bluuebunny/crossref_metadata_2025 huggingface dataset:
Diversity of β-Lactam Resistance-Conferring Amino Acid Substitutions in Penicillin-Binding Protein 3 of
Haemophilus influenzae
Authors: Dabernat Henri , Delmas Catherine , Seguy Martine , Pelissier Roseline , Faucon Genevieve , Bennamani Safia , Pasquier Christophe
Published: July 2002

The sequences of the
ftsI
gene, encoding the transpeptidase domain of penicillin binding protein (PBP) 3A and/or PBP 3B, which are involved in septal peptidoglycan synthesis, were determined for 108 clinical strains of
Haemophilus influenzae
with reduced susceptibility to β-lactam antibiotics with or without β-lactamase production and were compared to those of the ampicillin-susceptible Rd strain and ampicillin-susceptible clinical isolates. The sequences have 18 different mutation patterns and were classified into two groups on the basis of amino acid substitutions deduced from the nucleotide sequences located between bp 960 and 1618 of the
ftsI
gene. In group I strains (
n
= 7), His-517 was substituted for Arg-517. In group II strains (
n
= 101), Lys-526 was substituted for Asn-526. In subgroup IIa (
n
= 5;
H. influenzae
ATCC 49247), the only observed substitution was Lys-526 for Asn-526; in subgroup IIb (
n
= 56), Val-502 was substituted for Ala-502 (
n
= 13), along with several other substitutions: Asn-350 for Asp-350 (
n
= 15), Asn-350 for Asp-350 and Glu-490 for Gly-490 (
n
= 14), and Asn-350 for Asp-350 and Ser-437 for Ala-437 (
n
= 5). In subgroup IIc (
n
= 25), Thr-502 was substituted for Ala-502. In subgroup IId, Val-449 was substituted for Ile-449 (
n
= 15). The MICs of β-lactam antibiotics for the 108 strains were to 8 to 16 times the MICs for susceptible strains. The strains, isolated from both adults and children, were analyzed for genetic relationship by pulsed-field gel electrophoresis and by determination of
ftsI
sequence phylogeny. Both analyses revealed the lack of clonality and the heterogeneity of the strains, but some clusters suggest the spread and/or persistence of a limited number of strains of the same pulsotype and pattern of amino acid substitutions. Reduced susceptibility to β-lactam, brought about by mutations of the
ftsI
gene, is becoming a frequent phenomenon, affecting both strains that produce β-lactamase and those that do not. The level of resistance remains low but opens the way to greater resistance in the future.
Alterations in Penicillin-Binding Protein 1A Confer Resistance to β-Lactam Antibiotics in
Helicobacter pylori
Authors: Gerrits M. M. , Schuijffel D. , van Zwet A. A. , Kuipers E. J. , Vandenbroucke-Grauls C. M. J. E. , Kusters J. G.
Published: July 2002

Most
Helicobacter pylori
strains are susceptible to amoxicillin, an important component of combination therapies for
H. pylori
eradication. The isolation and initial characterization of the first reported stable amoxicillin-resistant clinical
H. pylori
isolate (the Hardenberg strain) have been published previously, but the underlying resistance mechanism was not described. Here we present evidence that the β-lactam resistance of the Hardenberg strain results from a single amino acid substitution in HP0597, a penicillin-binding protein 1A (PBP1A) homolog of
Escherichia coli
. Replacement of the wild-type HP0597 (
pbp1A
) gene of the amoxicillin-sensitive (Amx
s
)
H. pylori
strain 1061 by the Hardenberg
pbp1A
gene resulted in a 100-fold increase in the MIC of amoxicillin. Sequence analysis of
pbp1A
of the Hardenberg strain, the Amx
s
H. pylori
strain 1061, and four amoxicillin-resistant (Amx
r
) 1061 transformants revealed a few amino acid substitutions, of which only a single Ser
414
→Arg substitution was involved in amoxicillin resistance. Although we cannot exclude that mutations in other genes are required for high-level amoxicillin resistance of the Hardenberg strain, this amino acid substitution in PBP1A resulted in an increased MIC of amoxicillin that was almost identical to that for the original Hardenberg strain.
Pressure of Zidovudine Accelerates the Reversion of Lamivudine Resistance-Conferring M184V Mutation in the Reverse Transcriptase of Human Immunodeficiency Virus Type 1
Authors: Diallo Karidia , Oliveira Maureen , Moisi Daniela , Brenner Bluma , Wainberg Mark A. , Götte Matthias
Published: July 2002

We cultured lamivudine-resistant human immunodeficiency virus type 1 (HIV-1) variants over an extended period of time in the presence of zidovudine and observed a premature reversion of the resistance-conferring M184V mutation. These data suggest that the presence of ZDV amplifies differences in replication capacity between wild-type HIV-1 and the mutant variant.
Antibiotic Susceptibilities of 96 Isolates of
Bacillus anthracis
Isolated in France between 1994 and 2000
Authors: Cavallo Jean-Didier , Ramisse Francoise , Girardet Monique , Vaissaire Josée , Mock Michelle , Hernandez Eric
Published: July 2002

Ninety-six isolates of
Bacillus anthracis
recovered in France between 1994 and 2000 were tested for their susceptibilities to 25 different antibiotics. Resistance to penicillin G and amoxicillin was 11.5%. All of the isolates were resistant to cotrimoxazole and susceptible to doxycycline, ciprofloxacin, pefloxacin, levofloxacin, teicoplanin, vancomycin, clindamycin, imipenem, and rifampin.
Alkoxyalkyl Esters of Cidofovir and Cyclic Cidofovir Exhibit Multiple-Log Enhancement of Antiviral Activity against Cytomegalovirus and Herpesvirus Replication In Vitro
Authors: Beadle James R. , Hartline Caroll , Aldern Kathy A. , Rodriguez Natalie , Harden Emma , Kern Earl R. , Hostetler Karl Y.
Published: July 2002

