bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Localization and Molecular Characterization of the
nonS
Gene Required for Macrotetrolide Biosynthesis in
Streptomyces griseus
DSM40695
Authors: Smith Wyatt C. , Xiang Longkuan , Shen Ben
Published: July 2002

The macrotetrolides are a family of cyclic polyethers derived from tetramerization, in a stereospecific fashion, of the enantiomeric nonactic acid (NA) and its homologs. Isotope labeling experiments established that NA is of polyketide origin, and biochemical investigations demonstrated that 2-methyl-6,8-dihydroxynon-2
E
-enoic acid can be converted into NA by a cell-free preparation from
Streptomyces lividans
that expresses
nonS
. These results lead to the hypothesis that macrotetrolide biosynthesis involves a pair of enantiospecific polyketide pathways. In this work, a 55-kb contiguous DNA region was cloned from
Streptomyces griseus
DSM40695, a 6.3-kb fragment of which was sequenced to reveal five open reading frames, including the previously reported
nonR
and
nonS
genes. Inactivation of
nonS
in vivo completely abolished macrotetrolide production. Complementation of the
nonS
mutant by the expression of
nonS
in
trans
fully restored its macrotetrolide production ability, with a distribution of individual macrotetrolides similar to that for the wild-type producer. In contrast, fermentation of the
nonS
mutant in the presence of exogenous (±)-NA resulted in the production of nonactin, monactin, and dinactin but not in the production of trinactin and tetranactin. These results prove the direct involvement of
nonS
in macrotetrolide biosynthesis. The difference in macrotetrolide production between in vivo complementation of the
nonS
mutant by the plasmid-borne
nonS
gene and fermentation of the
nonS
mutant in the presence of exogenously added (±)-NA suggests that NonS catalyzes the formation of (−)-NA and its homologs, supporting the existence of a pair of enantiospecific polyketide pathways for macrotetrolide biosynthesis in
S. griseus
. The latter should provide a model that can be used to study the mechanism by which polyketide synthase controls stereochemistry during polyketide biosynthesis.
In Vitro Antimicrobial Activities of Novel Anilinouracils Which Selectively Inhibit DNA Polymerase III of Gram-Positive Bacteria
Authors: Daly Jennifer S. , Giehl Theodore J. , Brown Neal C. , Zhi Chengxin , Wright George E. , Ellison Richard T.
Published: July 2002

The 6-anilinouracils are novel dGTP analogs that selectively inhibit the replication-specific DNA polymerase III of gram-positive eubacteria. Two specific derivatives, IMAU (6-[3′-iodo-4′-methylanilino]uracil) and EMAU (6-[3′-ethyl-4′-methylanilino]uracil), were substituted with either a hydroxybutyl (HB) or a methoxybutyl (MB) group at their N3 positions to produce four agents: HB-EMAU, MB-EMAU, HB-IMAU, and MB-IMAU. These four new agents inhibited
Staphylococcus aureus
, coagulase-negative staphylococci,
Enterococcus faecalis
, and
Enterococcus faecium
. Time-kill assays and broth dilution testing confirmed bactericidal activity. These anilinouracil derivatives represent a novel class of antimicrobials with promising activities against gram-positive bacteria that are resistant to currently available agents, validating replication-specific DNA polymerase III as a new target for antimicrobial development.
A Spectrum of Changes Occurs in Peptidoglycan Composition of Glycopeptide-Intermediate Clinical
Staphylococcus aureus
Isolates
Authors: Boyle-Vavra Susan , Labischinski Harald , Ebert Christine C. , Ehlert Kerstin , Daum Robert S.
Published: July 2002

The mechanism of glycopeptide resistance in
Staphylococcus aureus
is not known with certainty. Because the target of vancomycin is the
d
-Ala–
d
-Ala terminus of the stem peptide of the peptidoglycan precursor, by subjecting muropeptides to reversed-phase high-performance liquid chromatography, we investigated peptidoglycan obtained from glycopeptide-intermediate
S. aureus
(GISA) isolates for changes in composition and evaluated whether any peptidoglycan structural change was a consistent feature of clinical GISA isolates. GISA isolates Mu50 and Mu3 from Japan had the large glutamate-containing monomeric peak demonstrated previously, although strain H1, a vancomycin-susceptible MRSA isolate from Japan that was clonally related to Mu3 and Mu50, and a
femC
mutant that we studied, did also. For the U.S. GISA isolates, strain NJ had a large monomeric peak with a retention time identical to that described for the glutamate-containing monomer in strains H1, Mu3, and Mu50. However, a much smaller corresponding peak was seen in GISA MI, and this peak was absent from both GISA PC and a recent GISA isolate obtained from an adult patient in Illinois (strain IL). These data suggest that a uniform alteration in peptidoglycan composition cannot be discerned among the GISA isolates and indicate that a single genetic or biochemical change is unlikely to account for the glycopeptide resistance phenotype in the clinical GISA isolates observed to date. Furthermore, a large monomeric glutamate-containing peak is not sufficient to confer the resistance phenotype.
In Vitro Activities of Posaconazole (Sch 56592) Compared with Those of Itraconazole and Fluconazole against 3,685 Clinical Isolates of
Candida
spp. and
Cryptococcus neoformans
Authors: Pfaller M. A. , Messer S. A. , Hollis R. J. , Jones R. N.
Published: July 2002

