bluuebunny/crossref_metadata_2025 huggingface dataset:
Experimental Induction of Fluconazole Resistance in
Candida tropicalis
ATCC 750
Authors: Barchiesi Francesco , Calabrese David , Sanglard Dominique , Falconi Di Francesco Luigi , Caselli Francesca , Giannini Daniele , Giacometti Andrea , Gavaudan Stefano , Scalise Giorgio
Published: July 2002

Candida tropicalis
is less commonly isolated from clinical specimens than
Candida albicans
. Unlike
C. albicans
, which can be occasionally found as a commensal,
C. tropicalis
is almost always associated with the development of fungal infections. In addition,
C. tropicalis
has been reported to be resistant to fluconazole (FLC). To analyze the development of FLC resistance in
C. tropicalis
, an FLC-susceptible strain (ATCC 750) (MIC = 1.0 μg/ml) was cultured in liquid medium containing increasing FLC concentrations from 8.0 to 128 μg/ml. The strain developed variable degrees of FLC resistance which paralleled the concentrations of FLC used in the medium. The highest MICs of FLC were 16, 256, and 512 μg/ml for strains grown in medium with 8.0, 32, and 128 μg of FLC per ml, respectively. Development of resistance was rapid and could be observed already after a single subculture in azole-containing medium. The resistant strains were cross-resistant to itraconazole (MIC > 1.0 μg/ml) and terbinafine (MIC > 512 μg/ml) but not to amphotericin B. Isolates grown in FLC at concentrations of 8.0 and 32 μg/ml reverted to low MICs (1.0 μg/ml) after 12 and 11 passages in FLC-free medium, respectively. The MIC for one isolate grown in FLC (128 μg/ml) (128 R) reverted to 16 μg/ml but remained stable over 60 passages in FLC-free medium. Azole-resistant isolates revealed upregulation of two different multidrug efflux transporter genes: the major facilitators gene
MDR1
and the ATP-binding cassette transporter
CDR1
. The development of FLC resistance in vitro correlated well with the results obtained in an experimental model of disseminated candidiasis. While FLC given at 10 mg/kg of body weight/day was effective in reducing the fungal burden of mice infected with the parent strain, the same dosing regimen was ineffective in mice infected with strain 128 R. Finally, the acquisition of in vitro FLC resistance in strain 128 R was related to a loss of virulence. The results of our study elucidate important characteristics and potential mechanisms of FLC resistance in
C. tropicalis
.
Peptide Deformylase in
Staphylococcus aureus
: Resistance to Inhibition Is Mediated by Mutations in the Formyltransferase Gene
Authors: Margolis Peter S. , Hackbarth Corinne J. , Young Dennis C. , Wang Wen , Chen Dawn , Yuan Zhengyu , White Richard , Trias Joaquim
Published: July 2002

Peptide deformylase, a bacterial enzyme, represents a novel target for antibiotic discovery. Two deformylase homologs,
defA
and
defB
, were identified in
Staphylococcus aureus
. The
defA
homolog, located upstream of the transformylase gene, was identified by genomic analysis and was cloned from chromosomal DNA by PCR. A distinct homolog,
defB
, was cloned from an
S. aureus
genomic library by complementation of the arabinose-dependent phenotype of a P

BAD

-def Escherichia coli
strain grown under arabinose-limiting conditions. Overexpression in
E. coli
of
defB
, but not
defA
, correlated to increased deformylase activity and decreased susceptibility to actinonin, a deformylase-specific inhibitor. The
defB
gene could not be disrupted in wild-type
S. aureus
, suggesting that this gene, which encodes a functional deformylase, is essential. In contrast, the
defA
gene could be inactivated; the function of this gene is unknown. Actinonin-resistant mutants grew slowly in vitro and did not show cross-resistance to other classes of antibiotics. When compared to the parent, an actinonin-resistant strain produced an attenuated infection in a murine abscess model, indicating that this strain also has a growth disadvantage in vivo. Sequence analysis of the actinonin-resistant mutants revealed that each harbors a loss-of-function mutation in the
fmt
gene. Susceptibility to actinonin was restored when the wild-type
fmt
gene was introduced into these mutant strains. An
S. aureus
Δ
fmt
strain was also resistant to actinonin, suggesting that a functional deformylase activity is not required in a strain that lacks formyltransferase activity. Accordingly, the
defB
gene could be disrupted in an
fmt
mutant.
Effect of Polygodial on the Mitochondrial ATPase of
Saccharomyces cerevisiae
Authors: Lunde Christopher S. , Kubo Isao
Published: July 2002

The fungicidal mechanism of a naturally occurring sesquiterpene dialdehyde, polygodial, was investigated in
Saccharomyces cerevisiae
. In an acidification assay, polygodial completely suppressed the glucose-induced decrease in external pH at 3.13 μg/ml, the same as the fungicidal concentration. Acidification occurs primarily through the proton-pumping action of the plasma membrane ATPase, Pma1p. Surprisingly, this ATPase was not directly inhibited by polygodial. In contrast, the two other membrane-bound ATPases in yeast were found to be susceptible to the compound. The mitochondrial ATPase was inhibited by polygodial in a dose-dependent manner at concentrations similar to the fungicidal concentration, whereas the vacuolar ATPase was only slightly inhibited. Cytoplasmic petite mutants, which lack mitochondrial DNA and are respiration deficient, were significantly less susceptible to polygodial than the wild type, as was shown in time-kill curves. A
pet9
mutant which lacks a functional ADP-ATP translocator and is therefore respiration dependent was rapidly inhibited by polygodial. The results of these susceptibility assays link enzyme inhibition to physiological effect. Previous studies have reported that plasma membrane disruption is the mechanism of polygodial-induced cell death; however, these results support a more complex picture of its effect. A major target of polygodial in yeast is mitochondrial ATP synthase. Reduction of the ATP supply leads to a suppression of Pma1 ATPase activity and impairs adaptive responses to other facets of polygodial's cellular inhibition.
Ofloxacin-Loaded Liposomes: In Vitro Activity and Drug Accumulation in Bacteria
Authors: Furneri Pio M. , Fresta Massimo , Puglisi Giovanni , Tempera Gianna
Published: July 2002

