4
inclusion-forming units (IFU) of the
C. trachomatis
MoPn serovar. Mice that received MOMP, CpG, and alum had a strong immune response, as shown by a high titer of serum antibodies to
Chlamydia
and significant lymphoproliferation of T-cells following stimulation with
C. trachomatis
EB. After the i.n. challenge mice immunized with MOMP, CpG, and alum showed significantly less body weight loss than the corresponding control mice immunized with ovalbumin, CpG, and alum. Ten days after the challenge the animals were euthanized, their lungs were weighed, and the numbers of IFU in the lungs were determined. The average weight of the lungs of the mice immunized with MOMP, CpG, and alum was significantly less than average weight of the lungs of the mice immunized with ovalbumin, CpG, and alum. Also, the average number of IFU recovered per mouse immunized with MOMP, CpG, and alum was significantly less than the average number of IFU per mouse detected in the mice inoculated with ovalbumin, CpG, and alum. In conclusion, our data show that CpG sequences can be used as an effective adjuvant with the
C. trachomatis
MoPn MOMP to elicit a protective immune response in mice against a chlamydial respiratory challenge.