The incidence of cytomegalovirus (CMV) retinitis is declining in AIDS patients but remains a significant clinical problem in patients with organ transplants and bone marrow transplants. Prophylaxis with ganciclovir (GCV) or valganciclovir reduces the incidence of CMV disease but may lead to the emergence of drug-resistant virus with mutations in the UL97 or UL54 gene. It would be useful to have other types of oral therapy for CMV disease. We synthesized hexadecyloxypropyl and octadecyloxyethyl derivatives of cyclic cidofovir (cCDV) and cidofovir (CDV) and found that these novel analogs had 2.5- to 4-log increases in antiviral activity against CMV compared to the activities of unmodified CDV and cCDV. Multiple-log increases in activity were noted against laboratory CMV strains and various CMV clinical isolates including GCV-resistant strains with mutations in the UL97 and UL54 genes. Preliminary cell studies suggest that the increase in antiviral activity may be partially explained by a much greater cell penetration of the novel analogs. 1-
O
-Hexadecyloxypropyl-CDV, 1-
O
-octadecyloxyethyl-CDV, and their corresponding cCDV analogs are worthy of further preclinical evaluation for treatment and prevention of CMV and herpes simplex virus infections in humans.
Methods for Data Mining from Large Multinational Surveillance Studies
Authors: Poupard James , Brown James , Gagnon Robert , Stanhope Michael J. , Stewart Chad
Published: July 2002

Traditionally, large surveillance studies have been analyzed by the use of the MICs at which 90% of isolates tested are inhibited (MIC
90
s), MIC
50
s, frequency distributions, and percent susceptibility. In the past, these approaches have proved satisfactory for the monitoring of resistance. From these traditional uses, one can readily detect an increase in MICs for organism and drug combinations. Now that large surveillance studies have been conducted for a number of years and databases have grown to include a large number of datum points, new approaches to the extraction of useful information from these studies are needed. The present study proposes approaches, including the use of antibiotypes, principal components analysis, phylogenetics, and population genetic analysis, to the evaluation of data from large multinational surveillance studies. Application of these types of analyses can be used to describe genetic diversity, analyze changes in susceptibility patterns over time, and possibly, shed light on the origins and evolution of antimicrobial resistance. As global surveillance studies become more common and new questions concerning the evolution of resistance are raised, innovative approaches to analysis of the data will increase in importance.
Efficacy of Intravenous Liposomal Amphotericin B (AmBisome) against Coccidioidal Meningitis in Rabbits
Authors: Clemons Karl V. , Sobel Raymond A. , Williams Paul L. , Pappagianis Demosthenes , Stevens David A.
Published: July 2002

The efficacy of intravenously administered liposomal amphotericin B (AmBisome [AmBi]) for the treatment of experimental coccidioidal meningitis was compared with those of oral fluconazole (FLC) and intravenously administered conventional amphotericin B (AMB). Male New Zealand White rabbits were infected by intracisternal inoculation of arthroconidia of
Coccidioides immitis
. Starting 5 days postinfection, animals received one of the following: 5% dextrose water diluent; AMB given at 1 mg/kg of body weight; AmBi given at 7.5, 15, or 22.5 mg/kg intravenously three times per week for 3 weeks; or oral FLC given at 80 mg/kg for 19 days. One week after the cessation of therapy, all survivors were euthanatized, the numbers of CFU remaining in the spinal cord and brain were determined, and histological analyses were performed. All AmBi-, FLC-, or AMB-treated animals survived and had prolonged lengths of survival compared with those for the controls (
P
< 0.0001). Treated groups had significantly lower numbers of white blood cells and significantly lower protein concentrations in the cerebrospinal fluid compared with those for the controls (
P
< 0.01 to 0.0005) and had fewer clinical signs of infection (e.g., weight loss, elevated temperature, and neurological abnormalities including motor abnormalities). The mean histological scores for AmBi-treated rabbits were lower than those for FLC-treated and control rabbits (
P
< 0.016 and 0.0005, respectively); the scores for AMB-treated animals were lower than those for the controls (
P
< 0.0005) but were similar to those for FLC-treated rabbits. All regimens reduced the numbers of CFU in the brain and spinal cord compared with those for the controls (
P
≤0.0005). AmBi-treated animals had 3- to 11-fold lower numbers of CFU than FLC-treated rabbits and 6- to 35-fold lower numbers of CFU than AmB-treated rabbits. Three of eight animals given 15 mg of AmBi per kg had no detectable infection in either tissue, whereas other doses of AmBi or FLC cleared either the brain or the spinal cord of infection in fewer rabbits. In addition, clearance of the infection from both tissues was achieved in none of the rabbits, and neither tissue was cleared of infection in AMB-treated animals. Overall, these data indicate that intravenously administered AmBi is superior to oral FLC or intravenous AMB and that FLC is better than AMB against experimental coccidioidal meningitis. These data indicate that AmBi may offer an improvement in the treatment of coccidioidal meningitis. Additional studies are warranted.
Tetracycline Rapidly Reaches All the Constituent Cells of Uropathogenic
Escherichia coli
Biofilms
Authors: Stone G. , Wood P. , Dixon L. , Keyhan M. , Matin A.
Published: July 2002