Different ofloxacin-loaded unilamellar vesicles were prepared by the extrusion technique, and their antimicrobial activities were determined in comparison to those of the free drug by means of MIC determinations with both American Type Culture Collection standards and wild-type bacterial strains (six strains of
Enterococcus faecalis
, seven strains of
Escherichia coli
, six strains of
Staphylococcus aureus
, and six strains of
Pseudomonas aeruginosa
). The accumulation of ofloxacin and liposome-ofloxacin was measured by determining the amount of the drug inside the bacteria as a function of time. Encapsulated fluoroquinolone yielded MICs which were at least twofold lower than those obtained with the free drug. In particular, liposomes made up of dimyristoylphosphatidylcholine-cholesterol-dipalmitoylphosphatidylserine and dimyristoylphosphatidylcholine-cholesterol-dihexadecylphosphate (4:3:4 molar ratio) provided the best improvement in antimicrobial activity against the various bacterial strains investigated. The liposome formulation produced higher intracellular fluoroquinolone concentrations than those achieved simultaneously with the free drug in both
E. coli
and
P. aeruginosa
.
Trends in Antifungal Drug Susceptibility of
Cryptococcus neoformans
Isolates in the United States: 1992 to 1994 and 1996 to 1998
Authors: Brandt Mary E. , Pfaller Michael A. , Hajjeh Rana A. , Hamill Richard J. , Pappas Peter G. , Reingold Arthur L. , Rimland David , Warnock David W.
Published: July 2002

The antifungal drug susceptibilities of two collections of
Cryptococcus neoformans
isolates obtained through active laboratory-based surveillance from 1992 to 1994 (368 isolates) and 1996 to 1998 (364 isolates) were determined. The MICs of fluconazole, itraconazole, and flucytosine were determined by the National Committee for Clinical Laboratory Standards broth microdilution method; amphotericin B MICs were determined by the E-test. Our results showed that the MIC ranges, the MICs at which 50% of isolates are inhibited (MIC
50
s), and the MIC
90
s of these four antifungal agents did not change from 1992 to 1998. In addition, very small numbers of isolates showed elevated MICs suggestive of in vitro resistance. The MICs of amphotericin B were elevated (≥2 μg/ml) for 2 isolates, and the MICs of flucytosine were elevated (≥32 μg/ml) for 14 isolates. Among the azoles, the fluconazole MIC was elevated (≥64 μg/ml) for 8 isolates and the itraconazole MIC (≥1 μg/ml) was elevated for 45 isolates. Analysis of 172 serial isolates from 71 patients showed little change in the fluconazole MIC over time. For isolates from 58 patients (82% of serial cases) there was either no change or a twofold change in the fluconazole MIC. In contrast, for isolates from seven patients (12% of serial cases) the increase in the MIC was at least fourfold. For isolates from another patient there was a 32-fold decrease in the fluconazole MIC over a 1-month period. We conclude that in vitro resistance to antifungal agents remains uncommon in
C. neoformans
and has not significantly changed with time during the past decade.
The Structure, Function, and Origin of the Microcin H47 ATP-Binding Cassette Exporter Indicate Its Relatedness to That of Colicin V
Authors: Azpiroz Marı́a F. , Rodrı́guez Eliana , Laviña Magela
Published: July 2002

Microcin H47, a gene-encoded peptide antibiotic produced by a natural
Escherichia coli
strain, was shown to be secreted by a three-component ATP-binding cassette exporter which was revealed to be strongly related to that of colicin V. The results of sequence and gene fusion analyses, as well as heterologous complementation assays, are presented.
Accurate Prediction of Macrolide Resistance in
Helicobacter pylori
by a PCR Line Probe Assay for Detection of Mutations in the 23S rRNA Gene: Multicenter Validation Study
Authors: van Doorn Leen-Jan , Glupczynski Youri , Kusters Johannes G. , Mégraud Francis , Midolo Peter , Maggi-Solcà Nadia , Queiroz Dulciene M. M. , Nouhan Nathalie , Stet Els , Quint Wim G. V.
Published: July 2002

Helicobacter pylori
strains from 299 patients were tested in six laboratories in different countries. Macrolide susceptibility of the strains was determined by agar dilution (17.4%) or the epsilometer test (82.6%). Mutations in the 23S ribosomal DNA (rDNA) that are associated with macrolide resistance were analyzed by PCR and reverse hybridization (PCR-line probe assay [LiPA]). This method identifies A2115G, G2141A, A2142G, A2142C, A2142T, A2143G, and A2143C mutations in the 23S rDNA.
vacA
s-region (s1a, s1b, s1c, and s2) and m-region (m1, m2a, and m2b) genotypes and
cagA
status were also determined using another PCR-LiPA system. Of the 299 strains investigated by MIC testing, 130 (43.5%) were resistant and 169 (56.5%) were susceptible to clarithromycin. Of the 130 resistant strains, 127 (97.7%) contained 23S rDNA mutations, whereas 167 (98.8%) of the 169 susceptible strains contained wild-type sequences. The predominant mutations were A2143G (45.2%) and A2142G (33.3%). Twenty-eight (19.8%) strains contained multiple 23S rDNA mutations. Only five resistant strains contained the A2142C mutation (three of these in combination with the A2142G mutation), and the A2115G, G2141A, A2142T, and A2143C mutations were not found. MICs of clarithromycin for the A2142G mutant strains were significantly higher than MICs for the A2143G strains. Although there was no significant association between 23S rDNA mutations and the
vacA
and
cagA
status, clarithromycin-susceptible strains more often contained mixed
vacA
genotypes, indicating the presence of multiple
H. pylori
strains. In conclusion, our data confirmed the very strong association between 23S rDNA mutations and macrolide resistance and showed that the PCR-LiPA permits accurate and reliable diagnosis of macrolide resistance in
H. pylori
.
Genomic Profiling of the Response of
Candida albicans
to Itraconazole Treatment Using a DNA Microarray
Authors: De Backer Marianne D. , Ilyina Tatiana , Ma Xiao-Jun , Vandoninck Sandy , Luyten Walter H. M. L. , Vanden Bossche Hugo
Published: July 2002