Posaconazole is a new investigational triazole with broad-spectrum antifungal activity. The in vitro activities of posaconazole were compared with those of itraconazole and fluconazole against 3,685 isolates of
Candida
spp. (3,312 isolates) and
C. neoformans
(373 isolates) obtained from over 70 different medical centers worldwide. The MICs of the antifungal drugs were determined by broth microdilution tests performed according to the National Committee for Clinical Laboratory Standards method using RPMI 1640 as the test medium. Posaconazole was very active against all
Candida
spp. (MIC at which 90% of the isolates were inhibited [MIC
90
], 0.5 μg/ml; 97% of MICs were ≤1 μg/ml) and
C. neoformans
(MIC
90
, 0.5 μg/ml; 100% of MICs were ≤1 μg/ml).
Candida albicans
was the most susceptible species of
Candida
(MIC
90
, 0.06 μg/ml), and
Candida glabrata
was the least susceptible (MIC
90
, 4 μg/ml). Posaconazole was more active than itraconazole and fluconazole against all
Candida
spp. and
C. neoformans
. These results provide further evidence for the spectrum and potency of posaconazole against a large and geographically diverse collection of clinically important fungal pathogens.
Two New Point Mutations at A2062 Associated with Resistance to 16-Membered Macrolide Antibiotics in Mutant Strains of
Mycoplasma hominis
Authors: Furneri Pio Maria , Rappazzo Giancarlo , Musumarra Maria Pia , Di Pietro Patrizia , Catania Lucrezia S. , Roccasalva Lucia Silvana
Published: July 2002

We describe two mutants of
Mycoplasma hominis
PG-21 which show resistance to 16-membered macrolides but susceptibility to lincosamides, obtained by in vitro exposure to increasing doses of josamycin. The 23S rRNA gene showed that each had a mutation (A2062G and A2062T) corresponding to nucleotide 2062 in
Escherichia coli
, which was associated with the acquired phenotype.
Two-Year Surveillance of Antibiotic Resistance in
Streptococcus pneumoniae
in Four African Cities
Authors: Benbachir Mohamed , Benredjeb Saida , Boye Cheick Saadbouh , Dosso Mireille , Belabbes Houria , Kamoun Aouatef , Kaire Omar , Elmdaghri Naima
Published: July 2002

Worldwide spread of antibiotic resistance in
Streptococcus pneumoniae
is a major problem. However, data from West and North African countries are scarce. To study the level of resistance and compare the situations in different cities, a prospective study was conducted in Abidjan (Ivory Coast), Casablanca (Morocco), Dakar (Senegal), and Tunis (Tunisia), from 1996 to 1997. The resistances to eight antibiotics of 375 isolates were studied by
E
test, and the results were interpreted using the breakpoints recommended by the National Committee for Clinical Laboratory Standards. Overall, 30.4% of the isolates were nonsusceptible to penicillin G (25.6% were intermediate and 4.8% were resistant). Amoxicillin (96.3% were susceptible) and parenteral third-generation cephalosporins (92.7%) were highly active. Resistance to chloramphenicol was detected in 8.6% of the isolates. High levels of resistance were noted for erythromycin (28%), tetracycline (38.3%), and cotrimoxazole (36.4%). Resistance to rifampin was rare (2.1%). There were significant differences in resistance rates between individual countries. Multiple resistance was more frequent in penicillin-nonsusceptible isolates than in penicillin-susceptible isolates. Recommendations for treatment could be generated from these results in each participating country.
Penicillin Pharmacodynamics in Four Experimental Pneumococcal Infection Models
Authors: Erlendsdottir Helga , Knudsen Jenny Dahl , Odenholt Inga , Cars Otto , Espersen Frank , Frimodt-Møller Niels , Fuursted Kurt , Kristinsson Karl G. , Gudmundsson Sigurdur
Published: July 2002

Clinical and animal studies indicate that with optimal dosing, penicillin may still be effective against penicillin-nonsusceptible pneumococci (PNSP). The present study examined whether the same strains of penicillin-susceptible pneumococci (PSP) and PNSP differed in their pharmacodynamic responses to penicillin by using comparable penicillin dosing regimens in four animal models: peritonitis, pneumonia, and thigh infection in mice and tissue cage infection in rabbits. Two multidrug-resistant isolates of
Streptococcus pneumoniae
type 6B were used, one for which the penicillin MIC was 0.016 μg/ml and the other for which the penicillin MIC was 1.0 μg/ml. Two additional strains of PNSP were studied in the rabbit. The animals were treated with five different penicillin regimens resulting in different maximum concentrations of drugs in serum (
C
max
s) and times that the concentrations were greater than the MIC (
T
>MIC
s). The endpoints were bacterial viability counts after 6 h of treatment in the mice and 24 h of treatment in the rabbits. Similar pharmacodynamic effects were observed in all models. In the mouse models bactericidal activity depended on the
T
>MIC
and to a lesser extent on the
C
max
/MIC and the generation time but not on the area under the concentration-time curve (AUC)/MIC. Maximal bactericidal activities were similar for both PSP and PNSP, being the highest in the peritoneum and blood (∼6 log
10
CFU/ml), followed by the thigh (∼3 log
10
CFU/thigh), and being the lowest in the lung (∼1 log
10
CFU/lung). In the rabbit model the maximal effect was ∼6 log
10
CFU/ml after 24 h. In the mouse models bactericidal activity became marked when
T
>MIC
was ≥65% of the experimental time and
C
max
was ≥15 times the MIC, and in the rabbit model bactericidal activity became marked when
T
>MIC
was ≥35%,
C
max
was ≥5 times the MIC, and the AUC at 24 h/MIC exceeded 25. By optimization of the
C
max
/MIC ratio and
T
>MIC
, the MIC of penicillin for pneumococci can be used to guide therapy and maximize therapeutic efficacy in nonmeningeal infections caused by PNSP.
Ganciclovir and Cidofovir Treatment of Cytomegalovirus-Induced Myocarditis in Mice
Authors: Lenzo Jason C. , Shellam Geoffrey R. , Lawson Cassandra M.
Published: July 2002