We have developed a method for visualizing
Escherichia coli
cells that are exposed to tetracycline in a biofilm, based on a previous report that liposomes containing the
E. coli
TetR(B) protein fluoresce when exposed to this antibiotic. By our method, cells devoid of TetR(B) also exhibited tetracycline-dependent fluorescence. At 50 μg of tetracycline ml
−1
, planktonic cells of a uropathogenic
E. coli
(UPEC) strain developed maximal fluorescence after 7.5 to 10 min of exposure. A similar behavior was exhibited by cells in a 24- or 48-h UPEC biofilm, as examined by confocal laser microscopy, regardless of whether they lined empty spaces or occupied densely packed regions. Further, a comparison of phase-contrast and fluorescent images of corresponding biofilm zones showed that all the cells fluoresced. Thus, all the biofilm cells were exposed to tetracycline and there were no pockets within the biofilm where the antibiotic failed to reach. It also appeared unlikely that niches of reduced exposure to the antibiotic existed within the biofilms.
Pharmacokinetics and Safety of Voriconazole following Intravenous- to Oral-Dose Escalation Regimens
Authors: Purkins L. , Wood N. , Ghahramani P. , Greenhalgh K. , Allen M. J. , Kleinermans D.
Published: July 2002

In this study, the safety, tolerability, and pharmacokinetics of intravenous (i.v.)- to oral-dose regimens of voriconazole were evaluated with a group of 42 healthy men, 41 of whom completed the study. Two cohorts of subjects participated in the study. Cohort 1 (
n
= 28) took part in two study periods, each consisting of 14 days separated by a minimum 7-day washout. In one of the periods, 14 subjects received 6 mg/kg i.v. twice a day (b.i.d.) on day 1 followed by 3 mg/kg i.v. b.i.d. on days 2 to 7 and were then switched to 200 mg orally b.i.d. for days 8 to 14. In the other period, subjects received 6 mg/kg i.v. b.i.d. on day 1 followed by 5 mg/kg i.v. b.i.d. on days 2 to 7and were then switched to 400 mg orally b.i.d. for days 8 to 14. The remaining 14 subjects in cohort 1 received a matching placebo throughout the study. In cohort 2 (
n
= 14), 7 subjects received 6 mg/kg i.v. b.i.d. on day 1 followed by 4 mg/kg i.v. b.i.d. on days 2 to 7 and were then switched to 300 mg orally b.i.d. for days 8 to 14. The remaining seven subjects in cohort 2 received a matching placebo. Blood samples were taken prior to dosing on days 1 to 6 and on days 8 to 13. Blood samples were drawn prior to dosing and at frequent intervals up to 12 h following the morning dose on days 7 and 14 of each study period. The samples were assayed for voriconazole by a high-performance liquid chromatography method. The maximum concentration in plasma (
C
max
) occurred at the end of the 1-h i.v. infusion and between 1.4 and 1.8 h after oral administration. Voriconazole exhibited nonlinear pharmacokinetics, possibly due to saturable metabolism. For cohort 1, both
C
max
and the area under the concentration-time curve within a dosage interval (AUC
τ
) increased disproportionately with dose for both i.v. and oral dosing. For i.v. dosing, a 1.7-fold increase in dose resulted in 2.4- and 3.1-fold increases in
C
max
and AUC
τ
, respectively. Similarly, a 2-fold increase in oral dosing resulted in 2.8- and 3.9-fold increases in
C
max
and AUC
τ
, respectively. The mean values for
C
max
observed following oral dosing were lower than those obtained after i.v. administration, ranging from 62.7 to 89.6% of the i.v. value. After the switch from i.v. to oral dosing, most subjects achieved steady state by day 4, and mean minimum concentrations in plasma remained above clinically important MICs. The pharmacokinetic profiles for saliva followed a pattern similar to those observed for plasma; there was a highly significant correlation between plasma and saliva voriconazole concentrations (
P
< 0.0001). Voriconazole was well tolerated; the most commonly reported adverse events in voriconazole-treated subjects were mild to moderate headache, rash, and abnormal vision. Visual function tests detected no further abnormalities during voriconazole treatment.
Transport of an Antifungal Trypsin Inhibitor Isolated from Corn across the Blood-Brain Barrier
Authors: Banks William A. , Niehoff Michael L. , Brown Robert L. , Chen Zhi-Yuan , Cleveland Thomas E.
Published: July 2002

We determined whether an antifungal 14-kDa protein trypsin inhibitor isolated from corn is able to cross the blood-brain barrier. We found that it completely crossed the blood-brain barrier by means of a saturable mechanism at a rate of 0.153 μl/g · min, with about 0.082% of the intravenously injected dose being taken up per gram of brain.
Induction of Apoptosis by a Nonnucleoside Human Immunodeficiency Virus Type 1 Reverse Transcriptase Inhibitor
Authors: Pilon A. A. , Lum J. J. , Sanchez-Dardon J. , Phenix B. N. , Douglas R. , Badley A. D.
Published: July 2002

Inhibition of human immunodeficiency virus type 1 reverse transcriptase (RT) by both nucleoside and nonnucleoside RT inhibitors profoundly inhibits virus replication. Nucleoside RT inhibitors are known to be toxic, but there is little information regarding the toxicities of nonnucleoside RT inhibitors (NNRTI). We demonstrate that efavirenz (an NNRTI) induces caspase- and mitochondrion-dependent apoptosis of Jurkat T cells and human peripheral blood mononuclear cells. The clinical relevance of these observations is not yet clear.
Two Novel Vaginal Microbicides (Polystyrene Sulfonate and Cellulose Sulfate) InhibitGardnerella vaginalisand Anaerobes Commonly Associated with Bacterial Vaginosis
Authors: Simoes Jose A. , Citron Diane M. , Aroutcheva Alla , Anderson Robert A. , Chany Calvin J. , Waller Donald P. , Faro Sebastian , Zaneveld Lourens J. D.
Published: July 2002