The application of genome-wide expression profiling to determine how drugs achieve their therapeutic effect has provided the pharmaceutical industry with an exciting new tool for drug mode-of-action studies. We used DNA chip technology to study cellular responses to perturbations of ergosterol biosynthesis caused by the broad-spectrum antifungal agent itraconazole. Simultaneous examination of over 6,600
Candida albicans
gene transcript levels, representing the entire genome, upon treatment of cells with 10 μM itraconazole revealed that 296 genes were responsive. For 116 genes transcript levels were decreased at least 2.5-fold, while for 180 transcript levels were similarly increased. A global upregulation of
ERG
genes in response to azole treatment was observed.
ERG11
and
ERG5
were found to be upregulated approximately 12-fold. In addition, a significant upregulation was observed for
ERG6, ERG1, ERG3, ERG4, ERG10, ERG9, ERG26, ERG25, ERG2, IDII, HMGS, NCP1,
and
FEN2,
all of which are genes known to be involved in ergosterol biosynthesis. The effects of itraconazole on a wide variety of known metabolic processes are discussed. As over 140 proteins with unknown function were responsive to itraconazole, our analysis might provide—in combination with phenotypic data—first hints of their potential function. The present report is the first to describe the application of DNA chip technology to study the response of a major human fungal pathogen to drug treatment.
Antibiotic Susceptibility and Mechanisms of Erythromycin Resistance in Clinical Isolates of
Streptococcus agalactiae
: French Multicenter Study
Authors: De Mouy Dany , Cavallo Jean-Didier , Leclercq Roland , Fabre Roland ,   The Aforcopi-Bio Network
Published: July 2002

Among 126
Streptococcus agalactiae
isolates collected in 10 French laboratories in 1999, 27 (21.4%) had macrolide resistance related to the presence of
erm
(B) (11 strains),
erm
(A) subclass
erm
(TR) (10 strains), and
mef
(A) genes (2 strains) and the presence of combinations of
erm
(B) and
erm
(A) genes or
mef
(A) genes (3 strains).
Detection of Intracellular Bacteria in the Buds of Scotch Pine (
Pinus sylvestris
L.) by In Situ Hybridization
Authors: Pirttilä Anna Maria , Laukkanen Hanna , Pospiech Helmut , Myllylä Raili , Hohtola Anja
Published: July 2002

Bacterial isolates were obtained from pine (
Pinus sylvestris
L.) tissue cultures and identified as
Methylobacterium extorquens
and
Pseudomonas synxantha
. The existence of bacteria in pine buds was investigated by 16S rRNA in situ hybridization. Bacteria inhabited the buds of every tree examined, primarily colonizing the cells of scale primordia and resin ducts.
Microbial Biofilm Formation and Contamination of Dental-Unit Water Systems in General Dental Practice
Authors: Walker James T. , Bradshaw David J. , Bennett Allan M. , Fulford Martin R. , Martin Michael V. , Marsh Philip D.
Published: July 2002

Dental-unit water systems (DUWS) harbor bacterial biofilms, which may serve as a haven for pathogens. The aim of this study was to investigate the microbial load of water from DUWS in general dental practices and the biofouling of DUWS tubing. Water and tube samples were taken from 55 dental surgeries in southwestern England. Contamination was determined by viable counts on environmentally selective, clinically selective, and pathogen-selective media, and biofouling was determined by using microscopic and image analysis techniques. Microbial loading ranged from 500 to 10
5
CFU · ml
−1
; in 95% of DUWS water samples, it exceeded European Union drinking water guidelines and in 83% it exceeded American Dental Association DUWS standards. Among visible bacteria, 68% were viable by
Bac
Light staining, but only 5% of this “viable by
Bac
Light” fraction produced colonies on agar plates.
Legionella pneumophila
,
Mycobacterium
spp.,
Candida
spp., and
Pseudomonas
spp. were detected in one, five, two, and nine different surgeries, respectively. Presumptive oral streptococci and
Fusobacterium
spp. were detected in four and one surgeries, respectively, suggesting back siphonage and failure of antiretraction devices. Hepatitis B virus was never detected. Decontamination strategies (5 of 55 surgeries) significantly reduced biofilm coverage but significantly increased microbial numbers in the water phase (in both cases,
P
< 0.05). Microbial loads were not significantly different in DUWS fed with soft, hard, deionized, or distilled water or in different DUWS (main, tank, or bottle fed). Microbiologically, no DUWS can be considered “cleaner” than others. DUWS deliver water to patients with microbial levels exceeding those considered safe for drinking water.
Accumulation of Poly[(
R
)-3-Hydroxyalkanoates] in
Pseudomonas oleovorans
during Growth with Octanoate in Continuous Culture at Different Dilution Rates
Authors: Durner Roland , Witholt Bernard , Egli Thomas
Published: July 2002