The cardiovascular disease myocarditis is characterized by inflammation and necrosis of cardiac muscle. This disease has been associated with various viral etiologies, including cytomegalovirus (CMV). Murine CMV (MCMV) infection of adult BALB/c mice produces a disease with acute and chronic phases similar to that found in humans. In our murine model, we have investigated the therapeutic efficacy of antiviral drug administration on myocarditis. Two drugs commonly used for CMV treatment, ganciclovir and cidofovir, were subjected to trials, with both drugs showing potent antiviral activity against MCMV both in vitro and in vivo. The acute phase of myocarditis was significantly reduced when antiviral therapy commenced 24 h postinfection. Such treatment also reduced the severity of the chronic phase of myocarditis. In contrast, antiviral treatment commencing after the acute phase had no effect on chronic myocarditis. Reinfection of mice with MCMV caused exacerbation of myocardial inflammation. Such an increase in severity of myocarditis could be prevented with either ganciclovir or cidofovir treatment, but the preexisting inflammation and necrosis of the myocardium persisted. These data highlight possible therapeutic uses of antiviral drugs in viral myocarditis as well as further elucidating the pathogenic nature of the disease.
Absence of Mutations in
marRAB
or
soxRS
in
acrB
-Overexpressing Fluoroquinolone-Resistant Clinical and Veterinary Isolates of
Escherichia coli
Authors: Webber Mark A. , Piddock Laura J. V.
Published: July 2002

The amount of
acrB, marA
, and
soxS
mRNA was determined in 36 fluoroquinolone-resistant
E. coli
from humans and animals, 27 of which displayed a multiple-resistance phenotype.
acrB
mRNA was elevated in 11 of 36 strains. A mutation at codon 45 (Arg→Cys) in
acrR
was found in 6 of these 11 strains. Ten of the 36 isolates appeared to overexpress
soxS
, and five appeared to overexpress
marA
. A number of mutations were found in the
marR
and
soxR
repressor genes, correlating with greater amounts of
marA
and
soxS
mRNA, respectively.
Novel Bifunctional Inhibitor of Xylanase and Aspartic Protease: Implications for Inhibition of Fungal Growth
Authors: Dash Chandravanu , Ahmad Absar , Nath Devyani , Rao Mala
Published: July 2002

A novel bifunctional inhibitor (ATBI) from an extremophilicBacillussp. exhibiting an activity against phytopathogenic fungi, includingAlternaria, Aspergillus, Curvularia, Colletotricum, Fusarium, andPhomopsisspecies, and the saprophytic fungusTrichodermasp. has been investigated. The 50% inhibitory concentrations of ATBI ranged from 0.30 to 5.9 μg/ml, whereas the MIC varied from 0.60 to 3.5 μg/ml for the fungal growth inhibition. The negative charge and the absence of periodic secondary structure in ATBI suggested an alternative mechanism for fungal growth inhibition. Rescue of fungal growth inhibition by the hydrolytic products of xylanase and aspartic protease indicated the involvement of these enzymes in cellular growth. The chemical modification of Asp or Glu or Lys residues of ATBI by 2,4,6-trinitrobenzenesulfonic acid and Woodward's reagent K, respectively, abolished its antifungal activity. In addition, ATBI also inhibited xylanase and aspartic protease competitively, withKivalues 1.75 and 3.25 μM, respectively. Our discovery led us to envisage a paradigm shift in the concept of fungal growth inhibition for the role of antixylanolytic activity. Here we report for the first time a novel class of antifungal peptide, exhibiting bifunctional inhibitory activity.
Role of Penicillin-Binding Protein 5 in Expression of Ampicillin Resistance and Peptidoglycan Structure in
Enterococcus faecium
Authors: Sifaoui Farid , Arthur Michel , Rice Louis , Gutmann Laurent
Published: July 2002

The contribution of penicillin-binding protein 5 (PBP 5) to intrinsic and acquired β-lactam resistance was investigated by constructing isogenic strains of
Enterococcus faecium
producing different PBP 5. The
pbp5
genes from three
E. faecium
clinical isolates (BM4107, D344, and
H80721
) were cloned into the shuttle vector pAT392 and introduced into
E. faecium
D344S, a spontaneous derivative of
E. faecium
D344 highly susceptible to ampicillin due to deletion of
pbp5
(MIC, 0.03 μg/ml). Immunodetection of PBP5 indicated that cloning of the
pbp5
genes into pAT392 resulted in moderate overproduction of PBP 5 in comparison to wild-type strains. This difference may be attributed to a difference in gene copy number. Expression of the
pbp5
genes from BM4107 (MIC, 2 μg/ml), D344 (MIC, 24 μg/ml), and
H80721
(MIC, 512 μg/ml) in D344S conferred relatively low levels of resistance to ampicillin (MICs, 6, 12, and 20 μg/ml, respectively). A methionine-to-alanine substitution was introduced at position 485 of the BM4107 PBP 5 by site-directed mutagenesis. In contrast to previous hypotheses based on comparison of nonisogenic strains, this substitution resulted in only a 2.5-fold increase in the ampicillin MIC. The reversed-phase high-performance liquid chromatography muropeptide profiles of D344 and D344S were similar, indicating that deletion of
pbp5
was not associated with a detectable defect in cell wall synthesis. These results indicate that
pbp5
is a nonessential gene responsible for intrinsic resistance to moderate levels of ampicillin and by itself cannot confer high-level resistance.
In Vitro Activities of New and Conventional Antifungal Agents against ClinicalScedosporiumIsolates
Authors: Meletiadis Joseph , Meis Jacques F. G. M. , Mouton Johan W. , Rodriquez-Tudela Juan Luis , Donnelly J. Peter , Verweij Paul E. , the EUROFUNG Network
Published: July 2002