This is the first report demonstrating the in vitro inhibitory activity of two novel microbicides (cellulose sulfate and polystyrene sulfonate) against bacterial vaginosis (BV)-associated bacteria. Vaginal application of these microbicides not only may reduce the risk of acquisition of human immunodeficiency virus and other sexually transmitted infection-causing organisms but may also decrease the incidence of BV.
In Vitro Susceptibilities of Zygomycetes to Combinations of Antimicrobial Agents
Authors: Dannaoui Eric , Afeltra Javier , Meis Jacques F. G. M. , Verweij Paul E.
Published: July 2002

Combinations of antimicrobial agents were tested against 35 strains of zygomycetes. The interaction between amphotericin B and rifampin was synergistic or additive. Flucytosine alone was inactive and, upon combination with amphotericin B, synergy was not achieved. The combination of amphotericin B with terbinafine was synergistic for 20% of strains, and the interaction between terbinafine and voriconazole was synergistic for 44% of strains. Antagonism was not observed.
Hematologic Effects of Linezolid: Summary of Clinical Experience
Authors: Gerson Stanton L. , Kaplan Sheldon L. , Bruss Jon B. , Le Vu , Arellano Felix M. , Hafkin Barry , Kuter David J.
Published: July 2002

Linezolid has been associated with reversible myelosuppression. Clinical trial data were evaluated for anemia, thrombocytopenia, and neutropenia. Thrombocytopenia and a slight increased risk for anemia were evident at ≥2 weeks of linezolid treatment. Hematologic abnormalities were consistent with mild, reversible, duration-dependent myelosuppression. Appropriate monitoring is warranted with linezolid use.
The Genetic Properties of the Primary Endosymbionts of Mealybugs Differ from Those of Other Endosymbionts of Plant Sap-Sucking Insects
Authors: Baumann Linda , Thao MyLo Ly , Hess Justin M. , Johnson Marshall W. , Baumann Paul
Published: July 2002

Mealybugs (Hemiptera, Coccoidea, Pseudococcidae), like aphids and psyllids, are plant sap-sucking insects that have an obligate association with prokaryotic endosymbionts that are acquired through vertical, maternal transmission. We sequenced two fragments of the genome of
Tremblaya princeps,
the endosymbiont of mealybugs, which is a member of the β subdivision of the
Proteobacteria
. Each of the fragments (35 and 30 kb) contains a copy of 16S-23S-5S rRNA genes. A total of 37 open reading frames were detected, which corresponded to putative rRNA proteins, chaperones, and enzymes of branched-chain amino acid biosynthesis, DNA replication, protein translation, and RNA synthesis. The genome of
T. princeps
has a number of properties that distinguish it from the genomes of
Buchnera aphidicola
and
Carsonella ruddii,
the endosymbionts of aphids and psyllids, respectively. Among these properties are a high G+C content (57.1 mol%), the same G+C content in intergenic spaces and structural genes, and similar G+C contents of the genes encoding highly and poorly conserved proteins. The high G+C content has a substantial effect on protein composition; about one-third of the residues consist of four amino acids with high-G+C-content codons. Sequence analysis of DNA fragments containing the rRNA operon and adjacent regions from endosymbionts of several mealybug species suggested that there was a single duplication of the rRNA operon and the adjacent genes in an ancestor of the present
T. princeps.
Subsequently, in one mealybug lineage
rpS15,
one of the duplicated genes, was retained, while in another lineage it decayed. These results extend the diversity of the types of endosymbiotic associations found in plant sap-sucking insects.
Parallel Characterization of Anaerobic Toluene- and Ethylbenzene-Degrading Microbial Consortia by PCR-Denaturing Gradient Gel Electrophoresis, RNA-DNA Membrane Hybridization, and DNA Microarray Technology
Authors: Koizumi Yoshikazu , Kelly John J. , Nakagawa Tatsunori , Urakawa Hidetoshi , El-Fantroussi Saïd , Al-Muzaini Saleh , Fukui Manabu , Urushigawa Yoshikuni , Stahl David A.
Published: July 2002

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family
Desulfobacteriaceae
. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.
Molecular Characterization of the S-Layer Gene,sbpA, ofBacillus sphaericusCCM 2177 and Production of a Functional S-Layer Fusion Protein with the Ability To Recrystallize in a Defined Orientation while Presenting the Fused Allergen
Authors: Ilk Nicola , Völlenkle Christine , Egelseer Eva M. , Breitwieser Andreas , Sleytr Uwe B. , Sára Margit
Published: July 2002