Pseudomonas oleovorans
ATCC 29347 was grown in chemostat culture at different dilution rates with mineral media varying in their ratios of octanoate to ammonia (C
0
/N
0
ratio). At all dilution rates tested, three distinct growth regimes were observed: (i) carbon limitation with NH
4
+
in excess at low C
0
/N
0
ratios, (ii) purely nitrogen-limited growth conditions at high C
0
/N
0
ratios with residual octanoate in the culture supernatant, and (iii) an intermediate zone of dual-nutrient-limited growth conditions where both the concentration of octanoate and that of ammonia were very low. The dual-nutrient-limited growth zone shifted to higher C
0
/N
0
ratios with decreasing dilution rates, and the extension of the dual-nutrient-limited growth zone was inversely proportional to the growth rate. The cells accumulated the storage compound medium-chain-length poly[(
R
)-3-hydroxyalkanoate] (mcl-PHA) during dual (C and N)-nutrient-limited and N-limited growth conditions. Within the dual-nutrient-limited growth zone, the cellular mcl-PHA contents increased when the C
0
/N
0
ratio in the feed was increased, whereas the cellular mcl-PHA level was independent from the feed C
0
/N
0
ratio during N-limited growth. The monomeric composition of the accumulated mcl-PHA was independent of both the dilution rate and the feed C
0
/N
0
ratio and consisted of 12 mol% 3-hydroxyhexanoic acid and 88 mol% 3-hydroxyoctanoic acid. Accumulation of mcl-PHA led to an increase in the cellular C/N ratio and to changes in elemental growth yields for nitrogen and carbon.
Antigenic Characterization of the Fish Pathogen
Flavobacterium psychrophilum
Authors: Crump Elizabeth M. , Perry Malcolm B. , Clouthier Sharon C. , Kay William W.
Published: July 2002

Flavobacteria are a poorly understood and speciated group of commensal bacteria and opportunistic pathogens. The psychrotroph
Flavobacterium psychrophilum
is the etiological agent of rainbow trout fry syndrome and bacterial cold water disease, septicemic diseases that heavily impact salmonids. Consequently, two verified but geographically diverse isolates were characterized phenotypically and biochemically. A facile typing system was devised which readily discriminated between closely related species and was verified against a pool of recent prospective isolates.
F. psychrophilum
was found to be enveloped in a loosely attached, strongly antigenic outer layer comprised of a predominant, highly immunogenic, low-molecular-mass carbohydrate antigen as well as several protein antigens. Surface-exposed antigens were visualized by a combination of immunoflourescence microscopy, immunogold transmission, and thin-section electron microscopy and were discriminated by Western blotting using rabbit antisera, by selective extraction with EDTA-polymyxin B agarose beads, and by extrinsic labeling of amines with sulfo–
N
-hydoxysuccinimide–biotin and glycosyl groups with biotin hydrazide. The predominant ∼16 kDa antigen was identified as low-molecular-mass lipopolysaccharide (LPS), whereas high-molecular-mass LPS containing O antigen was not as prevalent on whole cells but was abundant in culture supernatants. Rainbow trout convalescent antisera recognized both molecular mass classes of LPS as well as a predominant ∼20-kDa protein. This study represents the first description at the molecular level of the surface characteristics and potential vaccine targets of confirmed
F. psychrophilum
strains.
Bacterial Populations Colonizing and Degrading Rice Straw in Anoxic Paddy Soil
Authors: Weber Sabine , Stubner Stephan , Conrad Ralf
Published: July 2002

Rice straw is a major substrate for the production of methane, a greenhouse gas, in flooded rice fields. The bacterial community degrading rice straw under anoxic conditions was investigated with molecular methods. Rice straw was incubated in paddy soil anaerobically for 71 days. Denaturing gradient gel electrophoresis (DGGE) of the amplified bacterial 16S rRNA genes showed that the composition of the bacterial community changed during the first 15 days but then was stable until the end of incubation. Fifteen DGGE bands with different signal intensities were excised, cloned, and sequenced. In addition, DNA was extracted from straw incubated for 1 and 29 days and the bacterial 16S rRNA genes were amplified and cloned. From these clone libraries 16 clones with different electrophoretic mobilities on a DGGE gel were sequenced. From a total of 31 clones, 20 belonged to different phylogenetic clusters of the clostridia, i.e., clostridial clusters I (14 clones), III (1 clone), IV (1 clone), and XIVa (4 clones). One clone fell also within the clostridia but could not be affiliated to one of the clostridial clusters. Ten clones grouped closely with the genera
Bacillus
(3 clones),
Nitrosospira
(1 clone),
Fluoribacter
(1 clones), and
Acidobacterium
(2 clones) and with clone sequences previously obtained from rice field soil (3 clones). The relative abundances of various phylogenetic groups in the rice straw-colonizing community were determined by fluorescence in situ hybridization (FISH). Bacteria were detached from the incubated rice straw with an efficiency of about 80 to 90%, as determined by dot blot hybridization of 16S rRNA in extract and residue. The number of active (i.e., a sufficient number of ribosomes)
Bacteria
detected with a general eubacterial probe (Eub338) after 8 days of incubation was 61% of the total cell counts. This percentage decreased to 17% after 29 days of incubation. Most (55%) of the active cells on day 8 belonged to the genus
Clostridium
, mainly to clostridial clusters I (24%), III (6%), and XIVa (24%). An additional 5% belonged to the
Cytophaga-Flavobacterium
cluster of the
Cytophaga-Flavobacterium-Bacteroides
phylum, 4% belonged to the α, β, and γ
Proteobacteria
, and 1.3% belonged to the
Bacillus
subbranch of the gram-positive bacteria with a low G+C content. The results show that the bacterial community colonizing and decomposing rice straw developed during the first 15 days of incubation and was dominated by members of different clostridial clusters, especially clusters I, III, and XIVa.
Diversity and Specificity of
Frankia
Strains in Nodules of Sympatric
Myrica gale, Alnus incana
, and
Shepherdia canadensis
Determined by
rrs
Gene Polymorphism
Authors: Huguet Valerie , Batzli Janet Mccray , Zimpfer Jeff F. , Normand Philippe , Dawson Jeffrey O. , Fernandez Maria P.
Published: July 2002