The susceptibilities of 13 clinical isolates ofScedosporium apiospermumand 55 clinical isolates ofS. prolificansto new and conventional drugs belonging to three different classes of antifungal agents, the azoles (miconazole, itraconazole, voriconazole, UR-9825, posaconazole), the polyenes (amphotericin B, nystatin and liposomal nystatin), and allylamines (terbinafine), were studied by use of proposed standard M38-P of NCCLS. Low growth-inhibitory antifungal activities were found in vitro for most of the drugs tested againstS. prolificansisolates, with the MICs at which 90% of isolates are inhibited (MIC90s) being >8 μg/ml; the MIC90s of voriconazole and UR-9825, however, were 4 μg/ml.S. apiospermumisolates were more susceptible in vitro, with the highest activity exhibited by voriconazole (MIC90s, 0.5 μg/ml), followed by miconazole (MIC90s, 1 μg/ml), UR-9825 and posaconazole (MIC90s, 2 μg/ml), and itraconazole (MIC90s, 4 μg/ml). The MICs of terbinafine, amphotericin B, and the two formulations of nystatin (for which no statistically significant differences in antifungal activities were found for the two species) forS. apiospermumisolates were high. Cross-resistance was observed among all the azoles except posaconazole and among all the polyenes except the lipid formulation. A distribution analysis was performed with the MICs of each drug and for each species. Bimodal and skewed MIC distributions were obtained, and cutoffs indicating the borders of different MIC subpopulations of the distributions were determined on the basis of the normal plot technique. These cutoffs were in many cases reproducible between 48 and 72 h.
Potent Efficacy of Entecavir (BMS-200475) in a Duck Model of Hepatitis B Virus Replication
Authors: Marion Patricia L. , Salazar Felix H. , Winters Mark A. , Colonno Richard J.
Published: July 2002

The ability of entecavir (ETV) to inhibit
Duck hepatitis B virus
(DHBV) infection in duck hepatocytes and ducklings was examined using lamivudine (3TC) as a comparator drug. ETV exhibited antiviral activity (50% effective concentration [EC
50
], 0.13 nM) in DHBV-infected duck hepatocytes that was >1,000-fold more potent than that of 3TC (EC
50
, 138 nM). A 21-day treatment of ducklings with 1 mg of ETV per kg of body weight per day by oral gavage resulted in a mean reduction of log
10
3.1 in serum DHBV DNA levels. Daily treatment with 0.1 mg of ETV/kg was nearly as effective, achieving an average viral DNA level decrease of log
10
2.1. Reducing the daily dose of ETV to only 0.01 mg/kg resulted in an average viral DNA level decrease of log
10
0.97. Daily treatment with 25 mg of 3TC/kg resulted in an average viral DNA level decrease of log
10
0.66, compared to the log
10
0.20 drop seen for ducklings given the vehicle alone. ETV was also more effective in decreasing the DHBV DNA levels in duck livers after 21 days of treatment, causing average drops of log
10
1.41, log
10
0.76, and log
10
0.26 for dose levels of 1.0, 0.1, and 0.01 mg/kg, respectively, compared to a decrease of log
10
0.06 for 3TC at a dose level of 25 mg/kg. Levels of viral covalently closed circular DNA in the treatment group receiving 1 mg of ETV/kg were reduced compared to those in the vehicle-treated group. ETV and 3TC were both well tolerated in all treated animals. These results show that ETV is a highly potent and effective antiviral in the DHBV duck model.
Grepafloxacin, a Dimethyl Derivative of Ciprofloxacin, Acts Preferentially through Gyrase in
Streptococcus pneumoniae
: Role of the C-5 Group in Target Specificity
Authors: Morris Julia E. , Pan Xiao-Su , Fisher L. Mark
Published: July 2002

Grepafloxacin, a 5-
methyl
-7-piperazinyl-3"-
methyl
analogue of ciprofloxacin, was used to obtain stepwise-selected mutants of
Streptococcus pneumoniae
7785. Analysis of the quinolone resistance-determining regions of the
gyrA
,
gyrB
,
parC
, and
parE
genes in these mutants revealed that
gyrA
mutations preceded those in
parC
. Given that ciprofloxacin (5-
H
,7-piperazinyl) and AM-1121 (5-
H
,7-piperazinyl-3"-
methyl
) both act through topoisomerase IV, we conclude that the 5-
methyl
group of grepafloxacin favors gyrase in
S. pneumoniae
.
Doxycycline Induces Expression of P Glycoprotein in MCF-7 Breast Carcinoma Cells
Authors: Mealey Katrina L. , Barhoumi Rola , Burghardt Robert C. , Safe Stephen , Kochevar Deborah T.
Published: July 2002

P-glycoprotein (P-gp) overexpression by tumor cells imparts resistance to multiple antineoplastic chemotherapeutic agents (multiple drug resistance). Treatment of tumor cells with chemotherapeutic agents such as anthracyclines, epipodophyllotoxins, and
Vinca
alkaloids results in induction of P-gp expression. This study was performed to determine if clinically relevant antimicrobial drugs (i.e., drugs that are used to treat bacterial infections in cancer patients) other than antineoplastic agents can induce expression of P-gp in MCF-7 breast carcinoma cells. Expression of P-gp and
MDR1
mRNA was determined in samples from MCF-7 cells that were treated in culture with doxorubicin (positive control) and the antimicrobial drugs doxycycline, piperacillin, and cefoperazone. The functional status of P-gp was assessed using laser cytometry to determine intracellular doxorubicin concentrations. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was used to determine if the cytotoxicity of experimental drugs was related to their ability to induce P-gp expression. MCF-7 cells treated with doxycycline (MCF-7/doxy) were stimulated to overexpress P-gp, whereas cells treated with piperacillin and cefoperazone did not overexpress P-gp. MCF-7/doxy cells were compared to a positive-control subline, MCF-7/Adr, previously selected for doxorubicin resistance, and to MCF-7 cells treated with doxorubicin (MCF-7/doxo). All three sublines overexpressed P-gp and
MDR1
mRNA and accumulated less intracellular doxorubicin than did control MCF-7 cells. P-gp expression was induced only by experimental drugs that were cytotoxic (doxorubicin and doxycycline). Doxycycline, a drug that has been used for treatment of bacterial infections in cancer patients, can induce functional P-gp expression in cancer cells, resulting in multidrug resistance.
Expression of Efflux Pump Gene
pmrA
in Fluoroquinolone-Resistant and -Susceptible Clinical Isolates of
Streptococcus pneumoniae
Authors: Piddock Laura J. V. , Johnson Maggie M. , Simjee S. , Pumbwe L.
Published: July 2002