The nucleotide sequence encoding the crystalline bacterial cell surface (S-layer) protein SbpA ofBacillus sphaericusCCM 2177 was determined by a PCR-based technique using four overlapping fragments. The entiresbpAsequence indicated one open reading frame of 3,804 bp encoding a protein of 1,268 amino acids with a theoretical molecular mass of 132,062 Da and a calculated isoelectric point of 4.69. The N-terminal part of SbpA, which is involved in anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, comprises three S-layer-homologous motifs. For screening of amino acid positions located on the outer surface of the square S-layer lattice, the sequence encodingStrep-tag I, showing affinity to streptavidin, was linked to the 5′ end of the sequence encoding the recombinant S-layer protein (rSbpA) or a C-terminally truncated form (rSbpA31-1068). The deletion of 200 C-terminal amino acids did not interfere with the self-assembly properties of the S-layer protein but significantly increased the accessibility ofStrep-tag I. Thus, the sequence encoding the major birch pollen allergen (Bet v1) was fused via a short linker to the sequence encoding the C-terminally truncated form rSpbA31-1068. Labeling of the square S-layer lattice formed by recrystallization of rSbpA31-1068/Bet v1 on peptidoglycan-containing sacculi with a Bet v1-specific monoclonal mouse antibody demonstrated the functionality of the fused protein sequence and its location on the outer surface of the S-layer lattice. The specific interactions between the N-terminal part of SbpA and the secondary cell wall polymer will be exploited for an oriented binding of the S-layer fusion protein on solid supports to generate regularly structured functional protein lattices.
Monocyclic Aromatic Hydrocarbon Degradation by
Rhodococcus
sp. Strain DK17
Authors: Kim Dockyu , Kim Young-Soo , Kim Seong-Ki , Kim Si Wouk , Zylstra Gerben J. , Kim Young Min , Kim Eungbin
Published: July 2002

Rhodococcus
sp. strain DK17 was isolated from soil and analyzed for the ability to grow on
o-
xylene as the sole carbon and energy source. Although DK17 cannot grow on
m
- and
p
-xylene, it is capable of growth on benzene, phenol, toluene, ethylbenzene, isopropylbenzene, and other alkylbenzene isomers. One UV-generated mutant strain, DK176, simultaneously lost the ability to grow on
o
-xylene, ethylbenzene, isopropylbenzene, toluene, and benzene, although it could still grow on phenol. The mutant strain was also unable to oxidize indole to indigo following growth in the presence of
o
-xylene. This observation suggests the loss of an oxygenase that is involved in the initial oxidation of the (alkyl)benzenes tested. Another mutant strain, DK180, isolated for the inability to grow on
o
-xylene, retained the ability to grow on benzene but was unable to grow on alkylbenzenes due to loss of a
meta
-cleavage dioxygenase needed for metabolism of methyl-substituted catechols. Further experiments showed that DK180 as well as the wild-type strain DK17 have an
ortho
-cleavage pathway which is specifically induced by benzene but not by
o
-xylene. These results indicate that DK17 possesses two different ring-cleavage pathways for the degradation of aromatic compounds, although the initial oxidation reactions may be catalyzed by a common oxygenase. Gas chromatography-mass spectrometry and 300-MHz proton nuclear magnetic resonance spectrometry clearly show that DK180 accumulates 3,4-dimethylcatechol from
o
-xylene and both 3- and 4-methylcatechol from toluene. This means that there are two initial routes of oxidation of toluene by the strain. Pulsed-field gel electrophoresis analysis demonstrated the presence of two large megaplasmids in the wild-type strain DK17, one of which (pDK2) was lost in the mutant strain DK176. Since several other independently derived mutant strains unable to grow on alkylbenzenes are also missing pDK2, the genes encoding the initial steps in alkylbenzene metabolism (but not phenol metabolism) appear to be present on this approximately 330-kb plasmid.
Heterologous Expression of the
Lactococcus lactis
Bacteriocin, Nisin, in a Dairy
Enterococcus
Strain
Authors: Li Haiping , O'Sullivan Daniel J.
Published: July 2002

The bacteriocin nisin is produced only by some strains of
Lactococcus lactis
, and to date production in other lactic acid bacteria has not been achieved.
Enterococcus
sp. strain N12β is a nisin-immune transconjugant obtained from a nisin-producing donor (
L. lactis
ATCC 11454) and a dairy recipient (
Enterococcus
sp. strain S12β), but it does not produce nisin. In this study, using PCR amplification, we confirmed that the whole nisin operon is likely present in
Enterococcus
sp. strain N12β. Northern hybridization of total RNA from strain N12β with a
nisA
probe and the results of reverse transcriptase PCR showed the lack of
nisA
transcription in this strain. However,
nisA
transcription was partially restored in strain N12β upon growth in the presence of exogenous nisin, and the
nisA
transcription signal was intensified after an increase in the external nisin level. Furthermore, bioassays showed that active nisin was produced in a dose-dependent fashion by strain N12β following induction by exogenous nisin. These results indicated that expression of the nisin genes in
Enterococcus
sp. strain N12β depended on autoinduction via signal transduction. However, the amount of external inducing signal required was significantly greater than the amount needed for autoinduction in
L. lactis
.
Improving the Pathogenicity of a Nematode-Trapping Fungus by Genetic Engineering of a Subtilisin with Nematotoxic Activity
Authors: Åhman Johan , Johansson Tomas , Olsson Maja , Punt Peter J. , van den Hondel Cees A. M. J. J. , Tunlid Anders
Published: July 2002