The identity of
Frankia
strains from nodules of
Myrica gale, Alnus incana
subsp.
rugosa
, and
Shepherdia canadensis
was determined for a natural stand on a lake shore sand dune in Wisconsin, where the three actinorhizal plant species were growing in close proximity, and from two additional stands with
M. gale
as the sole actinorhizal component. Unisolated strains were compared by their 16S ribosomal DNA (rDNA) restriction patterns using a direct PCR amplification protocol on nodules. Phylogenetic relationships among nodular
Frankia
strains were analyzed by comparing complete 16S rDNA sequences of study and reference strains. Where the three actinorhizal species occurred together, each host species was nodulated by a different phylogenetic group of
Frankia
strains.
M. gale
strains from all three sites belonged to an
Alnus-Casuarina
group, closely related to
Frankia alni
representative strains, and were low in diversity for a host genus considered promiscuous with respect to
Frankia
microsymbiont genotype.
Frankia
strains from
A. incana
nodules were also within the
Alnus-Casuarina
cluster, distinct from
Frankia
strains of
M. gale
nodules at the mixed actinorhizal site but not from
Frankia
strains from two
M. gale
nodules at a second site in Wisconsin.
Frankia
strains from nodules of
S. canadensis
belonged to a divergent subset of a cluster of
Elaeagnaceae
-infective strains and exhibited a high degree of diversity. The three closely related local
Frankia
populations in
Myrica
nodules could be distinguished from one another using our approach. In addition to geographic separation and host selectivity for
Frankia
microsymbionts, edaphic factors such as soil moisture and organic matter content, which varied among locales, may account for differences in
Frankia
populations found in
Myrica
nodules.
Biochemical Characterization of Ochratoxin A-Producing Strains of the Genus
Penicillium
Authors: Larsen Thomas Ostenfeld , Svendsen Anne , Smedsgaard Jørn
Published: July 2002

In order to explore the biochemical scope of ochratoxin A-producing penicillia, we screened 48
Penicillium verrucosum
isolates for the production of secondary metabolites. Fungal metabolites were analyzed by high-pressure liquid or gas chromatography coupled to diode array detection or mass spectrometry. The following metabolites were identified: ochratoxins A and B, citrinin, verrucolones, verrucines, anacines, sclerotigenin, lumpidin, fumiquinazolines, alantrypinones, daldinin D, dipodazine, penigequinolines A and B, 2-pentanone, and 2-
methyl
-isoborneol. By use of average linking clustering based on binary (nonvolatile) metabolite data, the 48 isolates could be grouped into two large and clearly separated groups and a small outlying group of four non-ochratoxin-producing isolates. The largest group, containing 24 isolates, mainly originating from plant sources, included the type culture of
P. verrucosum
. These isolates produced ochratoxin A, verrucolones, citrinin, and verrucines and had a characteristic dark brown reverse color on yeast extract-sucrose agar medium. Almost all of a group of 20 isolates mainly originating from cheese and meat products had a pale cream reverse color on yeast extract-sucrose agar medium and produced ochratoxin A, verrucolones, anacines, and sclerotigenin. This group included the former type culture of
P. nordicum
. We also found that
P. verrucosum
isolates and three
P. nordicum
isolates incorporated phenylalanine into verrucine and lumpidin metabolites, a finding which could explain why those isolates produced relatively lower levels of ochratoxins than did most isolates of
P. nordicum
.
Transfer of the Pheromone-Inducible Plasmid pCF10 among
Enterococcus faecalis
Microorganisms Colonizing the Intestine of Mini-Pigs
Authors: Licht Tine Rask , Laugesen Dorthe , Jensen Lars Bogø , Jacobsen Bodil Lund
Published: July 2002

A new animal model, the streptomycin-treated mini-pig, was developed in order to allow colonization of defined strains of
Enterococcus faecalis
in numbers sufficient to study plasmid transfer. Transfer of the pheromone-inducible pCF10 plasmid between streptomycin-resistant strains of
E. faecalis
OG1 was investigated in the model. The plasmid encodes resistance to tetracycline. Numbers of recipient, donor, and transconjugant bacteria were monitored by selective plating of fecal samples, and transconjugants were subsequently verified by PCR. After being ingested by the mini-pigs, the recipient strain persisted in the intestine at levels between 10
6
and 10
7
CFU per g of feces throughout the experiment. The donor strain, which carried different resistance markers but was otherwise chromosomally isogenic to the recipient strain, was given to the pigs 3 weeks after the recipient strain. The donor cells were initially present in high numbers (10
6
CFU per g) in feces, but they did not persist in the intestine at detectable levels. Immediately after introduction of the donor bacteria, transconjugant cells appeared and persisted in fecal samples at levels between 10
3
and 10
4
CFU per g until the end of the experiment. These observations showed that even in the absence of selective tetracycline pressure, plasmid pCF10 was transferred from ingested
E. faecalis
cells to other
E. faecalis
organisms already present in the intestinal environment and that the plasmid subsequently persisted in the intestine.
An Exported Rhodanese-Like Protein Is Induced during Growth of
Acidithiobacillus ferrooxidans
in Metal Sulfides and Different Sulfur Compounds
Authors: Ramírez Pablo , Toledo Héctor , Guiliani Nicolas , Jerez Carlos A.
Published: July 2002