Thirty-four ciprofloxacin-resistant (MIC ≥ 2 μg/ml) and 12 ciprofloxacin-susceptible clinical isolates of
Streptococcus pneumoniae
were divided into four groups based upon susceptibility to norfloxacin and the effect of reserpine (20 μg/ml). The quinolone-resistance-determining regions of
parC
,
parE
,
gyrA
, and
gyrB
of all ciprofloxacin-resistant clinical isolates were sequenced, and the activities of eight other fluoroquinolones, acriflavine, ethidium bromide, chloramphenicol, and tetracycline in the presence and absence of reserpine were determined. Despite a marked effect of reserpine upon the activity of norfloxacin, there were only a few isolates for which the activity of another fluoroquinolone was enhanced by reserpine. For most isolates the MICs of acriflavine and ethidium bromide were lowered in the presence of reserpine despite the lack of effect of this efflux pump inhibitor on fluoroquinolone activity. The strains that were most resistant to the fluoroquinolones were predominantly those with mutations in three genes. Expression of the gene encoding the efflux pump PmrA was examined by Northern blotting (quantified by quantitative competitive reverse transcriptase PCR) and compared with that of
S
.
pneumoniae
R6 and R6N. Within each group there were isolates that had high-, medium-, and low-level expression of this gene; however, increased expression was not exclusively associated with those isolates with a phenotype suggestive of an efflux mutant. These data suggest that there is another reserpine-sensitive efflux pump in
S
.
pneumoniae
that extrudes ethidium bromide and acriflavine but not fluoroquinolones.
Human Immunodeficiency Virus (HIV) Type 1 Reverse Transcriptase Resistance Mutations in Hepatitis B Virus (HBV)-HIV-Coinfected Patients Treated for HBV Chronic Infection Once Daily with 10 Milligrams of Adefovir Dipivoxil Combined with Lamivudine
Authors: Delaugerre Constance , Marcelin Anne-Geneviève , Thibault Vincent , Peytavin Gilles , Bombled Tony , Bochet Marie-Valérie , Katlama Christine , Benhamou Yves , Calvez Vincent
Published: July 2002

Adefovir dipivoxil (ADV) at a suboptimal concentration for human immunodeficiency virus type 1 (HIV-1) (10 mg once daily) can be used to treat hepatitis B virus (HBV) infection in HIV-1-HBV-coinfected patients and does not, even in the case of uncontrolled HIV-1 replication, select for either ADV mutations at codons 65 and 70 or any other particular HIV-1 reverse transcriptase resistance profile.
Identification of Major Glucan-Associated Cell Wall Proteins of
Candida albicans
and Their Role in Fluconazole Resistance
Authors: Angiolella Letizia , Micocci Mia M. , D'Alessio Simona , Girolamo Antonietta , Maras Bruno , Cassone Antonio
Published: July 2002

Identification of major glucan-associated proteins (GAPs) of the cell wall of a number of
Candida albicans
isolates susceptible or resistant to fluconazole (FLC) was addressed by direct sequencing of the protein bands resolved by unidimensional gel electrophoresis. Changes in the GAP compositions of the different strains grown in the presence of the drug were also investigated. In the FLC-susceptible strains, the major (more abundant) GAPs were enolase (46 kDa), two isoforms of phosphoglyceromutase (32 and 29 kDa), and two β-(1-3)-exoglucanases (44 and 34 kDa), one of which (the 34-kDa component) was glycosylated. When these strains were grown in the presence of FLC there were substantial decreases in the intensities of the two enzymes of the glycolytic pathway (enolase and the phosphoglyceromutases), which were apparently replaced by enhancement of the exoglucanase constituents, particularly the 44-kDa one. This GAP pattern closely mimicked that observed in the FLC-resistant strains whether they were grown in the presence or in the absence of the drug. Both the enolase and the exoglucanase constituents were detected in the culture supernatants of FLC-treated cells, together with substantial amounts of highly glycosylated, probably mannoprotein secretory material, suggesting that FLC may cause marked alterations of GAP incorporation into the cell wall. Altogether, we were able to identify all major GAP constituents and monitor their distributions in the cell wall of
C. albicans
during treatment with FLC. The near equivalence of the GAP profile for the FLC-susceptible strain grown in the presence of FLC to that for the FLC-resistant strain suggests that the effects of the drug on GAPs may be stably incorporated into the cell wall of the fungus upon acquisition of resistance.
Concentration and Detection of Caliciviruses in Water Samples by Reverse Transcription-PCR
Authors: Huang P. W. , Laborde D. , Land V. R. , Matson D. O. , Smith A. W. , Jiang X.
Published: July 2002

Human caliciviruses (HuCVs) cause waterborne outbreaks of gastroenteritis. Standard indicators of a safe water supply do not adequately predict contamination of water by viruses, including HuCVs. We developed a method to concentrate and detect HuCVs in water samples by using a cultivable primate calicivirus (Pan-1) as a model. Viable Pan-1 was seeded in different types of water and then filtered with a 1MDS filter, eluted with beef extract (BE), and reconcentrated by polyethylene glycol (PEG) precipitation. The viruses in the final samples were tested by plaque assay or by reverse transcription (RT)-PCR following extraction of the RNA with Trizol. Pan-1 was more sensitive to high-pH treatment than poliovirus was; a pH 9.0 BE solution was found to recover 35% more viable Pan-1 than a pH 9.5 BE solution recovered. Pan-1 was recovered from small volumes of deionized, finished, ground, and surface waters at efficiencies of 94, 73, 67, and 64%, respectively, when samples were assayed after elution without further concentration. When larger volumes of water (up to 40 liters) were tested after elution and concentration with PEG, 38, 19, and 14% of the seeded Pan-1 were recovered from finished, ground, and surface waters, respectively. The limit of detection of Pan-1 by RT-PCR was estimated to be 0.75 to 1.5 PFU in 40 liters of finished water. This method may be adapted for monitoring HuCVs in drinking water and other types of water for public health safety.
Phenylacetyl-Coenzyme A Is the True Inducer of the Phenylacetic Acid Catabolism Pathway in
Pseudomonas putida
U
Authors: García Belén , Olivera Elías R. , Miñambres Baltasar , Carnicero David , Muñiz Carmen , Naharro Germán , Luengo José M.
Published: July 2002