Nematophagous fungi are soil-living fungi that are used as biological control agents of plant and animal parasitic nematodes. Their potential could be improved by genetic engineering, but the lack of information about the molecular background of the infection has precluded this development. In this paper we report that a subtilisin-like extracellular serine protease designated
PII
is an important pathogenicity factor in the common nematode-trapping fungus
Arthrobotrys oligospora
. The transcript of
PII
was not detected during the early stages of infection (adhesion and penetration), but high levels were expressed concurrent with the killing and colonization of the nematode. Disruption of the
PII
gene by homologous recombination had a limited effect on the pathogenicity of the fungus. However, mutants containing additional copies of the
PII
gene developed a higher number of infection structures and had an increased speed of capturing and killing nematodes compared to the wild type. The paralyzing activity of PII was verified by demonstrating that a heterologous-produced PII (in
Aspergillus niger
) had a nematotoxic activity when added to free-living nematodes. The toxic activity of PII was significantly higher than that of other commercially available serine proteases. This is the first report showing that genetic engineering can be used to improve the pathogenicity of a nematode-trapping fungus. In the future it should be possible to express recombinant subtilisins with nematicidal activity in other organisms that are present in the habitat of parasitic nematodes (e.g., host plant).
Degradation of Humic Acids by the Litter-Decomposing Basidiomycete
Collybia dryophila
Authors: Steffen Kari Timo , Hatakka Annele , Hofrichter Martin
Published: July 2002

The basidiomycete
Collybia dryophila
K209, which colonizes forest soil, was found to decompose a natural humic acid isolated from pine-forest litter (LHA) and a synthetic
14
C-labeled humic acid (
14
C-HA) prepared from [U-
14
C]catechol in liquid culture. Degradation resulted in the formation of polar, lower-molecular-mass fulvic acid (FA) and carbon dioxide. HA decomposition was considerably enhanced in the presence of Mn
2+
(200 μM), leading to 75% conversion of LHA and 50% mineralization of
14
C-HA (compared to 60% and 20%, respectively, in the absence of Mn
2+
). There was a strong indication that manganese peroxidase (MnP), the production of which was noticeably increased in Mn
2+
-supplemented cultures, was responsible for this effect. The enzyme was produced as a single protein with a pI of 4.7 and a molecular mass of 44 kDa. During solid-state cultivation,
C. dryophila
released substantial amounts of water-soluble FA (predominantly of 0.9 kDa molecular mass) from insoluble litter material. The results indicate that basidiomycetes such as
C. dryophila
which colonize forest litter and soil are involved in humus turnover by their recycling of high-molecular-mass humic substances. Extracellular MnP seems to be a key enzyme in the conversion process.
Bovine Enteroviruses as Indicators of Fecal Contamination
Authors: Ley Victoria , Higgins James , Fayer Ronald
Published: July 2002

Surface waters frequently have been contaminated with human enteric viruses, and it is likely that animal enteric viruses have contaminated surface waters also. Bovine enteroviruses (BEV), found in cattle worldwide, usually cause asymptomatic infections and are excreted in the feces of infected animals in large numbers. In this study, the prevalence and genotype of BEV in a closed herd of cattle were evaluated and compared with BEV found in animals in the immediate environment and in environmental specimens. BEV was found in feces from 76% of cattle, 38% of white-tailed deer, and one of three Canada geese sharing the same pastures, as well as the water obtained from animal watering tanks, from the pasture, from streams running from the pasture to an adjacent river, and from the river, which emptied into the Chesapeake Bay. Furthermore, BEV was found in oysters collected from that river downstream from the farm. These findings suggest that BEV could be used as an indicator of fecal pollution originating from animals (cattle and/or deer). Partial sequence analysis of the viral genomes indicates that different viral variants coexist in the same area. The possibility of identifying the viral strains found in the animals and in the contaminated areas by sequencing the RNA genome, could provide a tool to find the origin of the contamination and should be useful for epidemiological and viral molecular evolution studies.
Identification of Proteins Involved in the Heat Stress Response of
Bacillus cereus
ATCC 14579
Authors: Periago Paula M. , van Schaik Willem , Abee Tjakko , Wouters Jeroen A.
Published: July 2002

To monitor the ability of the food-borne opportunistic pathogen
Bacillus cereus
to survive during minimal processing of food products, we determined its heat-adaptive response. During pre-exposure to 42°C,
B. cereus
ATCC 14579 adapts to heat exposure at the lethal temperature of 50°C (maximum protection occurs after 15 min to 1 h of pre-exposure to 42°C). For this heat-adaptive response, de novo protein synthesis is required. By using two-dimensional gel electrophoresis, we observed 31 heat-induced proteins, and we determined the N-terminal sequences of a subset of these proteins. This revealed induction of stress proteins (CspB, CspE, and SodA), proteins involved in sporulation (SpoVG and AldA), metabolic enzymes (FolD and Dra), identified heat-induced proteins in related organisms (DnaK, GroEL, ClpP, RsbV, HSP16.4, YflT, PpiB, and TrxA), and other proteins (MreB, YloH, and YbbT). The upregulation of several stress proteins was confirmed by using antibodies specific for well-characterized heat shock proteins (HSPs) of
B. subtilis
. These observations indicate that heat adaptation of
B. cereus
involves proteins that function in a variety of cellular processes. Notably, a 30-min pre-exposure to 4% ethanol, pH 5, or 2.5% NaCl also results in increased thermotolerance. Also, for these adaptation processes, protein synthesis is required, and indeed, some HSPs are induced under these conditions. Collectively, these data show that during mild processing, cross-protection from heating occurs in pathogenic
B. cereus
, which may result in increased survival in foods.
Laccase-Catalyzed Oxidation of Mn
2+
in the Presence of Natural Mn
3+
Chelators as a Novel Source of Extracellular H
2
O
2
Production and Its Impact on Manganese Peroxidase
Authors: Schlosser Dietmar , Höfer Christine
Published: July 2002