By proteomic analysis we found a 21-kDa protein (P21) from
Acidithiobacillus ferrooxidans
ATCC 19859 whose synthesis was greatly increased by growth of the bacteria in pyrite, thiosulfate, elemental sulfur, CuS, and ZnS and was almost completely repressed by growth in ferrous iron. After we determined the N-terminal amino acid sequence of P21, we used the available preliminary genomic sequence of
A. ferrooxidans
ATCC 23270 to isolate the DNA region containing the
p21
gene. The nucleotide sequence of this DNA fragment contained a putative open reading frame (ORF) coding for a 23-kDa protein. This difference in size was due to the presence of a putative signal peptide in the ORF coding for P21. When
p21
was cloned and overexpressed in
Escherichia coli
, the signal peptide was removed, resulting in a mature protein with a molecular mass of 21 kDa and a calculated isoelectric point of 9.18. P21 exhibited 27% identity and 42% similarity to the
Deinococcus radiodurans
thiosulfate-sulfur transferase (rhodanese; EC 2.8.1.1) and similar values in relation to other rhodaneses, conserving structural domains and an active site with a cysteine, both characteristic of this family of proteins. However, the purified recombinant P21 protein did not show rhodanese activity. Unlike cytoplasmic rhodaneses, P21 was located in the periphery of
A. ferrooxidans
cells, as determined by immunocytochemical analysis, and was regulated depending on the oxidizable substrate. The genomic context around gene
p21
contained other ORFs corresponding to proteins such as thioredoxins and sulfate-thiosulfate binding proteins, clearly suggesting the involvement of P21 in inorganic sulfur metabolism in
A. ferrooxidans
.
Role of Acetate in Production of an Autoinducible Class IIa Bacteriocin in
Carnobacterium piscicola
A9b
Authors: Nilsson Lilian , Nielsen Michael K. , Ng Yin , Gram Lone
Published: July 2002

Carnobacterium piscicola
strain A9b isolated from cold smoked salmon inhibits growth of the food-borne pathogen
Listeria monocytogenes
partly due to the production of a proteinaceous compound (L. Nilsson, L. Gram, and H. H. Huss. J. Food Prot. 62:336-342, 1999). The purpose of the present study was to purify the compound and describe factors affecting its production, with particular emphasis on food-relevant factors. Amino acid sequencing showed that the compound is a class IIa bacteriocin with an N-terminal amino acid sequence identical to that of carnobacteriocin B2. The production of the bacteriocin was autoinducible, and the threshold level for induction was 9.6 × 10
−10
M. We also report, for the first time, that acetate acts as an induction factor, with a threshold concentration of 0.3 to 12 mM. Acetate could not act as an inducer during the late exponential phase of
C. piscicola
A9b. The induction of bacteriocin production showed a dose-dependent relationship at acetate concentrations of up to 10 to 20 mM (depending on the growth medium) and at a concentration of 1.9 × 10
−8
M for the bacteriocin itself; a saturation level of bacteriocin specific activity was reached at these concentrations of induction factors. The combined use of both inducers did not enhance the saturation level of bacteriocin production compared to that seen with the use of each inducer alone. Increasing NaCl and glucose concentrations negatively influenced the efficiency of acetate as an induction factor. Based on the results, carnobacteriocin B2 was used as an induction factor to manipulate the production of bacteriocin in cold smoked salmon juice and thus improve the ability to inhibit
L. monocytogenes
.
Variation of Branched-Chain Fatty Acids Marks the Normal Physiological Range for Growth in
Listeria monocytogenes
Authors: Nichols David S. , Presser Kirsty A. , Olley June , Ross Tom , McMeekin Tom A.
Published: July 2002

The fatty acid composition of
Listeria monocytogenes
Scott A was determined by close-interval sampling over the entire biokinetic temperature range. There was a high degree of variation in the percentage of branched-chain fatty acids at any given temperature. The percentage of branched C
17
components increased with growth temperature in a linear manner. However, the percentages of iso-C
15:0
(i15:0) and anteiso-C
15:0
(a15:0) were well described by third-order and second-order polynomial curves, respectively. There were specific temperature regions where the proportion of branched-chain fatty acids deviated significantly from the trend established over the entire growth range. In the region from 12 to 13°C there were significant deviations in the percentages of both i15:0 and a15:0 together with a suggested deviation in a17:0, resulting in a significant change in the total branched-chain fatty acids. In the 31 to 33°C region the percentage of total branched-chain components exhibited a significant deviation. The observed perturbations in fatty acid composition occurred near the estimated boundaries of the normal physiological range for growth.
Naturally Acquired Antibody Responses to the C-Terminal Region of Merozoite Surface Protein 1 ofPlasmodium vivaxin Korea
Authors: Park Jae-Won , Moon Seung-Hwan , Yeom Joon-Sup , Lim Kook-Jin , Sohn Mi-Jin , Jung Woo-Chul , Cho Young-Jung , Jeon Ki-Won , Ju Woong , Ki Chang-Seok , Oh Myoung-Don , Choe Kangwon
Published: July 2002

We expressed a protein inSaccharomyces cerevisiaein order to evaluate the humoral immune responses to the C-terminal region of the merozoite surface protein 1 ofPlasmodium vivax. This protein (Pv20018) had a molecular mass of 18 kDa and was reactive with the sera of individuals with patent vivax malaria on immunoblotting analysis. The levels of immunoglobulin M (IgM) and IgG antibodies against Pv20018were measured in 421 patients with vivax malaria (patient group), 528 healthy individuals from areas of nonendemicity (control group 1), and 470 healthy individuals from areas of endemicity (control group 2), using the indirect enzyme-linked immunosorbent assay (ELISA) method. To study the longevity of the antibodies, 20 subjects from the patient group were also tested for the antibody levels once a month for 1 year. When the cutoff values for seropositivity were determined as the mean + 3 × standard deviation of the antibody levels in control group 1, both IgG and IgM antibody levels were negative in 98.5% (465 of 472) of control group 2. The IgG and IgM antibodies were positive in 88.1% (371 of 421) and 94.5% (398 of 421) of the patient group, respectively. The IgM antibody became negative 2 to 4 months after the onset of symptoms, whereas the IgG antibody usually remained positive for more than 5 months. In conclusion, indirect ELISA using Pv20018expressed inS. cerevisiaemay be a useful diagnostic method for vivax malaria.
Specificities and Sensitivities of Four Monoclonal Antibodies for Typing of
Borrelia burgdorferi
Sensu Lato Isolates
Authors: Bretz A. G. , Ryffel K. , Hutter P. , Dayer E. , Péter O.
Published: July 2002