Aerobic degradation of phenylacetic acid in
Pseudomonas putida
U is carried out by a central catabolism pathway (phenylacetyl-coenzyme A [CoA] catabolon core). Induction of this route was analyzed by using different mutants specifically designed for this objective. Our results revealed that the true inducer molecule is phenylacetyl-CoA and not other structurally or catabolically related aromatic compounds.
Photosynthetic Bradyrhizobia Are Natural Endophytes of the African Wild Rice
Oryza breviligulata
Authors: Chaintreuil Clémence , Giraud Eric , Prin Yves , Lorquin Jean , Bâ Amadou , Gillis Monique , de Lajudie Philippe , Dreyfus Bernard
Published: July 2002

We investigated the presence of endophytic rhizobia within the roots of the wetland wild rice
Oryza breviligulata
, which is the ancestor of the African cultivated rice
Oryza glaberrima
. This primitive rice species grows in the same wetland sites as
Aeschynomene sensitiva
, an aquatic stem-nodulated legume associated with photosynthetic strains of
Bradyrhizobium
. Twenty endophytic and aquatic isolates were obtained at three different sites in West Africa (Senegal and Guinea) from nodal roots of
O. breviligulata
and surrounding water by using
A. sensitiva
as a trap legume. Most endophytic and aquatic isolates were photosynthetic and belonged to the same phylogenetic
Bradyrhizobium/Blastobacter
subgroup as the typical photosynthetic
Bradyrhizobium
strains previously isolated from
Aeschynomene
stem nodules. Nitrogen-fixing activity, measured by acetylene reduction, was detected in rice plants inoculated with endophytic isolates. A 20% increase in the shoot growth and grain yield of
O. breviligulata
grown in a greenhouse was also observed upon inoculation with one endophytic strain and one
Aeschynomene
photosynthetic strain. The photosynthetic
Bradyrhizobium
sp. strain ORS278 extensively colonized the root surface, followed by intercellular, and rarely intracellular, bacterial invasion of the rice roots, which was determined with a
lacZ
-tagged mutant of ORS278. The discovery that photosynthetic
Bradyrhizobium
strains, which are usually known to induce nitrogen-fixing nodules on stems of the legume
Aeschynomene
, are also natural true endophytes of the primitive rice
O. breviligulata
could significantly enhance cultivated rice production.
Improved Direct Viable Count Procedure for Quantitative Estimation of Bacterial Viability in Freshwater Environments
Authors: Yokomaku Daisaku , Yamaguchi Nobuyasu , Nasu Masao
Published: July 2002

A direct viable count (DVC) procedure was developed which clearly and easily discriminates the viability of bacterial cells. In this quantitative DVC (qDVC) procedure, viable cells are selectively lysed by spheroplast formation caused by incubation with antibiotics and glycine. This glycine effect leads to swollen cells with a very loose cell wall. The viable cells then are lysed easily by a single freeze-thaw treatment. The number of viable cells was obtained by subtracting the number of remaining cells after the qDVC procedure from the total cell number before the qDVC incubation. This improved procedure should provide useful information about the metabolic potential of natural bacterial communities.
Interspecific Transfer of
Streptomyces
Giant Linear Plasmids in Sterile Amended Soil Microcosms
Authors: Ravel Jacques , Wellington Elizabeth M. H. , Hill Russell T.
Published: July 2002

The interspecific transfer of two giant linear plasmids was investigated in sterile soil microcosms. Plasmids pRJ3L (322 kb) and pRJ28 (330 kb), both encoding mercury resistance, were successfully transferred in amended soil microcosms from their streptomycete hosts, the isolates CHR3 and CHR28, respectively, to a plasmidless and mercury-sensitive strain,
Streptomyces lividans
TK24. Transconjugants of
S. lividans
TK24 were first observed after 2 to 3 days of incubation at 30°C, which corresponded to the time taken for the formation of mycelia in soil. Transfer frequencies were 4.8 × 10
−4
and 3.6 × 10
−5
CFU/donor genome for pRJ3L and pRJ28, respectively. Transconjugants were analyzed by pulsed-field gel electrophoresis for the presence of plasmids, and plasmid identity was confirmed by restriction digests. Total genomic DNA digests confirmed that transconjugants were
S. lividans
TK24. The mercury resistance genes were shown to be on the plasmid in the transconjugants by hybridization analysis and were still functional. This is the first demonstration of transfer of giant linear plasmids in sterile soil microcosms. Giant linear plasmids were detected in many
Streptomyces
spp. isolated from mercury-contaminated sediments from Boston Harbor (United States), Townsville Harbor (Australia), and the Sali River (Tucuman, Argentina). Mercury resistance genes were shown to be present on some of these plasmids. Our findings that giant linear plasmids can be transferred between
Streptomyces
spp. and are common in environmental
Streptomyces
isolates suggest that these plasmids are important in gene transfer between streptomycetes in the environment.
Biocatalytic Synthesis of Vanillin
Authors: Li Tao , Rosazza John P. N.
Published: July 2002

The conversions of vanillic acid and
O
-benzylvanillic acid to vanillin were examined by using whole cells and enzyme preparations of
Nocardia
sp. strain NRRL 5646. With growing cultures, vanillic acid was decarboxylated (69% yield) to guaiacol and reduced (11% yield) to vanillyl alcohol. In resting
Nocardia
cells in buffer, 4-
O
-benzylvanillic acid was converted to the corresponding alcohol product without decarboxylation. Purified
Nocardia
carboxylic acid reductase, an ATP and NADPH-dependent enzyme, quantitatively reduced vanillic acid to vanillin. Structures of metabolites were established by
1
H nuclear magnetic resonance and mass spectral analyses.
Selective Inhibition of the Oxidation of Ferrous Iron or Sulfur in
Thiobacillus ferrooxidans
Authors: Harahuc Lesia , Lizama Hector M. , Suzuki Isamu
Published: July 2002