A purified and electrophoretically homogeneous blue laccase from the litter-decaying basidiomycete
Stropharia rugosoannulata
with a molecular mass of approximately 66 kDa oxidized Mn
2+
to Mn
3+
, as assessed in the presence of the Mn chelators oxalate, malonate, and pyrophosphate. At rate-saturating concentrations (100 mM) of these chelators and at pH 5.0, Mn
3+
complexes were produced at 0.15, 0.05, and 0.10 μmol/min/mg of protein, respectively. Concomitantly, application of oxalate and malonate, but not pyrophosphate, led to H
2
O
2
formation and tetranitromethane (TNM) reduction indicative for the presence of superoxide anion radical. Employing oxalate, H
2
O
2
production, and TNM reduction significantly exceeded those found for malonate. Evidence is provided that, in the presence of oxalate or malonate, laccase reactions involve enzyme-catalyzed Mn
2+
oxidation and abiotic decomposition of these organic chelators by the resulting Mn
3+
, which leads to formation of superoxide and its subsequent reduction to H
2
O
2
. A partially purified manganese peroxidase (MnP) from the same organism did not produce Mn
3+
complexes in assays containing 1 mM Mn
2+
and 100 mM oxalate or malonate, but omitting an additional H
2
O
2
source. However, addition of laccase initiated MnP reactions. The results are in support of a physiological role of laccase-catalyzed Mn
2+
oxidation in providing H
2
O
2
for extracellular oxidation reactions and demonstrate a novel type of laccase-MnP cooperation relevant to biodegradation of lignin and xenobiotics.
Purification and Characterization of an Extracellular Protease from
Penicillium chrysogenum
Pg222 Active against Meat Proteins
Authors: Benito María J. , Rodríguez Mar , Núñez Félix , Asensio Miguel A. , Bermúdez María E. , Córdoba Juan J.
Published: July 2002

An extracellular protease from
Penicillium chrysogenum
(Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60°C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45°C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and
Aspergillus oryzae
protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from
Penicillium citrinum
. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.
Silencing of the Aspergillopepsin B (
pepB
) Gene of
Aspergillus awamori
by Antisense RNA Expression or Protease Removal by Gene Disruption Results in a Large Increase in Thaumatin Production
Authors: Moralejo Francisco J. , Cardoza Rosa Elena , Gutierrez Santiago , Lombraña Marta , Fierro Francisco , Martín Juan F.
Published: July 2002

Aspergillopepsin B was identified in culture broths of
Aspergillus awamori
by in situ detection of its proteolytic activity and by immunodetection with anti-aspergillopepsin B antibodies. Severe thaumatin degradation was observed after in vitro treatment of thaumatin with purified aspergillopepsin B. The
pepB
gene encoding aspergillopepsin B of
A. awamori
was cloned and characterized. It is located in chromosome IV of
A. awamori
, as shown by pulsed-field gel electrophoresis, and encodes a protein of 282 amino acids with high similarity to the aspergillopepsin B of
Aspergillus niger
var.
macrosporus
. The
pepB
gene is expressed at high rates as a monocistronic 1.0-kb transcript in media with casein at acidic pH values. An antisense cassette constructed by inserting the
pepB
gene in the antisense orientation downstream from the
gpdA
promoter resulted in a good level of antisense mRNA, as shown by reverse transcription-PCR. Partial silencing of the
pepB
gene by the antisense mRNA resulted in a 31% increase in thaumatin yield. However, significant residual degradation of thaumatin still occurred. To completely remove aspergillopepsin B, the
pepB
gene was deleted by double crossover. Two of the selected transformants lacked the endogenous
pepB
gene and did not form aspergillopepsin B. Thaumatin yields increased by between 45% in transformant APB 7/25 and 125% in transformant 7/36 with respect to the parental strain. Reduction of proteolytic degradation by gene silencing with antisense mRNA or total removal of the aspergillopepsin B by directed gene deletion was a very useful method for improving thaumatin production in
A. awamori
.
New Chromogenic Agar Medium for the Identification ofCandidaspp
Authors: Cooke Venitia M. , Miles R. J. , Price R. G. , Midgley G. , Khamri W. , Richardson A. C.
Published: July 2002

A new chromogenic agar medium (Candidadiagnostic agar [CDA]) for differentiation ofCandidaspp. is described. This medium is based on Sabouraud dextrose agar (Oxoid CM41) and contains (per liter) 40.0 g of glucose, 10.0 g of mycological peptone, and 15.0 g of agar along with a novel chromogenic glucosaminidase substrate, ammonium 4-{2-[4-(2-acetamido-2-deoxy-β-d-glucopyranosyloxy)-3-methoxyphenyl]-vinyl}-1-(propan-3-yl-oate)-quinolium bromide (0.32 g liter−1). The glucosaminidase substrate in CDA was hydrolyzed byCandida albicansandCandida dubliniensis, yielding white colonies with deep-red spots on a yellow transparent background after 24 to 48 h of incubation at 37°C. Colonies ofCandida tropicalisandCandida kefyrwere uniformly pink, and colonies of otherCandidaspp., includingCandida glabrataandCandida parapsilosis, were white. CDA was evaluated by using 115 test strains ofCandidaspp. and other clinically important yeasts and was compared with two commercially available chromogenic agars (Candida ID agar [bioMerieux] and CHROMagar Candida [CHROMagar Company Ltd.]). On all three agars, colonies ofC. albicanswere not distinguished from colonies ofC. dubliniensis. However, for the group containingC. albicansplusC. dubliniensis, both the sensitivity and the specificity of detection when CDA was used were 100%, compared with values of 97.6 and 100%, respectively, with CHROMagar Candida and 100 and 96.8%, respectively, with Candida ID agar. In addition, for the group containingC. tropicalisplusC. kefyr, the sensitivity and specificity of detection when CDA was used were also 100%, compared with 72.7 and 98.1%, respectively, with CHROMagar Candida. Candida ID agar did not differentiateC. tropicalisandC. kefyrstrains but did differentiate members of a broader group (C. tropicalis,C. kefyr,Candida lusitaniaeplusCandida guilliermondii); the sensitivity and specificity of detection for members of this group were 94.7 and 93.8%, respectively. In addition to the increased sensitivity and/or specificity ofCandidadetection when CDA was used, differentiation of colony types on CDA (red spotted, pink, or no color) was unambiguous and did not require precise assessment of colony color.
Diagnosis of Visceral Leishmaniasis by Enzyme-Linked Immunosorbent Assay Using Urine Samples
Authors: Islam Mohammad Zahidul , Itoh Makoto , Shamsuzzaman S. M. , Mirza Rusella , Matin Farzana , Ahmed Iftikhar , Shamsuzzaman Choudhury A. K. M. , Hossain M. Akram , Qiu Xu-Guang , Begam Nilufar , Furuya Masato , Leafasia Judson L. , Hashiguchi Yoshihisa , Kimura Eisaku
Published: July 2002