Borrelia burgdorferi
, the agent of Lyme borreliosis, is genetically more heterogeneous than previously thought. In Europe five genospecies have been described from the original
B. burgdorferi
sensu lato (sl):
B. burgdorferi
sensu stricto (ss),
B. garinii, B. afzelii, B. lusitaniae
, and
B. valaisiana
. In the United States,
B. burgdorferi
ss as well as
B. bissettii
in California and
B. andersonii
on the East Coast were differentiated. In Asia,
B. japonica
has been identified along, with
B. garinii, B. afzelii
, and
B. valaisiana
. In order to evaluate sensitivity and specificity of four species-specific monoclonal antibodies, we analyzed 210
B. burgdorferi
sl isolates belonging to eight genospecies by immunoblot and confirmed genospecies by restriction fragment length polymorphism (RFLP) of
rrf
(5S)-
rrl
(23S) intergenic spacer amplicon. Monoclonal antibody H3TS had 100% sensitivity for 55
B. burgdorferi ss
isolates but showed reactivity with all four isolates belonging to
B. bissetii
. Monoclonal antibody I 17.3 showed 100% specificity and sensitivity for 45
B. afzelii
isolates. Monoclonal antibody D6 was 100% specific for
B. garinii
but missed 1 of 64 isolates (98.5% sensitivity). Monoclonal antibody A116k was 100% specific for
B. valaisiana
but was unreactive with 4 of 24 isolates (83.5% sensitivity). Genetic analysis correlated well with results of reactivity and confirmed efficacy of the phenotypic typing of these antibodies. Some isolates showed atypical RFLP. Therefore, both phenotypic and genotypic analyses are needed to characterize new
Borrelia
isolates.
Reverse Transcriptase-PCR Analysis of Bacterial rRNA for Detection and Characterization of Bacterial Species in Arthritis Synovial Tissue
Authors: Kempsell Karen E. , Cox Charles J. , Hurle Michael , Wong Anthony , Wilkie Scott , Zanders Edward D. , Gaston J. S. Hill , Crowe J. Scott
Published: July 2002

Onset of rheumatoid arthritis (RA) is widely believed to be preceded by exposure to some environmental trigger such as bacterial infectious agents. The influence of bacteria on RA disease onset or pathology has to date been controversial, due to inconsistencies between groups in the report of bacterial species isolated from RA disease tissue. Using a modified technique of reverse transcriptase-PCR amplification, we have detected bacterial rRNA in the synovial tissue of late-stage RA and non-RA arthritis controls. This may be suggestive of the presence of live bacteria. Sequencing of cloned complementary rDNA (crDNA) products revealed a number of bacterial sequences in joint tissue from each patient, and from these analyses a comprehensive profile of the organisms present was compiled. This revealed a number of different organisms in each patient, some of which are common to both RA and non-RA controls and are probably opportunistic colonizers of previously diseased tissue and others which are unique species. These latter organisms may be candidates for a specific role in disease pathology and require further investigation to exclude them as causative agents in the complex bacterial millieu. In addition, many of the detected bacterial species have not been identified previously from synovial tissue or fluid from arthritis patients. These may not be easily cultivable, since they were not revealed in previous studies using conventional in vitro bacterial culture methods. In situ hybridization analyses have revealed the joint-associated bacterial rRNA to be both intra- and extracellular. The role of viable bacteria or their nucleic acids as triggers in disease onset or pathology in either RA or non-RA arthritis controls is unclear and requires further investigation.
Relationship of Blood Group Determinants on
Helicobacter pylori
Lipopolysaccharide with Host Lewis Phenotype and Inflammatory Response
Authors: Heneghan Michael A. , McCarthy Ciaran F. , Moran Anthony P.
Published: July 2002

As Lewis a (Le
a
) and Lewis b (Le
b
) blood group antigens are isoforms of Lewis x (Le
x
) and Lewis y (Le
y
) and are expressed in the gastric mucosa, we evaluated whether the patterns of expression of Le
x
and Le
y
on
Helicobacter pylori
lipopolysaccharides reflected those of host expression of Le
a
and Le
b
. When 79 patients (secretors and nonsecretors) were examined for concordance between bacterial and host Le expression, no association was found (χ
2
= 5.734, 3 df,
P
= 0.125), nor was there a significant difference between the amount of Le
x
or Le
y
expressed on isolates from ulcer and chronic gastritis patients (
P
> 0.05). Also, the effect of host and bacterial expression of Le antigens on bacterial colonization and the observed inflammatory response was assessed. In ulcer patients, Le
x
expression was significantly related to neutrophil infiltration (
r
s
= 0.481,
P
= 0.024), whereas in chronic gastritis patients significant relationships were found between Le
x
expression and
H. pylori
colonization density (
r
s
= 0.296,
P
= 0.03), neutrophil infiltrate (
r
s
= 0.409,
P
= 0.001), and lymphocyte infiltrate (
r
s
= 0.389,
P
= 0.002). Furthermore, bacterial Le
y
expression was related to neutrophil (
r
s
= 0.271,
P
= 0.033) and lymphocyte (
r
s
= 0.277,
P
= 0.029) infiltrates. Thus, although no evidence of concordance was found between bacterial and host expression of Le determinants, these antigens may be crucial for bacterial colonization, and the ensuing inflammatory response appears, at least in part, to be influenced by Le antigens.
Tumor Necrosis Factor Receptor p55-Deficient Mice Respond to AcuteYersinia enterocoliticaInfection with Less Apoptosis and More Effective Host Resistance
Authors: Zhao Yi-Xue , Lajoie Ginette , Zhang Hongwei , Chiu Basil , Payne Ursula , Inman Robert D.
Published: July 2002