The oxidation of either ferrous iron or sulfur by
Thiobacillus ferrooxidans
was selectively inhibited or controlled by various anions, inhibitors, and osmotic pressure. Iron oxidation was more sensitive than sulfur oxidation to inhibition by chloride, phosphate, and nitrate at low concentrations (below 0.1 M) and also to inhibition by azide and cyanide. Sulfur oxidation was more sensitive than iron oxidation to the inhibitory effect of high osmotic pressure. These differences were evident not only between iron oxidation by iron-grown cells and sulfur oxidation by sulfur-grown cells but also between the iron and sulfur oxidation activities of the same iron-grown cells. Growth experiments with ferrous iron or sulfur as an oxidizable substrate confirmed the higher sensitivity of iron oxidation to inhibition by phosphate, chloride, azide, and cyanide. Sulfur oxidation was actually stimulated by 50 mM phosphate or chloride. Leaching of Fe and Zn from pyrite (FeS
2
) and sphalerite (ZnS) by
T. ferrooxidans
was differentially affected by phosphate and chloride, which inhibited the solubilization of Fe without significantly affecting the solubilization of Zn.
Highly Ordered Vertical Structure of
Synechococcus
Populations within the One-Millimeter-Thick Photic Zone of a Hot Spring Cyanobacterial Mat
Authors: Ramsing Niels Birger , Ferris Mike J. , Ward David M.
Published: July 2002

A variety of contemporary techniques were used to investigate the vertical distribution of thermophilic unicellular cyanobacteria,
Synechococcus
spp., and their activity within the upper 1-mm-thick photic zone of the mat community found in an alkaline siliceous hot spring in Yellowstone National Park in Wyoming. Detailed measurements were made over a diel cycle at a 61°C site. Net oxygenic photosynthesis measured with oxygen microelectrodes was highest within the uppermost 100- to 200-μm-thick layer until midmorning, but as the day progressed, the peak of net activity shifted to deeper layers, stabilizing at a depth of 300 μm from midday throughout the afternoon. Examination of vertical thin sections by bright-field and autofluorescence microscopy revealed the existence of different populations of
Synechococcus
which form discrete bands at different vertical positions. Denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA gene segments from horizontal cryosections obtained at 100-μm-thick vertical intervals also suggested vertical stratification of cyanobacterial, green sulfur bacterium-like, and green nonsulfur bacterium-like populations. There was no evidence of diel migration. However, image analysis of vertical thin sections revealed the presence of a narrow band of rod-shaped
Synechococcus
cells in which the cells assumed an upright position. These upright cells, located 400 to 800 μm below the surface, were observed only in mat samples obtained around noon. In mat samples obtained at other time points, the cells were randomly oriented throughout the mat. These combined observations reveal the existence of a highly ordered structure within the very thin photic zone of this hot spring microbial mat, consisting of morphologically similar
Synechococcus
populations that are likely to be differentially adapted, some co-occurring with green sulfur bacterium-like populations, and all overlying green nonsulfur bacterium-like populations.
Significance of Inoculum Size in the Lag Time of
Listeria monocytogenes
Authors: Augustin Jean-Christophe , Brouillaud-Delattre Agnès , Rosso Laurent , Carlier Vincent
Published: July 2002

The lag time of
Listeria monocytogenes
growing under suboptimal conditions (low nutrient concentrations, pH 6, and 6.5°C) was extended when the inoculum was severely stressed by starvation and the inoculum size was very small. Predictive microbiology should deal with bacterial stress and stochastic approaches to improve its value for the agro-food industry.
Prey Range Characterization, Ribotyping, and Diversity of Soil and Rhizosphere
Bdellovibrio
spp. Isolated on Phytopathogenic Bacteria
Authors: Jurkevitch E. , Minz D. , Ramati Barak , Barel Gili
Published: July 2002

Thirty new
Bdellovibrio
strains were isolated from an agricultural soil and from the rhizosphere of plants grown in that soil. Using a combined molecular and culture-based approach, we found that the soil bdellovibrios included subpopulations of organisms that differed from rhizosphere bdellovibrios. Thirteen soil and seven common bean rhizosphere
Bdellovibrio
strains were isolated when
Pseudomonas corrugata
was used as prey; seven and two soil strains were isolated when
Erwinia carotovora
subsp.
carotovora
and
Agrobacterium tumefaciens
, respectively, were used as prey; and one tomato rhizosphere strain was isolated when
A. tumefaciens
was used as prey. In soil and in the rhizosphere, depending on the prey cells used, the concentrations of bdellovibrios were between 3 × 10
2
to 6 × 10
3
and 2.8 × 10
2
to 2.3 × 10
4
PFU g
−1
. A prey range analysis of five soil and rhizosphere
Bdellovibrio
isolates performed with 22 substrate species, most of which were plant-pathogenic and plant growth-enhancing bacteria, revealed unique utilization patterns and differences between closely related prey cells. An approximately 830-bp fragment of the 16S rRNA genes of all of the
Bdellovibrio
strains used was obtained by PCR amplification by using a
Bdellovibrio
-specific primer combination. Soil and common bean rhizosphere strains produced two and one restriction patterns for this PCR product, respectively. The 16S rRNA genes of three soil isolates and three root-associated isolates were sequenced. One soil isolate belonged to the
Bdellovibrio stolpii-Bdellovibrio starrii
clade, while all of the other isolates clustered with
Bdellovibrio bacteriovorus
and formed two distantly related, heterogeneous groups.
Genes Expressed in
Pseudomonas putida
during Colonization of a Plant-Pathogenic Fungus
Authors: Lee Seon-Woo , Cooksey Donald A.
Published: July 2002