A diagnostic method has been developed to detect anti-
Leishmania donovani
immunoglobulin G (IgG) in urine by enzyme-linked immunosorbent assay (ELISA). In measuring anti-
L. donovani
IgG, IgA, and IgM in urine, the method performed best in the detection of IgG. The sensitivity and specificity of the assay were determined with panels of urine samples from 62 visceral leishmaniasis (VL) patients, 59 healthy controls from areas of endemicity, 53 healthy controls from areas of nonendemicity, 59 malaria patients, 13 tuberculosis patients, 23 cutaneous leishmaniasis patients, and 7 patients with other diseases. Using
L. donovani
promastigote crude antigen, the test had 93.5% sensitivity (58 positives of 62 VL patient samples) and 89.3% specificity (191 negatives of 214 non-VL patient samples). The ELISA with acetone-treated
L. donovani
promastigote antigen raised the sensitivity and specificity to 95.0 and 95.3%, respectively. Western blot analysis revealed that most of the samples that cross-reacted with crude antigen in ELISA did not recognize any antigenic component of
L. donovani
crude antigen. We also checked 40 serum samples from the same group of VL patients for anti-
L. donovani
IgG and got 90.0% sensitivity with both crude and acetone-treated antigens. As collection of urine is much easier than collection of serum, the detection of anti-
L. donovani
IgG in urine with acetone-treated antigen will be useful in epidemiological studies. It could be an adjunct of laboratory diagnosis.
Genetic Heterogeneity of Small Ruminant Lentiviruses Involves Immunodominant Epitope of Capsid Antigen and Affects Sensitivity of Single-Strain-Based Immunoassay
Authors: Grego Elena , Profiti Margherita , Giammarioli Monica , Giannino Laura , Rutili Domenico , Woodall Chris , Rosati Sergio
Published: July 2002

The
pol
and
gag
gene fragments of small ruminant lentivirus field isolates collected in the last decade in Italy were amplified, sequenced, and analyzed. Phylogenetic analysis revealed that the majority of ovine isolates form a distinct cluster more similar to caprine lentivirus prototypes than to the visna virus prototype. These findings confirm and extend those reported by Leroux et al. (Arch. Virol., 142:1125-1137, 1997). Moreover, we observed that a variable region of Gag, included in the fragment analyzed, corresponded to one of the three major capsid antigen epitopes, which suggests that the antibody response to this epitope may be type specific. To test this hypothesis, two recombinant peptides, derived from the Icelandic prototype K1514 and this novel genotype, were expressed and used in an enzyme-linked immunosorbent assay to screen a panel of ovine and caprine sera collected from different geographical locations in Italy. Several sera reacted in a type-specific manner, indicating that in a diagnostic setting the combination of at least these two type-specific peptides is necessary to cover a wide range of infections. Additionally, these results support the hypothesis of cross-species transmission based on the phylogenetic analysis described above. This has implications for the control and eradication of small ruminant lentivirus infections.
Identification, Molecular Cloning, and Evaluation of Potential Use of Isocitrate Dehydrogenase II of
Mycobacterium bovis
BCG in Serodiagnosis of Tuberculosis
Authors: Florio W. , Bottai D. , Batoni G. , Esin S. , Pardini M. , Maisetta G. , Campa M.
Published: July 2002

Diagnosis of tuberculosis is time-consuming and requires infrastructures which are often not available in countries with high incidences of the disease. In the present study, an 82-kDa protein antigen was isolated by affinity chromatography and was identified by peptide mass fingerprinting as isocitrate dehydrogenase II, which is encoded by the
icd2
gene of
Mycobacterium bovis
BCG. The
icd2
gene of BCG was cloned by PCR, and the product of recombinant gene expression was purified and analyzed by two-dimensional polyacrylamide gel electrophoresis. The recombinant protein, named rICD2, was tested for its recognition by immunoglobulin G (IgG) antibodies from the sera of 16 patients with tuberculosis (TB) and 23 healthy individuals by Western blotting. The results showed that rICD2 is recognized by IgG antibodies from the sera of all TB patients tested at serum dilutions of ≥1:640. At a serum dilution of 1:1,280, the sensitivity was 50% and the specificity was 86.9%. These results indicate that rICD2 might represent a candidate for use in a new assay for the serodiagnosis of TB.