Tumor necrosis factor (TNF) has generally been regarded as a protective cytokine in host defense against bacterial infections. In the present study, we evaluated the role of TNF in the acute phase of infection byYersinia enterocoliticaby using mice rendered genetically deficient in TNF receptor p55 (TNFRp55−/−). Unexpectedly, TNFRp55−/−mice showed more effective resistance to the bacteria, reflected in enhanced bacterial clearance and less tissue damage, than did control C57BL/6 mice. C57BL/6 mice showed evidence of extensive apoptosis in the spleen accompanied by a selective decrease in the CD4+-T-cell population of splenocytes, whereas TNFRp55−/−mice were spared these changes. The splenocytes from TNFRp55−/−mice also maintained a robust gamma interferon IFN-γ response to mitogenic stimulation, while the comparable response in C57BL/6 mice was impaired. In addition, splenocytes harvested from infected mice demonstrated lower production of interleukin-10 IL-10 in TNFRp55−/−mice than in C57BL/6 mice. These findings suggest thatYersiniacan induce TNFRp55-mediated apoptosis of splenocytes in the acute phase of the infection and that alteration of T-cell-generated cytokines can dramatically alter the early events in host defense against this pathogen.
Bordetella pertussisVirulence Factors Affect Phagocytosis by Human Neutrophils
Authors: Weingart Christine L. , Weiss Alison A.
Published: July 2002

The interaction between human neutrophils and wild-typeBordetella pertussisor mutants expressing altered lipopolysaccharide or lacking virulence factors—pertussis toxin, adenylate cyclase toxin, dermonecrotic toxin, filamentous hemagglutinin (FHA), pertactin, or BrkA—was examined. In the absence of antibodies, the wild-type strain and the mutants, with the exception of mutants lacking FHA, attached efficiently to neutrophils. The addition of opsonizing antibodies caused a significant reduction (approximately 50%) in attachment of the wild-type strain and most of the mutants expressing FHA, suggesting that bacterium-mediated attachment is more efficient than Fc-mediated attachment. Phagocytosis was also examined. In the absence of antibodies, about 12% of the wild-type bacteria were phagocytosed. Opsonization caused a statistically significant reduction in phagocytosis (to 3%), possibly a consequence of reduced attachment. Phagocytosis of most of the mutants was similar to that of the wild type, with the exception of the mutants lacking adenylate cyclase toxin. About 70% of the adenylate cyclase toxin mutants were phagocytosed, but only in the presence of opsonizing antibody, suggesting that Fc receptor-mediated signaling may be needed for phagocytosis. These studies indicate that FHA mediates attachment ofB. pertussisto neutrophils, but adenylate cyclase toxin blocks phagocytosis.
Listeria monocytogenes-Infected Human Dendritic Cells: Uptake and Host Cell Response
Authors: Kolb-Mäurer Annette , Gentschev Ivaylo , Fries Hans-Werner , Fiedler Franz , Bröcker Eva-Bettina , Kämpgen Eckhart , Goebel Werner
Published: July 2002

Dendritic cells (DCs) are potent antigen-presenting cells and play a crucial role in initiation and modulation of specific immune responses. Various pathogens are able to persist inside DCs. However, internalization of the gram-positive bacteriumListeria monocytogenesinto human DCs has not yet been shown. In the present study, we demonstrate that human monocyte-derived immature DCs can efficiently phagocytoseL. monocytogenes. This uptake is independent of listerial adhesion factors internalin A and internalin B but requires cytoskeletal motion and factors present in human plasma. A major portion of internalized bacteria is found in membrane-bound phagosomes and is rarely free in the cytosol, as shown by transmission electron microscopy and by using anL. monocytogenesstrain expressing green fluorescent protein when in the host cell cytosol. The infection caused maturation of the immature DCs into mature DCs displaying high levels of CD83, CD25, major histocompatibility complex class II, and the CD86 costimulator molecule. This effect appeared to be largely mediated by listerial lipoteichoic acid. AlthoughL. monocytogenesinfection is known to induce death in other cell types, infection of human DCs was found to induce necrotic but not apoptotic death in fewer than 20% of DCs. Therefore, the ability of DCs to act as effective antigen-presenting cells for listerial immunity is probably enhanced by their resistance to cell death, as well as their ability to rapidly differentiate into mature, immunostimulatory DCs upon encountering bacteria.
Haemophilus influenzaePorin Contributes to Signaling of the Inflammatory Cascade in Rat Brain
Authors: Galdiero Massimiliano , D'Amico Michele , Gorga Fernanda , Di Filippo Clara , D'Isanto Marina , Vitiello Mariateresa , Longanella Anna , Tortora Annalisa
Published: July 2002

In the present study we observed that theHaemophilus influenzaetype b (Hib) porin, among the different surface bacterial components, is involved in the pathophysiology of bacterial meningitis. This study demonstrates that inoculation of Hib porin into the fourth cerebral ventricle causes the simultaneous expression of interleukin-1α (IL-1α), tumor necrosis factor alpha (TNF-α), and macrophage inflammatory protein 2 (MIP-2) at 6 h after inoculation. At 24 h, the expression of MIP-2 decreases while the expression of IL-1α and TNF-α increases. The mRNA expression of IL-1α, TNF-α, and MIP-2 is correlated with injury to the blood-brain barrier as demonstrated by the appearance of serum proteins and leukocytes in cerebrospinal fluid and by the increase in brain water content.