In vivo expression technology (IVET) was employed to study colonization of
Phytophthora parasitica
by a biological control bacterium,
Pseudomonas putida
06909, based on a new selection marker. The
pyrB
gene, which encodes aspartate transcarbamoylase, an enzyme used for pyrimidine biosynthesis, was cloned from
P. putida
06909. A
pyrB
-disrupted mutant did not grow in pyrimidine-deficient media unless it was complemented with
pyrBC
′ behind an active promoter. Thirty clones obtained from
P. putida
06909 that were expressed on fungal hyphae but not on culture media were isolated by IVET based on the promoterless transcriptional fusion between
pyrBC
′ and
lacZ
. Nineteen of these clones were induced during late-stage bacterial growth in vitro, while 11 of the clones were expressed only when they were inoculated onto fungal hyphae. Restriction analysis of these 11 clones revealed that there were five unique clones. Sequence analyses of three of the five unique clones showed that the 3′ ends of the clones fused to
pyrB
were similar to genes encoding diacylglycerol kinase (DAGK), bacterial ABC transporters, and outer membrane porins. The sequences of the two other clones were not similar to the sequences of any of the genes in the database used. A LuxR family response regulator was found upstream of DAGK, and a LysR family response regulator was found upstream of the ABC transporter. The location of the inducible promoter of two clones suggested that DAGK and the ABC transporter are induced and may play a role in colonization of the fungus
P. parasitica
by
P. putida
06909.
Molecular Diversity of Plasmids Bearing Genes That Encode Toluene and Xylene Metabolism in
Pseudomonas
Strains Isolated from Different Contaminated Sites in Belarus
Authors: Sentchilo Vladimir S. , Perebituk Alexander N. , Zehnder Alexander J. B. , van der Meer Jan Roelof
Published: July 2002

Twenty different
Pseudomonas
strains utilizing
m
-toluate were isolated from oil-contaminated soil samples near Minsk, Belarus. Seventeen of these isolates carried plasmids ranging in size from 78 to about 200 kb (assigned pSVS plasmids) and encoding the
meta
cleavage pathway for toluene metabolism. Most plasmids were conjugative but of unknown incompatibility groups, except for one, which belonged to the IncP9 group. The organization of the genes for toluene catabolism was determined by restriction analysis and hybridization with
xyl
gene probes of pWW0. The majority of the plasmids carried
xyl
-type genes highly homologous to those of pWW53 and organized in a similar manner (M. T. Gallegos, P. A. Williams, and J. L. Ramos, J. Bacteriol. 179:5024–5029, 1997), with two distinguishable
meta
pathway operons, one upper pathway operon, and three
xylS
-homologous regions. All of these plasmids also possessed large areas of homologous DNA outside the catabolic genes, suggesting a common ancestry. Two other pSVS plasmids carried only one
meta
pathway operon, one upper pathway operon, and one copy each of
xylS
and
xylR
. The backbones of these two plasmids differed greatly from those of the others. Whereas these parts of the plasmids, carrying the
xyl
genes, were mostly conserved between plasmids of each group, the noncatabolic parts had undergone intensive DNA rearrangements. DNA sequencing of specific regions near and within the
xylTE
and
xylA
genes of the pSVS plasmids confirmed the strong homologies to the
xyl
genes of pWW53 and pWW0. However, several recombinations were discovered within the upper pathway operons of the pSVS plasmids and pWW0. The main genetic mechanisms which are thought to have resulted in the present-day configuration of the
xyl
operons are discussed in light of the diversity analysis carried out on the pSVS plasmids.
A New Method To Monitor Airborne Inoculum of the Fungal Plant Pathogens
Mycosphaerella brassicicola
and
Botrytis cinerea
Authors: Kennedy R. , Wakeham A. J. , Byrne K. G. , Meyer U. M. , Dewey F. M.
Published: July 2002

We describe a new microtiter immunospore trapping device (MTIST device) that uses a suction system to directly trap air particulates by impaction in microtiter wells. This device can be used for rapid detection and immunoquantification of ascospores of
Mycosphaerella brassicicola
and conidia of
Botrytis cinerea
by an enzyme-linked immunosorbent assay (ELISA) under controlled environmental conditions. For ascospores of
M. brassicicola
correlation coefficients (
r
2
) of 0.943 and 0.9514 were observed for the number of MTIST device-impacted ascospores per microtiter well and the absorbance values determined by ELISA, respectively. These values were not affected when a mixed fungal spore population was used. There was a relationship between the number of MTIST device-trapped ascospores of
M. brassicicola
per liter of air sampled and the amount of disease expressed on exposed plants of
Brassica oleracea
(Brussels sprouts). Similarly, when the MTIST device was used to trap conidia of
B. cinerea
, a correlation coefficient of 0.8797 was obtained for the absorbance values generated by the ELISA and the observed number of conidia per microtiter well. The relative collection efficiency of the MTIST device in controlled plant growth chambers with limited airflow was 1.7 times greater than the relative collection efficiency of a Burkard 7-day volumetric spore trap for collection of
M. brassicicola
ascospores. The MTIST device can be used to rapidly differentiate, determine, and accurately quantify target organisms in a microflora. The MTIST device is a portable, robust, inexpensive system that can be used to perform multiple tests in a single sampling period, and it should be useful for monitoring airborne particulates and microorganisms in a range of environments.
Roles of Fe Superoxide Dismutase and Catalase in Resistance of
Campylobacter coli
to Freeze-Thaw Stress
Authors: Stead Don , Park Simon F.
Published: July 2002

We demonstrated that oxidative stress plays a role in freeze-thaw-induced killing of
Campylobacter coli
following analysis of mutants deficient in key antioxidant functions. Superoxide anions, but not H
2
O
2
, were formed during the freeze-thaw process. However, a failure to detoxify superoxide anions may lead to spontaneous disproportionation of the radicals to H
2
O
2
.